Second International Symposium on Development of the EntericNervous System: Cells, Signals and Genes
UCL Institute of Child Health
London, UK
22–25 February 2009
These abstracts are published online as part of the February 2009 issue. Please visitwww3.interscience.wiley.com/journal/118498177/home to search and read the abstracts.
� 2009 Blackwell Publishing Ltd 223
Abstracts
Gene regulatory network underlying neural crest formation
m. bronner-fraser
Division of Biology, California Institute of Technology, Pasadena, CA, USA
The neural crest is a population of multipotent, migratory progenitor
cells that forms at the border of neural and non-neural ectoderm in
vertebrate embryos. These cells then migrate from the neural tube
along defined pathways, populate numerous sites and differentiate into
diverse cells types including melanocytes, sensory and autonomic
neurons, and mineralized matrices like bone and dentine. Data com-
piled from Xenopus, zebrafish, mouse and chick, suggest that a net-
work of interacting transcriptional regulators and downstream effector
genes confer properties like multipotency and migratory capacity to
nascent neural crest cells. These regulatory interactions can be divided
into distinct phases. The first involves inductive signals (e.g. Wnt,
BMP, FGF) that establish the neural plate border, by up-regulation of
border specifier genes like Msx1/2, Pax3/7, and Zic. These border genes
in turn up-regulate neural crest specifier genes like Slug/Snail, FoxD3
and the SoxE family. Finally, the neural crest specifiers turn on specific
downstream targets that render the neural crest migratory and multi-
potent. We are testing linkages in this hypothetical neural crest gene
regulatory network in chick and lamprey by systematically perturbing
a subset of the transcription factors involved in early neural crest
specification and examining the effect of these perturbations on likely
downstream genes in order to test the predicted interrelationships. In
addition, we are isolating cis-regulatory regions of genes in this puta-
tive neural crest regulatory network to identify neural crest enhancers,
determine additional inputs to the network and determine which
interactions are direct. The results suggest that a series of gene regu-
latory circuits are involved in the production of migratory neural crest
cells in the early vertebrate embryos and that many of these events
may be conserved to the base of vertebrates.
Characterization of the Stathmin SCG10 and its interactions with
Kinesin Binding Protein (KBP) in zebrafish
gm burzynski,* m alves,� e de graaf,� c hoogenraad,� bjl eggen,§
a brooks,� rmw hofstra� & i shepherd**Emory University, Altanta, GA, USA; �University Medical Center, Groningen,
the Netherlands; �Erasmus Medical Center, Rotterdam, the Netherlands; and
§University of Groningen, Groningen, the Netherlands
SCG10 (STMN2) is believed to be neuronal-specific stathmin that is
enriched in the growth cones of developing neurons and has a role in
neurite outgrowth. In all species so far examined SCG10 is expressed
in both the CNS and PNS. Recently we have shown that SCG10
interacts with the KBP (KIAA1279) protein. Mutations in kbp have
been shown to be responsible for human Goldberg-Shprintzen
(GOSHS) syndrome. Patients with this rare autosomal recessive
disorder manifest microcephaly, mental retardation, polymicrogyria,
facial dysmorphisms and Hirschsprung disease. The precise function of
KBP in nervous system development is currently not known. To begin
to understand the function of SCG10 we have determined the temporal
and spatial expression pattern of the two zebrafish SCG10 orthologues,
SCG10a and SCG10b, by RT-PCR and in situ hybridization. RT-PCR
shows that both transcripts are maternally deposited and are detectable
from 0 hpf through 5 days – the latest age we examined. In situ
hybridization analysis reveals that the pattern of expression of these
two genes is dynamic and spatially restricted but is nearly identical for
both homologues. SCG10a and b are principally restricted to the CNS
from 24–48 hpf. From 48 hpf onwards expression of both genes
becomes restricted to the anterior CNS and cranial ganglia. This pat-
tern of SCG10 expression at 48 hpf resembles the pattern of KBP gene
expression, which is limited to the anterior CNS from 48 hpf onwards.
Subsequently, both SCG10 genes are expressed additionally in the
developing gut from 72 hpf. To investigate the in vivo function of the
zebrafish SCG10 orthologues and their interactions with KBP, we
generated morpholinos (MOs) for all three genes. Morphant embryos
have a severe phenotype at relatively low MO concentration. It is
likely that there is redundancy between the two SCG10 genes as
double SCG10 morphants have a more severe phenotype when com-
pared to single morphants. Significantly, triple morphants injected
with sub-threshold doses of SCG10 and KBP MOs display a severe
phenotype, strongly suggesting an epistatic interaction between the
products of these genes. Our data suggests that SCG10 and KBP genes
are required for proper differentiation of pharyngeal arches, migration
of ENS precursors and later differentiation stages of the cranial ganglia.
Function of the RNA binding protein HERMES in gastrointestinal
morphology and motility
p de santa barbara, c rouleau, l le guen & c notarnicola
INSERM ERI25, �Muscle and Pathologies�, Montpellier, France
The motility of the digestive tract is ensured by the correct coordina-
tion of the autonomous enteric nervous system (ENS) and the visceral
smooth muscle cells (SMC). The ENS originates from neural crest cells
that migrate from the dorsal region of the neural tube and colonize the
whole gut to establish its innervation. The SMCs derive from the
splanchnopleural mesoderm that will form the undifferentiated vis-
ceral mesenchyme before their final differentiation. Motility disorders
in infants comprise a wide group of heterogeneous diseases. Hirsch-
sprung disease (HSCR) is a particular case due to an absence of the ENS
along certain lengths of the bowel. The SMC differentiation is also
often affected in patients with congenital gut malformations and
motility disorders such as Chronic Intestinal Pseudoobstruction (CIP).
However, few have investigated the status of SMC in physiopatho-
logical conditions. Our aim was to investigate the molecular mecha-
nisms that control the differentiation of the visceral mesenchyme into
SMCs in vertebrates. In order to identify these candidate pathways, we
developed and analyzed the gene expression profiles of undifferentiated
and differentiated avian stomach by microarray. We identified one new
candidate HERMES, a RNA binding protein and examined the function
of this factor during the development and the differentiation of the
SMC structure by performing exhaustive in vivo and in primary SMC/
ENS culture positive and negative approaches. We found that HERMES
control different aspects of the SMC differentiation and deregulation of
its expression and function alter the development of both SMC and
ENS systems. In addition, we analyzed the expression of homologous
HERMES gene in human physiopathological conditions. All these data
demonstrate the necessity of correct coordination between the
development/differentiation of the SMC and ENS and the importance
to focus on both motility effectors.
Development of the mucosal plexus of the human enteric nervous
system
m metzger,* as wallace,* kh schaefer,� aj burns* & n thapar**UCL Institute of Child Health, London, UK; and �Department of Biotechnology,
University of Applied Sciences, Kaiserslautern, Germany
Introduction: Knowledge regarding the structure and development of
the enteric nervous system (ENS) is crucial for the understanding of
gut function and its disorders. It is widely accepted that the ENS is
composed of two major plexus (myenteric and submucosal plexus)
arranged as concentric rings in the bowel wall. There is, however,
accumulating evidence from both animal studies, and our recent work
in humans, that nerve cells also reside within the post-natal intestinal
mucosa (mucosal plexus). The aim of this study was to examine the
development-dependent formation of an intrinsic mucosal plexus of
Neurogastroenterol Motil (2009)
� 2009 Blackwell Publishing Ltd i
the ENS in human gut. Material and methods: Human embryonic and
fetal gut tissue was obtained from the joint Medical Research Council
and the Wellcome Trust-funded Human Developmental Biology
Resource, and from the Department of Obstetrics, Clinical Faculty
Mannheim, University of Heidelberg under ethical approval. Human
post-natal tissue was obtained from patients of Great Ormond Street
Hospital, London, UK under ethical approval and with fully informed
consent. Immediately after preparation all tissues were fixed in 4%
paraformaldeyhde and processed for immunohistochemical analysis
using antibodies specific for enteric neurons and glia. Results: In
order to analyse the organization of the ENS in the mucosal plexus,
serial sections from human large intestine were studied by anti-Hu
(Anna-1), TuJ1, Sox10 and S100 immunohistochemistry. In addition,
expression of enteric neuronal subtype markers were investigated (VIP,
NOS). In contrast to the myenteric and submucosal plexus no neural
cells were observed within the mucosa of all analysed fetal gut stages.
Neural cell bodies were first detected in the newborn period and with
increasingly more prominent staining of individual cell bodies and
interconnected processes as post-natal age increased. The mucosal
plexus of large intestine most commonly contained isolated neural
cells at different topographical levels within the lamina propria and
around the mucosal crypts. On occasion small aggregates of cells akin
to true ganglia were observed. Conclusions: This study shows that
the ENS is not restricted to the myenteric and submucosal plexus, but
also exists as a mucosal plexus in post-natal gut. The development and
precise functional role of this plexus in post-natal human gut is
unclear, but it is likely that neural cells within the mucosa play
distinct and important roles perhaps in epithelial cross-talk and local
reflex activity of the gut. The regulatory mechanisms and importance
in mucosal functions need to be elucidated in detail in future studies.
Therapeutic potential of human enteric nervous system stem cells
derived from endoscopic gut mucosal biopsies
m metzger, c caldwell, aj barlow, aj burns & n thapar
UCL Institute of Child Health, London, UK
Introduction: There is increasing evidence that enteric nervous
system (ENS) stem cells may provide potential therapeutic tools for
disorders characterized by aganglionosis or defective ENS function.
Although full thickness human post-natal gut tissue has been used
successfully to generate such stem cells, its harvesting from surgical
resection poses significant practical limitations. Recent literature and
our own work supports the presence of a third distinct plexus of the
ENS within the intestinal mucosa. Such mucosa is routinely biopsied
during gastrointestinal endoscopy and provides a potential regenerat-
ing source of intestinal tissue. Objectives: The aim of this study was
to explore whether gut tissue obtained utilising minimally invasive
routine endoscopy techniques could be used to generate ENS stem
cells, which retain the potential to generate an ENS upon transplan-
tation into aganglionic gut. Methods: Post-natal human gut mucosal
tissue obtained from children undergoing gastrointestinal endoscopy
was used to generate cell cultures containing neurosphere like bodies
(NLBs), which have been shown to contain ENS stem cells. NLBs
generated from such mucosal biopsies were characterized by immu-
nostaining using a panel of established neural progenitor (Nestin) and
neural crest progenitor (p75 and Sox10) markers. The potential of
individual cells derived from NLBs to generate bi-potential colonies
was tested in clonogenic assays. The ability to generate mature neu-
ronal subtypes was examined by in-vitro differentiation of dissociated
NLBs and following their transplantation into models of aganglionic
gut. Results: Gut mucosal biopsies were obtained from 70 children
(aged 9 months 17 years). Immunostaining of the mucosal biopsies
showed they contained neuronal cells and cells positive for markers of
ENS stem cells (p75 and Sox10). On culturing of dissociated mucosal
biopsies characteristic NLBs were generated at all ages examined.
Post-natal mucosa-derived NLBs contained cells that were proliferat-
ing (Ki67), expressed ENS stem cell markers (p75, Sox10, Nestin), and
were capable of generating colonies containing neurons and glia in
clonogenic assays, and multiple ENS neuronal subtypes (CGRP, NOS,
Ser, VIP and ChAT). Upon transplantation into cultured recipient
aganglionic chick and human hindgut, cells from NLBs colonized re-
cipient gut, generated ganglia-like structures and enteric neurons and
glia, including mature neuronal subtypes. Conclusions: The results
show that ENS stem cells can be harvested from routine mucosal
biopsies of post-natal human gut using standard endoscopic proce-
dures. This represents a significant practical advance towards the
development of definitive cell replenishment therapies for aganglionic
gut disorders like Hirschsprung’s disease, or those characterized by
deficient or defective enteric neuronal function.
Interactions between L1cam and Sox10 during enteric nervous system
development
rb anderson & as wallace
Department of Anatomy & Cell Biology, University of Melbourne, Melbourne
Via, Australia
Introduction: Hirschsprung’s disease is a congenital disorder in which
there is an absence of enteric ganglia within the distal portion of the
gastrointestinal tract. There is increasing evidence to suggest that the
phenotypic variability and incomplete penetrance of Hirschsprung’s
disease is due to complex interactions between genes known to result
in Hirschsprung’s disease, such as Sox10, and unidentified modifier
genes. Human clinical and animal model studies have suggested that
the X-linked gene, L1CAM, may act as a modifier gene for the devel-
opment of Hirschsprung’s disease. Objectives: To examine whether
L1cam could interact with the transcription factor,
Sox10. Methods: To identify genetic interactions between L1cam and
Sox10, we used a two-locus complementation approach by crossing
female L1cam+/- mice with male Sox10+/- mice. To assay the effects of
any interaction, we examined whether the migration of enteric neural
crest-derived cells is altered in L1cam null mutant mice when com-
bined with a heterozygous mutation in Sox10. Results: We have
identified a significant genetic interaction between L1cam and Sox10.
In all ages examined, L1cam-/y; Sox10+/- mice showed a significant
delay in the colonization of the gut by enteric neural crest-derived
cells. To decipher the cellular basis of the enteric nervous system
(ENS) defect, we examined the rate of cell proliferation and cell death
of enteric neural crest-derived cells in the gut of single and double
mutant mice. The ENS defect observed in L1cam-/y; Sox10 +/- mice
does not appear to be due to changes in proliferation or cell
death. Conclusions: The results of this study show that the X-linked
gene, L1cam, can function as a modifier gene for the development of
Hirschsprung’s disease, and that the interactions between L1cam and
Sox10 are important for normal enteric nervous system development.
Development of organised motility patterns in the duodenum of the
mouse
jc bornstein,* rr roberts,*,� rm gwynne* & hm young�*Department of Physiology, University of Melbourne, Parkville, Vic, Australia;
and �Department of Anatomy and Cell Biology, University of Melbourne,
Parkville, Vic, Australia
Introduction: In adults, intestinal motility is regulated by interactions
between the enteric nervous system, pacemaker cells intrinsic to the
muscle layers, interstitial cells of Cajal (ICC), and the smooth muscle
itself. Both ICC and enteric neural circuits impose their own rhythms
on motility with their contributions differing between regions. In the
undistended mouse duodenum, most motor activity consists of small
circular muscle contractions that occur at ~50/min and propagate
anally. These may be due to pacemaker ICC. Distension triggers orally
propagating neurogenic motor patterns that manifest as low frequency
(1 every 2–5 min) waves of enhanced slow wave contractions. Anally
propagating contractile complexes (PCCs) are seen in mouse
colon. Objective: To determine when PCCs develop in relation to
enteric neurons and ICC. Methods: Spatiotemporal maps of intestinal
diameter were made from video recordings of embryonic and neonatal
mouse duodenum in vitro. This allows detailed analysis of spatial and
temporal relationships between contractions at neighbouring points.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdii
We undertook electrophysiological analysis of smooth muscle and
immunohistochemical studies of the appearance of enteric neurons
and ICC. Results: At embryonic day 12.5 (E12.5), the duodenum was
quiescent. By E14.5 spontaneous contractions propagating orally and
anally away from point sources were seen. These were like the ripples
seen in neonatal mouse colon and were insensitive to the neural
blocker tetrodotoxin. At E14.5, Kit-immunoreactivity (marker for ICC)
labelled undifferentiated cells between the serosa and myenteric
plexus, but there were no morphological ICC. By E16.5, ripples were
prominent; also seen in duodenum of Ret-/- mice, which lack enteric
neurons. ICC had not differentiated. At E18.5, some organized neural
activity was seen; mucosal stimulation evoked junction potentials in
the muscle; and electrical slow wave activity was seen. ICC networks
were identified immunohistochemically. Two high frequency patterns
were identified; whether either or both were ripples or slow wave
contractions was unclear. Mature PCCs were not present in the duo-
denum at P0. Conclusions: Mature motility does not appear until
well after arrival and initial differentiation of enteric neurons. The first
organized motility is independent of neurons or ICC and may be
intrinsic to the smooth muscle itself.
Analysis of talpid3 mutant chicken embryos reveals defects in gut and
enteric nervous system development
aj burns,* l bradshaw,* lj freem,* mg davey� & n thapar**Neural Development Unit, UCL Institute of Child Health, London, UK; and
�Division of Genetics and Genomics, Roslin Institute, Midlothian, UK
Introduction: Talpid3 is a classical chicken mutant with a wide range
of phenotypic defects including paddle-shaped limbs, and face, skele-
ton and vascular abnormalities. Although these defects were originally
described over 40 years ago, it was only recently determined that tal-
pid3 encodes a novel gene product that is required for the function of
Gli repressor and activator transcription factors in the Hedgehog (Hh)
signalling pathway. Objective: Since the Hh signalling pathway has
been implicated in the development of the gastrointestinal tract and
enteric nervous system (ENS), our aim was to examine talpid3 mutant
embryos to gain further insight into the role of the Hh pathway in the
development of these tissues. Methods: Fertilised eggs from the tal-
pid3 flock were incubated for up to 8 days. The eggs were windowed in
order to determine stage of development and phenotype, then embryos
were removed, fixed in 4% PFA and processed for frozen sectioning and
immunohistochemistry. Antibody staining was performed to identify
smooth muscle (aSMA), neural crest cells (HNK-1), and early neuronal
cells (bIII Tubulin – TuJ1). Results: Approximately 20% of embryos
were identified as being homozygous talpid3 mutants, based on visual
inspection of the blood vessels and limb buds. Upon dissection, the gut
was normally patterned along its length (esophagus, stomach,
pre-umbilical intestine, post-umbilical intestine, hindgut) but was
significantly shorter in talpid3 embryos compared with stage-matched
controls. In talpid3 gut, although the distribution of HNK-1 and TuJ1-
positive cells was similar to controls, they were reduced in number. At
E6, the distribution of SMA was considerably altered in talpid3
mutants. In control esophagus, concentric rings of SMA encircled the
gut, whereas SMA staining was absent in mutants. Although weak
SMA immunoreactivity was present in the stomach of mutants, in the
pre- and post-umbilical intestine SMA was distributed across the
entire gut wall, and abutted the epithelium. In the hindgut of talpid3
mutants SMA immunoreactivity was patchy and positive cells did not
encircle the gut as in controls. Conclusion: Preliminary data suggest
that although neural crest cells are able to migrate and differentiate in
talpid3 gut, they are reduced in number compared with controls. In
addition, overall gut length is significantly reduced and SMA-positive
cells are not organised into presumptive circular muscle. These find-
ings suggest that perturbation of Hh signalling in the talpid3 chick
embryo results in malformation of the gastrointestinal tract, lack of
gut smooth muscle patterning, and altered ENS development.
Divergence of enteric neuronal and glial lineages from uncommitted
progenitors: roles of bone morphogenetic proteins, neuregulins,
neuropoietic cytokines and glial cell line derived neurotrophic factor
a chalazonitis,* f d�autreaux,� td pham,* ja kessler� & md gershon**Columbia University, New York, NY, USA; and �Ecole Normale Superieure,
Paris, France; and �Northwestern University, Feinberg Medical School, Chicago,
IL, USA
The enteric nervous system (ENS) develops from a multipotent pop-
ulation of neural crest-derived cells (ENCDC). Factors in the enteric
microenvironment that induce common neural/glial progenitors to
differentiate selectively as neurons or glia remain to be identified.
BMPs-2 and -4, which are expressed in the enteric microenvironment,
enhance neuronal differentiation at the expense of proliferating pre-
cursors; moreover, common enteric neural/glial progenitors are driven
by glial cell derived neurotrophic factor (GDNF) to develop as neurons.
Neuropoietic cytokines (LIF and CNTF; NPC) have also been found to
increase numbers of glia developing in vitro. Because mice that lack
ErbB3, the binding receptor for neuregulin-1 (NRG-1/GGF2) lack
enteric glia, enteric gliogenesis must be NRG-1-dependent. We tested
the hypotheses that NPC and/or BMP-2 and -4 enhance glial differ-
entiation directly or by increasing the responsiveness of precursors to
NRG-1/GGF2. To analyze the role of BMPs in enteric gliogenesis
in vivo, the BMP antagonist, noggin (NSE-noggin mice), or BMP4 (NSE-
BMP4 mice), were overexpressed in the ENS of transgenic mice.
Overexpression of noggin in the ENS decreased the density of enteric
glia and their ratio to neurons while overexpression of BMP4 increased
glial density and the glia/neuron ratio. In vitro, BMPs promoted ErB3
expression and glial differentiation of isolated ENCDC in a concen-
tration and stage-dependent manner. BMPs also induced nuclear
translocation of phosphorylated receptor-activated SMAD proteins in
cells that expressed the glial marker, GFAP. In situ, expression in the
fetal gut of ErB3 was detected as early as E12. Isolated crest-derived
cells selectively expressed ErB3 and its immunoreactivity in situ was
coincident with that of GFAP at E17. NRG-1/GGF2 expression (E14)
followed that of ErbB3 and both continued to adulthood. NRG-1/GGF2
induced glial proliferation and decreased GDNF-induced neurogenesis.
In contrast, GDNF enhanced neurogenesis at the expense of NRG-1/
GGF2-induced gliogenesis. In contrast to NRG-1/GGF2 and GDNF,
NPC increased the development both of neurons and glia. BMP- and
NPC-induced gliogenesis were not additive. Exposure of ENCDC to
BMPs decreased NRG-1/GGF2-stimulated glial proliferation but
increased NRG-1/GGF2 dependence. Observations support the
hypotheses that NPC promote survival/differentiation of common
neural/glial precursors, BMPs promote differentiation without speci-
fying lineage. In contrast, differentiation is biased toward a glial
lineage by NRG-1/GGF2 and toward a neuronal lineage by GDNF.
Supported by NIH grants DK58056 (AC) NS15547 (MDG) NS20013,
NS20778 (JAK).
Sox2 expression provides a means to identify and isolate ENS
progenitors
ta heanue & v pachnis
Division of Molecular Neurobiology, MRC-National Institute for Medical
Research, London, NW7 1AA, UK
The enteric nervous system (ENS) derives from neural crest cell pro-
genitors that emigrate from the neural tube and invade the developing
foregut and migrate in a rostral to caudal direction to completely col-
onize the developing gut. These progenitors proliferate to generate
sufficient cell numbers and then progressively differentiate into neu-
rons and glial cells and become organized into a series of intercon-
nected ganglia. While most progenitors undergo differentiation, cells
exhibiting the properties of enteric progenitor cells (EPCs) can be iso-
lated from mouse gut tissue from E11.5 until post-natal stages. Our
previous microarray screen, conducted to identify markers of the
mammalian enteric nervous system, identified the neural progenitor
and stem cell marker Sox2 to be expressed in the embryonic ENS. We
now show that SOX2 is expressed from embryonic to post-natal stages,
and is co-expressed with known enteric progenitor cell markers such
as SOX10 and NESTIN. We have developed a strategy that uses
selection on the basis of Sox2 expression as an alternative method for
Abstracts
� 2009 Blackwell Publishing Ltd iii
isolating EPCs. Cells derived from gut tissue from a transgenic mouse
line in which the bgal-neomycin resistance gene fusion protein has
been knocked into the Sox2 locus (Sox2-bgeo) can be cultured under
G418 selection, thus allowing substantial enrichment of neomycin
resistant SOX2 expressing cells within the population. Cells can be
selected in this way from both embryonic and adult tissues and can
proliferate extensively in culture over a period of months. The
expression profile of Sox2-bgeo selected cells is consistent with EPCs;
selected cells express the progenitor marker SOX10 and do not express
differentiation markers such as TuJ1 and GFAP. The functional prop-
erties of Sox2-bgeo selected cells have also been tested. Selected cells
transplanted into the ENS progenitor cell migratory pathway of mouse
and chick embryos were capable of migrating along appropriate routes.
Moreover, we demonstrate that when transplanted into an E11.5
mouse gut environment, Sox2-bgeo selected cells can migrate, differ-
entiate, and form apparently normal connections with endogenous
neurons, thereby exhibiting important properties of enteric progeni-
tors. The relative ease of maintaining a population of enteric
progenitors using this technique lends itself well for use in stem cell
replacement studies currently underway in the lab that aim to restore
enteric neurons to aganglionic regions in mouse models of Hirsch-
sprung’s disease.
Colonisation of the developing lungs by tangential migration of
foregut-derived vagal neural crest cells
lj freem, s escot, n thapar & aj burns
Neural Development Unit, UCL Institute of Child Health, London, UK
Introduction: Neural crest cells (NCC) are a transient population of
multipotent cells that migrate extensively throughout the embryo and
give rise to a variety of cell types including neurons of the enteric
nervous system and intrinsic neurons that innervate the airway
smooth muscle of the developing lungs. We have previously shown
that the NCC that initially colonize the lungs arise from a subset of
cells that migrates from the developing foregut into the lung
buds. Objectives: To analyse the spatiotemporal colonization of the
lungs by NCC and to determine the signalling cues that influence their
migration, proliferation and differentiation. Specific objectives are to:
(i) analyse the spatiotemporal migration of NCC from the foregut into
the lung buds in mouse and human embryos, (ii) analyse the expres-
sion of candidate signalling molecules in NCC and within the
lung mesenchyme, and (iii) test the chemoattractive and trophic
capabilities of candidate signalling molecules. Methods: ROSA26Y
FPStop;Wnt1Cre transgenic mice, in which all NCC express yellow
fluorescent protein (YFP), were used to analyse the spatiotemporal
development of NCC in the developing foregut and associated lungs.
The roles of candidate signalling molecules in NCC and lung buds
were analysed using immunohistochemistry on control and mutant
(Ret-/-, Gfra1-/-, Dom-/-) embryonic mouse and human lung tissues.
The effects of putative chemoattractants on the directed migration of
NCC within the lung were tested using an in vitro model of lung
colonisation/growth. Results: Studies of ROSA26YFPStop;Wnt1Cre
transgenic mice clearly described the tangential migration of YFP-la-
belled NCC from the developing foregut, beginning at E10.5, into the
lungs (E11.5–13.5). YFP-positive cells were closely associated with
the airway smooth muscle of the branching bronchi. No differences
in the extent of lung innervation by NCC-derived precursors were
evident in E14.5 Ret-/-, Gfra1-/-, Dom-/- mutant mice compared
with controls. Experiments using cultured lung explants exposed
to candidate molecules delivered within agarose beads demonstrated
that YFP-positive NCC were attracted towards GDNF, the RET
ligand. Conclusion: The determination of the mechanisms underly-
ing development of the intrinsic innervation of the lung would be
a significant advance and further our understanding of lung innerva-
tion in particular, and mechanisms of NCC development in general.
The project focuses attention on identification of potential neural
crest-related defects in the lung that could have significance in health
and disease before and after birth.
Mode of gut colonization by enteric neural crest-derived cells
h enomoto,* c nishiyama,* df newgreen� & hm young�*RIKEN Center for Developmental Biology, Kobe, Japan; �The Murdoch Childrens
Research Institute, Parkville, Via, Australia; and �Department of Anatomy & Cell
Biology, University of Melbourne, Melbourne,Via, Australia
Introduction: Recent time-lapse studies on GFP-labeled migrating
enteric neural crest-derived cells (ENCCs) have revealed dynamic
patterns of ENCC migration and suggested that distinct regions or
subsets of ENCCs contribute differentially to the formation of the
enteric nervous system. These studies also suggested a high level of
cell-cell interaction among ENCCs during migration. However, the
use of a single-wavelength fluorescence limits visualization of a given
cell population against other cell populations, and it is often difficult
to follow the behaviors and fate of ENCCs by this cell labeling tech-
nique. Objective and Methods: To more fully understand the mode of
gut colonization by ENCCs, we have generated Ednrb-Kik mice in
which all ENCCs are engineered to express the photo-convertible
fluorescent protein, humanized Kikume. Gut explants were dissected
from Ednrb-Kik embryos (embryonic day 12.5, E12.5) and time-lapse
imaging of ENCCs was performed using an inverted confocal
microscope. Kikume changes chromophore emission wavelength from
green to red upon UV exposure, enabling us to label a subset of ENCCs
at desired time points within the migrating network of ENCCs.
Results: We have made the following observations. (i) At least two
morphologically distinct cell populations are distinguishable within
the network of migrating ENCCs; multipolar immature neural crest-
like cells and unipolar neuron-like cells with a single neurite (the latter
migrated at the slower speed than the former), (ii) the wavefront of
migrating ENCCs is composed largely of immature neural crest-like
cells, (iii) although the meshwork formed by strands of ENCCs
maintains its basic structure over several hours, ENCCs continuously
change their neighbors within those strands, (iv) at least some of
the wavefront-derived ENCCs keep the �wavefront position� through-
out migration and (v) the ability of ENCCs to expand along the rostral-
caudal axis in the gut is highest in cell populations located at
the wavefront. Conclusions: The behavior of individual ENCCs sug-
gests a high degree of cell-cell and cell-matrix interactions. At E12.5
and thereafter, ENCCs located at the wavefront play a primary role in
colonizing the colon. Ednrb-Kik mice provide a valuable platform for
better understanding of dynamic and complex behaviors of ENCCs.
Regeneration in ileal anastomosis
s holland-cunz,* m chmelnik,* s weih,* m klotz� & kh schafer�*Department of Pediatric Surgery, University Hospital, Heidelberg, Germany; and
�Faculty of Science, Microsystem technique, Zweibruecken, Germany
Aim: This study wanted to show the regenerative capacity of the
bowel wall after interrupted and anastomosed small bowel Material
and Method: We performed laparotomy, ileal dissection and anasto-
mosis in 32 male Sprague Dawley rats. The anastomotic technique
varied between a single knot hand sutured anastomosis, the by our
team new developed CINPA method with non perforating single metal
clips and one case with a clued anastomosis, were we used a collagen
and fibrin layer. The anastomoses were resected after sacrificing the
animals at day 3 respectively 14 after the anastomosing procedure.
X-rays were performed for grading the stenosis rate of the anastomosed
region and stability tests, as bursting pressure and lengthening force
were realised.We calculated the morphological data and compared
between the different anastomotic techniques and the different sur-
vival times. Beside the nerval regeneration we observed changes in the
protein cluster and performed 2D DIGE separating of proteins. With
MALDI TOF the proteins were dedicated Results: Of these 32
anastomoses only 18 were applicable for a histological analyse. Out of
these 18 anastomoses we used 34 slides for the interpretation. There
was no increase of cell amount in between day three and 14 after
anastomosis. In the group of the sutured anastomoses there were more
nerve cells in the submucosal plexus and nearly equal relations in the
myenteric plexus. The area of the ganglions was a little bit enlarged in
the sutured group in comparison to the clipped ones. The glued
anastomosis showed similar results to the other ones.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdiv
FGF16 (Fibroblast growth factor) und TRIP 13 (Thyreoid receptor
interactin protein) were isolated and dedicated by protein analysis and
showed regulative increase in dependence of postoperative state.
Discussion: Different methods of anastomosing the bowel result in
various scar forming. The influence of these mechanical forces on the
damaged bowel is nearly unknown with regard to the enteric nervous
system. If there is any functional consequence out of these observa-
tions is improbable, because the motility of the bowel seems only to be
deranged over a very short distance and not notable dependent of these
damaged nerves. To appreciate the differences in healing quality of
anastomoses it should be possible to determinate protein clusters that
are corresponding with the effectiveness of actual healing forces.
The clinical genetics of Hirschsprung’s disease
s lyonnet, as jannot, a tullio-pelet & j amiel
Department of Genetics and INSERM U-781, University Paris Descartes, Hopital
Necker-Enfants Malades, Paris, France
Hirschsprung disease (HSCR) is the most common congenital mal-
formation of the enteric nervous system. HSCR is characterized by the
congenital absence of intrinsic ganglion cells in the submucosal and
myenteric plexuses of the hindgut, leading to intestinal obstruction
and colonic distension in newborns. The length of aganglionosis is
variable and correlates with the severity of the disease, extending from
the rectum to the recto-sigmoid junction (short-segment, 80% of cases)
or beyond (long-segment, 20%). HSCR has an incidence of 1/5000 live
births, a circumstance which, together with the actual good surgical
prognosis, has allowed recruitment of quite a large series of patients
over long periods of time and also identification of familial cases.
Interestingly, in a significant number of cases (up to 30%), HSCR is
associated with other malformation or developmental anomaly,
including more complex neural crest defects, a fact that underscores
the great importance of a clinical and dysmorphological examination
of HSCR patients. Regarded as a single field neurocristopathy owing to
the embryonic origin of enteric neurons, HSCR is also a model of a
genetically complex inborn error of development. Despite the high
proportion of sporadic cases (80%), segregation analyses showed that
HSCR has a high heritability and is inherited as a sex-modified oligo-
genic trait with a variable and non-mendelian risk to siblings (3-22%
depending on the length of aganglionosis, the gender of the proband, and
the familiality). The major locus is clearly RET, probably involved in all
cases. In addition, a number of parametric and non-parametric linkage
analyses used to deconstruct the complex genetic multiplicative model
of inheritance underlying HSCR, suggest heterogeneous modifier loci
whose requirement might depend on the nature (coding or non-coding
sequence mutations), the �dose�, and the transmitting parent of mutant
alleles at the major locus RET, as well as possibly the genetic and ethnic
background. The purpose of the present talk will be to discuss these data
in a clinical setting and the interest for patients and families. In partic-
ular, this presentation will attempt to address some of the following
issues: (i) the clinical approach of syndromic HSCR, (ii) the links be-
tween HSCR, regarded as a developmental anomaly, and some inherited
predisposition to cancer, (iii) the clinical interest of genetic testing in
HSCR families, and (iv) the risk figures and genetic counselling issues in
HSCR families.
High-field magnetic resonance imaging: stem cell tracking and
phenotyping of transgenic mice
mf lythgoe
UCL Institute of Child Health, London, UK
In this seminar I will focus on two challenges in biomedical imaging
science (i) the tracking of stem cells to the sites of tissue damage and
(ii) non-invasive visualisation of abnormalities in embryo develop-
ment. Development of methods capable of labelling and tracking cells
in vivo would be of great benefit to the understanding of cell migration
under normal and pathological conditions. In a recent study, we
demonstrate that endogenous neuroblast migration cells can be la-
belled in vivo with an MRI contrast agent and that they can be visu-
alised using MRI, which indicates the feasibility of in vivo imaging of
cell migration using MRI. lMRI is now an emerging technique for
high-throughput phenotyping of transgenic mouse embryos, visualis-
ing abnormalities in development. The mouse is a key model for
research into human disease and is ideally suited to genetic study, as
the genome and techniques for manipulating its sequence are readily
available. One such technique is ENU mutagenesis, utilised by the
large-scale programmes underway to create mutant models for each
of the >25 000 genes to study their function. This increasing use of
genetically modified mice has highlighted the need for techniques to
rapidly characterise the new morphological phenotypes presented in
these models. lMRI may be an ideal high-throughput technique for the
imaging of multiple embryos.
Development of Neurons and the Role of Neural Activity in the
Developing Gut
mm hao,* re moore,* rb anderson,* jc bornstein,� ea jennings* &
hm young**Departments of Anatomy and Cell Biology, University of Melbourne, Mel-
bourne, Via, Australia ; and �Department of Physiology, University of Melbourne,
Melbourne, Via, Australia
Introduction: As they migrate through the developing gut, enteric
neural crest-derived cells (ENCCs) begin to differentiate into neu-
rons. In the developing CNS, neural activity has recently been
shown to affect the proliferation, differentiation and migration of
neighbouring neural precursor cells. Objectives: To examine the
timetable of neuronal differentiation in the developing gut and
establish whether neural activity affects the migration of
ENCCs. Methods: Immunohistochemistry was performed on
wholemount preparations of E10.5-E11.5 gut. Intact explants of mid-
plus hindgut from E11.5 RetTGM-GFP/+ mice, where all ENCCs
express GFP, were grown in culture with drugs that inhibit different
forms of neural activity: tetrodotoxin (TTX) to block neural action
potentials, nitro-L-arginine (NOLA) to inhibit nitric oxide produc-
tion, tetanus toxin to inhibit vesicle-dependent neurotransmitter
release, and clotrimazole to block IK channel activity. The distance
that ENCCs migrated was examined after 48 h. Whole cell patch-
clamp recordings were made from isolated neurons from the
embryonic gut of Ednrb-Kik mice, where all neurons express
the photo-convertible fluorescent protein, Kikume, to examine
the development of electrical properties. Results: As reported pre-
viously, around 15% of ENCCs in the gut from E10.5–E12.5 mice
expressed the pan-neuronal markers Hu, Tuj-1 and neurofilament-M,
and extended processes. Tuj-1+ and neurofilament-M+ nerve fibres
were present in close apposition to many of the ENCCs, including
those at the migratory wavefront. Commencing at E11.5, nitric
oxide synthase (NOS), calbindin and IK channel immunoreactivity
were present on a sub-population of ENCCs. In cultured explants of
embryonic gut, TTX, NOLA and clotrimazole did not affect ENCC
migration, but tetanus toxin significantly reduced ENCC migration
as reported previously1. In patch-clamp studies, action potentials
were recorded from isolated enteric neurons from E18.5
mice. Conclusions: Although neurons expressing pan-neuronal and
enteric neuron sub-type specific markers are in close association
with ENCCs while they are colonizing the gut, we found no evi-
dence that neuronal activity affects ENCC migration. Tetanus toxin
inhibited ENCC migration but it is unclear whether this is due to
inhibition of neurotransmitter release or inhibition of other mech-
anisms such as membrane re-cycling.
Reference:
1 Vohra BP et al. Differential gene expression and functional analysis
implicate novel mechanisms in enteric nervous system precursor
migration and neuritogenesis. Dev Biol 2006; 298: 259–71.
Abstracts
� 2009 Blackwell Publishing Ltd v
Vitamin A facilitates enteric nervous system precursor migration by
reducing PTEN accumulation
m fu,* y sato,* a lyons-warren,* bin zhang,� ma kane,� jl napoli� & ro
heuckeroth*,§,–*Department of Pediatrics, Washington University School of Medicine, St Louis,
MO, USA; �Department of Pathology and Immunology, Washington University
School of Medicine, St Louis, MO, USA; �Department of Nutritional Science and
Toxicology, University of California, Berkeley, CA, USA; §Department of
Developmental Biology, Washington University School of Medicine, St Louis,
MO, USA; and –HOPE Center for Neurological Disorders, Washington University
School of Medicine, St Louis, MO, USA
Aims: To test the hypothesis that retinoid signaling is important for
enteric nervous system precursor migration and to identify the molec-
ular machinery that responds to RA. Methods: We used several dif-
ferent culture techniques to investigate the effect or RA signaling on
ENS precursor migration including gut organ culture, slice culture, and
Boyden chamber assays. We also investigated the effect or RA in vivo
using mice deficient in serum retinol binding protein (Rbp4).
These animals are dependent on daily vitamin A ingestion to meet
tissue needs and can be rapidly depleted on circulating
retinoids. Results: Vitamin A depleted Rbp4 -/- mice have striking
distal bowel aganglionosis, but these mice have a normal appearing ENS
if they are maintained on Vitamin A containing food. Rbp4 -/- Ret +/-
double mutant mice have increased distal bowel aganglionosis even
when fed Vitamin A containing food substantiating an important role
for vitamin A as a non-genetic modifier of EN\S development. In vitro
studies demonstrate that RA signaling is critical for ENS precursor
migration and lamellipodia formation. This effect is attributable to
reductions in Pten protein levels in the most actively migrating cells.
Pten overexpression also reduces GDNF induced ENS precursor
migration in vitro consistent with the hypothesis that RA induced Pten
suppression is required for efficient ENS precursor migra-
tion. Conclusion: This study suggests the intriguing possibility is that
Vitamin A deficiency is a preventable cause of Hirschsprung’s disease.
Correlated expression of S100 and GFAP in enteric glia in rodent and
human gut
kh schafer,* s maas-omlor,* aj burns� & u rauch**University of Applied Sciences, Kaiserslautern, Germany; and �UCL, Institute of
Child Health, London, UK
Introduction: For years, enteric glia have played a �wallflower�-like
role, although their importance was already recognised by various
authors, including Michael Gershon, Georgio Gabella and Kristjan
Jesssen, who demonstrated that enteric glia contain the glial fibrillary
acidic protein GFAP, like reactive astrocytes in the central nervous
system. In recent years, the key role of enteric glia in supplying neu-
rotrophic support or responding to inflammation has become increas-
ingly obvious. However, there is still little known about the
distribution or the relationship between the two glial markers S100, as
a general marker, and GFAP, as a marker for reactive
glia. Objectives: To deliver a baseline for further studies on the role of
enteric glia in different diseases and species. To that end S100 and
GFAP immunoreactivity was assessed during development as well as
in various diseases or disease models such as the DSS (dextran sulfate
sodium) colitis. Methods: Tissue from all parts of the gstrointestinal
tract from esophagus to distal colon were collected from human and
rodent sources. In humans, tissue from the 7th week of gestation to old
age, and in rat from postnatal day 1 to adult was collected and
embedded in paraffin. Additional tissues were obtained from cases of
appendicitis, colon cancer, or from mice where a DSS colitis was
induced. 5 lm sections were cut and doublestained for S100 and
GFAP. Results: Both glial markers appear to be developmentally reg-
ulated independently from each other. In humans S100 is first seen in
samples from week 12 of gestation. Here it appears in the myenteric
plexus only and with a slight gradient from esophagus to the distal
colon. During development the S100 signal gets stronger and is fully
developed in the middle of the second trimester. In our sections GFAP
does not appear prenatally. After birth the GFAP signal is strongly
correlated to diseases. Its signal gets stronger when the tissue is
inflamed or also in colon cancer tissue. In the rat, both S100 and GFAP
do not appear before birth. At the first postnatal day S100 is only
present in the esophagus and stomach and gets stronger with the age of
the animal. At postnatal day (P)14 the signal is fully developed
throughout the gut, while GFAP reactivity cannot be seen all over the
full length of the gut before P28, although it appears partially from P7
onwards. In general, there seem to be glial cells in the muscle layer which
never show GFAP reactivity. Conclusion: There is evidence for at least
two distinct glial populations in the enteric glia which may react in
various amounts to external influences such as inflammation.
Neurospheres are not like neurospheres
kh schafer,* ma micci�,� & pj pasricha�*Universitiy of Applied Sciences, Kaiserslautern, Germany; �UTMB, Galverston,
USA; and �University Stanford, Stanford, CA, USA
Introduction: The transplantation of suitable neural stem cells seem
more and more to be a challenging alternative for the restoration of
gastrointestinal dysfunctions, such as nitrergic deficits in pylorus ste-
nosis, gastroparesis, achalasia or dysganglionosis. Individual attempts
were performed to open up new resources for auto- or heterologous
stem cell supply which can be used in clinical treatments. Potential
sources are the subventricular zone from fetal tissue or the enteric
nervous system itself. Depending on the site and time of isolation
of the neural stem cells, there might be a significant difference in
the yield of cells, as well as their potential to differentiate into
all cell types needed for the restoration of gastrointestinal func-
tion. Objectives: To demonstrate the problems which have to be faced
by using varying sources for neural stem cells, we compared the growth,
the needs and the differentiation of neurospheres derived from either
the central nervous system or the gastrointestinal tract from the same
animal. Methods: Stage pregnant mice at the embryonic age E13 were
sacrificed by an overdose of isoflurane. The embryos were immediately
removed and stored on ice. From each embryo the subventricular zone
as well as the whole gastrointestinal tract was removed. Both tissues
were processed identically. After incubation in a mixture of collagenase
and dispase, the tissues were dissociated, cell number counted and cells
plated at equivalent cell numbers in Neurobasal medium containing
the neurotrophic factors EGF (epidermal growth factor), FGF (fibroblast
growth factor) and GDNF (glial cell line derived neurotrophic factor).
The cultures were kept for several days until neurospheres could
be harvested in significant numbers. Neurosphere and sizes were
measured. After 5 days in culture, the neurospheres were harvested
and explanted in a three dimensional gel (extracellular
matrix). Results: There were significant differences concerning the
trophic supply, the growth and the differentiation of the two types of
neurospheres. While SVZ spheres could be cultured in EGF and FGF
alone, the ENS spheres needed additional GDNF for sufficient growth.
In general the SVZ spheres grew faster, bigger and more numerous.
Within the gel, the two types of neurospheres grew differentially. The
SVZ spheres differentiated slower, starting with a glial migration, fol-
lowed by a neurite outgrowth after three days in vitro, while the ENS
spheres started immediately with a very dense neurite network and
only few migrating glial cells. Conclusion: Before using individual
stem cell sources for clinical trıals, the quality of the isolated neural
stem cells have to be assessed of being usable in an appropriate way.
Interaction of the enteric nervous system and enterocytes in vitro is
increased by growth hormone treatment
l schwarz, d griesemer, u rauch, m klotz & kh schafer
Universitiy of Applied Sciences, Kaiserslautern, Germany
Introduction: Growth and regeneration of the mucous layer is a very
important prerequisite for the undisturbed function of the gastroi-
ntestinal tract. Lacking, insufficient or inaccurate regeneration and
proliferation might lead to severe disturbances of food resorption,
deficiencies or even malformations of the mucous layer. While in most
in vitro studies merely enterocyte cell lines were used to model the
mucosal barrier, the enormous influence of the intrinsic nervous sys-
tem, the enteric nervous system, upon the gastrointestinal regenera-
tion is mostly neglected. In this study the interaction of ENS and
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdvi
mucosal cells in vitro is investigated, especially under the influence of
growth hormone treatment. Objectives: The use of growth hormone
to increase the mucosal surface for a better resorption i.e. in short
bowel disease is still controversial. In the presented study the indirect
influence of GH upon mucosal proliferation via the enteric nervous
system is investigated. Methods: In vitro experiments were per-
formed using enterocytic cell lines alone and in coculture with isolated
myenteric plexus from the newborn rat. Both cell types were cultured
under the influence of growth hormone. Supernatants were collected
and fed to either one of the two cell types. Enterocyte proliferation was
measured using BrdU-ELISAs under or without the influence of neu-
ronal and glial secreted factors. Enterocyte cultures were fixed and
processed for scanning electron microscopy to evaluate the variation in
microvilli density. Results: The enterocyte proliferation in vitro
could significantly be increased by using supernatants from dissociated
myenteric plexus cultures, especially when treated with GH. These
supernatants where measured concerning neurotrophin content and
various neurotrophic factors such as NT-3 or CNTF could be detected.
Scanning electron evaluations of the enterocyte surfaces revealed
changes in cell size and microvilli density depending on the treat-
ment. Conclusion: The enteric nervous system and the enterocytes
do strongly influence each other. This influence might be used to
stimulate mucosal growth and regeneration by influencing the ENS
directly using various neurotrophic factors. The use of co-culture
models where several compounds of the gut wall such as mucosal
barrier and the ENS are represented might lead to a much better
understanding of the mucosa-nervous-system interaction.
Stage-specific control of neural crest stem cell proliferation
l sommer
University of Zurich, Zurich, Switzerland
During vertebrate development, neural crest (NC) cells emigrate from
the neural tube to generate a variety of neural and non-neural tissues.
Both migratory NC cells and their target structures contain so-called
neural crest-derived stem cells (NCSCs) that display multipotency and
self-renewal capacity. However, maintenance and proliferation of
these NCSC populations are differentially regulated. Indeed, Cdc42 or
Rac1 inactivation reduces self-renewal and proliferation of later stage
NCSCs, including those isolated from the enteric nervous system,
while self-renewal of early migratory NCSCs is Cdc42/Rac1-indepen-
dent. Accordingly, deletion of either Cdc42 or Rac1 in the NC results
in size reduction of multiple NC target structures because of increased
cell cycle exit. In contrast, NC cells emigrating from the neural tube
are not affected. This stage-specific requirement for small Rho GTP-
ases is due to changes in NCSCs that during development acquire
responsiveness to mitogenic EGF acting upstream of both Cdc42 and
Rac1. Thus, our data reveal distinct mechanisms for growth control of
NCSCs from different developmental stages.
Development and maintenance of interstitial cells of Cajal networks
in health and disease
sm ward, sj hwang, y bayguinov & km sanders
Department of Physiology and Cell Biology, University of Nevada, Reno, USA
Interstitial cells of Cajal (ICC) play critical roles in several GI motility
functions including: (i) generation of pacemaker activity (ii) propagation
of pacemaker slow waves (iii) mediation of excitatory and inhibitory
enteric motor inputs (iii) providing stretch-dependent responses within
the smooth muscle syncytium and (iv) guidance of vagal intramuscular
afferent nerves. It is well accepted that the development and mainte-
nance of ICC networks is a necessary pre-requisite for normal gut motor
function and their loss contributes to several GI dysmotilities. How the
development and maintenance of ICC networks is achieved within the
GI tract is quite controversial. It has been reported that ICC networks
develop from Kit(+) mesenchymal precursors between E15-E17.1
However, the importance of Kit signaling for the development of ICC
networks prior to birth is contentious.2,3 ICC may be replaced after
birth from Kit(low)CD44(+)CD34(+) Insr(+)Igf1r(+) progenitor cells.4
More recently it has also been demonstrated that Kit(+) ICC possess the
capability to undergo mitosis.5 Using a pulse-chase experimental
approach, tissues from animals of different ages were exposed to BrdU
and Edu for varying durations, to determine the capability of Kit(+) ICC
networks to undergo mitosis prior to and after birth. We further eval-
uated whether ICC undergo apoptosis and whether dividing ICC expand
networks to provide replacement of ICC in adult animals. We also
evaluated whether expansion of ICC networks during growth of the GI
tract occurs by cell division of mature Kit(+) ICC. In E17 tissues, nuclei
of Kit(+) ICC incorporated BrdU or EdU and this continued until at least
P30, suggesting that mature ICC within networks possess the capability
of cellular division. The number of ICC undergoing mitosis decreased
with age and dropped significantly after P10. We found little evidence
for apoptosis in Kit(+) ICC in GI tissues over the same time period, but
Kit immunoreactivity decreased and pacemaker activity ceased in tis-
sues following application of the pro-apoptotic agent camptothecin
(5lg ml-1; for 24 h). Acute application of camptothecin had no affect on
pacemaker activity. In conclusion Kit(+) ICC possess the capability to
undergo mitosis prior to and after birth and this process is likely to
ensure growth of ICC networks during development of the gut wall.
References:
1 Torihashi et al. Development of c-Kit-positive cells and the onset of
electrical rhythmicity in murine small intestine. Gastroenterology
1997; 112: 144–55.
2 Kluppel et al. Developmental origin and Kit-dependent development
of the interstitial cells of cajal in the mammalian small intestine.
Dev Dyn 1998, 211: 60–71.
3 Beckett et al. Kit signaling is essential for development and main-
tenance of interstitial cells of Cajal and electrical rhythmicity in the
embryonic gastrointestinal tract. Dev Dyn 2007, 236: 60–72.
4 Lorincz et al. Progenitors of interstitial cells of cajal in the postnatal
murine stomach. Gastroenterology 2008, 134: 1083–93.
5 Mei et al. An age-dependent proliferation is involved in the postnatal
development of interstitial cells of Cajal in the small intestine of
mice. Histochem Cell Biol 2008; Oct 3, Epub.
SOX10 genetic interactions: Waardenburg syndrome and mouse
double mutant studies
l stanchina,* y watanabe,* v baral,* m goossens,*,� v pingault*,� &
n bondurand**INSERM U955, IMRB, departement de genetique, Creteil, France; and �AP-HP,
Groupe Albert Chenevier-Henri Mondor, service de biochimie et genetique,
Creteil, France.
Study of the dominant megacolon mice and SOX10 involvement in
Waardenburg syndrome type 4 (WS4) first highlighted the crucial role
of this transcription factor for proper enteric nervous system and
melanocyte development. Recently, we described SOX10 deletions in
15% of patients presenting with WS2 (pigmentation defects without
HSCR). These results further documented the molecular complexity
and close relationship that link the different subtypes of WS, but still
leave 70% of WS2 and 25%–35% of WS4 with no molecular explana-
tion, and suggest that other genes might be involved, or that mutations
within the known genes still escape screening performed so far. Based
on these observations, we looked for point mutations and rearrange-
ments within the EDN3/EDNRB and MITF/SNAI2 genes in both WS2
and WS4 cases, and show that the molecular overlap between WS2 and
4 is mostly restricted to SOX10. We also considered the possibility that
mutations or deletions within the known regulatory sequences of
identified genes could explain some WS4 and WS2 cases. Results of
this ongoing work will be presented. In parallel, we pursued our effort
aiming at better apprehend SOX10 function during ENS development
and its interaction with other WS or HSCR genes. We recently focused
on ZEB2, a transcriptional repressor of the TGF-b/BMP signaling
pathway. Its alteration in human and mice causes severe defects of
neural crest development including ENS defects, but little is known on
its mechanism of action. Expression studies revealed that it is present
in SOX10+ enteric progenitor cells, and switches off during neuronal
differentiation in a manner reminiscent to what is observed for SOX10.
Based on these observations, we tested the hypothesis that interactions
between SOX10 and ZEB2 are essential to control ENS progenitor
maintenance and differentiation. To this end, we crossed Sox10 and
Zfhx1b knockout mice and compared the enteric phenotype of single
Abstracts
� 2009 Blackwell Publishing Ltd vii
and double heterozygotes. TuJ1 and Xgal immunostaining revealed a
more severe ENS phenotype in double mutants at all stages observed.
Experiments are ongoing to determine the cellular and molecular origin
of these observations.
Glutamate affects intestinal motility in zebrafish larvae
c olsson, c bergstrom & i wronski
Department of Zoology/Zoophysiology, University of Gothenburg, Goteborg,
Sweden
Glutamate is known to act as a neurotransmitter in the mammalian
gut. It is found in myenteric inter- and sensory nerons and in extrinsic
afferents and may affect gastrointestinal motility mainly by acting on
excitatory motorneurons. There are several receptor subclasses, many
of which are also present in the gut. Zebrafish (Danio rerio) larvae
provide an excellent model for in vivo studies on the development of
gut motility. Anally and orally propagating contraction waves are seen
from 3 days post fertilization (dpf), using video microscopy on the
intact transparent larvae (e.g. Holmberg et al 2003). Furthermore, the
effects of both inhibitory and excitatory transmitters have been
demonstrated using this method. In the present study, glutamate was
added to zebrafish larvae and the effect on propagating contraction
frequency was recorded. In addition, the effects of three different glu-
tamate receptor antagonists were examined: the NMDA-receptor
antagonist MK801, the AMPA-receptor antagonist CNQX and the
metabotropic receptor antagonist CPPG. The response to glutamate
itself was inconsistent. Most preparations did not show any effect on
contraction frequency, except a small increase in retrograde contrac-
tions at 7 dpf and a decrease at 9 dpf. In contrast, MK801 and CNQX
caused either a direct reduction in anterograde contraction frequency,
or a reduction was seen as a response to a subsequent glutamate
application at 4, 7 and 9 dpf. CPPG showed a reduction at 9 dpf only.
The effects on retrograde contractions were less consistent but fol-
lowed a similar pattern. The results indicate that glutamate can affect
gut motility very early in zebrafish development. The direct effect of
CNQX seen at 4 dpf further indicates that glutamate may be endoge-
nously released already at that stage. The results also indicate that
glutamate has both excitatory and inhibitory effects and this could be
the reason for the apparent lack of response to exogenous glutamate.
However, by blocking NMDA- and AMPA-receptors, an inhibitory
response to glutamate could be evoked. This suggests the involvement
of several receptor types in the glutamatergic control, but the exact
nature of the zebrafish receptors needs further investigations.
Reference:
1 Holmberg et al. Ontogeny of intestinal motility in correlation to
neuronal development in zebrafish embryos and larvae. J Fish Biol
2003; 63: 318–31.
A full mutation spectrum in Hirschsprung disease: copy number
analysis
bq doan,* c stewart,� c kashuk,* sm arnold,* a chakravarti* & inter-
national hscr consortium*,�,§,–,**
*JHMI, Baltimore, MD USA; �Boston College, Boston, MA, USA; �INSERUM,
Paris, France §Istituto Gaslini, Italy; –U.Groningen, Netherlands; and
**H.U.Virgen del Rocıo, Spain
Introduction: Multiple genes with rare and common risk variants and
gene dosage sensitivity have been identified in Hirschsprung disease
(HSCR). Using genome-wide Affymetrix 500K SNP arrays, we initially
performed a family-based association analysis on 233 trios from the
International HSCR Consortium, and a novel set of genes were identified
on 7q: the semaphorins. Objective: Given that only one new set of
genes were identified and successfully replicated from our association
results, we subsequently performed a copy number analysis using SNP
intensity data to identify gene dosage sensitive regions. Methods: We
performed a copy number variant (CNV) analysis applying the ITALICS
copy number analysis tool to identify CNVs. ITALICS uses an aCGH
segmentation algorithm and controls for non-biological variation across
arrays. We also used the 270 HapMap samples to determine expected
levels of CNVs. Results: We identified a total of ~5000 CNVs (72 bp–
110 mb) in HSCR, with an average length per individual of 500 kb
(32SNPs : Nsp) or 270 kb (22SNPs : Sty). Multiple regions were identi-
fied that were biologically interesting. A de novo deletion of 1.4 mb on
6q25.1 was the most statistically significant variant, although present in
only one affected offspring. Corresponding genotype data revealed high
proportions of missing data, homozygous calls, and Mendelian errors
supporting the presence of a true deletion. Examining all CNVs within
this region, five more individuals with 1.3–62.9 kb deletions were
identified, as compared to only one 33 kb deletion in HapMap. This
suggests that although rare, the 6q25.1 region is potentially associated
with HSCR. Of great interest is the identification of 665 bp–112 kb
deletions (both de novo and segregating) in 29 individuals at 9q31, which
resides in the middle of a known linkage peak shown to be a genetic
modifier, and a 28 kb amplification in 11 individuals on 1q32.2, which
encompasses the PlexinA2 gene, the co-receptor for semaphorin3A.
Focusing on only recurrent de novo events reveals additional
variants of interest: a 128 kb region on 8p22 that occurs in four trios, and
a 203 kb amplification on 21q21.3 that occurs in three trios. The 21q21
region has also been previously shown to be a genetic modifier
locus. Conclusions: We have identified several gene dosage sensitive
regions, a few which reside in regions with previously known linkage
evidence, and a region that overlaps a gene identified as the co-receptor
for a gene recently identified from a genome-wide association study. To
minimize follow-up of false positives, we are reassessing these CNVs in
the context of rare and common disease variants identified from our
medical resequencing and genome wide association analysis on the
same study population. Prioritized regions are being experimentally
validated.
A zebrafish view of progressive fate restriction in neural crest
development
rn kelsh,* x yang,* ss lopes� & m nikaido**Department of Biology and Biochemistry, University of Bath, Claverton Down,
Bath, UK; and �Instituto Gulbenkian de Ciencia, Oeiras, Portugal
A fundamental issue in stem cell biology concerns the mechanisms
whereby cells adopt individual derivative fates. Neural crest cells con-
sist of multipotent progenitors for an astonishing diversity of different
cell-fates, including enteric neurons and glia, but also encompassing
multiple other peripheral neuronal and pigment cell types, as well as
varied skeletogenic fates. A major research objective in the field is to
uncover the mechanisms of fate specification from multipotent neural
crest (stem) cells. In vivo studies in the zebrafish model system strongly
suggest a progressive fate restriction model of neural crest cell devel-
opment, whereby progenitors become gradually restricted in the fates
that they can adopt. We will summarise briefly this evidence, before
describing our genetic and cell biological studies of the shady/leukocyte
tyrosine kinase (ltk) mutants (Lopes et al., 2008). Shady mutants lack
iridophores, a widespread pigment cell-type in cold-blooded vertebrates
and this results from gene function within the neural crest. Expression of
ltk at late stages [30 h post fertilisation (hpf) onwards] itself is an
excellent marker for differentiated iridophores and using this, together
with newly characterised markers for this cell-type, we show that dif-
ferentiation of iridophores fails in mutants. However, at c. 20 hpf ltk
expression is also absent, suggesting that ltk functions either in multi-
potent progenitors to mediate iridophore specification or very early in
iridoblasts to mediate their ongoing development.We show that in
mutants cell death is elevated, but only from later stages (c. 35 hpf on-
wards). We show that this phenotype parallels that in the well-charac-
terised sox10 mutants, in which fate specification of neural and pigment
cell fates is a common feature. Strikingly, early expression of ltk is
strongly increased in sox10 mutants. Importantly, this expression is
non-overlapping with that of very early neural crest cell markers like
snail2. We conclude that ltk expression may be a marker for a partially-
restricted neural crest progenitor, perhaps the hypothetical chromato-
blast; we propose that ltk function is critical for iridophore lineage
specification. We are now trying various approaches to test this
hypothesis and will present our progress to date.
Reference:
1 Lopes, SS, et al. Leucocyte tyrosine kinase functions in pigment cell
development. PLoS Genetics, 2008; 4: e1000026.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdviii
Identification of mammalian neural crest enhancers through
transgenesis in Zebrafish
as mccallion,* wj pavan,� a chakravarti,* s johnson,� a antonellis,�jl huynh,* ze stine,* dm mcgaughey,* d gorkin,* mk prasad,*
es emison* & sk loftus�*McKusick–Nathans Institute of Genetic Medicine, The Johns Hopkins Univer-
sity School of Medicine, Baltimore, USA; �National Human Genome Research
Institute, National Institutes of Health, Bethesda, USA; and �Washington Uni-
versity, St Louis, USA
Introduction: Gene regulation is the framework on which cellular
diversity is built. The substantial cellular diversity that characterizes
embryonic development in vertebrates such as humans must therefore
require immense regulatory complexity. Although regulatory control
acts at many levels, we will focus on the roles played by cis-regulatory
elements (REs) in controlling the timing, location and levels of
neural crest transcripts. However, the biological relevance of non-coding
sequences cannot be inferred by examination of sequence alone.
Perhaps the most commonly used indicator of non-coding REs is
evolutionary sequence conservation. Although conservation can
uncover functionally constrained sequences, it cannot predict biological
function; regulatory function is also not confined to conserved se-
quences. At its simplest level, regulatory instructions are inscribed in
transcription factor binding sites (TFBS) within REs. Yet, while many
TFBS have been identified, TFBS combinations predictive of specific
regulatory control have not yet emerged for vertebrates. The absence of
such vocabularies serves as a significant impediment to correlations of
non-coding sequence variation and disease phenotypes. A role for such
non-coding variation in Hirschsprung Disease (HSCR) risk is now
well established; we will describe our efforts to identify HSCR
relevant REs and to assay the pathological significance of variants
therein. Objectives: We posit that motif combinations accounting for
tissue-specific regulatory control can be identified in REs of genes ex-
pressed in those cell types. We have set out to uncover NCREs at critical
NC genes, focusing on major HSCR and related loci. Our long-range goal
is to begin to identify TFBS combinations that can predict neural crest
(NC) REs – a first step in developing a regulatory lexicon. This data is
being integrated with ongoing human genetic efforts to evaluate the role
that non-coding variation may play in disease risk, in particular HSCR
risk. Methods: We have developed a highly efficient reporter transgene
system in zebrafish that can accurately evaluate the regulatory control of
mammalian sequences, enabling characterization of reporter expression
during development at a fraction of the cost in mice. We are actively
developing novel strategies to further increase the efficiency of
these assays and to enable assays of the in vivo functional consequences
of disease associated non-coding variation within identified
REs. Results: We have already identified many REs based on their
sequence conservation among vertebrates, although we have recently
demonstrated that they can also be embedded in non-conserved
sequence space. We are systematically evaluating the regulatory activity
of putative REs identified at all major HSCR loci. To date we have
identified ‡75 novel REs including numerous NCREs at genes including,
but not restricted to, RET, GFRA1, PHOX2B, SOX10, EDNRB
and ZFHX1B. Identified NCRE control ranges from discrete NC
subpopulations e.g. the enteric nervous system or melanocyte
populations, to pan neural crest control consistent with their varied and
often pleiotropic roles in development. We will describe our ongoing
efforts to determine the biological relevance of putative REs at these
and other genes; to build functional datasets large enough to begin the
search for regulatory vocabularies of specific NC populations and to
determine the pathological relevance of disease-associated identified
therein. Conclusions: The development of regulatory vocabularies
for specific NC cell lineages will potentially require the identification
ofhundreds of elements with overlapping regulatory control in
those cell types as well and those known to control expression
outside the NC. We have already cloned and analyzed in excess of
150 putative REs in vivo; we will discuss our progress in establishing
this functional sequence substrate and in illuminating pathological
variation therein.
Social controls of migration and proliferation of neural crest-derived
cells in the gut
df newgreen,* dc zhang,* b binder,� mj simpson,� ka landman,�h enomoto� & hm young§*The Murdoch Childrens Research Institute, Parkville, Via, Australia; �Depart-
ment of Mathematics and Statistics, University of Melbourne, Melbourne,
Melbourne, Via, Australia; �RIKEN Center for Developmental Biology, Kobe,
Japan; and §Department of Anatomy and Cell Biology, Melbourne, Via, Australia
Introduction: Enteric neural crest-derived cells (ENCC) colonize the
gut in a rostro-caudal wave. In Hirschsprung’s disease (HSCR), this
wave does not reach the distal gut, which remains aneuronal. Defects
in many genes predispose to HSCR, but a notable feature of HSCR
genes is the lack of penetrance. Objectives: To develop formal models
that replicate the colonization of the gut by ENCC, and illuminate the
HSCR phenotype. Methods: We developed avian and mouse models
via electroporation and transgenesis to enable the migration of ENCC
to be observed in 3D intestinal tissue by timelapse microscopy. We
also developed cellular automata (CA) models of ENCC behaviour
incorporating contact-influenced motility and proliferation as well as
simultaneous growth of the gut tissue. Results: Migrating ENCC did
not retain contacts with previous ENCC neighbours, and their trajec-
tories were unpredictable. Proliferation of ENCC was frequent at the
wavefront, but behind the wavefront proliferation was notable only
when the gut was also growing. Labelling few pre-migratory ENCC
with GFP via electroporation gave ENCC groups (probably clones) of
sizes and positions that varied widely between different individuals,
and which overlapped ENCC of different clonal origins. All these
conditions spontaneously emerged from CA models with few and
simple rules governing cell movement and cell proliferation. In CA
models the growth of the gut determined success or HSCR-like failure
of colonization. Interestingly, in CA simulations in which ENCC
invasion parameters were reduced, invasive outcomes (successful
colonization of the entire gut or a distal aneuronal zone) varied sto-
chastically between simulation runs with identical starting condi-
tions. Conclusions: We conclude that the ENCC behave as
individuals, yet they require interactions between the cells which
exerts a decisive overall control on migration outcomes. We propose
that population-scale directional invasion is driven by cell prolifera-
tion, crucially at the invasive front (�frontal expansion�). Growth of the
gut enables ENCC proliferation behind the front (�in-filling�) and
smearing of clonal boundaries. We suggest that events acting within
simple migration and proliferation rules but with stochastic compo-
nents at the cell level govern much of the colonisation process. In
addition, we raise the possibility that lack of penetrance of HSCR, as
observed even in highly inbred animal models, could also have a
stochastic element.
Netrin biosynthesized by enteric neurons mediates the attraction of
vagal sensory axons to the fetal gut
em ratcliffe,* a chalazonitis� & md gershon�*Department of Pediatrics, McMaster University, Hamilton, ON, Canada; and
�Department of Pathology & Cell Biology, Columbia University, New York, USA
Vagal sensory axons innervate the gut during fetal life. By applying DiI
to the nodose ganglia at a range of gestational ages in fetal mice, we
have found that vagal sensory axons descend to the stomach and small
bowel by E12 and E14 respectively, following the rostral-caudal
migration of crest-derived cells. DiI-labeling has furthermore revealed
that vagal sensory axons fail to innervate the aganglionic gut of mice
lacking the tyrosine kinase receptor, Ret. Previous work has shown
that netrin is expressed in the bowel wall and, by acting on its receptor
Deleted in Colorectal Cancer (DCC), mediates the guidance of vagal
sensory axons to the developing gut. We therefore tested the hypo-
thesis that developing enteric ganglia express netrin. Crest-derived and
non-crest-derived cells of E15 rat gut were separated by positive and
negative immunoselection with antibodies to p75NTR, a marker for
crest-derived cells in the fetal bowel. The p75NTR-immunoreactive
cells were then cultured for 6 days in media supplemented with the
Abstracts
� 2009 Blackwell Publishing Ltd ix
Ret ligand, glial cell derived neurotrophic factor (GDNF; 10 ng ml-1) to
promote neuronal differentiation. Transcripts encoding netrins-1 and -3
were found by RT-PCR in the cultured enteric neurons and in the non-
crest-derived cells, but not in the immunoselected crest-derived pre-
cursors. To test whether enteric neurons take up, as well as synthesize
netrin, the p75NTR-immunoreactive cells were cultured for 6 days in
3-dimensional collagen gels with stably transfected 293-EBNA cells
expressing c-Myc-tagged netrin-1. Controls included crest-derived cells
cultured alone or with parental 293-EBNA cells. In all three types of
culture, neurons (identified by their expression of PGP9.5) were found
to be netrin-immunoreactive. Cultures were immunostained with
antibodies to c-Myc to identify netrin secreted by the transfected
293-EBNA cells. Although the netrin-1-expressing 293-EBNA cells
were c-Myc-immunoreactive, the neurons that developed from the
crest-derived cells were not. Neurons thus did not take up secreted
netrin. To further investigate the role of netrin synthesized by enteric
ganglia, as opposed to from the enteric mesenchyme, protein was
extracted from the bowel of E14 Ret -/-, Ret +/- and Ret +/+ fetal mice.
Quantitative analysis of netrin-1 identified through Western blotting
revealed no significant difference in total amount of netrin-1 protein
per gut. We propose that the source of secreted netrin may play a more
important role than the aggregate amount of enteric netrin in guiding
vagal axons to their correction locations in the developing bowel.
Supported by AGA, CIHR and NS 15547/12969.
A functional approach to understand the role of the Kinesin Binding
Protein (KBP) in Goldberg-Shprintzen syndrome
mm alves,*,� as brooks,�,§ e de graaff,�,§ c hoogenraad,�,§ bjl eggen� &
rmw hofstra**Deptartment of Genetics University Medical Center Groningen, Groningen, The
Netherlands; �Deptartment of Developmental Genetics, Faculty of Mathematics
and Natural Sciences, University of Groningen, Haren, The Netherlands;
�Deparment of Clinical Genetics, Erasmus Medical Center, Rotterdam, The
Netherlands; and §Deparment of Neuroscience, Erasmus Medical Center,
Rotterdam, The Netherlands
Goldberg-Sphrintzen syndrome is a rare autosomal recessive disorder
characterized by polymicrogyria, mental retardation and Hirschsprung
disease. Recently, we showed that all patients suffering from this disease
have inactivating mutations in the KBP/KIAA1279 gene (1). However,
the function of its encoded protein is unknown. Considering that
mutations in KBP are associated with both enteric and central nervous
system defects, clarifying its function will lead to new insights about the
protein network associated with neuronal development. Based on its
primary sequence, KBP is predicted to have two tetratricopeptide repeats
(TPRs). Since the basic function of TPR motifs is to mediate protein-
protein interactions, a yeast two-hybrid screen was performed to identify
possible KBP interacting proteins. We obtained various possible inter-
actors, of which the two major categories were kinesin like proteins
involved in the transport of vesicles and organelles towards the plus end
of the microtubules, and microtubule destabilizing proteins implicated
in axonal growth. These interactions were further confirmed
by co-localization studies, co-immunoprecipitation assays and
expression studies in mice primary cortical neurons. A probable inter-
action with microtubules was also explored, both by co-localization and
in vitro binding assays. Based on the results obtained, we postulate that
KBP plays an important role in microtubule organization/stability
and may also be involved in vesicle transport. In conclusion, our
results bring new insights about Hirschsprung disease and polymicro-
gyria development pointing towards a microtubule associated problem
and present KBP as a new/major player involved in neuronal
development.
Reference:
1 Brooks AS et al. Homozygous nonsense mutations in KIAA1279 are
associated with malformations of the central and enteric nervous
systems. Am J Hum Genet 2005; 77 (1): 120–6.
The association of Bardet-Biedl Syndrome and Hirschsprung disease
highlights the role of the primary cilium in ENS development
j amiel,*,� l de pontual,*,1s thomas,*,1
na zaghloul,�,1dm mcgaughey,�
h dollfus,– c baumann,** ee davis,� a munnich,*,� h etchevers,*
m vekemans,*,� s lyonnet,*,� a mccallion,�,§ t attie-bitach*,� &
n katsanis�*Universite Paris Descartes, INSERM U-781, Paris, France; �Departement de
Genetique, Faculte de Medecine; AP-HP, Hopital Necker-Enfants-Malades, Paris,
France; �McKusick-Nathans Institute of Genetic Medicine Johns Hopkins
University School of Medicine, Baltimore, MD, USA; §Department of Molecular
and Comparative Pathobiology, Johns Hopkins University School of Medicine,
Baltimore, MD, USA; –Service de Genetique Medicale, Hopital de Haute-Pierre,
Strasbourg, France; and **Service de Genetique Medicale, Hopital Robert Debre,
Paris, France
Hirschsprung disease (HSCR) is a model neurocristopathy for under-
standing genetic diseases with a multigenic mode of inheritance.
Roughly 30% of the time, HSCR is associated with other congenital
anomalies as the result of chromosomal rearrangements (mostly Down
syndrome), monogenic Mendelian disorders of all modes of inheritance,
or other, unknown causes. In such associations, penetrance for the
HSCR trait is always incomplete (5–70%), suggesting additional
predisposing genetic factor(s). Bardet–Biedl syndrome (BBS, MIM
209900) is a genetically heterogeneous multisystemic disorder charac-
terized by postaxial polydactyly, progressive retinal dystrophy, obesity,
hypogonadism, renal dysfunction, variable learning difficulties and, in
about 5% of cases, HSCR. BBS proteins are involved in the assembly
and function of primary cilia or the function of basal bodies, affecting
both intraflagellar transport (IFT) and planar cell polarity (PCP). We
previously demonstrated the role of RET as a modifier gene for the
enteric phenotype in certain HSCR-predisposing syndromes (congenital
central hypoventilation, Down, and Bardet-Biedl syndromes) but not all
(Mowat-Wilson and Waardenburg type 4 due to SOX10 gene muta-
tions). This situation suggests that some HSCR cases are RET-depen-
dent and others, RET-independent. Surprisingly, the greatest RET
dependence was observed in the group of syndromic patients with both
BBS and HSCR, although functional interactions between RET and BBS
proteins are not known. We here report the co-segregation of mutations
at one BBS locus and at the RET locus for the HSCR trait to occur in BBS
familial and sporadic cases. We showed that human NCC possess a
primary cilium and express BBS genes as well as many components of
the PCP pathway. Genetic interactions between the BBS and RET
pathways were supported by zebrafish double Ret and Bbs4 morphants
in which enteric neurons were completely absent from the distal gut.
These data emphasize that the role of a primary cilium in NCC
migration and enteric nervous system development has been underes-
timated.
Semaphorin Family 3 Members Are Associated With Hirschsprung
Disease
s arnold,* m guy,* k kashuk,* y li,� g abecasis,� a chakravarti*
& international hscr consortium*,�*IGM, Johns Hopkins Univ Sch Med, Baltimore, MD, USA; and �Ctr for Stat Gen,
Department of Biostatistics, School of Public Health, University of Michigan, Ann
Arbor, MI, USA
Hirschsprung Disease (HSCR) is a complex disorder for which a
growing number of candidate genes and chromosomal regions hypo-
thetically modify the function of the major HSCR gene, the receptor
tyrosine kinase RET. HSCR varies in the length of colon affected by
aganglionosis, from the least severe and most common short segment
disease (S-HSCR) to the least common and most severe form, total
colonic agangionosis (TCA). The transmitted allele for a common RET
enhancer variant, RET+9.7, has greater penetrance with decreasing
severity of disease, while coding RET mutations are more common
with increasing severity of disease. In order to identify other common
variants that might contribute to the majority of HSCR cases, 220
1These authors contributed equally to the work.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdx
S-HSCR trios were analyzed on the Affymetrix 500K SNP array plat-
form. Although RET displayed the highest degree of association by the
Transmission Disequilibrium Test (TDT), a significant cluster of SNPs
was identified in a region on chromosome 7 that fell downstream from
the axonal guidance protein SEMA3D and upstream from additional
SEMA family 3 members (3A, 3E, and 3C, in order of proximity). The
two SNPs with highest significance (SNP1, with P = 6.12 x 10-6 and
SNP2 with P = 5.15 x 10-6) were genotyped in an independent sample
of 420 HSCR trios that represented all segment lengths: both SNPs
maintained significance (P = 2.8 x 10-2 and 3.6 x 10-4, respectively).
Imputation, which added ~2 million SNPs to the analysis, strength-
ened the significance of the SEMA cluster and refined the location of
its peak, which included SNP2. The transmitted SNP2 allele displayed
greatest penetrance in males and in homozygous individuals, analo-
gous to the RET+9.7 enhancer variant. The four SEMA family three
members demonstrated very similar temporal and spatial patterns of
expression throughout the gut and were coexpressed with RET in these
tissues, supporting the possibility that one or all might modify RET
function in the developing enteric nervous system. Coding exons from
SEMA3D and SEMA3A, the two genes closest to the association peak,
were sequenced in 143 individuals with long segment disease or TCA
to identify deleterious mutations. While no novel variant was pre-
dicted to be deleterious by PolyPhen or SIFT analysis, the SEMA3D
variant S65P had a minor allele frequency that was five fold greater in
the HSCR population than in Caucasian controls (0.043 vs 0.009).
TCOF1 mutation affects the susceptibility to Hirschsprung’s Disease
aj barlow & pa trainor
Stowers Institute for Medical Research, Kansas City, MO 64110, USA
Hirschsprung’s disease (HSCR), or congenital aganglionic megacolon,
is a disorder of the digestive tract affecting 1 : 5000 live births. HSCR
is characterised by an absence of enteric ganglia from variable regions
of the colon. These ganglia comprise the enteric nervous system (ENS)
that is derived mainly from a migratory progenitor cell population
called the neural crest (NCC) which arises from the vagal level of the
neural tube at somites 1-7. At embryonic day (E) 9.0–9.5, ventrally
migrating NCC enter the foregut and over the next 4 days colonise the
entire length of the gut. The establishment of a proper functioning
ENS requires the coordination of NCC survival, migration, prolifera-
tion and differentiation. Data from animal models of HSCR reveal that
there is tight regulation of the ncc numbers that initially enter the
foregut and also that the maintenance of a sufficient pool of neural
crest progenitor cells is necessary during migration to ensure complete
colonisation (Barlow et al., 2003; 2008; Bondurand et al., 2006). Tcof1
encodes a nucleolar phosphoprotein known as treacle, that interacts
with upstream binding factor (UBF), and RNA polymerase I (Valdez
et al., 2004) to control ribosome biogenesis, a process vital for normal
cell growth and proliferation. Recently we demonstrated that Tcof1 is
essential for neural crest formation and proliferation during craniofa-
cial morphogenesis. Mutations in Tcof1 lead to a deficiency in the
number of migrating neural crest which is responsible for the cranio-
facial anomalies characteristic of Treacher Collins Syndrome (Dixon
et al., 2006). In addition to its role in governing cranial neural crest cell
development, we have investigated roles for Tcof1 both as an intrinsic
regulator and as a modifier of Hirschsprung’s disease. Using whole guts
isolated from E10.5 to 18.5 Tcof1± embryos immunostained with the
neuronal marker TuJ1, we observed a reduction in neural crest number
and delayed migration until E14.5. However, during later development,
the gut becomes completely colonised revealing that haploinsuffi-
ciency of Tcof1 alone is insufficient to cause HSCR. Interestingly, the
loss of Tcof1 was able to sensitize other mutant backgrounds to HSCR.
Pax3 heterozygote mice for example do not endogenously exhibit an
abnormal ENS phenotype, but do present HSCR at E18.5 when com-
bined with Tcof1 heterozygosity. Our data thus identifies Tcof1 as a
.new gene involved in HSCR susceptibility.
Effects of tissue age, presence of neurons and endothelin-3 on the
ability of enteric neuron precursors to colonize recipient gut:
Implications for cell-based therapies
aj bergner,* r hotta,* rb anderson,* df newgreen� & hm young**Department of Anatomy and Cell Biology, The University of Melbourne,
Victoria, Australia; and �Murdoch Children’s Research Institute, Royal
Children’s Hospital, Victoria, Australia
Introduction: There is currently great interest in the power of cell
therapy to replace diseased or absent enteric neurons in post-natal
humans. However, it is unclear whether neural crest stem/progenitor
cells will be able to colonize colon (i) in which the mesenchyme has
differentiated into distinct layers, (ii) that already contains enteric
neurons or (iii) that lacks a gene expressed by the gut mesenchyme,
such as endothelin-3 (Et-3). Objectives: To examine the effects of age,
presence of neurons and Et-3 on the ability of enteric neural crest-
derived cells (ENCCs) to colonize recipient gut. Methods: Co-cultures
were used to examine the ability of enteric ENCCs from E11.5 mouse
gut to colonize a variety of recipient hindguts. The area or the distance
colonized by donor ENCCs within the explants were compared. To
investigate whether premature neuronal differentiation affects the
ability of ENCCs migration, the proportion of ENCCs expressing
neuronal markers within the recipient gut was also deter-
mined. Results: ENCCs migrated in E14.5 and E16.5 aneural (lacking
enteric neurons) recipient colon in which the external muscle layers
had differentiated, but they did not migrate as far as they did in
younger colon. The reduced migratory ability of ENCCs in older
recipient gut did not appear to be due to premature neuronal differ-
entiation as the proportion of ENCCs expressing the neuronal marker,
Hu, was not significantly different in recipient gut of different ages.
ENCCs failed to enter most recipient E14.5 and E16.5 colon explants
containing enteric neurons, and when they did, they showed very
limited migration. Finally, ENCCs migrated a shorter distance in
recipient E11.5 Et-3-/- colon compared to wild-type recipient
colon. Conclusions: (1) Colonization of older aneural colon by intro-
duced cells is possible, but occurs more slowly than at earlier ages. (2)
A genetic defect in the colon mesenchyme slows but does not prevent
colonization by introduced cells. (3) Compared to colonization of
aneural colon, restitution of neuronal sub-populations by introduced
cells may be difficult to achieve as ENCCs failed to enter most
recipient explants containing enteric neurons.
Transcriptomics approach of the human enteric nervous system
c besmond,* m clement-ziza,* jp jaıs,� s thomas,* h etchevers,* f joubert,�e sarfati,§ c de chaizematin§ & s lyonnet**Hospital Necker, Inserm U781, Paris, France; �School of Medecine, University
Paris-Descartes, Paris, France; �Pathology Department, Hospital Necker, Paris,
France; and §Surgery Department, Hospital Saint-Louis, Paris, France
Objectives: Our aim was to establish the transcriptomic repertoire of
the human enteric nervous system cells, i.e. neuronal and glial cells
from myenteric plexi, as well as of embryonic neural crest
cells. Methods: Starting from adult surgical colon samples, we per-
formed laser capture microdissection of individual neurons and pat-
ches of glial cells. Neural crest cells were recovered from cultured
neural tubes. As maintaining the highest RNA quality is critical when
performing expression studies on microarrays, we worked out an
alcoholic staining protocol and a heavier than air neutral gas, i.e. argon,
laser capture microscopy procedure. The goal was to prevent rehydra-
tion and subsequent activation of RNases in sample sections from
aqueous staining and air humidity during microdissection time. These
protocols allowed us to unambiguously identify the cells to be
captured and preserve RNA from degradation during up to 90 min of
microdissection time. RNA quantities recovered from these experi-
ments were estimated to range from 100 to 500 picograms. Same
quantities of RNA from the neural crest cells, neuronal and
glial cells were amplified using Nugen WT-amplification Pico
protocol. Results: Data were GC-RMA normalized and principal
component analysis showed that the data were grouped according to
the corresponding biological structures. After Range2 filtering
Abstracts
� 2009 Blackwell Publishing Ltd xi
(fdr<1%), 9866 probesets were selected and aggregated through non-
supervised classification into three sets of overexpressed probesets in
neural crest cells, glial cells and neurons. GO annotations for genes
overexpressed in neural crest cells featured transcription, translation,
replication, mitosis, embryonic development, neural tube formation,
morphogenesis, cardiac development and neural tube closure whereas
glial cells listed immunity, inflammation, cellular adhesion, extra-
cellular matrix, nervous system development and anion transport.
Annotations for neurons featured neurotransmitters metabolism,
synaptic transmission, axonal and dendritic growth and ionic trans-
port. As an example of superposing our differential analysis results to
Kegg networks, we saw that TGF-ß/BMP and WNT canonical path-
ways were activated in neural crest cells. This is consistent with the
fact that Smads, that we found overexpressed in neural crest cells,
transduce signals from the TGF-ß family and associate to transcription
factors to modulate expression of target genes. BMPs are essential to
development of neural crest cells because they influence their migra-
tion and WNT plays a role in induction and differentiation of these
cells.Conclusion: This transcriptomics study should allow us to
highlight the molecular specificities related to the cell types of the
enteric nervous system and putative candidate genes to neurocristop-
athies.
Stronger inhibitory neuromuscular transmission in the ileal
longitudinal muscle of neonatal guinea pigs
x bian
Michigan State University, East Lansing, MI, USA
Neurotransmission to smooth muscle is crucial for normal gut
motility but little is known about postnatal maturation of neuro-
muscular transmission. Postnatal development of neuromuscular
transmission in the gut is an important issue for understanding pae-
diatric motility disorders in the gastrointestinal tract. We investigated
excitatory and inhibitory neuromuscular transmission in vitro using
ileal nerve-muscle preparations from neonatal (£ 48 h postnatal) and
adult (~ 4 months postnatal) guinea pigs. Drugs and electrical field
stimulation (EFS, 20 V, 0.3 ms) were used to evoke cholinergic con-
tractions and non-adrenergic, non-cholinergic (NANC) relaxations in
longitudinal muscle. Immunohistochemistry was used to localise
substance P (SP) and nitric oxide synthase (NOS) in longitudinal
muscle with attached myenteric plexus (LMMP) preparations. In both
adult and neonatel tissues, nicotine (0.3–30 l mol L-1) contracted the
longitudinal muscle in a concentration dependent and tetrodotoxin
(TTX; 0.3 l mol L-1)-sensitive manner. Scopolamine (1 l mol L-1),
a cholinergic muscarinic receptor antagonist, largely inhibited the
nicotine-induced contractions in adult tissues and completely blocked
the nicotinic contractions in neonatal tissues. Neuronal fibers con-
taining SP were abundant in tertiary interganglionic strands in adults,
however, much fewer SP fibers were found in tertiary interganglionic
strands in neonates. In LMMP preparations in both adult and neonatal
animals, NOS immunoreactivity (IR) was present mainly in enteric
neurons in ganglia but very limited NOS-IR in neuronal intergangl-
ionic fibers. Quantitative studies demonstrated that the intensity of
NOS-IR in nerve fibers was higher in neonates than adults. In
the presence of the NOS inhibitor Nx-nitro-L-arginine (NLA;
100 l mol L-1), scopolamine (1 l mol L-1)-resistant nicotine-induced
contractions were revealed in neonatal tissues. NLA (100 l mol L-1)
also enhanced the nicotinic muscle contractions in neonates but not in
adults. The scopolamine (1 l mol L-1)-resistant nicotinic contractions
in adult tissues were enhnced by NLA (100 l mol L-1). Trains (5–25
Hz, 1 s) of EFS evoked frequency-dependent, NLA (100 l mol L-1) and
TTX (0.3 l mol L-1)-sensitive NANC relaxations. Frequency-response
curves in neonates were left-shifted compared to adults. Conclusions:
The neonatal gut is predominantly under the influence of inhibitory
neuromuscular transmission. It is likely that this inhibitory neuro-
transmission in the gut matures earlier than excitatory neurotrans-
mission. Delayed maturation of excitatory motor pathways may
contribute to paediatric motility disturbances.
Ret, caspases, cleavage and cell polarity: the complexity of being a
dependence receptor
jr cabrera, j bouzas, l giraud, s tauszig-delamasure & p mehlen
Laboratory Apoptosis Cancer and Development, CNRS UMR 5238, Center Leon
Berard, University of Lyon 1, Lyon, France
Introduction: Ret is a tyrosine kinase receptor activated by GDNF
Family Ligands (GFLs) that controls proliferation, differentiation and
migration of neural crest cells and derived cells. However we believe
Ret does not act only as a typical tyrosine kinase receptor but also as a
dependence receptor: this means that in the absence of trophic signal
Ret is cleaved by caspases, and this cleavage permits the release of
peptides with different biological activity from Ret full length recep-
tor. Objectives and Methods: Here we characterize a caspase-trun-
cated form of Ret detected in primary culture of neurons and in
transfected cell-lines. Results and Conclusions: We hypothesize
about a putative function of caspase-truncated form of Ret on cell
polarity during migration and axonal growth.
Migration of mouse sacral neural crest cells from their origin to the
hindgut
x wang,* lh bao,* m li,* aj burns� & wy chan**Department of Anatomy, Faculty of Medicine, The Chinese University of Hong
Kong, Hong Kong; and �Neural Development Unit, UCL Institute of Child
Health, London, UK
Enteric neurons and glia in the hindgut are derived from neural crest
cells (NCCs) from vagal and sacral regions. While it has been known
that vagal NCCs undergo extensive migration from the caudal hind-
brain to colonize the entire gastrointestinal tract, information on the
migratory pathways and behaviours of sacral NCCs is still scarce,
especially in rodents. In the present study, we sought to map the entire
migratory pathway of sacral NCCs from the caudal neural tube to the
hindgut in mouse embryos by using cell labelling, whole embryo
culture and immunohistochemical methods, and then examined their
migratory behaviours within the hindgut with time-lapse fluorescent
microscopy. Sacral NCCs caudal to somite 24 began their migration
from the dorsal neural tube at 27–28 somite stage (E9.5) and migrated
to regions around the dorsal aorta at 35–36 somite stage (E10.5). Most
of migrating NCCs expressed both Sox10 and p75. Double immuno-
fluorescence staining showed that these Sox10/p75 double positive
NCCs started to aggregate on two sides of the hindgut at E11.5 to form
pelvic ganglia. At E14.0, sacral NCCs entered the hindgut from pelvic
ganglia through the ventrolateral side of the hindgut at S2 and S3
levels. At this stage, sacral NCCs were closely associated with neuritis
extending from pelvic ganglia. By E14.5, sacral NCCs intermingled
with vagal NCCs migrating craniocaudally within the hindgut
and became morphologically indistinguishable from them. To
further examine their migratory behaviours within the hindgut, we
established an organ culture system, in which movements of green
fluorescent protein (GFP)-labelled sacral NCCs were observed with
time-lapse imaging. Consistent with the observations from immuno-
fluorescence staining, GFP-labelled sacral NCCs from pelvic ganglia
migrated into the hindgut via its ventrolateral side, while the dorsal
region of the distal hindgut seemed to be inaccessible to sacral NCCs.
Upon entering the hindgut, most of sacral NCCs dispersed within the
intestinal mesenchyme, in contrast to vagal NCCs which migrated
mostly in chains along cellular strands. Following arrival of vagal
NCCs to the caudal hindgut, sacral NCCs joined the cellular network
of the presumptive myenteric plexus formed by vagal NCCs. Our data
therefore showed that sacral NCCs were able to migrate from the
neural tube over long distances to enter the hindgut to contribute to
the enteric nervous system.
Acknowledgment: The work was supported by General Research
Funds from the Research Grants Council of the Hong Kong Special
Administrative Region, China (Project nos. CUHK4418/03M and
461808).
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxii
Beta1 integrins are required for the invasion of the caecum and
proximal hindgutby enteric neural crest cells
s dufour, ma breau,1 a dahmani, f broders-bondon & jp thiery2
UMR144 CNRS-Institut Curie, Paris, France
Integrins are major adhesive receptors for extracellular matrix with
various roles in development. We used the Ht-PA-Cre mouse strain to
target the b1 gene disruption in neural crest cell (NCC) derivatives. In
the mutant, b1-null enteric NCCs fail to colonize the gut completely,
leading to an aganglionosis of the descending colon. Moreover, mutant
enteric NCCs form abnormal aggregates in the gut wall, giving rise to a
severe alteration of the ganglia network organisation. Mice producing a
fluorescent protein in b1-null ENCC were generated using the
R26EYFP mouse strain with constitutive expression of the YFP-
encoding gene upon Cre-recombination at the R26REYFP locus. Live
imaging experiments showed that the migration defect occurs pri-
marily during the invasion of the caecum, where b1-null ENCC stop
their normal progression before invading the caecum and proximal
hindgut by keeping abnormaly aggregated. Whilst entering in the
caecum, the speed and the persistence of leading cells movements
were significantly reduced in the mutant. Unlike controls, the
migratory front in mutants was not organised in chains of cells in the
proximal hindgut. The caecum and the proximal hindgut express high
levels of fibronectin and tenascin-C, two well-known ligands of inte-
grins. In vitro, we showed that b1-null enteric neural crest cells
exhibited an enhanced response to the inhibitory effect of tenascin-C
on adhesion and migration, whereas they were unsensitive to the
stimulatory effect of fibronectin. These findings showed that b1 inte-
grins play crucial role at various steps of the enteric nervous system
development. They suggest that b1-integrins are required to overcome
the tenascin-C-mediated inhibition of migration within the caecum
and proximal hindgut and further fibronectin-dependent migration in
these regions.
Functional analysis of enteric nervous system progenitor cells
d edgar,* r lindley,*,� d hawcutt,� s theocharatos* & s kenny�*University of Liverpool School of Biomedical Sciences, Liverpool, UK; and
�Institute of Child Health, Royal Liverpool University Children’s Hospital, Alder
Hey, UK
Neural crest-derived progenitor cells of the enteric nervous system
(ENS) are a potential source of neurons that could be used in future
transplant therapies to help restore normal gut function in patients with
Hirschspung’s disease (congenital aganglionosis of the bowel). We and
others have previously developed techniques to isolate ENS progenitor
cells both from the embryonic mouse and neonatal human gut,
including samples obtained at surgery from Hirschsprung patients.
These cells may be maintained and amplified in culture as neuro-
spheres-aggregates of ENS stem cells and their differentiating neuronal
and glial cell progeny. Subsequently we have gone on to exploit a model
assay system using cultured explants of aganglionic embryonic mouse
gut to demonstrate that after justaposition of neurospheres on the gut
explants, cells migrate out of the neurosphere along the bowel wall and
differentiate into glia and neurons characteristic of the developing ENS.
Importantly, by observing the contractility and calcium fluxes that
develop in the differentiating smooth muscle of the gut explants, we
could show that while the endogenous contractility of the smooth
muscle requires the presence of interstitial cells of Cajal, it is inde-
pendent of the ENS. However, if these cultures are maintained so that
they reach term-equivalent stages of gut development, the neurosphere
transplants exert a regulatory role to coordinate calcium flux and
decrease the frequency of endogenous contractions in the distal colon,
resulting in slower but more powerful contractions. The characteristics
of these contractions are similar to those seen in normoganglionic colon
that has developed to term in vivo. Pharmacological analysis was used
to demonstrate that the regulatory role of the transplanted neurosphere-
derived cells is dependent on neuronal activity and is mediated largely
by nitrergic neurons. Current work is aimed at understanding the
mechanisms that regulate ENS stem cell proliferation and differentia-
tion in situ and when isolated from the neonatal gut. To this end we
have developed a lentiviral transduction technique to permanently label
with green fluorescent protein essentially all cells in cultured neuro-
spheres. This labelling allows us to follow the behaviour of ENS stem
cells and their progeny both after manipulation in culture and after
transplantation into bowel in vitro and in vivo.
Guts, germs, and the enteric nervous system: regulatory interactions
in the developing intestine
js eisen,* j kuhlman,* e mittge� & k guillemin�*Institute of Neuroscience, University of Oregon, Eugene, OR, USA; and
�Institute of Molecular Biology, University of Oregon, Eugene, OR, USA
Normal development and function of the intestinal tract requires
appropriate signalling amongst several different cell types, including
neural crest-derived neurons and glia of the enteric nervous system,
endoderm-derived cells of the gut epithelium, and mesenchyme-
derived muscle cells and interstitial cells of Cajal. The mesenchyme and
gut epithelium produce signals important for enteric nervous system
differentiation. A consortium of intestinal microbes, the gut microbiota,
is necessary for normal development of the gut epithelium, and thus may
have effects on differentiation of the enteric nervous system. One of our
goals is to understand the interactions among host cells and the micro-
biota, which regulate intestinal development and function, and what
happens when those interactions go awry. Here we describe several
mutations isolated in forward genetics screens in zebrafish that affect the
number and distribution of enteric nervous system neurons and how
changes inneuronnumberaffect intestinalmotility.Wealsodescribe the
role of the enteric nervous system in maintaining the integrity of the
intestinal microbial community and how changes in this community are
associated with an inflammatory response in which immune cells infil-
trate the intestine.
Regulation of Sox10 expression and function
m wegner
Institut fur Biochemie, Erlangen University, Erlangen, Germany
The HMG-box containing transcription factor Sox10 is essential for
enteric nervous system (ENS) development. Accordingly, Sox10
heterozygosity in human patients leads to Hirschsprung disease, and
deletion in the mouse causes complete ENS loss. During ENS devel-
opment, Sox10 is first expressed in vagal and enteric neural crest cells
where it is important for survival and maintenance of the undifferen-
tiated state. Later, Sox10 continues to be expressed in enteric glia
arguing that there may be an additional role for Sox10 at this later
stage. To understand the role of Sox10 in the developing ENS, it is
important to know how Sox10 expression is regulated and how its
activity is adjusted to different tasks during consecutive phases of ENS
development. My group has recently identified conserved enhancers
that drive Sox10 expression in different tissues and at different times of
development. These also include two enhancers that drive Sox10
expression in the developing ENS with very similar kinetics.
Additionally, we have obtained some evidence how Sox10 activity is
modulated in vivo and how its function can be adapted in glial cells to
serve stage-specific functions. In addition to functional interactions
with transcriptional activators that exhibit stage-specific expression,
we also detected crosstalks with transcriptional repressors including
proteins of the SoxD family. These results will be presented and dis-
cussed in light of their relevance for ENS development.
Congenital Short Bowel Syndrome
cs van der werf, jbgm verheij & rmw hofstra
Department of Genetics, University Medical Center Groningen, The Netherlands
Introduction: Congenital Short Bowel Syndrome is a rare gastrointes-
tinal disease. The main features patients share are inherited short
small intestines and malrotation. Biopsy specimen show an abnormal
1Present address: National Institute for Medical Research, Division of
Developmental Neurobiology, Mill Hill, London, UK.2Present address: IMCB A*STAR, Singapore.
Abstracts
� 2009 Blackwell Publishing Ltd xiii
appearance of the neuronal cells in the bowel wall in around 10% of
the patients described in literature. Sixty per cent of the cases are
familial. Because boys and girls are both affected and in 25% of
the cases the parents are consanguineous, an autosomal recessive
pattern of inheritance is suspected. Objectives: The aim of
our study was to identify the disease-mutation containing
gene. Methods: Homozygosity mapping was performed using 250K
SNP arrays from Affymatrix on a small consanguineous family with
two affected sibs, one of them was also diagnosed with Hirschsprung’s
disease. We searched for the longest homozygous shared haplotype, as
we hypothesised that this haplotype must contain the disease causing
gene. Using the NCBI database we determined which genes are present
in the shared region and subsequently the genes from this region were
sequenced. Moreover, DNA samples from three published CSBS pa-
tients were requested from the authors that reported these patients and
the data of the SNP arrays of these samples were used to confirm
linkage finding. Results: Homozygosity mapping on the consanguin-
eous family resulted in a longest shared homozygous region of 12 cM
located on chromosome 13. In the shared region 24 genes were located
and sequencing of 9 genes totally and 6 genes partially did not result in
the identification of a disease causing mutation. Finding a heterozy-
gous polymorphism in one of the genes in one of the patients helped us
to narrow down the shared homozygous region. We could not confirm
our linkage finding with the data of the SNP arrays performed on the
three additional families. Discussion and conclusion: We successfully
applied homozygosity mapping to a small family with CSBS and
identified the possible disease-mutation containing region on chro-
mosome 13. So far, sequencing did not result in the identification of
the mutated gene, however, it did make the shared homozygous region
slightly smaller. We could not confirm our linkage finding in the
additional families. Because these patients are from different parts of
the world, we do not expect that they will share the same disease-
mutation. Different mutations in the same gene or multiple genes
might be involved in this gastrointestinal disease.
RET promotor and genetic variation between the diverse ethnic groups
of South African Hirschsprung’s disease
sw moore & mg zaahl
University of Stellenbosch, Tygerberg, South Africa
Introduction: Reported associations between specific RET promoter
and intron 1 variations associated with other common variants and
haplotypes have been shown to interfere with RET function and
increasing the risk to HSCR pathogenesis. A paucity of information
exists about the nature of the promotor region in African populations
although some evidence of variation has been proposed. This study
focuses on specific RET mutations in the promotor region and other
related variations in a population sample of the diverse South African
population groups. Patients and methods: Ethical permission was
obtained and DNA extracted from whole blood samples in 30 patients
with histologically proven HSCR from the three main South African
ethnic groups. PCR products were screened for genetic variation of the
RET by direct sequencing analysis. Results: A sample of 30 HSCR pa-
tients from the three main ethnic groups [viz: Caucasian (10), mixed
(coloured) (10) Black African (10)] were studied. HSCR phenotypes did
not appear to differ between the ethnic groups. RET promotor variations
were detected in 13 patients (43%) situated at -1782A/G (11/30) -1697C/
G (3/30) -1479G/A (1/30) -1449G/A (1/30) -1285G/A (1/30) -1239C/T (2/
30) and -719T/C (8/30). Three patients had a combination of these vari-
ants. All these variants were identified in the heterozygous state except
for 1782A/G (5) and 719T/C (2) which were also identified in the
homozygous state. Four of these variants were identified only in the
Black African population. The intronic variants [SNP1 (rs 2506004) and
SNP2 (rs 2435357)] occurred at a high frequency in the South African
population. Conclusions: Potential disease-related RET mutations
identified in the promotor region in 43% of HSCR patients investigated.
The novel mutations identified in the promotor region adds to our
understanding of HSCR in African patients.
Regulation of proliferation of interstitial cells of Cajal
g farrugia
Mayo Clinic, Rochester, MN, USA
Normal gastrointestinal motility requires intact networks of inter-
stitial cells of Cajal (ICC). Loss of ICC is associated with several
motility disorders. Recent work strongly suggests that the number of
ICC at any given time point is not static. Rather, ICC networks
appear to be maintained by a dynamic, tightly regulated process,
involving, on one hand, growth factors, trophic factors and replen-
ishment from precursor cells and on the other, transdifferentiation
and pro-death factors. When the balance between cell loss and
replenishment is altered the result is a change in the number of
ICC. This presentation will focus on regulation of proliferation of
ICC. The stem cell factor/Kit pathway was the first pathway shown
to be critical in the regulation of ICC development and mainte-
nance. Loss of signaling is associated with decreased ICC numbers
and upregulation of the pathway associated with gastrointestinal
stromal tumors. Nitric oxide has been recently shown to be a
trophic factor with increased numbers of ICC in the presence of
nitric oxide and a decreased number of ICC when neuronally derived
nitric oxide or nNOS are absent. 5-HT has also emerged as an ICC
proliferation factor. ICC express 5HT2B receptors. Activation of the
5HT2B receptor in vitro in ICC cultures induces an increase in
numbers of proliferating ICC via a signaling cascade that involves
activation of PLC, IP3 receptor mediated intracellular calcium
release and activation of the calcium dependent PKCc. More recent
work has focused on determining if there is endogenous activation
of the ICC 5HT2B receptor and whether proliferation occurs in adult
ICC networks in vivo utilizing PKCc knockout mice and adult mice
with gene targeted deletion of the 5-HT2B receptor. The data suggest
that ICC proliferate in adult mouse ICC networks. In primary
culture, ICC derived from PKCc knockout mice do not show a
proliferative response to 5HT2B receptor agonists suggesting that
this PKC is required for the proliferative effect of activation of the
5HT2B receptor. Also, in the absence of the 5-HT2B receptor the
numbers of proliferating ICC in the myenteric plexus region of
whole mount preparations were significantly lower compared with
wild type controls. In conclusion, there appears to be ongoing
proliferation of ICC. This proliferation is regulated by a number of
growth and trophic factors. Alteration in these factors or in their
ligands result in abnormal ICC numbers and therefore modulation of
the pathways may serve to regulate ICC numbers.
Development redux: stimulated neurogenesis in the enteric nervous
system of adult mice
md gershon & m liu
Department of Pathology and Cell Biology, Columbia University, NY, USA
The enteric nervous system (ENS) develops from precursors that
migrate to the bowel from the neural crest. These precursors prolifer-
ate but cease dividing when they terminally differentiate as neurons
(glia remain capable of mitosis). Although a functioning ENS is
obligatorily present at birth to accommodate feeding, enteric neurons
continue to be born through P21. Growth of the bowel, however,
persists and enteric neurons increase in number. Evidence suggests
that the postnatal gut retains neuron-competent stem cells; moreover,
neuronal replacement, which occurs in the CNS, would seen to be
equally necessary in the ENS. Neurogenesis, however, has been diffi-
cult to demonstrate in adult ENS. Conceivably, stem cells might
normally, in adults, be quiescent and persist only in microenviron-
mental niches that are extraganglionic. These hypotheses were tested.
5-Bromo-2¢-deoxyuridine (BrdU) was continuously infused for 1 week
so as to label even infrequently dividing cells; BrdU infusion was fol-
lowed by 2 weeks to 6 months of chase to permit BrdU to be dissipated
in continually dividing cells and migration of BrdU-labelled neuro-
blasts to be detected. Under control conditions, no BrdU incorporation
into neurons was detected; however, if animals were simultaneously
treated with a 5-HT4 agonist (tegaserod or RS RS67506) or colitis was
induced with dextran sodium sulphate (DSS), then cells appeared in
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxiv
which BrdU was co-expressed with markers of mature (HuC/D) or
developing neurons (doublecortin, Sox10, Phox2b, and nestin). 5-HT4
agonists and inflammation stimulated proliferation, detected immu-
nocytochemically with antibodies to Ki67, in �germinal niches�between myenteric ganglia and the longitudinal muscle, where the
doubly labelled HuC/D+BrdU-immunoractive cells were initially
located. After extended periods of chase, BrdU+HuC/D-labelled neu-
rons approached, and by 4–6 months, entered myenteric ganglia. Nei-
ther tegaserod nor RS RS67506 caused HuC/D+BrdU-immunoractive
cells to become manifest in 5-HT4-/- mice; however, such cells were
seen during DSS-induced inflammation. Peripherin-immunoreactive
swellings, which lacked synaptic vesicle markers, were observed at the
ends of neurites in 5-HT4-/- mice and after DSS-induced inflamma-
tion. Electron microscopy confirmed that these swellings represented
nerve terminal degeneration and was accompanied by increased neu-
ronal autophagy. We suggest that enteric neurogenesis occurs in
retained, but extraganglionic �germinal niches� in the adult ENS.
Autophagy in neurites accompanies inflammation and may be linked
to the initiation of neurogenesis. 5-HT4 receptors appear to be neces-
sary for ENS maintenance. Supported by NIH NS12969 and NS15547.
Endothelial cells promote migration and proliferation of enteric neural
crest cells via ß1 integrin signaling
am goldstein,* o mwizerwa* & n nagy�*Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA;
and �Semmelweis University, Budapest, Hungary
Introduction: Enteric neural crest-derived cells (ENCCs) migrate along
the intestine and form an organized network of ganglia that comprises
the enteric nervous system (ENS). The signals directing the migration
of ENCCs, and their patterning into two concentric rings of ganglia,
are largely unknown. Objectives: The purpose of this study was
to identify cellular and molecular signals responsible for regulating
migration and patterning of ENCCs in the developing ENS.
Methods: Neurovascular development was examined during avian gut
development using immunohistochemistry and electron microscopy.
The role of the intestinal microvasculature in ENS formation was
tested in gut organ cultures grown on a variety of cellular and matrix
protein surfaces. ENCC migration, proliferation, and apoptosis were
analysed in these cultures. Results: Endothelial cells are present in
the gut prior to ENCC arrival and are patterned in concentric rings
predictive of the patterning of enteric ganglia. During ENS develop-
ment, ENCCs migrate adjacent to endothelial cells as they colonize
the gut. Inhibiting endothelial cell development using SU5416, a VEGF
receptor inhibitor, leads to intestinal aganglionosis, supporting an
essential role for the intestinal vasculature in ENCC migration. When
intestine is explanted onto cultured endothelial cells, ENCCs migrate
out of the gut and onto the surrounding cells, which promote both
migration and proliferation of ENCCs. This activity is inhibited in the
presence of a ß1 integrin function-blocking antibody. Laminin, which
is strongly expressed by the basement membrane of vessels, also serves
as an excellent substrate for ENCC migration, an activity inhibited by
the same antibody. Conclusions: These results support the existence
of important interactions between integrins on the ENCC surface and
the vascular basement membrane in regulating cell migration and
patterning in the developing ENS.
Sox10± mice as Hirschsprung model for cell replacement therapy with
embryonic stem cell-derived enteric neurons
a. kempe*, h. reichmann*, o. brustle� & g. gossrau**Department of Neurology, University of Dresden Medical Center, Dresden,
Germany; and �Institute of Reconstructive Neurobiology, University of Bonn
Medical Center, Bonn, Germany
The transcription factor Sox10 regulates early neural crest develop-
ment and specification of neural crest-derived lineages. Sox10± mice
reveal an aganglionosis in the intestines comparable to the human
Hirschsprung’s disease (HSCR), the most common developmental
disorder of the enteric nervous system. Because of limits associated
with the current surgical treatment of HSCR, we investigate whether
regenerative tissue repair of the gut using embryonic stem cell-derived
enteric neurons based on enrichment of Phox2b-expressing neural
precursors, represents an attractive alternative therapy for this disease.
Transplantation of ES cell-derived donor neurons in the intestines of
the Sox10 mouse mutants, a model of HSCR, indicates survival and
integration of the transplanted cells. Studies are ongoing to assess the
ability of these donor cells to restore a physiological motility pattern
following their transplantation into dysganglionotic intestines. The
in vitro differentiated putative enteric neurons can potentially serve as
donor cells for regenerative therapy in HSCR.
Genetic and non-genetic mechanisms of enteric nervous system
development
ro heuckeroth
Washington University School of Medicine, St. Louis, Mo, USA
Introduction: Hirschsprung disease is a complex multigenic disorder
with incomplete penetrance and variable expressivity. Many indi-
viduals with Hirschsprung disease also have associated anomalies.
This genetic complexity is a reflection of the elaborate biochemical
machinery required to facilitate ENS precursor migration through
the bowel, control cell proliferation and influence differentiation.
Even after ENS precursors colonize the entire bowel, specific
molecular signals guide neurite growth, control axon specification,
regulate neurotransmitter choice, and determine cell-cell interaction.
Defects in molecular signals controlling this developmental process
cause not only Hirschsprung disease, but also other intestinal
motility disorders. Objectives: To identify new ways to prevent
Hirschsprung disease and other intestinal motility disorders.
Methods: To identify mechanisms critical for ENS development and
for preventing human disease, we have systematically studied gene
expression patterns in the developing bowel, and are actively pur-
suing biochemical pathways controlled by known Hirschsprung
disease susceptibility genes. We are also focused on molecules crit-
ical for cell migration and neurite growth. Results: These studies
led to the observation that many of the regulatory molecules
required for ENS development are influenced by commonly used
medications or by specific micronutrients. We therefore now
hypothesize that non-genetic factors could critically influence dis-
ease severity and penetrance. To test this hypothesis, we have ini-
tiated studies to identify novel gene-environment interactions that
might influence ENS development. Conclusions: These approaches
have the potential to reduce the severity or the penetrance of
Hirschsprung disease and other intestinal motility disorders.
Goldberg–Shprintzen syndrome and microtubules
rmw hofstra,* m alves,* g burzynski,� e de graaf,� c hoogenraad,§
bjl eggen,– i shepherd� & a brooks�*Department of Genetics, University Medical Center Groningen, The Nether-
lands; �Department of Biology, Emory University, Altanta, GA, USA; �Depart-
ment of Clinical Genetics, Erasmus Medical Center, Rotterdam, The Netherlands;
§Department of Neuroscience Erasmus Medical Center, Rotterdam, The Nether-
lands; and –Department of Developmental Genetics, University of Groningen,
The Netherlands
Introduction: The identification of genes involved in syndromic-Hir-
schsprung (HSCR) might give us new and unexpected knowledge on
the pathology of HSCR. We, therefore, focussed on such a syndromic
form of HSCR, Goldberg–Shprintzen syndrome (GOSHS). GOSHS is a
rare, autosomal recessive disorder characterized by polymicrogyria,
mental retardation and HSCR. Recently, we showed that Goldberg–
Shprintzen is caused by inactivating mutations in the KBP gene (pre-
viously called KIAA1279). Since then, we have identified several new
patients with more mutations in KBP, confirming our initial find-
ing Objectives: As both the enteric and the central nervous system
are involved in the disease, the KBP protein seems to play an important
role in neuronal development in general, although the precise function
of KBP is still largely unknown. This study aims at elucidating the
function of KBP. Methods: Based on its primary sequence, KBP is
predicted to have two tetratricopeptide repeats (TPRs). Since the basic
function of TPR motifs is to mediate protein-protein interactions, a
yeast two-hybrid screen was performed to identify possible proteins
interacting with KBP. Results: We determined various possible
Abstracts
� 2009 Blackwell Publishing Ltd xv
proteins, including five kinesin-like ones. These proteins are involved
in the transport of vesicles and organelles towards the plus end of the
microtubules. The most frequently found interactor was a stathmin
protein named SCG10, an important regulatory protein of microtubule
dynamics. Conformation of the two-hybrid data and further studies on
KBP’s functioning are being undertaken. All our data point towards
KBP being involved in microtubule organisation, and possibly in
vesicle transport. Conclusions: Our results show that indeed studying
syndromic HSCR does give new and unexpected knowledge on the
pathologyofGOSHS,andthustoHSCRandpolymicrogyriadevelopment,
with everything pointing towards a microtubule-associated problem.
Small molecule induction of neural crest stem cells from human
embryonic stem cell-derived neural progenitor cells
r hotta,* l pepdjonovic,� rb anderson,* d zhang,� aj bergner,* j leung,�a pebay,�,§ m dottori,�,§ hm young* & df newgreen�*Department of Anatomy and Cell Biology, The University of Melbourne,
Victoria, Australia; �Centre for Neuroscience, The University of Melbourne,
Victoria, Australia; �Murdoch Children’s Research Institute, Royal Children’s
Hospital, Victoria, Australia; and §Department of Pharmacology, The University
of Melbourne, Victoria, Australia
Introduction: Cell replacement therapy using human embryonic stem
cells (hESCs) holds great promise for treating diseases, including neu-
rocristopathies involving a deficit of neural crest (NC) cells such as
Hirschsprung’s disease. Objectives: To develop a robust system for
producing NC-like cells and enteric neurons from hESC-derived neural
progenitors. Methods: A hESC line, in which GFP is expressed by all
stem cells and their differentiated progeny, was used (Costa M et al.
2005). Neural progenitor cells were induced from hESCs using the
BMP inhibitor, noggin, as previously described (Reubinoff BE et al.
2001). To induce NC-like cells, hESC-derived neurospheres were cul-
tured on mouse embryonic fibroblasts (MEFs) for 24 hours and then
exposed to the ROCK1/2 inhibitor, Y27632. Co-cultures between the
neurospheres and explants of aneural embryonic mice gut, and in vivo
transplantation, were used to assess the ability of hESCs-derived
NC-like cells to migrate and differentiate. Results: A small number of
cells migrated from hESC-derived neurospheres grown on fibronectin
or MEFs, but only migratory cells exposed to MEFs expressed the NC
markers, p75 and Sox10. The number of p75+/Sox10+ cells migrating
from hESC-derived neurospheres grown on MEFS was dramatically
increased by exposure to Y27632. hESC-derived cells exposed to MEFs
and Y27632 migrated into explants of aneural embryonic mouse gut
and gave rise to NC-like cells expressing Sox10 or Hu. Some of the Hu+
neurons within the gut explants also expressed nitric oxide synthase,
which is expressed by sub-types of enteric neurons. Cells derived from
segments of hESC-derived neurospheres that had been exposed to
MEFs and Y27632 were implanted into quail embryos. GFP+ cells
migrated along NC pathways and differentiated into neural (Hu+) and
glial (S100b+) cells. Conclusions: We have developed a robust system of
producing NC-like cells from hESC-derived neural progenitors using the
smallmoleculeROCK1/2inhibitor,Y27632.Cellsderivedbythismethod
are able to migrate along NC pathways in avian embryos and within
murine bowel, and to differentiate into cells with neuronal markers.
The bHLH transcription factor Hand2 is necessary for specification and
differentiation of neurons in the enteric nervous system
mj howard, j lei, tj henderhot & p pugh
Medical University of Ohio, Toledo, OH, USA
The basic helix-loop-helix DNA binding protein Hand2 is expressed in
neural crest-derived precursors of enteric neurons and has been shown
to affect both neurogenesis and neurotransmitter specification in both
avian and murine embryos. Expression of Hand2 decreases with
increased developmental age in murine embryos prompting us to
determine the effect of loss of Hand2 following neural specification.
The objectives of this study were to more fully understand the role of
Hand2 in supporting differentiation of neurons, to determine whether
Hand2 has a role in gliogenesis, and to identify molecular mechanisms
of Hand2 function(s) in the developing enteric nervous system (ENS).
We selectively deleted Hand2 in neural precursor cells by crossing our
floxed Hand2 line of mice to mice in which Cre recombinase expres-
sion is under control of the Nestin promoter. Based on in-situ
hybridization, transcript encoding Hand2 is completely absent in the
gut but only partially deleted in the sympathetic chain ganglia. Loss of
Hand2 after neurons have been specified results in death at around
P20. Analysis of the ENS between P8 and P10 demonstrated that tar-
geted deletion of Hand2 in neural precursor cells resulted in a loss of
neurons with the proximal bowel being more severally affected than
the distal bowel. Differentiation of glial cells was also impacted by
deletion of Hand2. Interestingly, targeted deletion of Hand2 in neural
precursor cells results in patterning defects along the entire gastro-
intestinal tract with the distal bowel being more severely affected than
the proximal bowel. Our data indicate that Hand2 is essential to
support neurogenesis and gliogenesis. Proper patterning of the myen-
teric ganglia is dependent upon Hand2. Additionally, Hand2 has a
maintenance function since the number of enteric neurons decreases
with increasing developmental age. We conclude that Hand2 is a
multifunctional transcriptional regulator affecting specification,
differentiation and patterning in the ENS.
Density of nerve fibres changes in the colon of Hirschsprung disease
i jester,* m klotz,� s maas-omlor,� c hagl,� s holland-cunz§ &
kh schafer�*Department of General Surgery, Birmingham Children’s Hospital, Birmingham,
UK; �Department of Biotechnology, University of Applied Sciences Kaiserslau-
tern, Zweibrucken, Germany; �Department of Paediatric Surgery, University
Hospital Mannheim gGmbH, Medical Faculty of University Heidelberg, Mann-
heim, Germany; and §Department of Paediatric Surgery, University of Heidel-
berg, Heidelberg, Germany
Purpose: Various approaches are used to analyse ganglia in Hirsch-
sprung disease (HSCR). The direct relation between muscle tissue
and nerve fibre density was not addressed. Aim of the study was to
evaluate the nerve fibres density (NFD) in the circular muscle layer
in the ganglionic, transitional and aganglionic zone of HSCR.
Methods: Fifteen patients underwent pull-through surgery for HSCR.
The age of children at the time of pull-through surgery was
5.67 ± 5.97 months. Samples were taken along the longitudinal gut
axis and stained (PGP 9.5 (Ultraclone)). NFD were measured in three
regions from the myenteric plexus (mpl) towards the submucous
plexus (spl) individually. Additionally in three of these 15 patients
specimens were analysed for neurotrophic factor GDNF using ELISA.
Control specimens were obtained from four patients at surgical
resection of a colon descendens enterostomy not related to HSCR.
Results: NFD increased significantly (P < 0.01) 30% (mean) within the
circular muscle layer from the myenteric plexus (mpl) towards the
submucous layer (spl) in the ganglionic gut (1759/mm2 (mpl); (2638/
mm2 (spl)) and throughout the transitional zone (proximal: (2267/mm2
(mpl); (3084/mm2 (spl); distal: (1444/mm2 (mpl); (1900/mm2 (spl)).
Additionally nerve fibre density increased 20% between the ganglionic
bowel (2254/mm2) to the beginning of the transitional zone (2697/
mm2) (P < 0.02). In distal regions of the transitional zone (1742/mm2,
as well as in the aganglionic region (1601/mm2), there was a 40%
decrease (P < 0.05). With the increase of nerve fibre density, GDNF
concentration increased from the proximal resection margin
(44.3 ± 29.9 pg ml-1) to the proximal transition zone (62.6 ± 32.4 pg ml-
1) and decreased within the transition zone to the distal end of the gut
(46.7 ± 33.4 pg ml-1). In the control group no graduation of NFD within
the muscle layer was found. Conclusion: The findings demonstrate
an unknown pattern of NFD in the gut of HSCR. The clinical relevance
of the increased NFD and GDNF concentration of the transition zone
is unclear and might reflect a repair mechanism.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxvi
A chemical screen in zebrafish for compounds impairing enteric
nervous system development
ji lake, o tusheva, bl graham & ro heuckeroth
Washington University in St Louis School of Medicine, St Louis, USA
Hirschsprung’s disease is a multigenic and partially penetrant disorder
of the ENS. While great strides have been made in determining the
roles of specific genes in the pathogenesis of the disease, less is known
about the effect of the environment on the developing ENS. We
propose that exposure to clinically used compounds may impair the
development of the ENS and affect disease penetrance in patients
with genetic risk. Such compounds may also provide tools and
suggest targets for further study of the development of the ENS.
Objectives: To screen chemicals in a high-throughput, in vivo system
and identify clinically relevant compounds that impair the develop-
ment of the enteric nervous system and test their roles as enviro-
mental contributors to disease penetrance. Methods: We screened the
Johns Hopkins Clinical Compound Library of 1520 compounds on
developing wild-type zebrafish embryos. Healthy embryos were
exposed to compounds in the library after the formation of the
embryonic gut and the extent of colonization of the distal gut was
measured by immunostaining for neuronal markers and micros-
copy. Results: We present several interesting compounds that impair
the development of the ENS, including sodium valproate. Valproate is an
anticonvulsant in wide clinical use, and is a known teratogen with well-
studied effects on the development of the CNS. Validation and charac-
terization of the results of the screen in zebrafish, in vitro, and murine
systems are ongoing.
Common regulatory mechanisms in enteric lymphoid and neuronal
organogenesis
h veiga-fernades,* a patel,* p pachnis,* m coles,* k foster,* v pachnis�& d kioussis**Division of Molecular Immunology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London, NW7 1AA, UK; and �Division of Molecular
Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, UK
Utilizing the GFP transgenic and knock-in mice has permitted an
analysis of cellular interactions, movement and function in lymphoid
organ development and function. The development of lymphoid organs
occurs during embryonic life as a result of interactions between lymph
node inducing cells and stromal cells. Utilizing human CD2-GFP
transgenic mice we have been able to analyse the molecular require-
ments for the development of the intestinal immune system. The
intestinal immune system consists of Peyers patches, cryptopatches,
intestinal lymphoid follicles and intraepithelial lymphocytes. The
development of Peyers patches follows the aggregation in the gut wall
of Peyers patch inducing cells during embryonic development.
Utilizing imaging technology we have been able to observe the role of
cell movement in the aggregation of Peyers patches. Utilizing a com-
bination of gene expression analyses of Peyers patch inducing cell
populations and genetic approaches we have studied molecules, such
as RET tyrosine kinase receptor and its ligands, which have an
important role in Peyers patch formation. Interestingly RET is a
molecule that is also essential for the mammalian enteric nervous
system (ENS) formation.
Differential proteomics of cultured enteric neural precursor cells
m klotz, u rauch & k-h schaefer
University of Applied Sciences Kaiserslautern/Zweibruecken, Germany
Introduction: The cultivation of isolated enteric plexus and neural
precursor cells obtained from it is important to understand the devel-
opment of the enteric nervous system and its ability for regeneration.
Monitoring the activation and differentiation of neural precursors
grown out of cultured myenteric plexus is a basic step towards using
them as a tool for the treatment of neuronal defects. Objectives: To
characterize the neural progenitors and their behaviour in culture, we
used differential proteomics to find the proteins regulated in neuro-
spheres collected after seven and ten days in culture compared to
isolated myenteric plexus. Methods: We used the 2-dimensional
fluorescence difference gel electrophoresis (DIGE) technique to search
for regulated proteins. Dissected and digested gut samples of newborn
rats were used to collect the myenteric plexus. These samples were
pooled and either used for the culture of which the neurospheres grew
out within 10 days, or directly frozen in liquid nitrogen for proteomics.
Seven and 10 days after their first appearance, neurospheres were
collected, pooled and frozen alike. Proteins of each pool were isolated,
labelled with fluorescent dye and then separated together in
2-dimensional electrophoresis. The overlay images of differentially
labelled myenteric plexus and neurospheres (day 7 or 10) were analysed
for regulated proteins by the Decyder software (GE) and proteins of
interest automatically picked out of the gel. Results: In the DIGE-gel
of plexus vs. neurospheres (day7) we found 55 differentially expressed
proteins out of over 1300 detected in total, plexus vs. neurospheres
(day10) showed 43 regulations. Several of these proteins could be
identified yet, among those ARHGAP17, which is down-regulated in
day 7 and day 10 neurospheres and Dihydrogenpyrimidine dehydro-
genase, which is down-regulated in day 7 and up-regulated in day 10
neurospheres compared to myenteric plexus. Conclusions: The dif-
ferential-proteomics approach is a promising way to characterize
neural progenitors in culture and tissue. It could help to understand
the effects of stimulation or environment on progenitors by providing
information of the reaction s of these cells on a protein level.
Watching Neural Crest Migration: How cells respond to embryonic
growth and microenvironmental signals to assemble the peripheral
nervous system
pm kulesa, m smith, r alexander, da stark, jc kasemeier & r mclennan
Stowers Institute for Medical Research, Kansas City, USA
Since the inception of intravital imaging, a fundamental problem has
been how the decisions of individual cells lead to stereotypical cell
migratory patterns in the vertebrate embryo. We combine our expertise
in the stem cell-like neural crest and high-resolution optical micros-
copy to study the mechanisms that regulate the programmed invasion
of the neural crest in the chick embryo. We show that there is a
coordination of molecular signals that regulate the attraction and
inhibition of neural crest behaviors to sculpt cells from the neural tube
towards specific peripheral targets. Using multicolour cell labelling
and multispectral imaging to accurately identify individual neural
crest cells, we correlate distinct cell morphometric characteristics
with position along a migratory route. Time-lapse analysis of neural
crest cell behaviours and photoactivation cell labelling of migratory
front and trailing cells reveal complex cell interactions that lead to
follow-the-leader behaviour and differences in local cell proliferation.
Our results suggest a model in which cell proliferation at the migratory
front drives neural crest expansion towards peripheral target sites, the
precise invasion of which is regulated by specific receptor-ligand
relationships.
Clonal deletion of RET using mosaic analysis with double markers
(MADM) in the enteric nervous system
r lasrado & v pachnis
National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7
1AA, UK
The enteric nervous system (ENS) is composed of a network of inter-
connected ganglia which reside within the gut wall and control its
motility, secretions and microcirculation. The ENS is one of the most
complex parts of the nervous system containing a vast number of neu-
rons whose axonal trajectory and connectivity is usually distinct from
that of its neighbours. The lack of obvious CNS-type �nuclei� composed
of a homogeneous neuronal population makes the tracing and study of
individual classes of neurons a daunting task. Therefore, the develop-
ment of tools that will allow the identification and study of individual
enteric neurons and their processes is fundamental for uncovering the
logic governing formation of enteric neuronal networks. Ret is a receptor
tyrosine kinase that is critical for the development of the mammalian
ENS. Despite extensive analysis of the developmental role of this sig-
nalling pathway, key questions relating to the in vivo role of Ret in
Abstracts
� 2009 Blackwell Publishing Ltd xvii
neuronal differentiation, connectivity and gangliogenesis remain
unanswered. Recently, we have demonstrated that, in addition to their
cell autonomous effects, mutations of Ret can also affect the early stages
of enteric neurogenesis in a non cell autonomous manner, raising the
possibility that the behaviour of individual neurons is influenced by
their immediate neighbours. To combine morphological and genetic
analysis of individual enteric neural crest cells/neurons, we have adop-
ted a novel approach called Mosaic Analysis with Double Markers
(MADM) which is based on Cre-loxP dependent inter-chromosomal
mitotic recombination. This approach requires the separate knock-in at
identical loci (such as Rosa26) on homologous chromosomes of two
reciprocally chimeric reporter genes (such as GFP and RFP), each con-
taining the N terminus of one marker and the C terminus of the other
interrupted by a loxP-containing intron. Cre-mediated inter-chromo-
somal recombination during mitosis results in two daughter cells
expressing one of the two fluorescent proteins upon G2-X segregation.
Introduction of a null mutation of Ret (which maps distal to the Rosa26
locus) allows the generation of genetic mosaics composed of wild-type
daughter cells labelled with one colour and homozygous mutant sibling
cells with another. We will be reporting on recent progress to study the
morphology, distribution and differentiation of enteric neurons in wild-
type and Ret-deficient MADM clones.
Condition Ptc1 knockout in vagal neural crest cells causes intestinal
hypoganglionosis
thc poon,* esw ngan,* fyl sit,* cc hui,� bj wainwright,� mh sham,§
pkh tam* & vch lui**Department of Surgery, LKS Faculty of Medicine, The University of Hong Kong,
21 Sassoon Road, Pokfulam Hong Kong; �Program in Development & Stem Cell
Biology, The Hospital for Sick Children, University of Toronto, Toronto Medical
Discovery Towers, 101 College Street, Toronto, Ontario, Canada; �Institute for
Molecular Bioscience, The University of Queensland, Brisbane, Queensland,
Australia; and §Department of Biochemistry, LKS Faculty of Medicine, The
University of Hong Kong, 21 Sassoon Road, Pokfulam Hong Kong
Introduction: In vertebrates, vagal neural crest cells (NCC) colonize
the developing gut rostro-caudally giving rise to the neuron and glia
progenitors of the enteric nervous system (ENS). Hh-/- mice showed
patterning and colonization defects and reduction of neurons
(hypoganglionosis) of the ENS. However, the molecular mecha-
nisms by which Hh regulate ENS development are largely
unclear. Objecives: To investigate the cell autonomous functions of
Ptc1 on the vagal NCC/ENS progenitors. Methods: We have gener-
ated special mouse strains in which Ptc1 is deleted in vagal NCC by
Cre-loxP approach, and analysed the development of ENS progenitors
and ENS in embryos and adults. Mice with homozygous knockout of
Ptc1 in vagal NCC were generated by intercrossing of Ptc1flox (floxed
Ptc1) mice with two vagal NCC cre expressing mouse lines (Wnt1-cre
and b3-IIIa-cre mice). Results: In both of these conditional Ptc1
knockout mice, Ptc1-/- ENS progenitors colonized the intestine nor-
mally and displayed no patterning defect, suggesting that the coloni-
zation and patterning defects of ENS in Hh-/- mice was attributed to
the abnormally developed gut mesenchyme in Hh-/- mice. Instead, we
observed reduction of proliferative ENS progenitors in the mutant
embryonic guts, and reduction of enteric neurons in the mutant adult
intestines. Ptc2 was only weakly expressed by the ENS progenitors,
and that Ptc2 was not upregulated in Ptc1-/- ENS progenitors, indi-
cating that loss of Ptc1 activity in vagal NCC/ENS progenitors was not
compensated by an upregulation of Ptc2. Conclusions: Our data
indicated that a deletion of Ptc1 in vagal NCC resulted in a reduced
proliferation of the progenitors in embryonic gut giving rise to hypo-
ganglionosis in adult mice, suggesting a specific cell-autonomous
regulatory function of Ptc1 on the proliferation of ENS progeni-
tors. This work was supported by research grants from the Hong Kong
Research Grants Council (HKU7339/04M; HKU7528/06M) to VCH
Lui.
Contact inhibition of locomotion controls neural crest directional
migration
r mayor, c carmona-fontaine & hk matthews
Department of Cell and Developmental Biology, University College London,
London, UK
The Neural Crest cells have been called the �explorers of the embryos�because they migrate all over the embryo where they differentiate into
a variety of diverse kinds of cells. It has been found in recent years
that many of the factors that control neural crest migration work as
negative signals, such as semaphorins, ephrins, Slit/Robo, etc. How-
ever no positive signal that works as a chemoattractant for the neural
crest has been found so far. Here we propose a novel mechanism for
directional migration of neural crest cells, based on contact inhibition
of locomotion, where no chemoattractants are required. Contact
inhibition of locomotion was discovered by Abercrombie more than
50 years ago to describe the behaviour of fibroblast cells confronting
each other in vitro, where they retract their protrusions and change
direction upon contact. Its failure was suggested to contribute to
malignant invasion. However, the molecular basis of Contact inhibi-
tion of locomotion and whether it also occurs in vivo are still
unknown. Here we show that neural crest cells, a highly migratory
and multipotent embryonic cell population, whose behaviour has
been likened to malignant invasion, exhibit contact inhibition of
locomotion both in vivo and in vitro, and that this accounts for their
directional migration. When two migrating neural crest cells meet,
they stop, collapse their protrusions and change direction. By contrast,
when a neural crest cell meets another cell type, it fails to display
contact inhibition of locomotion; instead, it invades the other tissue,
like metastatic cancer cells. We show that inhibition of non-canonical
Wnt signalling abolishes both contact inhibition of locomotion and
the directionality of neural crest migration. Wnt signalling members
localize at the site of cell contact, leading to activation of RhoA in
this region. These results provide the first example of Contact
Inhibition of Locomotion in vivo, present an explanation for coherent
directional migration of group of cells and establish a novel role for
non-canonical Wnt signalling.
Abnormal enteric nervous system development in a Sox10N-GFP
mouse mutant
m zhang,* c leung,* vch lui,� pkh tam� & mh sham**Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Pokfulam, Hong Kong SAR, China; and �Department of Surgery, Li Ka
Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong
SAR, China
Introduction: SOX10 mutations have been identified in human
Waardenburg–Hirschsprung (WS4) patients who displayed a varied
degree of intestinal aganglionosis. Studies on mouse mutants have
suggested that the Sox10 gene is implicated in modulating the prolif-
eration, maintenance, differentiation and migration of enteric neural
crest cells, but the mechanisms for regulating these processes remain
unclear. Objectives: To elucidate the roles of Sox10 in different
aspects of enteric nervous system development, in particular to visu-
alize the migration behaviour of abnormal enteric neural crest cells
and their derivatives, we have generated a novel mouse mutant
Sox10N-GFP in which the HMG DNA-binding domain and the
transactivation domain of Sox10 have been replaced by the EGFP
reporter. Methods: We analysed abnormal phenotypes of the Sox10N-
GFP mutants by immunohistochemical staining, NADPH-diaphorase
and acetylcholine esterase assays. We isolated and examined mutant
enteric neural crest cells by neurosphere cultures. We also visualized
the migration of the mutant enteric neural crest cells by time-lapse
imaging of gut explants in culture. Results: The heterozygous
Sox10N-GFP mutants displayed WS4 phenotypes including pigmen-
tation defects and megacolon. In Sox10N-GFP/+ mutants, the enteric
neural crest cells entered the midgut by E10.5, but migration of the
enteric neural crest cells was delayed by E12.5 and failed to populate
the full length of the gut at E14.5. Homozygous Sox10N-GFP/N-EGFP
mutants could survive till E13.5, at this stage enteric neurons could be
detected in the mutant oesophagus, but total aganglionosis was
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxviii
observed in the stomach and the gut. We have isolated the Sox10N-
GFP/+ mutant enteric neural crest cells and cultured them at clonal
density in neurosphere cultures. We are currently studying the differ-
entiation potential of the mutant neural crest cells and the abnormal
migration behaviour of Sox10N-GFP enteric neural crest cells by time-
lapse imaging.Conclusions: The Sox10N-GFP mutant serves as a no-
vel mouse model for Waardenburg–Shah Syndrome with abnormal
enteric nervous system development. This project was supported by a
research grant from the Research Grants Council of Hong Kong
(HKU7705/05M) to MHS.
Expansion and differentiation of neural progenitors derived from the
human adult enteric nervous system
m metzger, *,� pm bareiss, � t danker, � t skutella� & l just�*UCL Institute of Child Health, London, UK; �Institute of Anatomy, Tubingen,
Germany; and �Natural and Medical Sciences Institute, Tubingen, Germany
Objectives: A number of age-related gastrointestinal motility disor-
ders are caused by abnormalities of the enteric nervous system (ENS).
Stem or progenitor cell-based therapies may offer the potential of
replacing defective, damaged, or missing neural elements within the
bowel. Here, we describe a method suitable for the preparation of ENS
progenitor cells from human adult gut tissue and assess their trans-
plantation potential in vitro. Material and methods: Human enteric
spheroids from adult small and large gut were generated, expanded and
differentiated dependent on the applied cell culture conditions. For
implantation studies spheres were grafted into foetal slice cultures and
embryonic aganglionic hindgut explants of murine gut. Differentiating
enteric neural progenitors were characterized by BrdU labelling assay,
in situ hybridization, immunocytochemistry, quantitative RT-PCR
and electrophysiological studies. Results: We were able to generate
proliferating neurospheres from adult human gut tissues in vitro.
Spheroid derived cells could be differentiated into neuronal and glial
cells as demonstrated by the expression of a variety of neural markers
and clearly distinguishable sodium currents. Implantation experi-
ments into organotypic intestinal cultures showed the differentiation
capacity of neural progenitors in a three-dimensional environ-
ment. Conclusions: This study provides first evidence that adult
enteric neural progenitors persist in adult human gut tissue and that
they can be propagated in cell culture. Our findings open new possi-
bilities for enteric stem cell research and future development of
autologous cell-based therapies especially in the field of age-related
neurogastrointestinal disorders.
Development and physiology of enteric neurons in Drosophilai miguel-aliaga,* p cognigni,* s groenke� & ap bailey�*Department of Zoology, University of Cambridge, Cambridge, UK; �Institute of
Healthy Ageing, Department of Genetics, Evolution and Environment, UCL,
London, UK; and �MRC National Institute for Medical Research, London, UK
Invertebrate model systems such as Drosophila have greatly
contributed to our understanding of how somatic motor neurons
develop and interact with their target muscles. By contrast, the
power of Drosophila genetics has not yet been harnessed to inves-
tigate the development and physiology of visceral innervation. We
have investigated how and why the Drosophila digestive tract is
innnervated in both larvae and adult flies. In addition to the previ-
ously described innervation of the foregut and anterior midgut by
the stomatogastric nervous system,1 we have found peptidergic and
motor innervation of the hindgut by neurons with cell bodies
located in the central nervous system. These neuronal lineages
express Drosophila homologues of brain-gut peptides such as insu-
lin/IGF/relaxins. Developmental analysis reveals that several of
these fly visceral neurons are initially specified as pioneer neurons
that help form the longitudinal tracts of the embryonic central
nervous system. In the larva, they differentiate as visceral and
neurosecretory .2,3 Interestingly, some of the transcription factors
regulating their identity are similar to those involved in specifying
vertebrate visceral motor neurons. We are currently investigating the
mechanisms regulating targeting of visceral axons to the hindgut
muscles. In parallel, we have developed simple behavioural and
physiological assays to assess gut function in both wild-type flies
and mutants lacking specific peptides, or in which specific visceral
neurons have been genetically �silenced�. This allows us to deter-
mine the contribution of the peptidergic and motor innervation to
the functions of the digestive tract.
References:
1 Budnik et al. The Journal of Neuroscience 1989; 9: 2866–77.
2 Miguel-Aliaga et al. PLoS Biology 2008; 11; 6(3): e58.
3 Miguel-Aliaga and Thor Development 2004; 131(24): 6093–105.
Laminin, acetylcholinesterase and redundancy in ENS development
sw moore & ga johnson
University of Stellenbosch, Matieland, South Africa
Introduction: Laminin-111 and acetylcholinesterase (AChE) are both
involved in the promotion of cell adhesion and neurite outgrowth in
the developing ENS, a secondary function in AChE. We have
previously observed that AChE is able to bind laminin-
111 Objectives: In this study, we investigated the interaction sites of
AChE and Laminin. We also investigated the possibility that AChE
might be mimicking the function of other molecules during the
migration and differentiation stages of ENS develop-
ment. Methods: Preliminary localisation of the AChE molecule to
peripheral anionic site (PAS) region and the G4 domain of the laminin
alpha1 chain. Peptides representing various parts of these sequences
were synthesized and binding investigated by ELISA. Epitope analysis
of seven anti-AChE monoclonal antibodies blocking both cell adhe-
sion in neural cells and the AChE–laminin interaction was conducted
using a microarray of 500 peptides of varying lengths and degrees of
constraint. In silico docking of mouse AChE (1J06.pdb) and
mouse laminin (2JD4.pdb) was done on Hex 4.5. Bioinformatic anal-
ysis of structures was done on ProSite. Results: AChE was observed
on docking to bind to a discontinuous structure, 2718VRKRL, 2738YY,
2789YIKRK and 2817VGRK in the laminin alpha1 G4 domain. ELISAs
using synthetic peptides confirmed this. The major component of the
interaction site on AChE was found to be the sequence 90RELSED,
with additional contributions by 40PPV, 46R (linked to 94E by a salt
bridge) and 61DATTFQ. Epitope analysis of the antibodies showed
their major recognition site to be 40PPMGPRRFL; also cross-reaction
with Proline sequences 78PGFEGTE and 88PNRELSED. Antibodies
without 90RELSED motif recognition interfered with neither lami-
nin binding nor cell adhesion. The laminin site overlaps with the
heparin-binding site, and AChE competed with heparan sulfate
for laminin binding. This suggests possible functional redundancy
between AChE and HSPGs, [specifically, the syndecans, glypicans and
perlecan]. Search on molecules with 90RELSED and subsidiary motifs
yielded the receptor tyrosine kinase Mer, perlecan and the LDL
receptor. Conclusions: By defining sites for the interaction of laminin
and AChE, suggests an active role in developmental ENS cell adhe-
sion. The strong possibility of functional redundancy between AChE
and other molecules is also shown. Evidence suggests likely candi-
dates to include the syndecans, the glypicans, the receptor tyrosine
kinase Mer, the LDL receptor and, especially, perlecan.
Exploring aspects of chromosome 21 genes ITGB2 and SOD-1 in
Hirschsprungs disease
sw moore & mg zaahl
University of Stellenbosch, Matieland, South Africa
Introduction: Established associations between enteric nervous system
(ENS) pathology (e.g. Hirschsprungs disease (HSCR) and Chromosome
21 (e.g. Down’s syndrome, DS-HSCR) suggest a modifying link between
this region and the pathogenesis of an abnormal ENS. In addition to the
known susceptibility genes RET and EDNRB the role of the so-called
Down’s critical region at 21q22.2 remains uncertain. Potential chro-
mosome 21 candidate genes involved in ENS development flanking this
region include SOD1 and ITGB2 (CD18) which may have a modulatory
Abstracts
� 2009 Blackwell Publishing Ltd xix
effect on RET. Objectives: To investigate the genetic background of DS–
HSCR and the influence of the flanking genes SOD-1 and ITGB2 as
possible modifiers of the ENS in DS–HSCR. Methods: DNA extracted
from colonic tissue samples of 16 DS–HSCR [without associated
enterocolitis/cardiac lesions] and 20 sporadic HSCR patients was
screened for mutations in the RET, EDNRB,ITGB2 and SOD1 genes.
Results were compared with 60 control samples (20/ethnic group)
including unaffected Down’s syndrome patients. Polymerase chain
reaction (PCR) amplification was performed on the promoter and coding
region of the genes as specified, followed by heteroduplex single-strand
conformation polymorphism (HEX-SSCP) analysis. PCR products
demonstrating variation in the HEX-SSCP gels were subjected to auto-
mated DNA sequencing analysis Results: HEX-SSCP analysis revealed
RET variations in exons 2B (A45) 13 and 15 predominantly in DS HSCR.
An increased incidence of 561C/T in exon 2 of EDNRB was detected. The
ITGB2 gene demonstrated two known (V367, V441) as well as several
novel polymorphisms (-111T/C, 24G/T, 295G/A, 892A/G). In addition,
seven additional variants were identified in the 3�UTR of the gene. The
ITGB2-111T/C promoter polymorphism was statistically significantly
increased in HSCR (6/20) in the coloured population compared to con-
trols (1/20) (P < 0.05). A variant (D77N) was identified in one of 20 HSCR
patients and variants G567A, A572S, C575S, R586 was identified in four
of 13 DS–HSCR patients. In addition, aberrant HEX-SSCP banding pat-
terns were detected in the promoter region of SOD1 which included
three novel variants with one identified in exon 1 and the other in and
exon 3. Conclusion: In this study we demonstrate a possible interact-
ing chromosome 21 mechanisms involving ITGB2 and SOD genes in
HSCR pathogenesis The identification of several significant genetic
variants in this study indicates its probable role, as modifier loci, in
DS–HSCR phenotypes and possibly HSCR itself. The novel variants
described here, although present in low frequencies, may alter RET
expression.
Characterization and origin of a novel cell type in the avian enteric
nervous system
n nagy,* g csikos,* d molnar,* am goldstein� & i olah**Department of Human Morphology and Developmental Biology, Faculty of
Medicine, Semmelweis University, Budapest, Hungary; and �Department of
Pediatric Surgery, Massachusetts General Hospital, Harvard Medical School,
Boston, MA, USA
The enteric nervous system (ENS) shares embryological, morphological,
neurochemical, and functional features with the CNS therefore, the
gastroenterologists and anatomists may be considered the ENS as the
�brain in the gut�. Both CNS and ENS are structured by neuronal
and supporting cells, namely glial and Schwann or satellite cells,
respectively. In the CNS the micro- or mesoglia contributes to innate
(phagocytosis) and acquire immunities. A cell with similar phenotype to
the microglia has not been identified in the peripheral nervous system
(PNS) including the ENS. Recently we have recognized a cell type in the
PNS, which share many features with the microglia of the CNS. For
collecting information of this type of cells in the ENS we have used
antibodies, which identify; neural crest derived cells, hemopoietic cells
including B and T lymphocytes, macrophages and dendritic cells. Anti-
MHC II antibody was also used to obtain preliminary information about
immune function. In the enteric and sensory ganglia the hemopoietic
marker CD45 recognized a highly ramified cell type. This cell type
express B cell, but not T cell, macrophage and dendritic cell markers. The
CD45+/Bu1b+/RCA (Ricinus communis agglutinin I) I+ phenotype
strongly supports their non-neural crest, but hemopoietic origin. The
presence of MHC class II antigen on their surface suggests their capa-
bility for antigen presentation. Their migratory capability and invasion
into the intestine were proved by chick-quail chimeras: quail hindgut
with nerve of Remak were isolated from 7 days old embryo and trans-
planted into the coelomic cavity of 3 days old chicken embryo. After
14 days of incubation the quail hindgut and nerve of Remak were tested
for chicken specific (CD45, Bu1, MHC II, RCA I) antigens, which re-
sulted in colonization of quail intestine and nerve of Remak by chicken
blood-borne cells, evidencing the immigration of hemopoietic cells into
the ENS. Our experiments indicate that there is a highly dendritic-like
cell in the PNS with identical phenotype of the microglia.
Development of the ENS in Ret51 animals: a mouse model for
understanding human Hirschsprung’s disease
d natarajan,* j dattani� & v pachnis**Division of Molecular Neurobiology, National Institute for Medical Research,
The Ridgeway, Mill Hill, London, UK; and �Department of Mathematics, Uni-
versity of Bath, Bath, UK
The gut motility disorder, Hirschsprung’s (HSCR) disease affects
1 : 4500 births and is characterized by failure of formation of the enteric
ganglia in the distal bowel. The enteric nervous system (ENS) is derived
from a relatively small number of (mostly) vagal neural crest cells which
invade the foregut and undergo extensive proliferation, migration and
differentiation into neurons and glia that make up a complex network of
interconnected ganglia distributed throughout the length of the gastro-
intestinal tract. Processes that disrupt normal development of the ENS
will often result in distal (colonic) aganglionosis. Several genes have
been identified as important regulators of ENS development and muta-
tions in these genes are often detected in patients with HSCR. Among
them, the gene encoding the receptor tyrosine kinase Ret is mutated in
approx. 50% of familial cases of HSCR, while changes in its activity are
thought to underlie all cases of this disorder. In mice, deletion of Ret
results in total intestinal aganglionosis, whereas failure to express the
Ret9 isoform can lead to colonic aganglionosis, a phenotype reminiscent
of the human condition (de Graaff et al, 2001) ). Here, we have used
monoisoformic Ret51 mice as an animal model for HSCR in order to
understand the pathogenesis of this condition, and assess the anatomical
and functional status of the ENS in the colonised (normoganglionic)
parts of the gut. To enable a more detailed analysis of enteric neuro-
genesis in mice homozygous for the Ret51 hypomorphic allele, we
introduced into this background the Rosa26YFPstop allele and the
Wnt1cre transgene, the combination of which marks all populations of
neural crest cells and their descendants, including those colonising the
gut. Our data so far indicate that normal activity of Ret is required for the
proliferation and migration of early neural crest cells. In addition, Ret
function is also required for the differentiation and axonogenesis of en-
teric neurons, as Ret51 homozygous animals have a smaller fraction of
neurons bearing significantly smaller processes. These findings suggest
that the functional abnormalities associated with HSCR are due to both
the lack of enteric ganglia in the colon and the presence of malfunc-
tioning neuronal networks in the ganglionated parts of the intestine.
This hypothesis is currently being tested by further analysis of the mu-
tant phenotype, including electrophysiological characterisation of con-
trol and Ret51 enteric neurons.
Forward Genetic Screen for Regulators of ENS Development
l niswander, t-h kim, y zhang & m hanson
HHMI and University of Colorado Denver, Denver, CO, USA
To gain insight into the complex process of enteric nervous system
development, we have used an unbiased approach of forward genetic
screening in mice to identify mutations that affect ENS development.
The screen was done at embryonic day 18.5 and we scored for the
presence of green or red material in the gut, in contrast to wildtype gut
that is yellow or light orange. We are currently working on three
mutants that derived from this screen. Our goal is to clone the genes
which when mutated cause ENS defects and to determine the mech-
anisms by which these genes act to regulate this critical embryonic
process. We are also characterizing the phenotype and using live
imaging to track the behaviours of the mutant cells to better under-
stand the cause of the ENS defects.
Ret/Gdnf signalling in vagal neural crest-derived neurons of the chick
embryo cloaca
am o�donnell & p puri
Children’s Research Centre, Oor Lady’s Childrens Hospital, Crumlin, Dublin,
Ireland
Introduction: The growth factor, �Glial cell line-Derived Neurotrophic
Factor� (GDNF), is involved in the development of enteric ganglia,
using the tyrosine kinase receptor �Rearranged during Transfection�(RET) to stimulate the proliferation and differentiation of neural crest-
derived precursor cells. To date, the presence of these signalling
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxx
molecules have not been studied in the developing cloaca, thus the aim
of this study was to investigate the distribution of RET and GDNF, and
analyse their co-localisation in vagal-derived neurons of the cloaca
using quail-chick chimera embryos. Materials and methods: Chicken
embryos were incubated until the 10–12 somite stage. The vagal neural
tube was microsurgically ablated in ovo and replaced with the vagal
neural tube from age-matched quail embryos. Quail-chick chimera
embryos were harvested at E12 and E14, fixed and embedded in paraffin
wax, and serially sectioned through the cloaca. Immunohistochemis-
try was performed using human natural killer-1 (HNK-1),
quail-cell-specific perinuclear (QCPN), GDNF and RET antibod-
ies. Results: HNK-1 labelled all ganglia in the myenteric and sub-
mucosal plexuses of the cloaca, while the quail-specific QCPN
antibody labelled all ganglia derived from the transplanted quail vagal
neural tube (Figure 1, A + B). RET and GDNF were found both
co-localised and expressed in separate ganglia in the cloaca (Figure 1,
C + D). The majority of QCPN-labelled vagal-derived neurons also
expressed RET and GDNF. Conclusion: Results show that GDNF and
RET signalling play a major role in ENS development in the chick
embryo cloaca. We have shown, for the first time, that the majority of
vagal neural crest-derived neurons co-express RET and GDNF, thus
highlighting the importance of these signalling factors in cloacal
development.
Effects of protein extracts from human HSD smooth muscle and
TGFß-family members on rat enteric nerve cell survival and
development in vitrou rauch,* m klotz,* e wink,� c hagl� & kh schafer**Department of Microsystems Technology/Biotechnology, University of Applied
Sciences, Zweibruecken, Germany; and �Department of Pediatric Surgery, Uni-
versity Hospital, Mannheim, Germany
The enteric nervous system (ENS) is a complex neuronal network which
is located within the muscle wall of the gastrointestinal tract (GIT).
Because of the importance of the microenvironment on cell survival,
development and function we studied the effects of gut smooth muscle
protein extracts from human Hirschsprung’s disease (HSD) patients on
isolated rat myenteric plexus cells. Due to the fact that sensory fibers
reaching from the gut to the CNS are also in contact with the same
microenvironment, we used DRG neurons to mimic that situation
in vitro. Furthermore we quantified the protein content and measured
the amount of GDNF and TGFbeta within the muscle wall. In parallel
we studied the effects of GDNF, Neurturin, Persephin, Artemin as well
as TGFbeta and bFGF alone and in combination on rat ENS plexus cells
in vitro. Most HSD-extracts induced an increase in neurite length and
neuron number and nearly half of them also an increase in the branching
pattern of isolated rat plexus neurons compared with control (defined
medium). Regarding the effects on DRG-fiber density, most extracts did
not induce a significant change. More than half of all extracts tested so
far induced significantly higher DRG cell numbers while the increase
induced by the other extracts was not statistical significant. The effects
of various neurotrophic factors on cell survival, neurite length and
neurite number per cell strongly depended on factors like age and origin
of cells (small intestine or colon) but especially on synergistic effects of
the factors. In this study we could clearly show that gut muscle extracts
from HSD patients did not have a negative effect on isolated rat plexus or
DRG neurons in vitro. This result is of particular importance, because it
shows that the aganglionic gut might provide a satisfactory micro-
environment for stem cell therapy in the future. Furthermore we could
show the importance of various neurotrophic factors, especially their
interaction, on rat ENS nerve cells in vitro.
MIND the gap: an astroglial perspective on intestinal barrier regulation
tc savidge
University of Texas Medical Branch, Galveston, TX, USA
The blood–brain barrier (BBB) is a specialized tissue-interface that
provides an important homeostatic and immunosurveillance role in
the central nervous system. Unlike most microvascular tissues that
readily promote paracellular passage of solutes and macromolecules,
the BBB is more analogous to polarized intestinal epithelia that restrict
such permeability in order to prevent disease-onset. Recent transgenic
ablation studies have demonstrated that BBB and intestinal tissues also
share a requirement for astroglial-regulated barrier-integrity. This
presentation highlights the emerging concept that astroglia regulate
barrier function at markedly different tissue-interfaces. It also explores
possible lessons that might be learnt by adopting epithelial model
paradigms of the BBB. For example, novel glial-derived S-nitrosylation
signals that regulate intestinal permeability in the digestive tract may
provide new mechanistic insights into barrier function at the BBB.
A better understanding of such universal mechanisms for barrier
regulation will facilitate novel therapeutic strategies targeting
permeability disorders at central nervous system and mucosal tissue
interfaces.
A novel Sox10-NGFP mouse model of Waardenburg–Hirschsprung
Syndrome
mh sham,* m zhang,* kh chu,* eym wong,* dmc chee,* mch cheung,*
vch lui� & pkh tam�Department of *Biochemistry, Li Ka Shing Faculty of Medicine, The University of
Hong Kong, Hong Kong SAR, China; and �Department of Surgery, Li Ka Shing
Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
Introduction: Waardenburg–Hirschsprung (WS4) syndrome is charac-
terized by symptoms including sensorineural hearing loss, intestinal
aganglionosis, and pigmentation defects. Mutations of the SOX10 gene
have been identified in WS4 patients; molecular and mouse mutant
studies have suggested that the neural crest-derived abnormalities are
due to haploinsufficiency of Sox10. Objectives: The aim of this study
is to elucidate the roles of Sox10 during inner ear and enteric nervous
system development by mouse mutant analyses, in order to under-
stand the underlying pathogenic mechanisms for the sensorineural
hearing loss and intestinal obstruction displayed in WS4
patients. Methods: We have generated a novel mouse mutant Sox10-
NGFP in which the N-terminal domain of Sox10 is fused with the
EGFP gene. Using the Sox10-NGFP mutant, and in combination with
the Sox10-Dom, Sox9 or Sox2-GFP mutants, we examined the abnor-
mal development of the inner ear and ENS by morphological and
functional analyses. Results: By cell transfection and chick in-ovo
electroporation, we confirmed that Sox10-NGFP fusion proteins can
be stably expressed and have no transactivation functions. Heterozy-
gous Sox10-NGFP mutants displayed WS4 phenotypes including pig-
mentation defects, megacolon and mild hearing deficiency as
indicated by a slightly impaired auditory evolved brain response (ABR).
The Sox10-NGFP mutant embryos (E10.5) had reduced vestibular
ganglia with fewer neuronal and glial cells. The morphology of the
cochlear and vestibular structures of the Sox10-NGFP mutant
embryos was abnormal, particularly in homozygous mutants (E13.5),
though differentiation of sensory hair cells was not affected. By dye-
tracing, we showed that the innervation pattern of the spiral ganglion
was abnormal in the Sox10-NGFP mutant. Analysis of Sox9; Sox10
compound mutant embryos demonstrated that the inner ear defects
were more severe, indicating that the Group E Sox genes may have
distinct functions in the developing inner ear. On the other hand, in
Sox10-NGFP mutants, the colonization of enteric neural crest cells in
the gut was delayed at E12.5 and aganglionosis of the distal hindgut
was evident by E14.5. Conclusions: Our results on analysis of the
Sox10-NGFP mutant mice show that Sox10 has consistent roles in the
survival and differentiation of the enteric neural crest cells as well as
the progenitor cells in the vestibular ganglia, which would explain the
aganglionosis and hearing defects in WS4 patients.
Microarray–based identification of differentially expressed genes in the
intestines of Zebrafish ENS mutant lessen
i shepherd,* g burzynski,* l petrova,* j ngai� & j-m delalande**Department of Biology, Emory University, Atlanta, GA, USA; and �Department
of Molecular and Cell Biology, University of California, Berkeley, CA, USA
In order to gain insights into the molecular mechanisms that are
involved in the formation of the vertebrate enteric nervous system
Abstracts
� 2009 Blackwell Publishing Ltd xxi
(ENS) we have undertaken a microarray experiment to identify dif-
ferentially expressed genes in the intestine of the previously iden-
tified zebrafish ENS mutant lessen (lsn). The lsn mutant phenotype
is caused by a null mutation in MED24 (TRAP100), a subunit of the
Mediator co-transcriptional activator complex. lsn mutants have
fewer enteric neurons than wild type embryos due to the reduced
proliferation of ENS precursors. MED24 however is not expressed in
the ENS precursors but is expressed in the intestinal endoderm.
Based on these findings we hypothesized that the lsn mutant phe-
notype is caused by the reduced expression of endoderm derived
and/or endoderm regulated factors that are necessary for ENS pre-
cursor proliferation. To test this hypothesis we undertook a micro-
array experiment to identify differentially expressed genes in the
intestines of lsn mutant embryos as compared to wild type embryos
at a critical stage during zebrafish ENS development. In addition to
the microarray approach we also took a candidate gene strategy to
identify potential endoderm secreted ENS precursor mitogens.
Through the microarray experiment we have identified a large
number of genes that have reduced expression in the mutant. Down-
regulated genes include a number of previously identified ENS
associated transcription factors and components of several cell sig-
nalling pathways some of which had been previously implicated in
ENS development. We are currently characterizing the expression
and in vivo function of many of these genes using genetic and
pharmacological approaches. We will present describing these stud-
ies as well as more focused studies of investigating the function of
the Hedgehog and BMP signalling pathways in Zebrafish ENS
development.
Investigation of neural crest stem cell therapy for Hirschsprung’s
disease
n shimojima,* s shibata,� r hotta,* r nishikawa,* n nagoshi,�hj okano,� y morikawa* & h okano�*Keio University School of Medicine, Department of Pediatric Surgery, Tokyo,
Japan; �Keio University School of Medicine, Department of Physiology, Tokyo,
Japan; and �Keio University School of Medicine, Department of Orthopedic
Surgery, Tokyo, Japan
Introduction: Pathogenesis of Hirschsprung’s disease is thought that
migration arrest of neural crest cells results in a congenital absence of
ganglion cells in the distal segment of gut. The current only thera-
peutic choice is a surgical resection of the non-functioning
bowel. Length of the aganglionic (lack of neurons) segment vary and
patients with extensive lesion may need small bowel transplantation.
Therefore, novel therapeutic strategy, alternative to the surgery, is
highly expected. Objectives: The objective of this study is to inves-
tigate a potential of neural crest stem cells (NCSCs) transplantation as
a novel therapy for Hirschsprung’s disease. Methods: To make neural
crest derived cells distinct from other cells, special transgenic mice in
which all neural crest derived cells are enhanced green fluorescent
protein (EGFP) positive were used. Nagoshi N, et al. (2008). Embryonic
gut was used as NCSCs source. After the dissociation of gut, neural
crest derived cells were sorted as GFP positive cells. Sphere-forming
neural crest derived cells were identified after incubation with the
specific culture condition. Multilineage differentiation potency of the
sphere was assessed by immunocytochemistry for b-III tubulin,
smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP)
as neuron, smooth muscle, and glia markers, respectively. For the
transplantation experiment, NCSCs sphere was co-cultured with
recipient embryonic colon. Migration, localization, and differentiation
of transplanted NCSCs were analysed. Results: In the differentiation
assay, neural crest derived cells from mice differentiated into neuron,
smooth muscle, and glia. Transplanted NCSCs have migrated toward
the recipient gut at 16 h from the transplantation and the migration
progressed chronologically. Immunocytochemistry demonstrated the
existence of GFP and neuronal marker PGP9.5 double-positive cells in
migrating cells. Conclusions: We have isolated sphere-forming
NCSCs as EGFP-positive cells from mice embryonic gut. Transplanted
NCSCs successfully localized in the recipient gut and differentiated
into neurons. Although it is still preliminary data, we have started
experiments using human gut samples and similar spheres have been
formed. These data support the possibility of the future NCSCs
transplant therapy for Hirschsprung’s disease. Reference Nagoshi N,
et al. Ontogeny and multipotency of neural crest-derived stem cells in
mouse bone marrow, dorsal root ganglia, and whisker pad. Cell Stem
Cell. 2008;10: 392–403.
The role of neuropilin-1 in neural crest cell invasion
r mclennan & pm kulesa
Stowers Institute for Medical Research, Kansas, KS, USA
During vertebrate development, neural crest cells (NCCs) delami-
nate from the neural tube and migrate in a stereotypical pattern to
specific destinations. In the cranial and post-otic regions, discrete
NCC migratory streams invade the branchial arches to form facial
structures and the gut to pattern the enteric nervous system, yet
signalling mechanisms that produce the migration pattern are still
unclear. Our aim is to explore the function of potential NCC
guidance factors and perform detailed analysis of NCC behaviours in
vivo. Here we investigate a putative NCC guidance cue, neuropilin-
1. Neuropilin-1 is a membrane co-receptor for ligands from the
Semaphorin and VEGF families. When a neuropilin-1 siRNA (Bron
et al, 2004) is used to knock down neuropilin-1 expression in ovo,
NCCs fail to fully invade the second branchial arch and the rostral
portion of branchial arch 3. Detailed cell morphometric and RT-PCR
expression analyses show non-invading neuropilin-1 siRNA
transfected NCCs exhibit a loss of cell polarity, shortened filopodia
and changes in expression of cell adhesion molecules. Motility
and directionality of these non-invading NCCs is rescued by trans-
plantation into the hindbrain (rhombomere 4) of younger host em-
bryos, however neuropilin-1 siRNA transfected NCCs fail to
contribute to cranial gangliogenesis and skeletal elements. Similar
experiments suggest that neuropilin-2 does not influence the initial
formation of the cranial NCC streams. We conclude that neuropilin-
1 is critical to NCC homing into the branchial arches by
maintaining an active motility state and responding to local
microenvironmental signals.
Genetic and genomic approaches to identify gene–gene interactions
that modify severity of aganglionosis in the sox10dom model of
Hirschsprung disease
em southard-smith, se owens, jt harris, lc walters, va cantrell,
km bradley, jr smith & dc airey
Vanderbilty University Medical Center, Nashville, USA
Hirschsprung disease (HSCR) arises from abnormal development of the
enteric nervous system (ENS) and presents as intestinal obstruction
secondary to the absence of enteric ganglia in a variable portion of the
distal intestine. Cumulative evidence suggests HSCR is the conse-
quence of multiple gene interactions that modulate the ability of
enteric neural crest – derived cells to populate the developing gut.
Multiple HSCR susceptibility genes including RET, GDNF, EDNRB,
EDN3, and SOX10 have been identified through patient studies and
additional gene mutations are known to impact ENS development and
function based on studies in mouse models. However, little is known
of how interactions between genes impact the penetrance and severity
of aganglionosis. Few reports of gene-gene interaction effects specifi-
cally on aganglionosis have been published. Limited success identify-
ing relevant gene interactions stems in large part from the inherent
difficulties of studying aganglionosis phenotypes in mouse models
where enteric defects are typically only seen in homozygotes. Fortu-
itously, dominant negative forms of the NC transcription factor Sox10
(Sox10Dom) exhibit aganglionosis with variable penetrance and
severity in heterozygotes analogous to that seen in HSCR patients.
Analysis of Sox10Dom mice across multiple inbred strain backgrounds
demonstrates that a significant proportion of this phenotypic variation
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxxii
results from differences in genetic background. Consequently, the
Sox10Dom model facilitates identification of gene interactions that
impact ENS development and function. We have established extensive
genetic resources that incorporate the Sox10Dom allele with the aim
of mapping modifier genes, natural variants that interact with Sox10 to
alter aganglionosis. Genome wide studies have been combined with
focused analyses of candidate genes in linkage regions that are asso-
ciated with increased severity of Sox10Dom aganglionosis. Comple-
mentation testcrosses, immunohistochemical co-localization within
enteric progenitors and allele-specific expression studies have identi-
fied specific genes that interact with Sox10 to alter migration of enteric
progenitors into the developing intestine. Our results establish a
mechanistic basis for the effect of modifier genes on variation of
phenotype in HSCR disease.
Functional studies on Ret non-coding region mutations and the
involvement of the Nxf/Arnt2; Sim2/Arnt2 heterodimers in the
regulation of RET expression
y sribudiani, j osinga, k bos & rmw hofstra
Department of Genetics, University Medical Center of Groningen, Groningen,
The Netherlands
Ret coding mutations are considered the major cause of Hirschprung
(HSCR) disease. Up to 50% of familial cases and 15–20% of sporadic
cases proved to have such mutations. However, previous studies indi-
cated that non-coding Ret mutations, in particular SNP rs2506004 and/
or SNP rs2435357 both located in intron 1 of the Ret gene, are also
involved in HSCR susceptibility. It was hypothesized that the mutant
variants of those SNPs have an inhibiting effect on the expression of
RET. To test this hypothesis, we generated reporter constructs con-
taining the intronic sequence in which both non-coding Ret mutations
reside (separately or in combination) coupled to Luciferase. Moreover,
two different promoters were used (SV40 and RET). By using the SV40
constructs we have shown that in human and mouse neuroblastoma cell
lines the wild type variants either separately or in combination
enhanced Luciferase expression and that in contrast, both mutants
decreased Luciferase expression. Constructs with the RET promoter
however demonstrated that only the two RET mutant SNPs in combi-
nation are able to decrease the Luciferase expression. By using bioin-
formatics tools, we found that the sequences surrounding SNP
rs2506004 and six copies of similar sequences in the RET promoter are
the binding site for the Nxf/Arnt2 and Sim2/Arnt2 heterodimers.
Binding affinity was checked by Electrophoretic Mobility Shift Assay
(EMSA) and we have shown that Nxf/Arnt2 only binds to the wild type
SNP rs2506004 (-Cgtg-) and loses its binding affinity to the mutant SNP
(-Agtg-). Co-transfection of Nxf/Arnt2 with RET promoter constructs
into mouse neuroblastoma cell lines enhanced Luciferase expression
level by 40-fold compared to its control, proving that the Nxf/Arnt2
heterodimer acts as a transcription activator for RET. By contrast,
co-transfection of Sim2/Arnt2 with RET promoter constructs decreased
the luciferase expression compared to its control. Our data suggest that
SNPs rs2506004 and the six copies of similar sequences in the RET
promoter are likely to be involved in regulating RET expression and this
regulation is controlled by Nxf/Arnt2 and Sim2/Arnt2. This molecular
mechanism might also be a partial explanation for the common associ-
ation of HSCR with Down syndrome as Sim2 is located on chromosome
21.
Functional analysis of RET tyrosine kinase as a dependence receptor
m takahashi,* n asai* & f costantini�*Nagoya University Graduate School of Medicine, Nagoya, Japan; and �Columbia
University, New York, USA
RET encodes a receptor tyrosine kinase essential for the development
of the enteric nervous system and the kidney. Recently, RET was
reported to function as a �dependence receptor� which induces apop-
tosis in the absence of its ligand, GDNF. Without GDNF stimulation,
RET is cleaved by caspase-3 in vitro, thereby releasing a pro-apoptotic
fragment. Amino acids 707 and 1017 in RET are cleavage sites by
caspase-3, and both D707N and D1017N mutations failed to induce
cell death in vitro. We confirmed fragmentation of RET in cultured
human neuronal cells in a caspase-dependent manner, and wild-type
RET induced cell death. To determine the importance of RET pro-
apoptotic activity in vivo, we generated Ret knock-in mice in which
D707N mutation was introduced in the mouse Ret gene. D707
homozygous mutant mice showed hyperplasia of neural cells in the
enteric, sympathetic and parasympathetic nervous systems. However,
the neuron number in the distal half of the colon rather decreased. This
appeared to be due to migration defect of enteric neural crest cells
during embryogenesis. In addition, the adenopituitary lobe in the
pituitary gland was hyperplastic in D707N homozygous mice. These
findings suggest the important roles of RET pro-apoptotic activity for
normal tissue development. I discuss the significance of the results
observed in Ret D707N knock-in mice.
Using a lentiviral vector to label enteric nervous system progenitor
cells
s theocharatos,* s kenny,� r lindley*,� & d edgar**University of Liverpool, School of Biomedical Sciences, Liverpool, UK; and
�Institute of Child Health, Royal Liverpool University Children’s Hospital, Alder
Hey, UK
Understanding the enteric nervous system development is a critical step
in order to develop novel therapies for treating congenital disorders such
as Hirschsprung’s disease (aganglionosis of the bowel). Previous studies
have shown that neural crest-derived progenitor cells (NCCs) of the
enteric nervous system (ENS) migrate rostrocaudally along the bowel
during development and differentiate to form mature neurons and glial
cells. NCCs can be isolated from both embryonic mouse and neonatal
human bowel, and in culture under poorly adherent conditions they give
rise to floating aggregates of both stem cells and their progeny, known as
neurospheres. We have previously shown that transplantation of neur-
ospheres into aganglionic colon explants in vitro can restore the bowel
contractility as neurosphere-derived cells migrate into the explants and
differentiate into mature neurons and glial cells. However, little is
known about the mechanisms that control the migration and differen-
tiation of ENS precursors either in vivo or in vitro. Our current work
aims to track the fate of neurosphere-derived cells both within the
neurosphere and after transplantation into aganglionic bowel using an
EGFP expressing lentiviral vector. This construct utilizes the spleen
focus-forming virus (SFFV) promoter to enable constitutive expression of
EGFP in all cells after transduction. To do this, NCCs were isolated from
E11.5 mouse embryos and cultured in non-adherent conditions for at
least 20 days in order to form neurospheres. These neurospheres were
then dissociated and the resulting single cell population was transduced
by the lentivirus. Preliminary results showed that >85% of the neuro-
sphere cells were EGFP positive and when labelled neurospheres were
allowed to differentiate by attachment to adhesive tissue culture sub-
strates, axon-like fibers expressing EGFP extended from the neuro-
spheres, together with labelled cells. After culturing the transduced cells
for a minimum of 15 further days, EGFP positive neurospheres were
formed and were transplanted into E11.5 mouse colon explants. Single
labelled cells began to migrate from the neurosphere into the explant
during the first 24 h after transplantation. At later time points, EGFP
positive cells had migrated along the entire length of the explant. The
above lentiviral model enables us to transduce both dividing and non-
diving cells and it can be applied in both mouse and human NCCs.
Further steps will involve live cell imaging and immunostaining in order
to examine changes in the morphology and the identity of the EGFP
positive migratory cells.
Abstracts
� 2009 Blackwell Publishing Ltd xxiii
Bcl-xL-mediated rescue of enteric nervous system formation in a
mouse model for Hirschsprung disease
t uesaka & h enomoto
NDR, RIKEN Center for Developmental Biology, Kobe, Japan
The peripheral neurons that control the gut form a network known as
the enteric nervous system (ENS). Mutations in the RET gene are the
primary cause of Hirschsprung disease (HSCR), also known as con-
genital intestinal aganglionosis, in which enteric neurons fail to
develop in the distal colon. We previously demonstrated that condi-
tional ablation of Ret caused neuronal death in the colon and that mice
with diminished Ret expression show features of HSCR. In this mouse
model, aganglionosis was associated with impaired migration and
decreased survival of ENS precursors. (Journal of clinical investigation
2008; 118: 1890–8.). Since Bcl-xL can block enteric neuronal death
triggered by impaired RET signalling in vitro (Development 2007; 134:
2171–81.), we engineered Ret locus to allow elevated levels of Bcl-xL
expression in enteric neural crest-derived cells, and assessed the degree
of contribution of cell death to the emergence of aganglionosis in the
HSCR model mice. Now, we have demonstrated that Bcl-xL
overexpression reduced enteric neuronal loss in the colon by condi-
tional inactivation of Ret and rescue enteric neurons from colonic
aganglionosis in the HSCR model mice. These results strongly suggest
that cell death principally causes HSCR associated with Ret
mutations.
Neurochemical identification of enteric neurons in the larval and adult
intestine of the zebrafish (Danio rerio)
l uyttebroek,* f harrisson,* g hubens,* it shepherd,� j-p timmermans� &
l van nassauw*,�*Laboratory of Human Anatomy & Embryology, University of Antwerp,
Belgium; �Department of Biology, Emory University, Atlanta, GA, USA; and
�Laboratory of Cell Biology & Histology, University of Antwerp, Belgium
In the last decade, the zebrafish has emerged as a leading model
organism for the study of vertebrate developmental biology and has
begun to be used in studies of gastrointestinal congenital diseases.
While the general morphology and development of the enteric nervous
system (ENS) of the zebrafish are already known, specific details
regarding the physiological function and morphological characteristics
of enteric neurons is still incomplete. The aim of the present study is
to unravel the neurochemical coding of zebrafish enteric neurons,
revealing specific subpopulations. Using immunoenzymatic and mul-
tiple immunofluorescent staining methods on isolated intestines from
adult and larval zebrafish, we demonstrated and quantified the
expression of different neurochemical markers representing presump-
tive excitatory, inhibitory and sensory innervation. Three markers
[tyrosine hydroxylase, vasoactive intestinal peptide (VIP), and pituitary
adenylate cyclase-activating polypeptide (PACAP)] were only observed
in enteric nerve fibres, while other markers [calretinin (CR), calbindin
(CB), choline acetyltransferase (ChAT), serotonin (5HT) and neuronal
nitric oxide synthase (nNOS)] were also detected in neuronal cell
bodies. In all segments of the adult intestine, ±50% of the neurons
expressed CR, while ±40% expressed CB, ±40% ChAT and ±20%
nNOS. The proportion of 5HT(+) neurons significantly and progres-
sively decreased from the anterior part (±23%) to the posterior part
(±11%) of the gut. No colocalization was observed between 5HT and
CR or CB, while all CR(+) neurons expressed CB. ChAT colocalized
with CR and CB, but not with 5HT. VIP and PACAP were present from
72 hpf on in the mid- and posterior-gut. nNOS was also found from 72
hpf in these parts, while CR and CB were expressed in the midgut.
From 96 hpf on 5HT was also expressed in the ENS. The present results
indicate that inhibitory neurons are the first to differentiate in the
zebrafish ENS and that they play a significant role in the spontaneous
motor activity of the gut observed between hatching and the onset of
feeding. The results support also previous data that the ENS is well-
developed before the start of feeding. In the adult intestine, the results
are indicative of the presence of several subpopulations of enteric
neurons, and of the existence of regional differences.
Allelic influence of gain-of-function mutant KITK641E in the
development of KIT+ interstitial cells of Cajal in the mouse antrum
jm vanderwinden,* s ralea,* p hague,* p gromova* & b p rubin�*Universite Libre de Bruxelles, Brussels, Belgium; and �Lerner Research Institute
and Taussig Cancer Center, Cleveland, OH, USA
Introduction: Kit, a receptor tyrosine kinase, is essential for the
development of interstitial cells of Cajal (ICC) in the gut wall. Loss-
of-function Kit mutants cause defects in Kit expressing (Kit+) cells,
including ICC, while gain-of-function (oncogenic) mutants cause
either proliferation or defects in various Kit+ lineages. Kit alleles are
known to be co-dominant. Here we have investigated the influence of
the gain-of-function Kit mutant K641E1 in the development of the
muscularis propria in the postnatal mouse antrum. Material and
methods: Mice carrying wild type (WT), gain-of-function (K641E), loss-
of-function (Wv) and null (WZsGreen2) Kit alleles were interbred. In
viable genotypes, Kit+ ICC and smooth muscles were studied by
immunofluorescence for Kit and for alpha smooth muscle actin
immunoreactivity, respectively. Results: Only the WT Kit allele led
to normal development of the two KIT+ ICC populations (ICC-MP &
ICC-IM) and longitudinal muscle layer (LM) in the mouse antrum.
ICC-MP, myenteric ganglia and nerve fibers in the muscularis propria
were present in all genotypes. The gain-of-function allele K641E pro-
moted the proliferation of Kit+ ICC but in absence of WT allele, K641E
was unable to develop ICC-IM and it inhibited differentiation of the
LM. Discussion: This study highlights the different effects of Kit in
differentiation and in proliferation/survival processes. The gain-of-
function allele Kit K641E has a strong proliferative effect on Kit+ ICC,
while it impacts negatively the differentiation of Kit+ ICC-IM and of
LM in absence of WT. The signaling pathways involved and the
interactions between WT and mutant KIT are currently being inves-
tigated.
References:
1 Rubin BP et al. Cancer Research 2005; 65: 6631–39.
2 Wouters MM et al. Physiological genomics 2005; 22: 412–21.
Effects of partial loss of zebrafish intestinal smooth muscle on
migration and differentiation of enteric neural precursors
kn wallace & t akhtar
Biology Department, Clarkson University, Potsdam, NY, USA
Enteric neural precursors migrate and differentiate within intestinal
smooth muscle upon arrival in the digestive system. As a result,
changes in smooth muscle development may alter enteric neural pre-
cursor migration and differentiation. To this end, we disrupted intes-
tinal smooth muscle differentiation to determine if there are inductive
signals provided by this tissue as enteric neurons develop. FKBP9 was
previously shown to be required for differentiation of avian intestinal
smooth muscle. We identified the zebrafish homologue of fkbp9 and
characterized expression. zfkbp9 is expressed within the developing
anterior smooth muscle during the third day of embryogenesis when
smooth muscle is differentiating. To address the role of FKBP9 in
intestinal smooth muscle differentiation we used both FK506, a gen-
eral inhibitor of FKBPs, and a 5� morpholino to the gene. We find that
injection of either FK506 or the morpholino inhibits differentiation of
smooth muscle primarily in the anterior intestine. A morpholino to a
closely related gene, fkbp10, which is also expressed within developing
intestinal smooth muscle, does not result in inhibition of smooth
muscle differentiation. Disruption of intestinal smooth muscle
development by down-regulation of fkbp9 reveals a similar number of
enteric neurons in the anterior intestine with comparable axon pro-
jection. This suggests that either anterior smooth muscle does not
provide inductive signals to enteric neural precursors or remaining
smooth muscle produces sufficient signals. Alternatively, fkbp9 may
not affect early development of smooth muscle and signals for enteric
neuron differentiation may still be produced.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxxiv
L1cam does not interact with Ret during enteric nervous system
development
as wallace, a bergner & rb anderson
Department of Anatomy and Cell Biology, University of Melbourne, Victoria,
Australia
Introduction: Hirschsprung’s disease is a congenital disorder affecting
1/5 000 live births in which there is an absence of enteric ganglia
within the distal gastrointestinal tract. The genetics of Hirschsprung’s
disease are highly complex and non-Mendelian with a male sex bias of
4 : 1. With only 50% of Hisrschsprung’s cases attributable to known
susceptibility genes there may be many unknown genes and gene–gene
interactions responsible for this condition. The X-linked gene L1CAM
has been implicated in several Hirschprung’s cases, making it an
excellent candidate as a modifier gene for the development of
Hirschsprung’s disease. Objective: To examine whether there is
genetic interaction between L1cam and Ret. Methods: We used a two-
locus complementation approach, crossing L1+/- female mice with
Ret+/- male mice. Using the pan-neuronal markers PGP9.5 and Tuj1
we assessed whether the migration of enteric neural crest cells and
their derivatives was altered in L1cam null mutant mice when com-
bined with a heterozygous mutation in Ret at E18.5 gut. Results: We
show that enteric neural crest-derived cell migration is not affected
in L1cam; Ret mutant mice at E18.5. Preliminary data also
suggests that neuronal cell density is not affected in these
animals. Conclusions: This study shows that L1cam does not interact
with Ret to alter neural crest-derived cell migration during the devel-
opment of the enteric nervous system. However, another study from
our laboratory has shown that L1cam can interact with the
transcription factor, Sox10, to disrupt enteric neural crest-derived cell
migration. Therefore, it is important to identify which Hisrschsprung’s
susceptibility genes interact with L1cam.
Permissive action of GDNF on HGF induced neurite growth from
enteric neuron precursor cells
h wang & ro heuckeroth
Washington University, St Louis, MO, USA
Introduction: Hepatocyte growth factor/scatter factor (HGF/SF) regu-
lates the development and function of neuronal precursors in the
central and sympathetic nervous system, but a role for HGF in the
enteric nervous system (ENS) has not been previously reported. Given
the complexity of the enteric nervous system and the number of dif-
ferent neuronal subtypes, the trophic factors currently known to sup-
port ENS development seem unlikely to adequately control neurite
growth. In particular, it may be necessary to selectively control neurite
growth in subsets of enteric neurons. Objectives: To determine if
HGF could influence ENS precursor development. Methods: E12.5
mouse ENS precursor cells were grown in defined medium in dispersed
cell culture or gut slice cultures in the presence or absence of HGF and
GDNF. Immunohistochemistry of the developing and mature ENS for
HGF and its c-met receptor were also performed. Results: Immuno-
histochemistry demonstrated the expression of c-met and HGF in
enteric neuronal precusor cells and in subsets of neurons of mature
ENS. Cell culture experiments demonstrated that although HGF alone
had no effect on neurite growth, HGF enhanced neurite growth when
ENS precursors were grown in media containing low levels of GDNF.
Similar effects of HGF on neurite outgrowth were also observed in
mid-gut slice culture experiments. Specifically, low levels of GDNF
that alone had no effect on neurite outgrowth from gut slices,
permissively increased neurite growth in the presence of HGF. HGF
receptor blocking antibody prevented HGF induced neurite
growth, consistent with a role for c-met and HGF in ENS develop-
ment. Conclusions: These data suggest that HGF signaling may sig-
nificantly influence subsets of ENS precursors and be important for
normal neurite network formation within the ENS.
Abnormalities of enteric nervous system structure and function in
proximal small intestine of the Ednrb model of Hirschrpung disease
kc williams,* g farrugia,� db polk* & em southard-smith**Vanderbilt University Medical Center, Nashville, TN, USA; and �Mayo Clinic,
Rochester, MN, USA
Background: Mutation of the gene that encodes for endothelin recep-
tor B can cause aganglionic megacolon, or Hirschsprung’s disease
(HSCR), in humans. Although surgical removal of the distal agangli-
onic segment rescues patients from life threatening intestinal
obstruction, up to 2/3 of HSCR survivors report ongoing gastrointes-
tinal sypmtoms suggestive of further motility abnormalities, such as
vomitting, abdominal pain, and constipation. Alterations of enteric
nervous system (ENS) proximal to the aganglionic segment of distal
colon may explain why many HSCR survivors continue to suffer
symptoms of gastrointestinal dysmotility. Previous studies of the
Endothelin Receptor B (Ednrb) mouse model of HSCR have demon-
strated that the structure of the ENS in the proximal small intestine
exhibits altered patterning in homozygous (Ednrb-/-) and heterozygous
(Ednrb-/+) mice as compared with wildtype littermates (Ednrb+/+).
Ednrb-/- mice develop aganglionic megacolon, while Ednrb-/+mice
develop ganglia in the distal colon and appear normal. Hypothesis: We
hypothesized that mutation of Ednrb alters the structure and function
of the ENS in the proximal small intestine even when agangliosis does
not develop in the distal colon. The Aim of this study was to evaluate
ENS morphology and function in the proximal small intestine of
Ednrb-/-, Ednrb-/+ and Ednrb+/+ littermates. Methods: Whole mount
preparations of the external duodenal muscle layers of 2-week-old mice
were studied. The volume of ganglia in the myenteric plexus region
was determined from 0.625 lm Zeiss apotome slices by 3D recon-
struction and volume rendering. Electrical field stimulation studies in
normal Krebs solution were utilized to evaluate the response of
smooth muscle to neurotransmitter release in duodenal seg-
ments. Results: The volume of the myenteric plexus was decreased in
homozygote and heterozygote littermates compared with wildtype
littermates. Contractile reponses induced by electrical field stimula-
tion were reduced in homozygote and heterozygote littermates as
compared with wildtype littermates. Conclusions: Our results indi-
cate that mutation of the Ednrb gene can alter proximal ENS structure
and function even when the mutation does not lead to aganglionosis in
the distal colon.
A sensitized mouse mutagenesis screen for modifiers of Sox10
neurocristopathies
wj pavan, de watkins-chow, i matera, k buac, d larson, sk loftus, l hou,
a incao, dl silver, c rivas, ec elliott & ll baxter
National Human Genome Research Institute, National Institutes of Health,
Bethesda, MD, USA
The neural crest is a multipotent cell population that arises during
mammalian development and gives rise to a variety of cell types
including cartilage, bone, melanocytes of the skin, and neurons and
glia of the peripheral nervous system. Disrupting the normal devel-
opment of these lineages can cause debilitating diseases, collectively
referred to as neurocristopathies, that present with a variety of phe-
notypes including deafness, blindness, cleft lip, congenital megacolon
and albinism. As genetic background is known to affect the severity of
neurocristophathies in both humans and mice, we have established an
enhancer screen to identify mutations that increase the phenotypic
severity of Sox10 haploinsufficient mice, a well-characterized mouse
model of human neurocristopathies. In analysis of 400 pedigrees, we
have identified four dominant modifiers of Sox10 neurocristopathies
(Mos1-4) and four recessive phenotypes affecting embryonic Sox10
expression (msp1-4). The causative mutations affect genes involved in
a variety of functions including hedgehog, neuregulin and semaphorin
signalling as well as ribosomal and RNA binding proteins. The phe-
notypes we have identified do not overlap previously known major
mouse spotting loci, thus demonstrating the feasibility of this screen
to provide a more detailed understanding of the critical genes regu-
lating mammalian neural crest development and to provide additional
disease models for human neurocristopathies.
Abstracts
� 2009 Blackwell Publishing Ltd xxv
Dynamic behaviour of immature enteric neurons
hm young,* mm hao,* rb anderson* & h enomoto�*Department of Anatomy & Cell Biology, University of Melbourne, 3010,
Australia; and �RIKEN Center for Developmental Biology, Kobe, Japan
Introduction: While they are migrating along the developing gut, a
sub-population of neural crest-derived cells starts to differentiate into
neurons. It is unknown whether neuronal differentiation halts their
migration. Objectives: To examine the dynamic behaviour of imma-
ture enteric neurons. Methods: Mice in which only immature enteric
neurons express GFP (TH-GFP mice) and mice in which all enteric
neural crest-derived cells express the photo-convertible fluorescent
protein, Kikume (Ednrb-Kik mice) were used. Neurons were imaged in
intact explants of embryonic gut. Results: Although neuronal migra-
tion is common in the developing CNS, it has been assumed that
neural crest cells cease migration at the onset of neural differentiation.
However, around 50% of immature neurons migrated during the
imaging period with an average speed of 15 lm h-. This is slower than
the speed at which the population of neural crest-derived cells
advances along the developing gut. Most migrating neurons migrated
caudally by extending a long leading process followed by translocation
of the cell body. In many migratory neurons, a swollen structure of
variable size would detach from the cell body and move along the
leading process in the direction in which the neuron was migrating.
The segment at the rear would subsequently translocate in the direc-
tion of the leading process and rejoin the swelling. This mode of
migration is different from that of non-neuronal enteric neural crest
cells and neural crest cells in other locations, which have multiple
transient processes that extend and retract rapidly. To determine if the
swollen structure along the leading process contains the centrosome,
antibodies to pericentrin were used. In all neurons examined, peri-
centrin staining was located exclusively in the cell body. Nerve fibres
are closely associated with the network of neural crest cells and their
processes. However, most nerve fibres project directly down the long
axis of the gut whereas neural crest cells migrate spirally around the
gut. Time-lapse imaging revealed that the leading processes of imma-
ture neurons sometimes initially extended in multiple directions
before growing in a caudal direction. Conclusions: Neuronal differ-
entiation seems to slow, but not halt, the caudal migration of neural
crest-derived cells along the gut. Axons follow more linear, longitu-
dinal pathways than the migrating crest cells.
Phactr4 regulates cytoskeletal dynamics in enteric nervous system cell
migration
y zhang, th kim & l niswander
Department of Pediatrics, Section of Developmental Biology, Howard Hughes
Medical Institute, University of Colorado at Denver and Health Sciences Center,
Aurora, CO, USA
Hirschsprung disease arises due to an embryonic defect in formation of
the enteric nervous system (ENS) leading to a reduction of ENS cells in
the gut and gastrointestinal blockage. Here we study the humdy mouse
mutant, with a disruption in the Phactr4 gene, and show there is an
embryonic gastrointestinal defect. Humdy mutant embryos have a
reduced number of ENS cells in the gut. This is due to an ENS
migration defect such that vagal neural crest cells do not migrate
normally. To visualize neural crest cell migration along the mouse gut,
heterozygous humdy mutant mice were crossed with pRet-GFP mice
in which the expression of GFP is under the control of Ret promoter. In
homozygous humdy mutant embryos, only a few cells migrate into the
stomach. Moreover, neural crest migration from the neural tube is also
aberrant. Previous study has shown that the humdy mutation
specifically disrupts interaction with Protein Phosphatase I (PP1).
Here, using neural crest (NC) cultures, inhibition of PP1 by
0.2 mmol L-1 okadaic acid (OA) does not show an obvious effect on cell
migration. This suggests there may be a redundancy of PP1 function in
the regulation of ENS progenitor cell migration.
HOXB5 synergizes TTF-1 in the transcription of human RET
jj zhu, tyy leon, esw ngan, pkh tam & vch lui
The University of Hong Kong, Hong Kong, China
Hirschsprung’s disease (HSCR) is an oligogenic disorder typified by its
complex pattern of inheritance with manifestation of incomplete
penetrance. The RET gene, which is crucial for the development of the
ENS, is reported to be the major gene in HSCR and the haploinsuffi-
ciency of the RET affects the disease penetrance. The other HSCR
genes codifying for transcription factors SOX10, PAX3, PHOX2B, TTF-
1 and HOXB5 have been shown to regulate RET expression. We have
previously reported that HOXB5 can induce RET transcription and
defects in the activation of RET by HOXB5 could lead to RET hap-
loinsufficiency. To investigate the contribution of HOXB5 in the reg-
ulation of RET expression and in the aetiology of HSCR, we sought to
(i) examine if HOXB5 cooperates with these factors in the transcription
of the human RET, and (ii) localize the regulatory element(s) respon-
sible for the HOXB5 induction. Using luciferase reporter assay with
the full length (3.7 kb) human RET promoter, HOXB5 showed a
threefold induction while TTF-1 displayed a 10-fold induction from the
RET promoter. In contrast, we observed a 19-fold induction when
HOXB5 and TTF-1 were co-transfected. However, such synergistic
interaction was not observed when HOXB5 was co-transfected with
either PAX3, PHOX2B or SOX10. We have also localized the regulatory
elements in the human RET promoter responsible for the HOXB5
induction. Taken together all these suggest that HOXB5 may act as a
transcriptional co-activator and synergizes TTF-1 to induce the RET
transcription. HOXB5 may function as a HSCR modifier locus, in
which defects in the activation of RET by HOXB5 co-operating with
TTF-1 lead to RET haploinsufficiency and HSCR.
Temporal patterning of gastroesophageal innervation is abnormal in
rat embryos and fetuses with experimental diaphragmatic hernia
f pederiva, r aras lopez, l martinez & ja tovar
Department of Pediatric Surgery and Research Laboratory, Hospital Universitario
La Paz, Madrid, Spain
Background: Gastroesophageal reflux (GER) has been often described in
babies with congenital diaphragmatic hernia (CDH). Since deficient
innervation of the esophagus has been demonstrated in babies with
CDH, we tested the hypothesis that the temporal patterning of gastro-
esophageal innervation might be abnormal in rat embryos and fetuses
with experimental CDH. Material and methods: Pregnant rats were
given either nitrofen or vehicle on E9.5. Embryos were recovered on E15,
E18 and E21 and 10 control and nitrofen/CDH pups were studied. E15
and E18 esophagi were stained for anti-protein gene product 9.5 (PGP 9.5)
antibody and the intrinsic neural network was examined under confocal
microscopy. The intramural ganglia of E18 and E21 whole mount prep-
aration of stomachs stained for AChE were counted and measured.
Transverse sections of esophagi were immunostained for antineurotro-
phin receptor (p75NTR) and anti-PGP 9.5 antibodies. The relative sur-
face occupied by neural structures over the smooth muscle surface was
measured in the esophagus. Comparison between groups was made with
non-parametric tests. Results: Esophageal neural structures were
underdeveloped on E15 and E18 in nitrofen exposed pups. The relative
neural/muscle surface was decreased in the esophagus of CDH embryos
on E15 and E18 at all levels and tended to normalize on E21. The neural
network of the stomach was similar in control and nitrofen-exposed
embryos on E15 but on both E18 and E21 the number and the
mean surface of ganglia were significantly smaller in CDH
fetuses. Conclusions: Neural structures of the esophagus are sparser
and the ganglia of the stomach are fewer and smaller in rat fetuses with
CDH. These findings confirm the value of this model for further inves-
tigating the mechanisms of upper g.i. dysmotility in patients with
CDH.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxxvi
How many genes for Hirschsprung diease?
a chakravarti
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University,
Baltimore, MD USA
Despite the diversity of genetic models for the inheritance of Hir-
schsprung disease (HSCR) it is clear that it is multifactorial with a
host of genes affecting its pathophysiology. Three features are evi-
dent: (1) The necessity of RET mutations: I shall describe studies
from the International HSCR consortium that demonstrates the RET
effect in all forms of HSCR, the variation in this effect based on
gender, familiality and segment length of aganglionosis and the
complemetary genetic properties of rare coding versus common non-
coding (enhancer) mutations. (2) The existence of many common
polymorphisms in HSCR: recent studies from Paul Tam’s group and
the International Consortium suggests that a common non-coding
variant in Neuregulin 1 (NRG1) and within the Semaphorin 3 cluster
(likely SEMA 3A) affect HSCR. Since statistical power for detecting
these effects were low this suggests many other such undetected
polymorphic factors. (3) Structural abnormalities and copy number
variants impact HSCR: I shall describe new studies from our group
and the International Consortium that show novel dosage abnor-
malities in HSCR including those for the SEMA3A co-receptor
PlexinA2. From a variety of genetic experiments over 70 genes can be
implicated in HSCR and even more discovered. However, most of
them have not been directly tested structurally or functionally. It is
important to create an extensive resource of samples from patients
and their families to test genes, unravel pathophysiology and provide
improved genetic testing to individuals.
Genome-wide association study identifies NRG1 as a susceptibility
locus for Hirschsprung’s disease
pk h tam,* csm tang,� esw ngan,* vch lui,* y chen,* mt so,* tyy leon,*
xp miao,*,� cky shum,* fq liu,* my yeung,� zw yuan,§ wh guo,– l liu,�xb sun,** lm huang,�� jf tou,�� yq song,§§ d chan,§§ kmc cheung,––
kky wong,* ss cherny,�,*** pc sham�,*** & mm garcia-barcelo**Department of Surgery, The University of Hong Kong, Hong Kong, China;
�Department of Psychiatry, The University of Hong Kong, Hong Kong, China;
�Department of Surgery, Shenzhen Children’s Hospital, Shenzhen, China;
§Department of Paediatric Surgery, Shengjing Hospital, China Medical Univer-
sity, Shenyang, China; –Department of Surgery, Beijing Children’s Hospital,
Beijing, China; **Department of Pediatric Surgery, Shandong Medical University,
Shandong, China; ��Department of Surgery, Beijing University, Beijing, China;
��Department of Surgery, Zhejiang Children’s Hospital, Zhejiang, China;
§§Department of Biochemistry, The University of Hong Kong, Hong Kong, China;
––Department of Orthopaedics and Traumatology of the Li Ka Shing Faculty of
Medicine, The University of Hong Kong, Hong Kong, China; and ***Genome
Research Centre. The University of Hong Kong, Hong Kong, China
Background and objectives: Hirschsprung’s disease (HSCR), or
aganglionic megacolon, is a congenital disorder characterized by the
absence of enteric ganglia in variable portions of the distal intestine.
RET is a well-established susceptibility locus, although existing
evidence strongly suggests additional loci contributing to sporadic
HSCR. To identify these additional genetic loci, we carried out
a genome-wide association study using the Affymetrix 500K marker
set. Methods: We genotyped 4 93 840 single-nucleotide polymor-
phisms (SNPs) in 200 Chinese subjects with sporadic HSCR and 306
ethnically matched control subjects. The SNPs most associated with
HSCR were genotyped in an independent set of 190 HSCR and 510
control subjects. Results: Aside from SNPs in RET, the strongest
overall associations were found for two SNPs located in intron 1 of the
neuregulin1 gene (NRG1) on 8p12, with rs16879552 and rs7835688
yielding odds ratios of 1.68 [CI 95%:(1.40, 2.00), P = 1.80 · 10–8] and
1.98 [CI 95%:(1.59, 2.47), P = 1.12 · 10–9], respectively, for the
heterozygous risk genotypes under an additive model. There was also a
significant interaction between RET and NRG1 (P = 0.0095),
increasing the odds ratio 2.3-fold to 19.53 for the RET rs2435357 risk
genotype (TT) in the presence of the NRG1 rs7835688 hetero-
zygote. Conclusions: Our highly significant association findings are
backed-up by the important role of NRG1 as regulator of the devel-
opment of the enteric ganglia precursors. The identification of NRG1
as a new HSCR susceptibility locus not only opens new fields of
investigation into the mechanisms underlying the HSCR pathology,
but also the mechanisms by which a discrete number of loci interact
with each other to cause disease.
A Sox10-Histone2BVenus BAC transgene enables imaging and
isolation of multipotent enteric neural crest-derived progenitors
jc corpening, va cantrell, s byers, kk deal & em southard-smith
Departments of Medicine and Cell & Developmental Biology, Vanderbilt
University, Nashville, TN, USA
Sox10 is a neural crest (NC) transcription factor required for develop-
ment of enteric ganglia and other NC derivatives. Current methods of
visualizing Sox10+ lineages during NC development rely either on
antibodies or histochemical reporters. These approaches restrict anal-
ysis to fixed tissues and are problematic due to cross-reactivity
between Sox gene family members. To enable dynamic imaging and
isolation of viable enteric progenitors (EPs) that express Sox10, we
generated mouse transgenic lines that drive expression of a His-
tone2BVenus (H2BVenus) reporter from Sox10 regulatory regions. The
Sox10-H2BVenus transgene construct relied on modification of a BAC
spanning a 218 kb interval at the Sox10 locus that has been shown to
contain elements necessary to recapitulate Sox10 expression in vivo.
This strategy does not alter the endogenous Sox10 locus and thus
facilitates studies of normal NC-derived progenitors in enteric nervous
system development. The H2BVenus moiety also allows clear dis-
crimination of individual cells as a consequence of nuclear–localized
fluorescence. Expression of our Sox10-H2BVenus BAC transgene
exhibits appropriate temporal and spatial patterns of Sox10 expression.
H2B reporter expression illuminates migration of individual Sox10+
progenitors as they leave the neural tube, traffic through vagal and
sacral pathways and populate the intestine. Immunohistochemical
co-localization of transgene expression with lineage markers demon-
strates appropriate cell-type specific restriction during development
and reveals maintenance of Sox10-H2BVenus in mature enteric glial
cells. H2BVenus+ EPs are readily isolated by flow cytometry and
exhibit the ability to give rise to multi-potent colonies in clonal
cultures. These EPs also readily form neurospheres and are capable of
self-renewal in vitro. Interestingly, differential levels of reporter
expression are evident by both confocal microscopy and flow cytom-
etry due to down regulation of the transgene among distinct popula-
tions as lineage divergence of enteric populations occurs. Analysis of
gene expression in purified populations of enteric Sox10-H2BVenus+
cells identifies transcription of multiple stem cell genes, some of
which have previously not been recognized in enteric lineages. The
ability to image, isolate and compare expression between NC lineages
based on Sox10-H2BVenus trangene expression opens multiple options
for investigating distinct lineages and the effects of mutant alleles
during NC development.
Neural stem cell transplantation in the enteric nervous system:
roadmaps and roadblocks
pj pasricha
Stanford University, Stanford, CA, USA
The use of neural stem cells for the restoration of function in the
aganglionic gut is becoming increasingly feasible with several experi-
mental studies showing that CNS- or ENS-derived neural stem cells
(NSC) can be successfully transplanted into the gut and are capable of
modulating neuromuscular activity. However, in order for the promise
of cell replacement therapy to become a clinical reality several
important questions remain to be answered, including: (i) does func-
tional restoration require faithful recreation of myenteric ganglia and
related structures? (ii) what is the ideal stem cell source for trans-
plantation? (iii) what is the most appropriate route of stem cell
administration? (iv) what is the best approach (including in vitro
preparation and post-transplantation manipulation) to achieve an
appropriate, functional, and long-lasting integration of transplanted
stem cells into the host tissue? (v) what should the first clinical targets
be? This review will suggest possible pathways to clinical trials as well
as highlight the many gaps in our knowledge.
Abstracts
� 2009 Blackwell Publishing Ltd xxvii
Role of Rac GTPases in the ENS network formation and patterning
v sasselli, s bogni & v pachnis
National Institute for Medical Research, Mill Hill, London, UK
The functional development of the enteric nervous system (ENS)
requires newly generated neurons and their progenitors to migrate to
their appropriate sites, extend neurites and dendrites to suitable loca-
tions and, finally, establish synaptic connections with the appropriate
targets. Very little is known about the molecular mechanism under-
lying these processes, however recent evidence points towards a
potential role of Rho GTPases as key components in ENS patterning
and circuitry formation. Our current study addresses the in vivo role
and possible genetic interactions of two members of the Rho GTPase
family, Rac1 and Rac3, in enteric neurogenesis. Taking advantage of
the Cre/loxP recombination system and a Rac1 conditional inactiva-
tion mouse strain (Rac1flox/flox), we generated a Sox10Cre; Rac1flox/
flox; R26StopYFP mouse line, where Rac1 gene is specifically ablated
in the neural crest population, while Cre recombinase activity is
monitored by YFP fluorescence. Secondly, we generated double
Rac1Rac3 mutant animals by crossing the Sox10Cre; Rac1flox/flox;
R26StopYFP mouse line to a constitutive Rac3 KO strain (Rac3-/-).
Rac3-/- mice are fertile, survive to adulthood and do not show any
apparent ENS phenotype. In this analysis we therefore compared single
Rac1 mutants and double Rac1Rac3 mutants for possible genetic
interaction between the two loci. Rac1 and Rac3 are expressed in
enteric neural crest cells (ENCCs) during development. Genetic abla-
tion of Rac1 results in a delayed migration of ENCCs detectable from
embryonic day (E) 11.5 and maintained at least until E15.5, when Rac1
deficiency causes embryonic lethality. This delay in migration could
not be attributed to impaired proliferation or survival of progenitor
cells. Nevertheless, Rac1 mutant ENCCs exhibited a clear deficit in
migration using in vitro assays, and shorter neurite length both in vitro
and in vivo. Interestingly, Rac1Rac3 mutants showed no additional
migratory defects compared to single Rac1 mutants, but axons of
enteric neurons had an abnormal projection pattern at E11.5 and E12.5.
At these stages longitudinally oriented tracts become obvious, espe-
cially at the migratory wavefront. In Rac1Rac3 mutant guts, single
processes, or bundles of them, seem to have lost their normal longi-
tudinal and caudal projection pattern and they now project either
circumferentially or randomly. Ongoing analysis is addressing the
migratory behaviour and morphological features of ENCCs and enteric
neurons in Rac1 and Rac1Rac3 mutants, which might help to dissect
the specific role of the Rac subfamily of Rho GTPases on enteric
neurogenesis.
Enteric nervous system: too complex to understand?
v pachnis
National Institute for Medical Research, Mill Hill, London, UK
The enteric nervous system (ENS) is one of the most complex parts of
the nervous system in vertebrates. The complexity of the ENS is due to
the vast number of neurons and glial cells it contains, the large number
of distinct neuronal subtypes and the difficulty to predict the position
or axonal projection of a given neuron within the enteric ganglia. As
the mechanisms underlying the development of the ENS become
clearer, a new challenge emerges, namely the understanding of the
logic governing the formation of a highly complex and integrated
neuronal network from a relatively small number of undifferentiated
neural crest progenitors. I will report on studies which address the
dynamic regulation of enteric neurogenesis and gliogenesis at prenatal
and postnatal stages and discuss experiments that identify genetic
regulators of axonal pathfinding during ENS development.
Temporal regulation of neurogenesis in the enteric nervous system
catia laranjeira,* nicoletta kessaris� & vassilis pachnis**Division of Molecular Neurobiology, MRC National Institute for Medical
Research, London, UK; and �The Wolfson Institute, UCL, London, UK
The enteric nervous system (ENS) is a complex network of neurons
and glia within the gut wall which originate from neural crest cells.
Recently, a number of cell culture studies have shown that self-
renewing multipotential progenitors of the ENS can be isolated from
the gut of foetal mice and rats as well as from newborn and adult
animals. Despite these studies, the identity of the multilineage pro-
genitor of the ENS and the regulation of its neurogenic potential in
vivo, are currently unknown. Sox10 is an HMG-containing transcrip-
tional regulator which is expressed in progenitors of the ENS and
in enteric glia but is absent from enteric neurons. To establish whether
Sox10 is expressed in multipotential progenitors of the ENS in vivo, we
have combined the Sox10-Cre transgene with the R26ReYFP reporter
allele to lineally mark the progeny of Sox10-expressing cells. Our
analysis shows that in adult animals both the Sox10- neurons and
Sox10+ glia cells are derived from a common pool of Sox10-expressing
progenitors. To examine the temporal regulation of the neurogenic
potential of Sox10+ ENS progenitors, we generated the Sox10iCreERT2
transgenic line, in which expression of a tamoxifen inducible Cre
recombinase is under the control of the Sox10 promoter. Analysis of
Sox10iCreERT2; R26ReYFP double transgenics exposed to tamoxifen
at different time points showed that the neurogenic potential of Sox10-
expressing progenitor cells decreases progressively during embryogen-
esis and is undetectable at some point between P30 and P84. These
findings raise the question of the origin of multilineage ENS progeni-
tors isolated from cultures of postneurogenic gut. To address the
possibility that such progenitors originate from Sox10-expressing glial
cells, we cultured dissociated myenteric plexus of Sox10iCreERT2,
R26ReYFP double transgenics exposed to tamoxifen at p84. In such
cultures, glial cells proliferate, and at least a subset of them can give
rise to nNos+, VIP+ and NPY+ neurons. Similar results were obtained
using the hGFAPCreERT2; R26ReYFP transgenic mice. Taken
together, our data suggest that, although Sox10-expressing cells in the
ENS of adult animals loose their neurogenic capacity in vivo, they can,
under certain conditions be activated to generate self renewing,
multipotential progenitors.
Ultrasound-guided grafting of ENS progenitor cells into murine
embryonic gut in vivok sandgren & v pachnis
National Institute for Medical Research, Division of Molecular Neurobiology, The
Ridgeway, Mill Hill, London, UK
Hirschsprung’s Disease (HSCR) is a congenital disorder in which
enteric neurones are absent from varying lengths of the distal bowel.
The defect causes tonic contraction of the affected bowel segment
resulting in severe intestinal obstruction. Current treatment is surgi-
cal resection of the aganglionic segment. Transplantation of ENS or
other neural progenitors into aganglionic or abnormally innervated
bowel to restore ENS function has been suggested by several groups.
Aim of this study is to examine the feasibility of ENS progenitor cell
(EPC) transplantation as a means of rescuing the neuronal deficit in
HSCR. Microinjection of EPCs into isolated embryonic guts or into
whole embryos kept in organotypic cultures in vitro is a valuable tool
to study the ability of grafted cells to survive, proliferate, migrate, and
differentiate and also their capacity to colonize both wild-type and
aganglionic gut. In the present study, we developed a new method for
specifically targeting the gut in mouse embryos in vivo using an
ultrasound-based injection system. Reasons for establishing this sys-
tem are that we want to follow the fate of grafted EPCs within
embryonic gut in vivo providing a �niche� for neural crest stem cells.
Further, when considering postnatal grafting many important muta-
tions that mimic HSCR will die within a few days to a couple of weeks
after birth. Therefore transplantation into embryonic gut in vivo in
mutants with congenital aganglionosis prolongs the time window for
evaluating the potential of grafted EPCs to colonize aganglionic gut. In
this study EPCs were isolated from embryonic guts of the double
transgenic TgWnt1Cre/R26YFPStop mice and grafted into embryonic wild-
type intestine using ultrasound backscatter bio-microscopy. The
transplanted guts were harvested after 2–8 days, and evaluation was by
immunostaining for green fluorescent protein (GFP) and the neuronal
marker TUJ1. The preparations were analysed using epifluorescence
and confocal microscopy. Murine embryonic gut could be visualized
and EPCs grafted from embryonic stage (E) 10.5 onwards. Grafted cells
could be detected in approx. 50–70% of the transplanted guts as shown
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxxviii
by immunostaining for GFP. The overall survival of the embryos
varied between 73% and 84%. The successful outcome of such
experiments will provide information regarding molecular and cellular
mechanisms that control the migration and differentiation of neural
crest cells and thereby improve our understanding of diseases affecting
the ENS, and help in the search for new treatment modalities.
Effects of genetic background on ENS development in the Sox10Dom
model of Hirschsprung disease
lc walters, va cantrell & em southard-smith
Departments of Medicine and Cell & Developmental Biology, Vanderbilt
University, Nashville, TN, USA
Abnormalities in development of enteric neural progenitors (ENPs) can
lead to aganglionosis in a variable portion of the distal intestine,
causing Hirschsprung disease (HSCR). Cumulative evidence suggests
that variation in HSCR is the consequence of gene interactions that
modulate the ability of ENPs to populate the developing gut. Sox10 is
an essential gene for enteric ganglion development. Sox10Dom mice on
a mixed genetic background exhibit variable aganglionosis. We have
established congenic lines of Sox10Dom mice on distinct inbred genetic
backgrounds, C57BL/6J (B6) and C3HeB/FeJ (C3Fe). These lines differ
in penetrance and extent of aganglionosis. To define the impact of
genetic background on processes during enteric nervous system
ontogeny, we assayed these congenic lines for differences in migration,
lineage potential and proliferative capacity of ENPs. We analyzed ENP
migration in E12.5 guts by whole-mount immunohistochemistry. Both
strains of Sox10Dom mice displayed deficits in migration and decreased
density of enteric NC. However, the phenotype of B6 Sox10Dom
embryos was more pronounced with approximately half the cell
density and one third the migration as their wild-type littermates.
Differences in developmental potential were assayed by isolating
enteric NC stem cells (eNCSC) from Sox10Dom congenic lines by flow
cytometry. eNCSC were purified on the basis of p75/HNK-1
immunolabeling and cultured at clonal density. Immunohistochem-
istry was applied to identify cell lineages within the resulting colonies.
We observed significant differences in lineage potential, capacity for
multipotency, and total number of viable colonies between the strains
and genotypic classes. In particular, eNCSC from B6 guts have a
greater potential to develop a glial fate, while those from C3Fe guts are
more inclined toward a neuronal fate. We analysed proliferative
capacity by immunohistochemistry on dissociated E12.5 ENPs. The
Sox10Dom mutation had no effect on proliferation in either strain,
regardless of the gut region analysed. Our analysis indicates that the
deficiencies of enteric NC development leading to aganglionosis are
not strictly due to migration defects. The significant differences in
developmental potential between the Sox10Dom congenic lines suggest
that abnormalities in lineage determination events within ENPs can
contribute significantly to aganglionosis. The study of the Sox10Dom
congenic lines is valuable for understanding mechanisms that
contribute to variation in HSCR disease phenotype.
Enteric nervous system developmental potential of vagal and sacral
neural crest cells
aj burns
Neural Development Unit, UCL Institute of Child Health, London, UK
Neural crest cells (NCC) are a transient population of multipotent
cells that migrate extensively throughout the embryo and give rise to a
wide variety of cell types including the neurons and glial cells that
comprise the enteric nervous system (ENS), the intrinsic innervation
of the gastrointestinal tract. The ENS is specifically formed from vagal
and sacral neural crest-derived precursors. Although vagal NCC
(adjacent to somites 1–7) give rise to most of the ENS along the entire
gut, sub-regions of vagal crest have intrinsic differences in their ability
to colonize the gut, as evidenced by neural crest ablation and hetero-
topic transplantation experiments. In addition, our recent studies have
demonstrated that a subpopulation of these �enteric� vagal NCC leaves
the gut and migrates into the developing lungs where they form
intrinsic neural ganglia that innervate airway smooth muscle. Sacral
NCC, which normally contribute neural cells to the postumbilical gut
only, are much less invasive of the gut than vagal NCC, a property
likely due to their lower levels of RET expression. The aim of my
laboratory is to better understand the molecular basis of the differences
in developmental potential of vagal and sacral-derived ENS precursors
using quail-chick chimeric grafting, gene electroporation, DNA
microarray analysis and proteomics approaches. Data suggest that
intrinsic differences, such as varying levels of gene expression, differ-
ent capacity for proliferation, and cell death within the precursor
population, may account for the variation in ENS developmental po-
tential of different NCC.
ENS stem cells: practical problems, practical solutions
n thapar
Gastroenterology and Neural Development Units, UCL Institute of Child Health,
30 Guilford Street, London, UK
Many congenital and acquired ENS disorders are characterized by
severe gut dysmotility. This translates to a life-time of recurrent epi-
sodes of intestinal obstruction and management of complications,
which mainly relate to sepsis and aspects of parenteral nutrition on
which many patients are reliant for their survival. Even Hirschsprung’s
disease, with its long established surgical therapy, carries a question-
able long-term prognosis irrespective of the length of gut agangliono-
sis. Therefore, not surprisingly, the last few years has seen tremendous
progress in the field of ENS stem cell biology and therapeutics. Our
group and many others have reported successes in the isolation of ENS
stem cells including from human postnatal gut and their transplanta-
tion into models of aganglionic gut with emerging evidence of func-
tional rescue. Yet there is no doubting the many enormous challenges
of the task in hand, such as scaling up to human gut, identification of
the optimal source of stem cells and their harvesting from tissue,
overcoming problems of defective allogeneic stem cells, unwelcome
environments of recipient gut and limited colonization capacity of
transplanted cells, and whether true functional rescue can be effected.
More fundamental questions include what diseases should be consid-
ered for ENS stem cell transplantation? Perhaps Hirschsprung’s disease
is not the best target for ENS stem cell treatments. The outcome of
short segment disease is arguably adequate and the prospect of colo-
nizing the gut in long-segment disease perhaps unrealistic. Further-
more, in addition to aganglionosis, Hirschsprung’s gut is characterized
by significant hypertrophy of presumed extrinsic innervation; can the
functional effects of this ever be overcome? Clearly many of these
issues need addressing before cell therapy for ENS disorders can be
applied in the clinical arena. Maybe all is not so bleak. The structurally
�hypoganglionic� gut seen with aging is not associated with functional
failure giving hope that restitution of a complete, normal ENS is per-
haps not needed. Gene therapy is routinely used in clinical therapies
and the genetic rescue of defective ENS stem cells derived from murine
models of Hirschsprung’s disease has been shown to be possible. Tis-
sue transplantation and its immunological management is well
established and unlikely to be a major obstacle. Our recent work
addressing practicalities of harvesting cells for therapy has shown that
minimally invasive procedures such as endoscopy can be used to iso-
late ENS stem cells from a regenerating source of intestinal tissue and
ultimately deliver them back into gut. Transplantation of such cells
into models of aganglionic gut has demonstrated they are capable of
colonizing gut and generating components of the ENS.
Abstracts
� 2009 Blackwell Publishing Ltd xxix
Hirschsprung’s disease associated enterocolitis is related to defects
in innate immunity
n alexander,*,� s eaton,* m haston,� as wallace,� vv smith,–
aj burns,§ j salomon,�,** n shah,� nj klein,� a pierro* & n thapar�*Academic Unit of Paediatric Surgery, UCL Institute of Child Health, London,
UK; �Infectious Disease and Microbiology Unit, UCL Institute of Child Health,
London, UK; �Gastroenterology Unit, UCL Institute of Child Health, London,
UK; §Neural Development Unit, UCL Institute of Child Health, London, UK;
–Histopathology Department, Great Ormond Street Hospital, London, UK; and
**current address: Hopital Necker-Enfants Malades, 75015 Paris, France
Background and aims: Patients with Hirschsprung’s disease (HSCR)
frequently suffer HSCR-associated enterocolitis (HAEC) from very
early in life. The etiology and nature of HAEC remain unclear and it is
most commonly diagnosed on clinical criteria including abdominal
distension, diarrhoea (±blood), vomiting and fever. There is evidence of
an overlap between the genetics of ENS development and gut associ-
ated immunity.1 The aims of this study were to evaluate the preva-
lence of HAEC and whether it is associated with defects in innate
immunity. Methods: The study consisted of a retrospective review of
gut histology of HSCR patients presenting over a 15-year period and a
prospective study examining the relationship between the diagnosis
and clinical spectrum of HAEC and innate immunity in HSCR
patients. No genotyping of the HSCR patients was carried
out. Results: Gut inflammation, present from early life, was evident
in more than 30% of all HSCR patients. HSCR patients had signifi-
cantly reduced monocyte MHC class II expression compared with
controls and those with clinical HAEC had a plasma mannose binding
lectin (MBL) deficiency in addition to the MHC class II defect. The
MBL deficiency was strongly correlated to recognized mutations in the
MBL gene. Conclusions: It is likely that HSCR is associated with
inherent immunodysregulation, resulting in a spectrum of clinical
disease states. These come under the umbrella label of HAEC and
range from local gut inflammation to more generalized clinical man-
ifestations of immune deficiency and its complications. Although the
exact aetiology of HAEC is unclear the novel finding of immunopa-
ralysis provides evidence of an underlying immune disorder. Impor-
tantly, additional defects in innate immunity, namely MBL deficiency
appear to predispose individuals to develop systemic and potentially
more severe clinical disease. It is unknown whether the immune de-
fects relate to abnormalities in gut-associated immunity.
Reference:
1 Veiga-Fernandes H, Coles MC, Foster KE, Patel A, Williams A,
Natarajan D, Barlow A, Pachnis V, Kioussis D. Tyrosine kinase
receptor RET is a key regulator of Peyer’s patch organogenesis.
Nature 2007; 446(7135):547–51.
Abstracts Neurogastroenterology and Motility
� 2009 Blackwell Publishing Ltdxxx