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Molecular Genetic Analysis of the Mouse anorexia Mutation
by
Dennis Kim
A thesis submitted in conformity with the requirements
For the degree of Master of Science
Graduate Department of Molecular and Medical Genetics
University of Toronto
© Copyright by Dennis Kim 2008
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Abstract Molecular Genetics of the Mouse anorexia Mutation Dennis Kim
Master of Science 2008
Department of Molecular and Medical Genetics, University of Toronto
The serotonergic system regulates numerous behaviours and disruptions in this system
have been associated with disorders of mood and mind. Although molecular genetic
analysis has dissected many of the genes involved in the specification of the
serotonergic system, relatively little is known about the mechanisms that promote
axonal outgrowth from serotonin-producing neurons and how these projections are
directed to innervate and form synapses with their appropriate targets. The mouse
anorexia mutation causes hypersprouting of serotonergic projections in target fields and
has provided us the unique opportunity to examine the crucial events that lead to the
establishment of these complex serotonergic networks. Through positional cloning, I
have identified a candidate gene that is upregulated during a time in which innervation
and synaptogenesis of serotonergic neurons are maximal. I have assessed the
expression of this candidate gene in the brain and have found striking differences in the
pattern of expression between the normal and the mutant mouse. Furthermore, by
using transgenic methods, I have partially rescued several hallmark behavioural
phenotypes in the mutant mouse. Thus, this candidate almost certainly represents the
“Anorexia” gene.
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Table of Contents Content Page Abstract ii Table of Contents iii List of Figures v List of Tables vi List of Key Abbreviations vii Chapter 1: Introduction 1.1 The serotonergic system 1 1.2 Development of the serotonergic system 1 1.3 The mature mammalian serotonergic system 10 1.4 The mouse anorexia mutation 12 1.5 Aberrations in anx/anx hypothalamic circuitry 13 1.6 Positional cloning and candidate genes of the anx mutation 16 1.7 The Eph receptors and ephrins 17 1.8 The Trk receptors and neurotrophins 19 1.9 The receptor tyrosine kinase Tyro3 22 1.10 Summary 25 Chapter 2: Materials and Methods 2.1 Introduction 27 2.2 Meiotic recombination map 28 2.3 Candidate gene testing i. Brain-derived neurotrophic factor (BDNF) 29 ii. Glycine-amidinotransferase 29 2.4. Sequencing candidate genes 29
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Content Page 2.5 Generation of transgenic animals i. Mouse GFP-tagged normal and R7W-Tyro3 transgenes 30 ii. Human full-length Tyro3 transgene 32 iii. Identifying transgenic founder mice 33 2.6 Expression analysis of Tyro3 in normal and anx/anx mice by 33
RNA in situ hybridization 2.7 Weight and survival data from the progeny of transgenic 35 anx/+ heterozygote intercrosses
Chapter 3: Results 3.1 Introduction 36 3.2 Meiotic recombination mapping and candidate gene sequencing 38
identified a point mutation in the signal sequence of Tyro3 3.3 Tyro3 expression in normal P21 mouse brain 46 3.4 Tyro3 expression is altered in numerous brain regions in anx/anx mice 49 3.5 Generation and identification of transgenic mice 58 3.6 Transgenic anx/anx homozygotes have improved body weight at 61
P21 and live longer than their non-transgenic anx/anx littermates Chapter 4: Discussion 4.1 Signal sequence mutations 68 4.2 Expression analysis 69 4.3 Partial rescue in transgenic mice 71 4.4 Evidence suggestive that Tyro3 is causative of the anx mutation 74 4.5 Possible roles of Tyro3 75 Conclusion 77 References 78
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List of Figures Figure Page 1. Meiotic recombination mapping and sequencing of candidate genes reveal a 40 point mutation in the anorexia critical interval 2. Comparison of signal sequence predictions from SignalP analysis 44 3. Analysis of Tyro3 expression in the cerebral cortex in P21 normal and anx/anx 47 mice by RNA in situ hybridization 4. Analysis of Tyro3 expression in the hippocampus in P21 normal and anx/anx 50 mice by RNA in situ hybridization 5. Analysis of Tyro3 expression in the hypothalamus in P21 normal and anx/anx 52 mice by RNA in situ hybridization 6. Analysis of Tyro3 expression in the cerebellum in P21 normal and anx/anx 54 mice by RNA in situ hybridization 7. Analysis of Tyro3 expression in the brainstem in P21 normal mouse sagittal 56 sections by RNA in situ hybridization 8. Construction of the Tyro3 transgenes for rescue for mimic analyses 59 9. Analysis of bodyweight of transgenic anx/+ progeny at P21 62 10. Analysis of survival rates of transgenic and non-transgenic anx/anx progeny 66
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List of Tables Table Page 1. Genes within the anorexia critical interval 31 2. Percentage bodyweight difference in transgenic mouse lines 64
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List of Key Abbreviations 5-HT Serotonin anx anorexia mutation Arc Arcuate nucleus BDNF Brain-derived neurotrophic factor ME Median eminence P (e.g. P21) Post-natal day (21) PVN Paraventricular nucleus Vmh Ventromedial hypothalamus
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Chapter 1: Introduction
1.1 The serotonergic system
The serotonergic system is of crucial importance for normal functioning of the
developing and mature mammalian nervous system. Aberrations in the serotonergic
system can negatively affect physiological processes, such as sleep rhythms and
appetite regulation, and behaviours, such as anxiety and aggression. Subtle genetic
alterations can have wide-ranging consequences on proper wiring, firing, and
maintenance of serotonergic neurons and their targets. Variations in genes important for
serotonergic neurotransmission, such as the serotonin transporter (5HTT) and
tryptophan hydroxylase 2 (Tph2), are associated with psychiatric disorders, such as
phobic disorders (Furmark et al., 2004) and unipolar depressive disorder (Zhang et al.,
2006), respectively. Thus, improper development and/or maintenance of the
serotonergic system may contribute to disorders of mood and mind. Finally, there is
increasing evidence that serotonin itself plays a neuromodulatory role on neuronal
development along with its classical role as a neurotransmitter. All of these
observations highlight how crucial it is to have the correct amount of serotonin in the
developing nervous system at the right place and time.
1.2 Development of the serotonergic system
Serotonergic (5-HT) neurons are born in two main clusters along the midline of
the embryonic hindbrain, first appearing between embryonic (E) 10.5 -11.5 in the
mouse. Within one day of their birth, 5-HT neurons are capable of synthesizing
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serotonin and project axons toward their intended target areas in order to form
functional serotonergic networks, which, in the rat and mouse, do not fully mature until
after birth (Lidov and Molliver, 1982; Vitalis and Parnavelas, 2003). Once born, 5-HT
neurons migrate along the dorso-ventral axis and, at times, mediolaterally to eventually
form 9 cell groups collectively known as the raphe nuclei, labeled B1 to B9 from the
posterior to the anterior end. These nuclei are distinguished by their pre-migration
origins. The posterior groups, B1-B4, correspond to the raphe pallidus, the raphe
obscurus, the raphe magnus, and the raphe pontis, respectively, and arise from the
posterior hindbrain, while the anterior groups, B5-B9 collectively form the dorsal (B6/B7)
and median (B5/B8/B9) raphe nuclei. The boundaries of the raphe nuclei are diffuse
(Molliver, 1987) and only about one-third of the neurons within the raphe nuclei produce
5-HT (Descarries et al., 1982), thus characterizing the discrete locations of individual
nuclei is more difficult. There is also some debate as to whether the raphe nuclei
produced by the anterior cluster should indeed be considered as 5 distinct groups or
represent 2 larger functional groupings (reviewed in Cordes, 2005).
While our understanding of 5-HT neuron migration into the raphe nuclei is still
nebulous, great strides have been made towards understanding the signals that first
induce 5-HT neurons to be born and the transcriptional hierarchies important for their
specification. Experiments done on hindbrain explants suggest that Sonic hedgehog
(Shh) signaling from the notochord and floorplate in conjunction with FGF4 establish 5-
HT precursor identity (Ye et al., 1998). In line with these observations, the Shh-
downstream transcription factor, Gli2, has also been shown to play a role in 5-HT
induction as Gli2 null mutants exhibit a 50% reduction in 5-HTergic neurons in the
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mouse hindbrain (Matise et al., 1998). Additionally, FGF8, which is present in the
anterior hindbrain prior to exclusive production and secretion from the midbrain-
hindbrain organizing center (MHO), is required for the development of the anterior raphe
nuclei but not the posterior clusters (Ye et al., 1998). Displacement of the MHO either
rostral or caudal to its normal position causes a corresponding displacement of
serotonergic and dopaminergic cell clusters (Brodski et al., 2003), and transgenic mice
bearing a constitutively active form of Smoothened, a Shh receptor, produce 5-HT
precursors dorsal to their normal positions (Hynes et al., 2000). Shh signaling can
therefore establish both antero-posterior and dorsoventral patterning of monoaminergic
neurons in combination with other factors, but already during their earliest development
5-HT neurons in the anterior and posterior clusters are distinguished by their
dependence or independence on FGF8 signaling.
After early induction of 5-HT precursors, the concerted activity of several
transcription factors is required for subsequent position-appropriate differentiation of 5-
HT neurons. Initially, Nkx2.2, Nkx6.1, and Mash1 expression in hindbrain tissues
identify 5-HT precursors, which are followed by Mash1, Gata2, Gata3, Lmx1b, and Pet1
expression to establish 5-HT subtype selection (reviewed in Cordes, 2005). The
homeodomain transcription factor, Nkx2.2, which acts downstream of Shh, is required
for all but the dorsal raphe nuclei neurons to specify a 5-HTergic cell fate (Briscoe et al.,
1999). In the dorsal raphe nuclei, compensatory activity of Nkx2.9 is likely to direct the
5-HT neurochemical phenotype in the absence of Nkx2.2. Nkx2.2 also drives Nkx6.1
expression, which acts in cooperation to direct Gata2 and Gata3 expression required for
5-HT specification. Additionally, Nkx2.2 likely suppresses Phox2b expression, which,
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together with the down-regulation of Nkx2.9, marks the onset of 5-HT neuron
specification.
Differentiation of 5-HT precursors into terminal subtypes is directed by the
proneural helix-loop-helix transcription factor Ascl1/Mash1. Ascl1/Mash1 acts in two
ways; first, Ascl1/Mash1 downregulates Phox2b to generate post-mitotic 5-HT
precursors, leading to its second role in specifying 5-HT neurons (Pattyn et al., 2003).
The actions of Ascl1/Mash1 occur after the Nkx genes have exerted their effects to
induce 5-HT precursors. Ascl1/Mash1 null embryos lack later acting Ets-domain Pet1,
Lim homeodomain-containing Lmx1b, zinc finger Gata2 and Gata3 transcription factors
as well as 5-HT neurons altogether, even though expression of the earlier-acting Nkx
genes and Phox2b are unaltered. The zinc finger transcription factor Gata2 has been
shown to be necessary and sufficient to activate Lmx1b and Pet1 (Craven et al., 2004),
and is likely required for global 5-HT neuron development. Cultured Gata2-/- embryos at
E13 lack Pet1 and 5-HT neurons, even though expression of its companion zinc-finger
transcription factor Gata3 is unchanged. Gata3 acts in a cluster-specific role on
posterior 5-HT precursors to establish 5-HT specification. Gata3-/- mutants have an
80% reduction in neurons within these clusters, which likely acts in parallel with Lmx1b
and Pet1, both of which are unaffected in these mutants.
The Lim homeodomain transcription factor Lmx1b is required for 5-HT
specification through the Ets transcription factor, Pet1, which is lost in Lmx1b-/- embryos
(Ding et al., 2003, Cheng et al., 2003). Interestingly, ectopic 5-HT precursors in the
dorsal and ventral hindbrains of these mice suggest a role for Lmx1b in restricting the
cell migration of 5-HT neurons (Ding et al., 2003). Pet1 is exclusively required for the
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specification and differentiation of most hindbrain 5-HT neurons (Hendricks et al., 1999,
Hendricks et al., 2003). Loss of Pet1 causes a 70% loss of hindbrain 5-HT neurons due
to a failure to differentiate, and the remaining 5-HT neurons have reduced expression in
5-HTergic genes (Hendricks et al., 2003). However, the few Pet1-/- mice that do survive
until adulthood exhibit increased anxiety and aggression, in agreement with the notion
that proper 5-HTergic system development even during these early stages is necessary
for proper functioning in the adult and with the postulated role of 5-HT neurons in
modulating these behaviours.
Having established 5-HT precursor identity, factors that promote proliferation and
survival of 5-HTergic outgrowth are required to initiate the complex cascade leading
towards a proper 5-HT network. Some clues as to which molecules are involved in
these processes have been examined in culture and by using several mouse mutants
specific for a 5-HT axonal phenotype. Brain-derived neurotrophic factor (BDNF) has
been shown to promote the sprouting and survival of serotonergic neurons and their
projections. BDNF exposure of primary cultures of E14 rat rostral raphe nuclei induces
a near 2-fold increase in 5-HTergic neurons and importantly, a dramatic extension of
neurites (Rumajogee et al., 2002). Intracortical infusions of BDNF cause an increase in
axon density of 5-HTergic projections in the neocortex of adult rats, which also acts to
prevent chemically-induced lesioning of these fibers (Mamounas et al., 1995).
Conversely, mice with constitutively reduced levels of BDNF have a concomitant
decrease in 5-HT innervation of the hippocampus at 18 months (Luellen et al., 2007).
Mice heterozygous for a BDNF null allele exhibit increased aggressive behaviour,
hyperphagia, and weight gain; behaviours associated with 5-HT dysfunction, which is in
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part caused by the premature decline in forebrain 5-HT levels and 5-HTergic fiber
density (Lyons et al., 1999). The role of BDNF to promote neuron proliferation and
subsequent axon survival may also, in part, be regulated by the expression of pro-
apoptotic Bax and anti-apoptotic Bcl-2 genes. Damage to neural tissue due to cerebral
ischemia is reduced by pretreatment with BDNF which reduces the number of Bax-
positive neurons and increases the number of Bcl-2-positive neurons at the site of injury
(Schabitz et al., 2000). In double BDNF/Bax null mutant mice, cells of cranial sensory
neurons which are lost in BDNF null mice are completely rescued (Hellard et al., 2004).
However, these mutants are eventually overcome by the lethality of the BDNF null
phenotype due to a failure to innervate target tissues despite successful navigation to
their terminal fields. Whether 5-HT neurons cannot innervate their targets in these
animals has not been examined, but seems possible if not likely. Taken together, these
results suggest that BDNF acting on central neuronal populations for survival and
outgrowth of projections may converge with locally-acting BDNF required for in-growth
into target tissues. BDNF-sensitive neuronal populations, such as 5-HT neurons, may
therefore respond to such signals at both ends to provide trophic support and establish
functional synaptic connections.
Subsequent to adopting the 5-HT neuronal phenotype and expressing the
necessary genes thereafter, correct axonal guidance from the raphe nuclei to their
target fields is required to establish a functional 5-HT network. These complex
processes towards proper innervation are temporally and spatially regulated
(Parnavelas and Papadopalous, 1989), and though these processes are initiated during
gestation, complete 5-HTergic connectivity is not fully established until the third
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postnatal week as evidenced in the visual and primary somatosensory cortex in the rat
(Dori et al., 1996). While at times in immunostaining experiments, the wide-spread and
complex 5-HT projections may appear to be in a somewhat chaotic disarray, their
trajectories are far from haphazard, and axonal tracing experiments as well as chemical
lesioning have identified distinct areas innervated by specific raphe nuclei. The majority
of axonal projections from the dorsal and median raphe nuclei that constitute the
anterior group of 5-HT neurons ascend through the median forebrain bundle, but
diverge to innervate non-overlapping and complementary regions of the rat forebrain as
visualized by the anatomical tracer Phaseolus vulgaris-leucoagglutinin (Vertes, 1991,
Vertes et al., 1999). These axons also differ in their axonal morphologies. The axons
projecting from the dorsal raphe nuclei appear fine, and are beaded with tiny,
pleiomorphic varicosities unlike median raphe nuclei axons which have coarse
varicosities (Descarries et al., 1975, Maley et al., 1990). Furthermore, within these
anterior clusters, only dorsal raphe nuclei neurons are sensitive to treatment with such
psychotropic drugs as MDMA and PCA (Mamounas and Molliver, 1988; Molliver et al.,
1989). Furthermore, it has been shown that a small population of 5-HT neurons
projecting from B8 can branch out and innervate two separate areas (Kohler et al.,
1982, Kohler and Steinbusch, 1982). Hence, a single 5-HTergic axon can
simultaneously act on multiple and distinct areas of the brain, and this increases the
complexity of axon guidance for 5-HT neurons, since a single neuron must coordinate
the targeting of two different processes through territories that express distinct guidance
cues.
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There appear to be general axonal repulsive cues that affect both ascending and
descending axonal projections, including 5-HTergic projections, mediated by the Slit
proteins (Bagri et al., 2002). In Slit2 mutants, 5-HTergic fibers are displaced ventrally
through the diencephalon, while in Slit1;Slit2 double mutants, a number of 5-HTergic
fibers descend into the hypothalamus, and abnormally cross the midline in the basal
telencephalon. Thus, these repulsive cues act to maintain the dorsal position of these
pathways by inhibiting growth into ventral regions and into the midline, and to correctly
direct axons to target forebrain regions. However, the molecules involved to direct 5-
HTergic axonal growth and target specificity remain largely unknown. In a series of
explant and co-culture experiments, Petit et al. (2005) have begun to unravel some of
the properties of these guidance cues and repulsive molecules. Serotonergic axons
project from the dorsal raphe in a contact-dependent manner mediated by a GPI-linked
membrane protein. As these axons project into either ventral midbrain or striatal
explants, they adopt a preference for explants or membranes derived from the same
brain region by the activity of a membrane-bound molecule in these tissues, since this
preference is disrupted in the presence of high potassium chloride concentration.
Hence, target pathfinding of 5-HT projections is a multistep process in which broad
fields of 5-HTergic innervation are sequentially refined. When primary axons traverse
into target areas, they adopt a preference for innervating that specific target tissue and
divergent projections are inhibited from branching into and innervating alternate regions.
However, the exact molecules and pathways that allow 5-HT axons to acquire this
target-induced growth-inhibitory response are yet to be identified.
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The thalamocortical tract, a major ascending axonal pathway in the mammalian
brain, is modulated by 5-HT itself, and the overlapping expression of 5-HT1B and 5-HT1D
receptor transcripts with the axon guidance receptors Deleted in Colorectal Cancer
(DCC) and Unc5c interact to change attractive and repulsive cues in response to Netrin-
1 (Bonnin et al., 2007). The 5-HT1B and 5-HT1D are Gi/o-coupled receptors and upon
activation inhibit adenylate cyclase, thereby decreasing intracellular levels of cAMP
(Raymond et al., 2001). Using E14.5 day explant cultures, the attraction of posterior
dorsal thalamus axons to Netrin-1 is converted into a repulsive signal in the presence of
5-HT, which is dependent on the 5HT1B/1D receptor mechanism controlling camp
(Bonnin et al., 2007). The modulatory effects of 5-HT were also evident via in utero
electroporation of siRNAs to reduce the expression of 5-HT1B/1D at E12.5. Dorsal
thalamus axons expand ventrolaterally by E18.5, and in contrast, overexpression of
these 5-HT receptors has the exact opposite growth pattern, in which thalamic axons
are more restricted dorsomedially in the palladium. These results indicate that classic
neurotransmitters can play neuromodulatory roles, especially in the thalamocortical tract
which is shared by ascending 5-HT projections, and add a further complexity to 5-HT
neurodevelopment and its interrelation with other systems and overall brain
development.
Another poorly understood factor that affects 5-HT connectivity is feedback from
other neurotransmitter systems. This phenomenon is nicely exemplified by studies of
mice homozygous for the brindled-mottled mutation which affects copper metabolism
and thereby inhibits dopamine-beta-hydroxylase (DBH), which uses copper as a
cofactor to convert dopamine to norepinephrine (Martin et al., 1994). In brindled mottled
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homozygotes, 5-HT fibers exhibit heterotypic sprouting in the cerebral cortex. By P14,
axonal fiber density is 70% higher in the mutant than in control animals. Interestingly,
the brindled-mottled mutant highlights the often critical interaction of multiple systems
within the developing and mature nervous system. The only other known spontaneous
mouse mutant to directly affect 5-HT innervation and cause hypersprouting is the
anorexia mutation, which is the basis for the following study and will be discussed in
greater detail.
1.3 The mature mammalian serotonergic system
The mature 5-HTergic system is able to synthesize, release, and regulate 5-HT
between 5-HTergic terminals and target areas. This is accomplished by the coordinated
activity of at least 14 different 5-HT receptor subtypes, the 5-HT transporter (SERT),
and tryptophan hydroxylase 2 (Tph2), the 5-HT synthesizing enzyme, which is more
predominantly expressed in the brain than its companion isoform, Tph1 (Patel et al.,
2004, Sakowski et al., 2006). Several disorders of mood and mind have been traced to
disruptions in the 5-HTergic system, hence, model organisms that have altered 5-
HTergic systems may serve as models of human disease.
Convergent evidence from genetic analyses, animal models, and
pharmacological studies has elucidated the roles of some 5-HTergic genes. Targeted
inactivation of the 5-HT1A receptor in mice causes an increase in anxiety-related
behaviours (Heisler et al., 1998) as well as a decrease in cognitive ability (Sarnyai et al.,
2000). A polymorphism in the promoter region of the same gene in humans has been
positively linked to anxiety and major depression, potentially leading to suicidal
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behaviour (Hong et al., 2006, Kraus et al., 2007). 5-HT2C receptor null mutant mice are
cognitively deficient and exhibit hyperphagia-evoked weight gain (Heisler and Tecott,
1999). Similarly, 2 polymorphisms in the same gene in humans have been linked to
eating disorders, deficits in learning and memory, and the development of psychoses
(Massat et al., 2007, Hu et al., 2003). The serotonin transporter (SERT) and
monoamine oxidase A (MAOA) play roles in recycling and reducing the amount of 5-HT;
the first by uptake from the synaptic cleft into the presynaptic terminal, and the second
by deamination of 5-HT at the outer mitochondrial membrane. SERT null mice fail to
clear 5-HT and therefore, have excess levels of extracellular 5-HT (Holmes et al., 2003).
These mice show increased anxiety-related behaviours, reduced aggression, and
exaggerated stress responses. Interestingly, a human 5-HTT polymorphism has been
linked to anxiety and abnormal stress reactivity potentially leading to depression (Otte et
al., 2007, Munafo et al., 2005). This polymorphism is also linked to alcoholism (Feinn et
al., 2005) suggesting a role for 5-HTT in addictive behaviour, and eating disorders
(Monteleone et al., 2006, Matsushita et al., 2004). The MAOA knockout mouse displays
increased aggression (Popova et al., 2001) and abnormal stress reactivity (Seif and De
Maeyer, 1999, Popova et al., 2006). In humans, a polymorphism in MAOA has been
linked to antisocial behaviour, which can pathologically manifest as antisocial
personality disorder (Eisenberger et al., 2006). So far only single gene associations
have begun to be examined, but, given the substantial success of these limited efforts, it
is not difficult to imagine the vast number of potential direct or indirect interactions
between “serotonergic“ genes and the consequences of variations within them which
may contribute to the spectrum and complexity of psychiatric disorders. Identification of
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such genes and understanding their mechanisms of action will ultimately lead to tailored
and far more effective treatments for affected patients.
1.4 The mouse anorexia mutation
The mouse anorexia (anx) mutation displays a 5-HT-specific aberration in which
normal 5-HT terminal fields are hyper-innervated by 5-HT projections (Son et al., 1994).
At 3 weeks, in mice homozygous for the anx mutation, the density of 5-HT
immunoreactive fibers is greatly increased in the olfactory bulb, the cerebral cortex,
hippocampus, and cerebellum, whereas brain regions not normally innervated by 5-HT
projections remain virtually unaffected. Unlike the brindled mottled phenotype, in which
5-HT hyper-innervation occurs secondary to a DBH defect, anx/anx mice have normal
catecholaminergic innervation and cell body density as determined by tyrosine
hydroxylase immunostaining, and anx/anx mice preserve normal nuclear boundaries as
determined by Cresyl violet staining. A previously unreported phenotype has also been
found in our lab in anx/anx homozygotes and anx/+ heterozygotes at birth. Anx/anx
homozygotes lack any 5-HT in cortical areas suggesting a delay in 5-HT axon guidance,
while anx/+ heterozygotes have an intermediate level relative to normal littermates
(Huynh, unpublished). These data suggest that the effects of the anx mutation occur
during gestation and have a dosage-dependent effect.
The behavioural phenotype of anx/anx homozygotes also reflects the changes in
normal 5-HT innervation in the brains of these animals. Between P15- P18, anx/anx
mice develop body tremors, headweaving, abnormal gait and hyperactivity; behaviours
that can also be elicited by pharmacological stimulation of 5-HT receptors (Maltais et al.,
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1984). Additionally, treating P20 anx/anx mice with the 5-HT antagonist 5,7-
dihydroxytryptamine alleviates the severity of the behavioural phenotype. Hence, the
anx mutation appears to affect the 5-HT system directly.
1.5 Aberrations in anx/anx hypothalamic circuitry
Anx/anx mice are distinguishable from their normal and heterozygous littermates
by P9 by their reduced size and become emaciated in appearance until death by P22
(Maltais et al., 1984). From our observations and previous analyses, mutant mice at
P20 weigh only 30-50% of their unaffected littermates. These mutant mice were
therefore categorized as exhibiting “anorexia” due to their decreased food intake. At
P15, these mutant mice were devoid of fat and glycogen in their livers, in line with a
food intake abnormality. A hypothesis proposed by this group suggested that the
overstimulation of the 5-HT system in anx/anx mice inhibited the suckling response of
newborn pups, ultimately leading to the overall anorexic phenotype leading to death.
Metabolites of the 5-HT were known to be involved with the suckling response and
physiological control of food intake behaviour (Spear and Ristine, 1982), and 5-HT
agonists and antagonist had been shown to modulate this response (Caza and Spear,
1982). However, subsequent studies in our lab of the neural systems involved in
energy balance indicate that the anx mutation affects the circuitry regulating food intake
in the hypothalamus. Whether 5-HT overstimulation or disrupted neural circuitry is the
primary cause will be discussed in later sections.
The hypothalamus is divided into twelve nuclei which are individually sensitive to
numerous physiological cues to regulate homeostasis. The arcuate nucleus, located in
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the ventral region of the hypothalamus, sends projections dorsally and anteriorally to the
paraventricular nucleus (PVN), an area critical to food intake behaviour. The PVN
receives signals from two distinct cell groups from the arcuate nucleus. A subset of
arcuate nucleus neurons produce and secrete Neuropeptide Y (NPY) (Allen et al., 1983,
Chronwall et al., 1985, DeQuidt and Emson, 1986) and Agouti-Related Protein (AgRP)
(Shutter et al., 1997, Hahn et al., 1998), which, when released, stimulate food intake.
Another subset of arcuate nucleus neurons counteracts the effects of these orexigenic
signals. These neurons instead release Pro-Opiomelanocortin (POMC) (Bloch et al.,
1978, Bloom et al., 1978, Watson et al., 1978) and Cocaine- and amphetamine-
regulated transcript (CART) (Elias et al., 1998, Kristensen et al., 1998) deemed
anorexigenic in nature since they act to suppress food intake. Both cell populations
respond to peripheral signals including leptin from adipose tissue (Elias et al., 1999,
Elmquist et al., 1999), insulin and ghrelin (Lawrence et al., 2002, Seoane et al., 2003,
Scott et al., 2007). At low energy states, the release of NPY strongly stimulates feeding
behaviour (Stanley and Leibowitz, 1985). AgRP is co-expressed in the majority of NPY
neurons, which, when co-released, acts as an antagonist to the anorexigenic
melanocortin peptides (Fan et al., 1997, Ollmann et al. 1997). Thus, stimulation of
these neurons heightens food intake by suppressing hypothalamic inhibition of this
behaviour.
In the arcuate nucleus, POMC is further processed to generate
adrenocorticotropic hormone (ACTH) and α-melanocyte stimulating hormone (α -MSH),
which are increased in response to fasting (Brady et al., 1990). Pharmacological
stimulation of the melanocortin receptors to which these hormones bind can also
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suppress food intake (Fan et al., 1997, Grill et al., 1998). CART, which is produced by
these neurons, actively blocks feeding behaviour induced by NPY (Kristensen et al.,
1998, Lambert et al., 1998). However, regulation of food intake is also mediated
between these arcuate nucleus clusters as well. Not only are the projections from these
subsets of neurons linked and run parallel to each other (Zhang et al, 1994, Fuxe et al.,
1997, Broberger et al., 1997b, 1998, Csiffary et al., 1990), but POMC/CART neurons
can receive inputs from neighboring NPY neurons via NPY Y1 and Y5 receptors on their
cell surface (Broberger et al., 1997b, Fuxe et al., 1997), which, when activated,
stimulate food intake behaviour (Gerald et al., 1996, Stanley et al., 1992). Thus,
stimulation of NPY neurons can inhibit melanocortin release by acting on these
receptors enhancing food intake behaviour.
In the anx/anx hypothalamus, NPY is mislocalized to the cell body of arcuate
nucleus neurons as determined by NPY immunostaining (Broberger et al., 1997a,
1998). NPY and AgRP normally reside in the presynaptic terminal of arcuate nucleus
projections, leaving little NPY and AgRP in the cell body. Anx/anx arcuate nucleus
neurons exhibit increased NPY and AgRP immunoreactivity in the cell body, although
the transcription levels of the mRNAs for both peptides were normal as determined by
RNA in situ hybridization. Hence, in the anx/anx hypothalamus, 3 possibilities arise.
First, arcuate nucleus neurons fail to send axonal projections to terminal fields, in which
case the anx mutation would affect axonal outgrowth and/or maintenance of these
projections. Second, axonal projections extend normally to target areas, but axonal
transport mechanisms are disrupted leading to a sequestering of NPY and AgRP in the
cell body. Third, the anx/anx hypothalamic phenotype is secondary to the defect in 5-
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HT innervation. Although the hypothalamus is normally inundated with 5-HT fibers, a
very slight increase in 5-HT innervation has been previously reported in anx/anx mouse
brains (Son et al., 1994), in which case, the mechanism causing the 5-HT innervation
defect may nonetheless affect hypothalamic circuitry.
1.6 Positional cloning and candidate genes of the anx mutation
The anorexia mutation arose spontaneously in the F2 generation of a cross
between DW/J and an inbred strain derived from a cross between M. m. poschiavinus
and an inbred Swiss stock, and maps closely to pallid (pa) on chromosome 2 (Maltais et
al., 1984) in a relatively gene rich region. However, in looking for candidate genes
causative of the anx mutation, the most logical approach would be to examine genes
with known neurodevelopmental roles, and secreted or transmembrane proteins that
are similar to known axon guidance or neuronal maintenance molecules. In the case of
nervous system development, tyrosine kinase receptors, such as the Eph receptors and
Trk receptors, play particularly intriguing roles in axon guidance, neuronal cell migration,
and synaptogenesis. Two receptor tyrosine kinase genes, Tyro3 and Leukocyte
Tyrosine Kinase (Ltk), reside within the anx critical interval. Interestingly, the anx
mutation was also initially mapped near the BDNF gene, which is known to play a role in
5-HT neurite outgrowth and cell survival as described above, but, as I shall report in the
next chapter, we eliminated BDNF as a candidate gene via non-complementation and
further genetic mapping experiments.
17
1.7 The Eph receptors and ephrins
Among receptor tyrosine kinases (RTKs), the roles of the Eph receptor family in
axon guidance and synaptogenesis has been studied most extensively and, thus,
present a good starting point for understanding the possible roles of other RTKs in
modulating these complex neurobiological processes. The Eph receptor family is the
largest subfamily of RTKs and is comprised of 16 members classed as “A” or “B” based
on their binding affinities to 9 ephrin ligands, also classed as “A” or “B” (reviewed in
Goldshmit et al., 2006). Each receptor is characterized by a highly-conserved N-
terminal globular domain, a cysteine-rich motif, and two fibronectin type III repeats in the
extracellular region; the fibronectin type III repeats mediating receptor dimerization
(Lackmann et al., 1998). Two tyrosine residues located in the juxtamembrane region
are the major sites of receptor autophosphorylation and subsequent receptor signaling
(Bruckner and Klein, 1998, Holland, 1998). The intracellular kinase domain has been
posited to act on small GTPases and, thereby, affect cytoskeleton organization
(Nimnual et al., 1998), while a sterile alpha motif (SAM) domain formed by the last 60-
70 residues of the carboxy-terminus tail regulates cell-cell signal transduction (Schultz
et al., 1997, Stapleton et al., 1999). The inclusion of a post-synaptic density protein
zona occludens (PDZ)-binding domain is necessary to form Eph or ephrin complexes to
affect regulatory molecules and to direct these complexes to specific locations within the
cell (Kullander and Klein, 2002). To initiate a signal via Eph receptors, higher order
Eph-Ephrin complexes are an absolute necessity. These not only direct signals
downstream of the Eph receptor-expressing cell, but may also initiate reverse signaling
via clustered ephrins attached in the apposed cell membrane (Cowan and Henkemeyer,
18
2002; Davy and Soriano, 2005). Hence, a dual mechanism, in which initial binding of a
cluster of ephrins attached to one cell binds with a cluster of Eph receptors on the
apposed cell reinforcing cellular adhesion, which may be further reinforced or overcome
by intracellular changes affecting downstream processes (Klein, 2004).
The predominant of expression of most of the Eph receptors and ephrin ligands
in the nervous system is in line with their significant roles in neurodevelopment
(Flanagan and Vanderhaeghen, 1998; Nakamoto, 2000; Kullander and Klein, 2002,
Murai and Pasquale 2003). The Eph receptors and their associate ephrin ligands play a
role in the retinotectal mapping of visual circuits in the chick (Cheng et al., 1995,
Drescher et al., 1995, Nakamoto et al., 1996) and the mouse (Feldheim et al., 1998,
Feldheim et al., 2000), and direct axonal projections from motoneurons to their correct
targets in the hindlimb (Dottori et al., 1998; Kullander et al., 2001). The expression of
Eph B1, B2, B3, and A4 receptors and the B1, B2, and B3 ephrins is particularly high in
migratory regions in post-natal and adult mouse brains, and in regions of high plasticity
including the olfactory bulb, the hippocampus, and the cerebellum (Liebl et al., 2003).
EphB2 and EphB3 mutant mice are known for midline guidance defects of the tracts
forming the corpus callosum, and defects of the anterior commissure (Henkemeyer et
al., 1996, Orioli et al., 1996). EphA4, which can bind both A-class and B-class ephrins
(Gale et al., 1996), is required to establish corticospinal tract axons from the motor
cortex (Kullander et al., 2001). Additionally, a knock-in mouse generated to express a
constitutively active form of this receptor, EphA4EE, is deficient in forming
thalamocortical projections despite normal midline axon guidance and hindlimb
19
locomotion (Egea et al., 2005). These results highlight the complex nature of the Eph
receptors and ephrins and their roles in axon guidance and topographic mapping.
1.8 Trk receptors and neurotrophins
Another class of RTKs that have roles in neural development is the tropomyosin-
related kinase (Trk) receptors and their activating ligands, the neurotrophins. The
discovery of neurotrophins, in particular, nerve-growth factor (NGF), validated earlier
ablation and transplantation experiments in which a trophic factor acting locally at target
fields was necessary for survival and/or maintenance of neuronal projections (reviewed
in Levi-Montalcini, 1987). This also demonstrated the essential role of intercellular
communication in developmental processes. These molecules have numerous
functions acting not only as survival/trophic factors for neuronal projections as
discussed above for BDNF, but the interactions between Trk receptors and
neurotrophins have been shown to promote and direct axonal outgrowth (Tucker, 2002),
affect synaptic plasticity (Schinder and Poo, 2000), and mediate responses to injury
(Blesch et al., 1998). Furthermore, the Trk receptors and neurotrophins extend outside
of the nervous system with roles in vascularization (von Schack et al., 2001) and
oncogenesis (Descamps et al., 2001), highlighting the importance of these molecules in
both neural and non-neural tissues.
There are three Trk receptors which specifically bind four neurotrophins
(reviewed in Patapoutian and Reichardt, 2001). TrkA binds NGF, TrkB binds BDNF and
NT4, and TrkC binds NT3. The expression of these receptors and ligands in the
nervous system indicate roles in neuronal development. Each receptor is characterized
20
by a cleaved signal sequence followed by two cysteine clusters which flank three,
twenty-four-residue leucine-rich motifs. Adjacent to the second cysteine cluster, two
immunoglobulin-like domains complete the extracellular domain, which is then affixed to
the membrane and attached to an intracellular tyrosine kinase domain. Binding of a
neurotrophin to its specific Trk receptor occurs at the second Ig-like domain, although
the subsequent downstream events mediated by receptor binding are complicated by
several factors. The specificity of TrkA and TrkB to their proper neurotrophins is altered
in certain isoforms and by the promiscuity of NT3. Both receptors, when lacking an
insert by the juxtamembrane region, bind only their specific partners. However, when
the insert is included, TrkA and TrkB are able to bind NT3 (Clary and Reichardt, 1994,
Strohmaier et al., 1996). TrkB and TrkC also have isoforms lacking the intracellular
kinase domain, which may therefore act to suppress downstream signaling by
sequestering excess neurotrophin, although their exact role is unknown (Klein et al.
1990, Tsouflas et al., 1993). Additionally, the pan-neurotrophin receptor p75NTR causes
alterations in receptor binding behaviour of the Trk receptors. This receptor has low
binding affinity for each neurotrophin (Rodriguez-Tebar et al., 1991), however, p75NTR
activation enhances TrkA binding to NGF (Hempstead et al., 1991) and TrkB binding to
BDNF (Bibel et al., 1999) p75NTR is a distant member of the tumour necrosis factor
(TNF) receptor (Bothwell, 1995) and contains a cytoplasmic “death” domain (Liepinsh et
al., 1997). Ligand binding to p75NTR can induce neuronal death directly by initiating
apoptosis (Frade and Barde, 1998, Friedman, 2000). However, the activities of p75NTR
are necessary for neural development, since absence of this receptor causes
21
perturbations in axon growth in vitro and axon growth and target innervation in vivo (Lee
et al., 1994, Yamashita et al., 1999, Bentley and Lee, 2000).
In vitro experiments of all the neurotrophins indicate that signaling events
mediated by Trk receptor activation promote neurite outgrowth (reviewed in Patapoutian
and Reichardt, 2001). When subjected to gradients of neurotrophins, growth cones can
respond by redirecting their advancing trajectory. However, the reaction of neuronal
projections to neurotrophins as a chemoattractant or a chemorepellant is dependent on
the levels of cyclic nucleotides within those neurons (Song et al., 1997, Song and Poo,
1999). Inhibitors of the cAMP signaling cascade convert NGF- and BDNF-mediated
chemoattraction to chemorepulsion via their respective receptors. The chemoattraction
of TrkA activation via binding of NGF is lost in TrkA mutants unable to bind PI-3 kinase
(Ming et al., 1999). Interestingly, TrkC-NT3 mediated chemoattraction is converted to
chemorepulsion in the presence of cGMP signaling inhibitors (Song and Poo, 1999).
Thus, the intricate balance between chemoattraction and chemorepulsion may be
modulated by the presence of certain Trk receptor-neurotrophin signaling cascades and
further refined by intracellular events.
The activities of neurotrophins at target sites to promote innervation and
establish synaptic connections has been exemplified in studies in which excess
neurotrophin levels in normal targets lead to increased innervation and the presence of
neurotrophins in non-targets leads to ectopic innervation. NGF expression in pancreatic
islets driven by the insulin promoter causes dense sympathetic innervation of these
tissues which normally lack innervation (Edwards et al., 1989). Furthermore, when
driven by the keratin-14 promoter, similar innervation is induced in the epidermis
22
(Guidry et al., 1998). Additionally, ectopic expression of BDNF by the nestin promoter
stalls sensory axons from reaching the gustatory papillae and they are instead restricted
to the base of the tongue (Ringstedt et al., 1999). Thus, the central model of
neurotrophic factor action, in which targets of neuronal innervation secrete specific
amounts of survival factors which act to balance the size of a target tissue with the
number or amount of innervation, is repeatedly satisfied and reflects the careful neural
connections that are necessary to form functional networks in the brain.
1.9 The receptor tyrosine kinase Tyro3
Tyro3 is a member of the “TAM” family of receptor tyrosine kinases along with
Axl and Mer (reviewed in Hafizi and Dahlback, 2006). This receptor has adopted many
names since its identification including Rse (Mark et al., 1994), Sky (Ohashi et al., 1994)
Brt (Fujimoto and Yamamoto, 1994), Tif (Dai et al., 1994), Dtk (Crosier et al., 1994), and
Etk-2 (Biesecker et al, 1995) owing to its identification in different organisms and
different tissues. Of the TAM family members, Tyro3 is most extensively expressed in
the central nervous system, as shown in the mouse and rat (Lai et al., 1994; Ohashi et
al., 1994; Funakoshi et al., 2002; Prieto et al., 2000), but is also highly expressed in the
Sertoli cells of the testes. Additionally, Tyro3 has been shown to play a role in bone
resorption in osteoclasts (Nakamura et al., 1998), immune regulation (Lu and Lemke,
2001; Lemke and Lu, 2003), and platelet activation in response to injury (Dahlback and
Villoutreix, 2005), further underscoring the many potential roles of this receptor.
The extracellular domain in the mature protein contains a putative signal
sequence domain, and 2 immunoglobulin-like domains followed by 2 fibronectin type III
23
repeats, similar to neural-cell adhesion molecules (Lai et al., 1994). The
transmembrane receptor is complete with an intracellular tyrosine kinase domain,
which, upon activation by its ligands: growth-arrest specific gene 6 (Gas6) or Protein S,
initiates homophilic dimerization, cross-phosphorylation, and downstream signaling
through PI-3K/AKT or Ras/ERK signaling pathways (reviewed in Hafizi and Dahlback,
2006). Both ligands are composed of on N-terminal domain consisting of multiple post-
translationally modified γ–carboxyglutamic acid residues, which can interact with
negatively charged membrane phospholipids (Mann and Lawson, 1992). Following this,
four epidermal growth factor (EGF)-like repeats and a C-terminal globular sex hormone
binding globulin (SHBG)-like region consisting of two laminin G-like (LG) domains
complete each ligand (Tisi et al., 2000). An intervening loop region between the EGF-
like repeats and the SHBG-like region can be cleaved in Protein S but not Gas6 by
thrombin, ideal for its role in coagulation (Dahlback and Villoutreix, 2005). There is
some debate as to the exact binding partner of mouse and human Tyro3 based on
biochemical analysis with species-specific ligands. Human Tyro3 is preferentially bound
by human Gas6 and bovine Protein S over human Protein S, while mouse Tyro3
preferentially binds human protein S over human Gas6 (Godowski et al., 1995, Nyberg
et al., 1997, Stitt et al., 1995). These discrepancies between intraspecies ligand binding
affinities must be clarified to ascertain the roles of each ligand and receptor regarding
their effects in different tissues affecting different signaling pathways.
Like many other RTKs, interest in Tyro3 was drawn from its potential roles in
oncogenesis, but additional studies indicate that Tyro3 may have a role in mammalian
brain development. Published expression analyses have observed that Tyro3 is
24
expressed in the olfactory epithelium at E17.5 in the mouse, and is steadily upregulated
from birth to its maximum levels in the adult in layers 2/3, and 5 of the cerebral cortex, in
the CA1 region and at reduced levels in the CA3 region of the hippocampus, and in the
granule cells comprising the molecular layer of the cerebellum (Lai et al., 1994). In
comparing published expression analysis between the mouse and the rat, there are
some discrepancies as to the exact levels in certain regions of the brain. In the rat,
Tyro3 mRNA is reported to be expressed in layers 2, 3, and 6 of the cortex and at very
low but detectable levels in the dentate gyrus of the hippocampus (Prieto et al., 2000).
Despite these differences, Tyro3 activity in the brain appears to be important due to its
region-specific expression in the brain and its upregulation during a period of post-natal
development in which axonal projections and synaptogenesis are occurring at
maximum.
Early Northern blot analysis of RNA from numerous organs indicated high levels
of Tyro3 mRNA in the testes (Stitt et al., 1995). From birth to P18, Tyuro3 mRNA is
maintained at a high level in the testes and decreases slightly by P24 (Matsubara et al.,
1996). Tyro3 is expressed in the Sertoli cells of the testes which are responsible for
providing trophic support to the seminiferous tubules in concert with additional factors
secreted by surrounding Leydig cells. However, both the brain and testes appear
unaffected in a Tyro3 null mutant mouse line generated by insertion of a neo-cassette
replacing the second fibronectin type III domain (Lu, et al., 1999). These mice are
viable and fertile, and the only “neural” phenotype appears to be activity-induced
seizures that first occur around seven months. Additionally, double mutants of
Tyro3/Axl and Tyro3/Mer were also viable and fertile, with some aberrations in overall
25
health. However, Triple Tyro3/Axl/Mer mutants had numerous defects including cellular
degeneration in the neocortex, the hippocampus, and the cerebellum, and of the rods
and cones in the retina. Furthermore, the epithelium of the prostate, the parenchyma
of the liver and the walls of the blood vessels were also compromised, and the spleens
of these animals were enlarged though they were populated by apoptotic cells. In the
testes, the seminiferous tubules lacked mature sperm due to the progressive death of
differentiating germ cells. Tyro3, Axl, and Mer are all expressed in Sertoli cells, which
do not undergo cell death as Gata-1, a positive marker for Sertoli cells, was evident in
the testes of young adult triple mutant mice. The authors conclude that the coordinated
activity of these three receptors may be required for the necessary production of trophic
factors to support spermatogenesis. However, there are several caveats to the results
and discussions from these mouse mutants, especially regarding Tyro3. At the genetic
level, the neo cassette replacing the second fibronectin type III repeat was in-frame in
the final polypeptide. Hence, alternate splicing mechanisms may have indeed left some
of the functional aspects of the receptor intact. From the published Western blot, the
Tyro3 null mutants may actually be Tyro3 severe hypomorphs, since a very faint but
noticeable signal is visible in this mouse line.
3.4 Summary
From this brief summary it is clear that RTKs have important roles in
neurodevelopment. Aside from the more complex roles of BDNF and the interactions
between Eph receptors and their ephrin ligands, no other RTK signaling system has
been shown to definitively play a role in 5-HT axon guidance. In the next chapter, I will
26
present my studies which clearly show that the Tyro3 RTK plays an important,
previously unknown role in establishing and maintaining the 5-HT system.
27
Chapter 2: Materials and Methods
2.1 Introduction
In spite of the vast importance of this neurochemical, there are but few mutations
known to directly affect the development of the serotonergic system. We have further
characterized a 5-HT-specific developmental mouse mutation, known as anx, which is
specific for a defect in 5-HTergic innervation. This mutation causes a delay in 5-HT
axon guidance (Huynh, unpublished) which ultimately culminates in serotonergic
hypersprouting in terminal fields and death (Son et al., 1994). By characterizing the
neurobiological effects at this early stage in mammalian development, we can contribute
to the growing knowledge of molecules and pathways that mediate the complex
interactions required for brain development, and importantly, how these early events
play a role in the materialization of later, psychological disorders.
Forward genetic approaches and positional cloning allow for the identification of
novel genes and processes that might not otherwise have been implicated in a process
of interest – in this case, 5-HT system development. Here, I describe the positional
cloning of the anx gene which has selectively allowed us to provide strong evidence of a
receptor tyrosine kinase that has been previously shown to be expressed in nervous
tissue, but has not been shown to play a definite role in neurodevelopment. In
comparison to known receptor tyrosine kinases, our molecule of interest supports a role
in neurodevelopment.
The use of transgenic mouse models has provided a wealth of knowledge
regarding many different molecules and processes and how they are related to
28
numerous areas or research. In the following, I will also describe the results from the
use of transgenic mice to characterize the anx mutation, and discuss how our results
suggest certain roles which are disrupted by the anx mutation. I will also describe some
of the caveats of this approach to characterize anx, and additional research that may
elucidate some of the functional roles affected by the anx mutation.
2. 2 Meiotic recombination map
The anx mutation arose spontaneously in the F2 generation of a cross between
DW/J and an inbred strain derived from a cross between M. m. poschiavinus and an
inbred Swiss stock, and is maintained on a nonagouti (a) hybrid background referred to
as B6C3Fe a/a-anx/J. Heterozygous carriers of the mouse anorexia (anx) mutation
were obtained from Jackson Laboratories (Bar Harbor, Maine, USA) and crossed onto
the Molossinus/Ei and C57Bl6/J strains. I identified simple sequence length
polymorphisms using the following markers: D2Mit207, D2Mit276, D2Mit484, D2Mit104,
D2Mit190, D2Mit397, D2Mit277, and D2Mit446 that distinguish anx/anx homozygotes
and anx/+ heterozygotes from normal mice on the C57Bl6/J, Molossinus, and anx
background strains. A polymorphism at D2Mit190 distinguished 21 anx/anx mice from
their unaffected littermates and was used thereafter to identify heterozygous carriers
and homozygous mutants. Carriers on the C57Bl6/J strain background produce a ~145
bp PCR product, distinguishable from normal animals that produce a PCR product of
123 base pairs using the D2Mit190 marker. Similar differences were present on the
Molossinus/Ei strain background. Progeny from heterozygous intercrosses on each
29
background were genotyped using the above list of markers surrounding D2Mit190
generating a meiotic recombination map.
2.3 Candidate gene testing
i. Brain-derived neurotrophic factor (BDNF)
To determine whether anx is an allele of the BDNF gene, we performed a
complementation test by intercrossing anx heterozygotes with BDNF+/- mice. BDNF+/-
mice were obtained from Jackson Laboratories and genotyped as described in
Henneberger et al., 2000. All progeny were assessed for phenotypes associated with
BDNF-/- mice and anx/anx homozygotes in the first 3 weeks after birth.
ii. Glycine-amidinotransferase (GatM)
To test for possible involvement of GatM mutations contributing to the anx
phenotype and to determine potential creatine metabolism defects, I performed a
nutritional rescue experiment. After intercrossing with anx heterozygote males and after
birthing their litters, anx/+ Dams were fed powdered-rodent chow diet containing 2%
creatine monohydrate (Prolab, Chatworth, California, USA) to supply creatine to
anx/anx and normal littermates via their milk. All progeny were assessed to determine if
creatine supplementation reduced or altered anx/anx phenotypes in the first 3 weeks
after birth.
2.4 Sequencing candidate genes
In the critical interval containing the anorexia mutation, the exons and at least 75
base pairs of flanking intronic sequence of candidate genes were amplified by
30
polymerase chain reaction (PCR) and sequenced to identify possible mutations. We
tested candidate genes with known or postulated neurodevelopmental roles and/or
encoding membrane-bound/secreted or secreted proteins first (listed in Table 1). A
cytosine to guanidine point mutation in the putative signal sequence of Tyro3 was
identified in anx homozygotes compared to normal littermates, resulting in an arginine to
tryptophan conversion at the seventh position of the translated protein. This mutation
will thus be referred to as R7W-Tyro3.
2.5 Generation of transgenic animals
i. Mouse GFP-tagged normal and R7W-Tyro3 transgenes
Whole brains from P21 anx/anx and normal littermates were homogenized in
TrizolTM (Invitrogen, Carlsbad, California, USA) and extracted according to
manufacturer’s instructions. cDNA was synthesized from polyA mRNA from normal and
anx/anx brains by using the Superscript First-Strand Synthesis RT System (Invitrogen).
Tyro3 cDNA was amplified with high-fidelity platinum Pfx (Invitrogen) yielding a 2.6kb
PCR product using primers containing attB sites flanking the open reading frame for
recombination into entry vectors using Gateway TechnologyTM (Invitrogen), and
truncating the 3’ stop codon for in-frame C-terminal fusion with green fluorescent protein
(GFP). Entry clones were generated by inserting attB-flanked PCR products into the
pDONR201 vector in the presence of BP clonase (Invitrogen) and transformed in DB3.1
cells. The sequence of normal and R7W-Tyro3 clones were verified with primers. To
allow for the expression of Tyro3 transgenes in endogenous Tyro3 expressing domains,
we generated the T3XPRSSN vector
31
Table 1. Genes within the anorexia Critical Interval
1. isovaleryl coenzyme A dehydrogenase 2. dermatan-4-sulfotransferase-1 3. gene model 631 4. RIKEN cDNA 4921503C21; CG6187-like 5. DNA repair protein RAD51 homolog 1 6. zinc finger, FYVE domain containing 19 7. PKC-dependent PP1 inhibitory protein subunit 14 8. Kunitz-type protease inhibitor 1 precursor (HGF activator inhibitor type 1) 9. ras homolog gene family, member V 10. Vacuolar protein sorting 18 11. Delta-like protein 4 precursor (Drosophila Delta homolog 4) 12. Calcium-binding protein p22 (CHP) (Calcineurin homologous protein) 13. nucleolar protein ANKT 14. Complex I intermediate-associated protein 30, mitochondrial precursor 15. inositol 1,4,5-trisphosphate 3-kinase A 16. RNA polymerase II associated protein 1 17. MAX-interacting protein; Max dimerization protein 5 18. mitogen activated protein kinase binding protein 1; JNK-binding protein 1 19. Similar to phospholipase A2, group IVB (Cytosolic) (Fragment) 20. EH-domain containing protein 4 (mPAST2) 21. phospholipase A2-like 22. Similar to phospholipase A2, group IVB 23. Vam6/Vps39-like protein (Vps39 protein) 24. Neutral alpha-glucosidase C (EC 3.2.1.-) 25. Calpain 3 (Calpain L3) (Calpain p94) (Calcium-activated neutral proteinase 3) 26. zinc finger protein 106 27. Synaptosomal-associated protein 23 (SNAP-23) 28. Motor domain of KIF16A 29. MKIAA1300 protein 30. Codanin 1 31. tau tubulin kinase 1; tau-tubulin kinase 32. ubiquitin protein ligase E3 component n-recognin 1 33. cyclin D-type binding-protein 1; maternal inhibitation of differentiation 34. Erythrocyte membrane protein band 4.2 (Erythrocyte protein 4.2) (P4.2) 35. transglutaminase 5 36. Gamma-tubulin complex component 4 (GCP-4) 37. transformation related protein 53 binding protein 1; murine p53-binding protein. 38. RIKEN cDNA B430315C20 gene; KIAA0377-like 39. Creatine kinase, ubiquitous mitochondrial precursor 40. Stereocilin precursor 41. sperm-associated cation channel 2 42. Protein disulfide isomerase A3 precursor (EC 5.3.4.1) (Disulfide isomerase ER-60) 43. RNA polymerase II elongation factor ELL3 44. Small EDRK-rich factor 2 (4F5rel) (h4F5rel) (Gastric cancer-related protein VRG107) 45. Huntingtin interacting protein K 46. microfibrillar-associated protein 1 47. RIKEN cDNA D130060C09 isoform 1 48. eukaryotic translation initiation factor 3, subunit 1 alpha; merged
32
consisting of the 9.5kb region upstream of exon 2c, sequence-specific attR
recombination sites for the insertion of Tyro3 cDNA, an in-frame C-terminal GFP, and
Sv40 IVS polyA. Because sequence comparison between the non-coding sequences of
human, mouse, dog, and rat Tyro3 showed the highest conservation in the regions
contained within the first 9.5kb upstream of exon 2c, we postulated that these regions
might be likely to direct Tyro3 and transgene expression in the endogenous expressing
domains. The translational start site for all known signal sequence containing forms of
Tyro3 is located in exon 2c. Normal Tyro3 and R7W-Tyro3 were inserted into the
T3XPRSSN vector in the presence of LR clonase (Invitrogen). Resulting normal and
R7W-Tyro3 expression constructs were digested with NruI (NEB, Ipswich,
Massachusetts, USA) creating a 14kb transgene product purified with GeneClean Kit
(Qbiogene, Irvine, California, USA), and used to generate transgenic animals by
standard pronuclear microinjection.
ii. Human full-length Tyro3 transgene
To generate a full-length, untagged version of Tyro3 which was distinguishable
from endogenous Tyro3, I purified human Tyro3 following EcoR1 (NEB) digestion of
cDNA-containing pUC SRα, which was directly inserted into the filled-in Sal1 (NEB)
sites of PDONR201 by blunt-end ligation. Insertion into T3XPRSSN, digestion and
subsequent purification was the same as outlined above.
I therefore generated the following T3XPRSSN transgenic constructs: mouse
Tyro3 with C-terminal GFP fusion, mouse R7W-Tyro3 with C-terminal GFP fusion, and
human-Tyro3 with an intact stop codon.
33
iii. Identifying transgenic founder mice
Positive transgenic carriers for the GFP-tagged normal and R7W-Tyro3
transgenes were identified by Southern blot analysis. Approximately 10µg of genomic
DNA was digested with EcoR1 (NEB), separated on a 1% agarose gel, depurinated in
0.1N HCl for 2 x 20 minutes, denatured for 1 hour, neutralized for 1 hour, and blotted in
10X SSC onto Biodyne B membrane (Pall Corporation, East Hills, New York, USA).
The membrane was probed with a 700 bp GFP probe labeled with P32 at 106 cpm/ml of
hybridization solution for 16 hours at 42OC. The membrane was washed as follows: 2 x
1 hour, 2XSSC, 0.1% SDS; 2 x 1 hour, 1XSSC, 0.1% SDS at 65OC. Positive transgenic
animals were identified by exposing the membrane to HyperfilmTM MP (Amersham
Pharmacia Biotech, Little Chalfont, Buckinghamshire, United Kingdom) for 16-24 hours
at -80 OC. Positive transgenic animals for the full-length transgene were genotyped
using probes for the SV40 polyA IVS yielding a product size of 500 bp.
2.6 Expression analysis of Tyro3 in normal and anx/anx mice by RNA in situ
hybridization
Normal and anx/anx mice at P21 and normal mice at P14 were anaesthetized
with intraperitonial injection of Avertin (1.25% at 0.025ml/g) and cardially-perfused with
Ringer’s solution containing 50µg/ml heparin followed by 4% paraformaldehyde (PFA) in
diethyl-pyrocarbonate (DEPC)-treated 1xPBS. Brains were removed and post-fixed in
4% PFA overnight, rinsed 3 x 5 minutes in DEPC 1xPBS, washed 3 x 1 hour in DEPC
1xPBS, and placed in 30% sucrose in 1xPBS overnight. When no longer floating, the
34
brains were transferred into a 50:50 solution of 30% sucrose:optimal cutting
temperature (OCT) medium (Sakura Finetek, Torrance, California, USA) for 3 hours,
then washed 3 times in OCT for 1 hour each. All washes were performed at 4 OC.
Brains were embedded in OCT on dry ice and stored at -80OC. 14µm coronal sections
were taken using the Leica VT1000 cryostat onto Superfrost Plus microscope slides
(VWR, West Chester, Pennsylvania, USA).
Sense and antisense Tyro3 probes were generated by cloning a 588 bp region
coding for part of the intracellular kinase domain using HindIII (NEB) into pBluescript
KSII. To generate the sense probe, the Tyro3-containing vector was digested with
Xho1 (NEB) and in vitro transcribed with T3 RNA polymerase (Roche, Basel,
Switzerland). To generate the antisense probe, the vector was digested with EcoR1
(NEB) and in vitro transcribed with T7 RNA polymerase (Roche). For a positive control,
a probe for Kreisler was generated alongside Tyro3-specific probes by digesting 4CKS
with Nco1 (NEB) and in vitro transcribing with T7 RNA polymerase (Roche). All
riboprobes were DIG-tagged as specified in the manufacturer’s instructions.
RNA in situ hybridization was performed as outlined in Storm and Kingsley.
Staining was discontinued after 16-24 hours by 3 washes in 1x PBS and mounted with
either Permount (Daido Sangyo Co., Tokyo, Chiyoda-ku, Japan) or Cytoseal (Richard-
Allan Scientific, Kalamazoo, Michigan, USA) following standard dehydration and
rehydration into xylene.
35
2.7 Weight and survival data from the progeny of transgenic anx/+ heterozygote
intercrosses
Transgenic founders identified by Southern Blot analysis or by PCR genotyping
were crossed with anx/+ heterozygotes to generate transgenic anx/+ heterozygote
carriers. Litters from 5 mouse Tyro3 transgenic lines, 1 mouse R7W-Tyro3 mimic
transgenic line, and 3 human full-length Tyro3 transgenic lines were monitored regularly
from birth. Progeny from selected litters were weighed at P21. To measure the lifespan
of the offspring, animals with abnormal or anx-like behaviours were allowed to expire,
and the number of days survived was recorded.
36
Chapter 3: Results
3.1 Introduction
The anx mutation has provided a unique opportunity for studying a 5-HT-specific
aberration and allows for examining the relationship between axon guidance and
synaptogenesis between 5-HT projections and their terminal fields. Our data suggest
that among the known tyrosine kinases that play roles in cell proliferation, survival, and
migration, neurite/axonal outgrowth, and innervation to and synaptogenesis with central
targets, Tyro3, a tyrosine kinase of the TAM family of tyrosine kinase receptors, likely
plays a part in establishing these far-reaching circuits of the raphe nuclei. Tyro3 is
expressed at late embryonic stages in the mouse and its expression level increases in
the first 3 weeks after birth during a period of development in which axonal pathfinding
and synaptogenesis is maximal for 5-HT neurons. And, as Tyro3 has been found to be
predominantly expressed within specific regions of the mammalian brain, suggesting the
actions of Tyro3 may therefore be cell autonomous to 5-HT projections, we have found
Tyro3 expression in the hindbrain, where it has not been previously examined. Whether
this newfound expression coincides with the raphe nuclei has not been verified.
However, the activities of Tyro3 may indeed begin within the hindbrain or even 5-
HTergic neurons themselves.
Numerous lines of evidence have suggested to us that the point mutation
identified in the Tyro3 gene is indeed causative of the anx mutation. RNA in situ
analysis of Tyro3 mRNA at P21 is markedly reduced in known Tyro3-expressing
domains in the mammalian brain, and, perhaps even more profound than this, Tyro3
37
mRNA is absent in hypothalamic domains responsible for food intake behaviour, to
which the initial characterization of this mutation owes its name. Michael Huynh, a
previous graduate student in our lab, generated a TPH1-promoter driven PLAP reporter
system that solely labels Tph1-expressing axons and crossed these transgenic mice
onto an anx mouse line to generate transgenic anx/anx mice. At P15, these mice
exhibit much greater PLAP staining of several brain regions, and an altered pattern of
expression within the cortical layers. Within deeper cortical layers and at the most
superficial layer, dense PLAP staining is present in anx/anx mice relative to controls.
This pattern of PLAP expression was reversed in P0 transgenic mice in which anx/anx
mice lacked PLAP staining altogether and +/+ mice exhibited moderate staining.
Interestingly, heterozygous anx/+ mice exhibited an intermediate level of staining. This
research is the first to show a neonatal and heterozygous phenotype in anx mice which
has never been previously reported. Currently, Joanna Yu, a Ph. D. student in the lab,
has shown that anx/anx mice exhibit a platelet aggregation defect compared to normal
mice as determined by FACS sorting. Tyro3 is present in platelets and has a known
role in platelet aggregation, and a posited Tyro3 null mouse has been shown to have a
clotting defect. Hence, several lines of evidence strongly suggest that the point
mutation in Tyro3 is causative of the anx mutation.
To examine whether the point mutation found in Tyro3 is indeed causative of the
anx mutation, we generated several transgenic mouse lines to either rescue the anx
phenotype by insertion of a normal Tyro3 construct, or to mimic the anx phenotype by
insertion of a mutant Tyro3 construct. Two normal rescue Tyro3 constructs were
designed. One construct was made with mouse Tyro3 with an in-frame C-terminal GFP
38
tag. In doing so, these mouse lines could be used to trace transgene expression in
vibratome sections of fresh tissues by GFP fluorescence or by immunohistochemistry
using a GFP antibody. The other construct was made using human Tyro3, which is
virtually identical to the mouse Tyro3 gene. We labeled this construct “full-length” since
it contained an intact stop codon and would remove any potential interference of the
GFP tag, and importantly, allow us to perform future experiments that could selectively
knockdown the expression of this transgene. Using these transgenes, we have been
able to partially rescue two hallmarks of the anx phenotype by nearly doubling the body
weight and lifespan of anx/anx mice. We have also delayed the onset and severity of
the anx behavioural phenotype, though it has left us with the question of why either
rescue transgene could not overcome the endogenous mutation. Recently, Dr. Sabine
Cordes has examined cerebellar cytoarchitecture using immunofluorescent probes in
anx/anx brains transgenic for the mimic construct. In lieu of Purkinje cells normally
residing above the granule cell layer, large areas completely devoid of neurons are
present in transgenic anx/anx cerebella; an outcome suggestive of a gain-of-function
mutation in Tyro3. The consequences of this mutation and its effects are further
elaborated on in the discussion.
3.2 Meiotic recombination mapping and candidate gene sequencing identified a
point mutation in the signal sequence of Tyro3
To locate the anx mutation more precisely, we analyzed 335 progeny (670
meioses) from heterozygous intercrosses on the Molossinus/Ei (n = 224) and C57Bl6/J
(n = 111) strain backgrounds using simple sequence length polymorphisms (SSLPs),
39
and, thereby reduced the anx critical interval to a 3.2Mb region on chromosome 2
between markers D2Mit484 and D2Mit190 (Fig. 1A). During recombinational mapping,
we were able to eliminate two candidate genes within the critical interval. In the
complementation experiment in which BDNF+/- mice were crossed to anx/+
heterozygotes, compound BDNF/anx (n = 7) were normal and did not show anx
phenotypes at 3 weeks. Further recombinational mapping also positioned BDNF
outside of the anx critical interval. To test if GatM was causative of the anx phenotype,
we performed a nutritional rescue experiment in which anx/+ Dams were fed powdered-
rodent chow containing 2% creatine monohydrate after birthing. While normal and
heterozygous littermates were unaffected (n = 18), anx/anx homozygous mouse pups (n
= 5) receiving the nutritional supplement through the mother’s milk did not show
improvement from the anx phenotype and died by P21. Subsequently, we sequenced
the exons and flanking intronic regions of candidate genes having neurodevelopmental
roles or producing secreted or transmembrane proteins and identified a C to T point
mutation in the signal sequence of the receptor tyrosine kinase Tyro3 (Fig. 1C). In the
signal sequence-containing isoform of Tyro3, the mutation leads to a conversion from a
hydrophilic arginine to a hydrophobic tryptophan at the seventh amino acid position (Fig.
1D), henceforth referred to as R7W-Tyro3. The elimination of an NlaIV restriction site
by this point mutation was used in a PCR-based assay to genotype an additional 995
animals. All of the 176 affected progeny were homozygous for the point mutation,
whereas 819 unaffected animals were either heterozygous for the point mutation or
were normal (Fig.1E). We analyzed the predicted consequences of the R7W-Tyro3
mutation by using the Signal P program (www.cbs.dtu.dk/services/SignalP) a web-
42
Figure 1. Meiotic recombination mapping and sequencing of candidate genes
reveal a point mutation in the anorexia critical interval. (A) Meiotic mapping was
performed on a total of 335 progeny from anx/+ intercrosses. Progeny were analyzed
with the simple sequence length polymorphisms (SSLP) shown. Two recombinants
helped refine the critical interval to a 3.2Mb region between D2Mit484 and D2Mit190.
(B) A schematic diagram shows the positions of the SSLPs used for mapping and of
three relevant genes eliminated as candidates. A line diagram shows the approximate
positions of coding regions and Tyro3 within the anx critical interval. (C) Sequencing of
candidate genes within this region identified a point mutation in the signal sequence of
Tyro3, leading to an arginine-to-tryptophan conversion at the seventh position in the
translated protein, referred to as R7W-Tyro3. (D) The elimination of an NlaIV
restriction enzyme recognition site, which can be used to unambiguously genotype anx
animals, was further confirmed by analysis of 995 progeny from heterozygous anx/+
intercrosses. (E) Mice that exhibit the anx phenotype are homozygous for the point
mutation (n = 176), while mice that are heterozygous for R7W-Tyro3 (n = 584) or normal
(n = 235) do not show any anx-like phenotypes and appear normal.
43
based program to determine whether a given amino acid sequence has characteristics
of established protein signal sequences. The program does so by comparing an input
sequence to known characteristics of signal sequences from eukaryotic and prokaryotic
proteins. In all cases, three domains, known as the n-, h-, and c-region, are analyzed
and a potential signal sequence cleavage site is produced. In eukaryotes, the n-region
is only slightly arginine rich compared to prokaryotes, the h-region is short and very
hydrophobic, and the c-region lacks any amino acid sequence pattern. Analysis of the
first 45 amino acids of R7W-Tyro3 indicated that the observed amino acid change might
cause a slight deviation between the n-region and h-region of the signal sequence (Fig.
2). However, the R7W-Tyro3 conversion was not predicted to alter the status of the
translated polypeptide from that of a secretory molecule, nor was there a change in the
predicted cleavage site in the final protein product. These analyses suggested that the
signal sequence cleavage site should not be perturbed, but the mutation might affect
the localization, membrane retention, and/or potential post-translational modifications of
the extracellular domain which may be apparent in vivo. To more accurately determine
whether post-translational modifications were altered in the mutant protein, Joanna Yu,
a current Ph. D. candidate in our lab, performed an in vitro endoglycosidase H assay to
assess potential alterations to glycosylation of the extracellular domain. There was no
difference in the extent of glycosylation in the R7W-Tyro3 protein compared to normal
Tyro3. Additional analysis using GFP and RFP-tagged normal and R7W-Tyro3 that
were transfected in equal amounts in COS7 (Joanna Yu), Neuro2A (Joanna Yu and Dr.
Sabine Cordes), and HEK293 cells (Dr. Sabine Cordes) showed, at best, a very subtle
difference in the localization and apparent intensity of the mutant protein, in line with the
Figure 2.A. Normal Tyro3
B. R7W-Tyro3
44
45
Figure 2. Comparison of signal sequence predictions from SignalP analysis. The
first 45 amino acids of the normal Tyro3 and R7W-Tyro3 were examined using SignalP
to determine any aberrations resulting from the arginine-to-tryptophan conversion.
Comparison of the plots of the (A) normal and (B) mutant protein reveals an extremely
slight deviation between the n- and h-region in the mutant protein marked with an arrow.
However, the predicted cleavage site at amino acid 31 was virtually unaffected in the
mutant.
46
bioinformatic analysis. However, due to the strength of our genetic analysis including
100% identification of anx/anx animals as being homozygous for the point mutation, and
the elimination of any mutations in other candidate genes, we explored the possibility
that the R7W-Tyro3 mutation was responsible for the anx phenotype further.
3.3 Tyro3 expression in normal P21 mouse brain
To test if Tyro3 was expressed in regions affected by the anx mutation, I
performed RNA in situ hybridization on sections from normal and anx/anx mouse brains
at P21 to determine if there were any differences in the level or location of Tyro3
expression. Previous analyses had reported Tyro3 expression in cortical layers 2/3 and
6 in the mouse, at high levels in the CA1 region of the hippocampus but low levels in the
adjacent CA3 region and negligible expression in the dentate gyrus, in the median
eminence of the hypothalamus, and in the granule cells and at low levels in the Purkinje
cells of the cerebellum. There have been no previous reports of Tyro3 expression in
other areas of the brain in the 3 week old mouse or rat.
In my analyses, Tyro3 was expressed throughout the cortex in what appear to be
large Pyramidal neurons. The highest expression was evident in cortical layers 2/3, and
layers 5 and 6. Tyro3 expression was barely detectable in layers 1 or 4 (Fig. 3A, C).
Within the hippocampus, Tyro3 expression was high in the CA1 region, the CA3 region
and the dentate gyrus. The strong expression of Tyro3 mRNA in the dentate gyrus is
not an artifact of increased cell density within this structure, as a sense probe against
Tyro3 yielded only minute signal strength above background in these neurons. The
pattern of expression in the CA1 and CA3 region appears as a uniform band that
48
Figure 3. Analysis of Tyro3 expression in the cerebral cortex in P21 normal and
anx/anx mice by RNA in situ hybridization. (A) Tyro3 expression in the brains of
P21 normal mice show widespread staining throughout the cortex. However, Tyro3
expression is layer-specific being mainly expressed in layers 2/3, 5 and 6. This is
particularly evident at higher magnification (C). (B) Conversely, anx/anx mice show
dramatically reduced levels of Tyro3 expression. Although the Tyro3 expression
appears to maintain its laminar expression, it appears to absent from layer 2 or shifted
into layer 3, as can be seen at higher magnification in (D).
49
stretches across the hippocampus in what are likely Pyramidal cells. At high
magnification, Tyro3 expression outlining the neurons of the dentate gyrus and into the
extension leading away from these neurons appears punctuate (Fig. 4A, C). In the
hypothalamus, Tyro3 was expressed in the median eminence as previously reported.
However, contrary to previous reports, I detected Tyro3 mRNA in regions important for
regulating energy balance: the arcuate nucleus, located dorsal and lateral to the median
eminence along the rostral-caudal axis, and in the large ventromedial nuclei of the
hypothalamus (Fig. 5A, C). In the cerebellum, Tyro3 is expressed at high levels in
granule cells and the Purkinje cells that separate the granule cell layer from the
molecular layer. At high magnification, Tyro3 forms a fine outline of the soma of
Purkinje cells. There are no previous reports of Tyro3 in other structures within the
central nervous system (Fig. 6A, C, E). However, in sagittal sections through the
brainstem of the adult mouse, Tyro3 is expressed in clusters which approximate the
location of neurons that form the raphe nuclei (Fig. 7).
Tyro3 expression in the arcuate nucleus, the hippocampus, and presumptive 5-
HT producing neurons is particularly notable as deficits in these neurons and these
regions have been observed in anx/anx mutants.
3.4 Tyro3 expression is altered in numerous brain regions in anx/anx mice
Next, I examined whether Tyro3 expression was affected in anx/anx mutants. At P21,
Tyro3 expression in anx/anx brains is altered in some regions. In the cortex, Tyro3
expression appears to maintain its laminar expression in layers 3, 5, and 6, but the
signal appears markedly reduced compared to normal cortices. Furthermore, Tyro3
51
Figure 4. Analysis of Tyro3 expression in the hippocampus in P21 normal and
anx/anx mice by RNA in situ hybridization. Tyro3 expression is present in the CA1
region, the CA3 region, and the dentate gyrus of P21 normal mice hippocampi. In
anx/anx hippocampi, Tyro3 expression is markedly reduced, but appears to remain high
in the dentate gyrus. In the CA1 region, the width of the band expression Tyro3 is
reduced compared to the normal Tyro3 expression pattern indicated by the boxed area
in (A) and (B) which is shown at higher magnification in (C) and (D), and the Tyro3
expressing region in the dentate gyrus appears condensed.
53
Figure 5. Analysis of Tyro3 expression in the hypothalamus in P21 normal and
anx/anx mice by RNA in situ hybridization. (A, B) In the hypothalamus of P21
normal brains, Tyro3 is expressed in the median eminence, the arcuate nucleus, and
the ventromedial hypothalamus. (C, D) In P21 anx/anx brains, Tyro3 expression is
absent in the arcuate nucleus and ventromedial hypothalamus, and is dramatically
reduced in the median eminence.
55
Figure 6. Analysis of Tyro3 expression in the cerebellum of P21 normal and
anx/anx mice by RNA in situ hybridization. (A) In the normal cerebellum, Tyro3 is
expressed in the granule cells; and can be seen to outline the soma of the large
Purkinje cells. In cerebellar white matter, fine Tyro3 expressing processes can be seen
at high magnification and appear beaded (marked with arrows in E).. In P21 anx/anx
brains, Tyro3 expression in granule cells is maintained, however, Tyro3 expression is
undetectable in Purkinje cells. Numerous Tyro3 positive cell bodies appear in the white
matter surrounding the cerebellum which are not present in normal brains, but Tyro3
expressing processes are not readily apparent.
57
Figure 7. Analysis of Tyro3 expression in the brainstem in P21 normal mouse
sagittal sections by RNA in situ hybridization. Tyro3 is expressed in the brainstem
of normal P21 mice in loose but noticeable clusters. (A) Tyro3 is expressed in neurons
just anterior and dorsal to the cerebellum. This is likely the dorsal raphe of the anterior
cluster of 5-HT neurons. (B-D) Tyro3 is also present in clusters located near the
ventral edge of the hindbrain.
58
expression in layer 2 is barely discernible from background. At high magnification,
Tyro3 expression forms a faint outline of individual neurons (Fig 3B, D). In the
hippocampus of anx/anx brains, Tyro3 expression is maintained in the CA1 region,
however, the signal strength is greatly reduced compared to that in normal brains. Also,
the thickness of the Tyro3 expressing band in this region is reduced. There is little to no
expression of Tyro3 in the CA3 region of mutant animals. In the dentate gyrus, Tyro3
expression is comparable to the signal strength in the CA1 region. However, within the
cell bodies of some neurons, additional large, spherical bodies are evident (Fig. 4B, D).
Tyro3 expression in the median eminence of the hypothalamus is intact in anx/anx
brains at P21. However, Tyro3 expression is absent in the arcuate and ventromedial
nuclei altogether (Fig. 5B, D). The cerebellum of anx/anx animals appears
morphologically normal, and at low magnification, Tyro3 expression in the granule cell
layer is intact. However, there is little to no Tyro3 expression in Purkinje cells lining the
granule cell layer. Surprisingly, cells expressing Tyro3 can be found in the most
superficial layer of the cerebellum (Fig. 6B, D, F).
3.5 Generation and identification of transgenic mice
Of all the transgenic mouse founders generated by pronuclear microinjection of
our constructs, five of six Rescue-GFP lines, two of three Rescue-Full Length, and one
of two Mimic-GFP lines were able to breed successfully. Mice were identified by
Southern Blot analysis using a GFP probe, and progeny were subsequently genotyped
in a PCR-based assay (Fig. 8).
60
Figure 8. Construction of the Tyro3 transgenes for rescue and mimic analyses.
(A) The Tyro3 transgenes were constructed using a 9.5kb BamH1-Not1 fragment just
upstream of the translational start site located in exon 2c (adapted from Biesecker et al.,
1995). The translational start site for all known signal sequence containing forms of
Tyro3 is located in exon 2c. The signal sequence of Tyro3 is indicated in black. (B)
The T3EXPRSSN vector was designed with sequence-specific attR sites to allow for the
insertion of Tyro3 or R7W-Tyro3 cDNA using the Gateway System (Invitrogen), two
selection agents, and a C-terminal GFP tag for future tracing experiments and a
stabilizing Sv40 IVS pA tail. (C) Positive clones for the Tyro3 transgenes were
digested with NruI yielding a 14kb transgene that was purified to generate transgenic
mice by pronuclear microinjection. Transgenic founders carrying the GFP-tagged Tyro3
(D) or R7W-Tyro3 (E) were identified by standard Southern blot analysis using a GFP
probe. (F) Transgenic founders for the Full-Length Tyro3 transgene were identified by
PCR using primers located in the Sv40 IVS pA region (F).
61
3.6 Transgenic anx/anx homozygotes have improved body weight at P21 and live
longer than their non-transgenic anx/anx littermates
Normal and anx/+ heterozygote mice transgenic for full-length human or GFP-
tagged mouse Tyro3 exhibited no obvious abnormal phenotypes, weight gain or loss or
impaired survival, and were indistinguishable from each other and their normal or
heterozygote littermates. However, anx/anx homozygote animals transgenic for either
the normal mouse or human Tyro3 transgene showed very mild or no anx-related
phenotypes at P21.
At P21, transgenic anx/anx animals had dramatic body weight increases
compared to their non-transgenic anx/anx littermates in all transgenic mouse lines (Fig
9). In GFP-tagged transgenic line Rescue-GFP#1, transgenic anx/anx mice (n = 15)
had an average increased body weight of 83.5% compared to their non-transgenic
anx/anx littermates (n = 5, p < 0.01, one-way ANOVA). Pooled body weight data of
transgenic anx/anx mice from the remaining GFP-tagged lines (n = 18) showed a 69.5%
increase in average body weight compared to their non-transgenic anx/anx littermates
(n = 5, p < 0.01, one-way ANOVA). In the Full-length Rescue line#1, transgenic
anx/anx mice (n = 4) weighed 92.8% more compared to their anx/anx littermates (n = 5,
p < 0.01, one-way ANOVA). When all transgenic anx/anx mice from all lines are
compared to all anx/anx littermates, there is an average body weight increase of 82.3%
at P21 (Table 2).
Normally, anx/anx mice exhibit anxiogenic behaviours including head weaving
and uncoordinated gait by P16-P17 and die by P21. In line Rescue-GFP#1, 23 anx/anx
transgenic mice showed little or no overt anx-like phenotypes at P21, and survived until
Rescue-GFP#1: Body Weight at P21
0
2
4
6
8
10
12
14
Genotype
Bo
dy
w
eig
ht (g
)
+/+ anx/+ anx/anx anx/anx, tg
Cumulate Body Weight Analysis: Transgenic Tyro3-GFP M ice at P21
0.00
2.00
4.00
6.00
8.00
10.00
12.00
14.00
Genotype
Bo
dy
w
eig
ht a
t P
21
(g
)
N o r m a l a n x / + a n x / a n x a n x / a n x ; t g
Body Weight of M ice from Full-Length Human Tyro3 at P21
0.00
2.00
4.00
6.00
8.00
10.00
12.00
Genotype
Bo
dy
W
eig
ht a
t P
21
(g
)
N o r m a l a n x / + a n x / a n x a n x / a n x ; t g
Body Weight of All Transgenic M ouse Lines at P21
0.00
2.00
4.00
6.00
8.00
10.00
12.00
G e n o t y p e
N o r m a l a n x / + a n x / a n x a n x / a n x ; t g
normal anx/+ anx/anx anx/anx;0
10.0
8.0
6.0
4.0
2.0
12.0
0
10.0
8.0
6.0
4.0
2.0
12.0Bo
dyw
eigh
t at P
21 (g
)
n = 7 37 4 15
0
10.0
8.0
6.0
4.0
2.0
12.0
Body
wei
ght a
t P21
(g)
0
10.0
8.0
6.0
4.0
2.0
12.0
Body
wei
ght a
t P21
(g)
normal anx/+ anx/anx anx/anx;
n = 32 64 5 33
normal anx/+ anx/anx anx/anx;
n = 7 12 5 4
normal anx/+ anx/anx anx/anx;
n = 39 76 10 37
Body
wei
ght a
t P21
(g)
0
5.0
4.0
3.0
2.0
1.0
6.0
Body
wei
ght a
t P21
(g)
Body Weight at P21: anx/anx vs. anx/anx;tg
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Bo
dy
we
igh
t a
t P
21
(g
)
anx/anx anx/anx anx/anxanx/anx;t
g
anx/anx;t
g
anx/anx;t
gRescue-GFP Full-Length #1 All Weight
Body Weight at P21: anx/anx vs. anx/anx;tg
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Bo
dy
we
igh
t a
t P
21
(g
)
anx/anx anx/anx anx/anxanx/anx;t
g
anx/anx;t
g
anx/anx;t
gRescue-GFP Full-Length #1 All Weight
Body Weight at P21: anx/anx vs. anx/anx;tg
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Bo
dy
we
igh
t a
t P
21
(g
)
anx/anx anx/anx anx/anxanx/anx;t
g
anx/anx;t
g
anx/anx;t
gRescue-GFP Full-Length #1 All Weight
Body Weight at P21: anx/anx vs. anx/anx;tg
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Bo
dy
we
igh
t a
t P
21
(g
)
anx/anx anx/anx anx/anxanx/anx;t
g
anx/anx;t
g
anx/anx;t
gRescue-GFP Full-Length #1 All Weight
Body Weight at P21: anx/anx vs. anx/anx;tg
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
Bo
dy
we
igh
t a
t P
21
(g
)
anx/anx anx/anx anx/anxanx/anx;t
g
anx/anx;t
g
anx/anx;t
gRescue-GFP Full-Length #1 All Weight
non-Tg Tg non-Tg Tg non-Tg Tgn = 4 15 5 4 10 35
Rescue-GFP #1 Full-Length #1 Cumulative
A B
C D
E
Figure 9. Bodyweight of P21 Transgenic Mouse Lines
* *
* *
* * *
Tyro3-GFP#1 all Tyro3-GFP
Tyro3-FL#1 All Tyro3-Tg
* *
* *
62
63
Figure 9. Analysis of bodyweight of transgenic anx/+ progeny at P21. Transgenic
anx/anx progeny from all lines had improved bodyweight at P21. (A) In Rescue-GFP
#1, P21 transgenic anx/anx mice weighed significantly more than their anx/anx
littermates (p < 0.01). (B) When all mice transgenic for the GFP-tagged rescue
transgene are pooled, this trend is maintained (p < 0.01). (C) In Full-Length #1, P21
transgenic anx/anx also weighed significantly more than their anx/anx littermates (p <
0.01), which is shown when all transgenic mice for either the GFP-tagged or Full-
Length Tyro3 transgene are pooled (D, p < 0.01). (E) Transgenic anx/anx mice weigh
almost twice as much as their non-transgenic littermates (p < 0.01 for all groups).
92.8%6.001g ± 0.520(n = 4)
3.113g ± 0.268(n = 5)Full-Length #1
69.5%5.672g ± 0.800(n = 18)
Rescue-GFP#2, 4, 6
83.5%6.157g ± 1.176(n = 15)3.355g ± 0.580
(n = 5)
Rescue-GFP#1
% WeightIncreaseanx/anx; Tganx/anxMouse Line
Table 2. Percentage Bodyweight Difference in TransgenicMouse Lines
62
65
or past P35, unlike 9 of their anx/anx littermates that died by P21 (fig 10, p < 0.01, Χ2).
This is approximately a 66.7% increase in the lifespan of anx homozygotes. When the
lifespan of all other GFP-tagged mouse lines are combined, 19 transgenic anx/anx
animals survived at least until P35 compared to 7 anx/anx littermates that died by P21.
In two full-length human Tyro3 transgenic lines, ten transgenic anx/anx animals
exhibited the same lifespan increase compared to nine anx/anx littermates (p <
0.01, Χ2). Several transgenic anx/anx animals survived beyond P42, essentially
doubling their expected lifespan.
Figure 10. Survival of anx/anx Animals from Transgenic LinesN
umbe
r of M
ice
25
20
15
10
5
0
n = 9 0 0 23 8 0 0 19 4 0 0 5 5 0 0 5
Rescue-GFP Rescue-GFP Full-Length Full-Length #1 #2, 4, 5, 6 #1
#2
0
5
10
15
20
25
1 2 3 4 5 6 7 8anx/anx anx/anx; Tyro3-GFP
anx/anx anx/anx; Tyro3-GFP
anx/anx anx/anx; Tyro3-FL
anx/anx anx/anx; Tyro3-FL
Do not survive past P21
Survived past P35
66
67
Figure 10. Analysis of survival rates of transgenic and non-transgenic anx/anx
progeny. anx/anx homozygous mice are characterized by head weaving and
uncoordinated gait appearing between P15-P18 and die by P21. In all mouse lines
transgenic for either the GFP-tagged or Full-Length Tyro3 transgene, transgenic
anx/anx progeny show no or mild anx phenotypes at P21, which progressively increase
until death after P35. Many transgenic progeny survive up to and past P42, essentially
doubling the lifespan of anx/anx homozygous littermates.
68
Chapter 4: Discussion
4.1 Signal sequence mutations
Here, I have identified a point mutation in the signal sequence of the receptor
tyrosine kinase Tyro3 that is almost certainly causative of the anx mutation. A number
of mutations have previously been identified in the signal sequence of certain genes
that have affected post-translational processing and manifested in disease. A point
mutation causing an arginine to glycine conversion in the signal peptide of the human
factor X, known as XSanta Domingo inhibits cleavage by signal peptidase, even though the
mutant form is normally transported to the endoplasmic reticulum (Racchi et al., 1993).
The mutation also inhibits further post-translational processing of the mutant form,
which is retained in the endoplasmic reticulum, and is neither glycosylated nor secreted,
which normally occurs in the unaffected protein. This mutation is directly responsible for
bleeding diathesis in affected individuals.
Although bioinformatic analysis did not predict an appreciable impact on Tyro3
processing and we did not observe dramatic changes in the gycosylation state of the
extracellular domain or localization of the mutant protein in cell culture, the absence of
noticeable changes assayed in vitro may not be able to uncover subtle changes
incurring dramatic effects in vivo. For example, Smad4 is a necessary component for
proper early mouse development and is shuttled constantly between the cytoplasm and
nucleus by the presence of a nuclear localization and nuclear export signal. When the
nuclear export signal is mutated, Smad4 has been shown to sequester in the nucleus of
embryonic stem cells and is therefore hypothesized to effect TGF-β signaling in these
69
cells and negatively affect embryonic development. However, mice engineered with
these mutations develop normally, indicating that Smad4 nucleocytoplasmic shuttling is
not essential for normal mouse embryonic development and that compensatory
mechanisms may exist (Biondi et al., 2007). Furthermore, the question remains as to
whether R7W-Tyro3 protein is indeed secreted in vivo, if it is stable, and if it’s tyrosine
kinase activity is retained.
4.2 Expression analysis
Consistent with previous results in the adult mouse brain, Tyro3 mRNA was
detected in layers 2/3, 5 and 6 of the cerebral cortex, in the CA1 region of the
hippocampus, in the median eminence of the hypothalamus, and in the granule cells of
the cerebellum (Lai et al., 1994, Schulz et al., 1995). However, in contrast to these
studies, I consistently detected Tyro3 mRNA at different expression levels in certain
areas and in other, previously unnoticed brain regions. These include the CA3 region
and dentate gyrus of the hippocampus, the arcuate nucleus and ventromedial nucleus
of the hippocampus, and the Purkinje cells of the cerebellum. These striking differences
can perhaps be attributed to several factors. In previous studies, radiolabeled Tyro3
cDNA riboprobes may not have had the necessary signal strength to be detected even
after extensive exposure to autoradiographic film in lower Tyro3 expressing brain
regions. This may be especially true for cerebellar Purkinje cells, in which the finer
Tyro3 expression outlining these neurons may be masked by the much stronger granule
cell expression. As well, the small region encompassing arcuate nucleus neurons in the
hypothalamus located at the dorsal edge of the brain may not produce an adequate
70
signal using radiolabeled probes that were consistently detected using DIG-labeled
riboprobes. Also, within the hypothalamus, the ventromedial nuclei are clustered very
closely to the midline of the brain. Sagittal sections that are not adjacent to the midline
may indeed “miss” this cluster of cells which are present in serial coronal sections
through the hypothalamus.
We detected Tyro3 mRNA consistently in the CA3 region and dentate gyrus in
the hippocampus at comparable levels to CA1, which would not be rectified by the
possible explanations given above for other brain regions. Why previous studies did not
detect as strong or virtually any signal in these regions remains a mystery. One
possibility is that Tyro3 expression specifically in the CA3 region and dentate gyrus is
reduced after P21. These previous studies only used “adult” mouse brains but did not
specify the exact age of the mice used. Another possibility is that our method of OCT
embedding our brains somehow preserves expression in these regions that are
negatively affected via paraffin embedding. However, this is very unlikely since other
areas expressing Tyro3 should also be affected in paraffin-embedded brains.
At P21, Tyro3 mRNA expression in anx/anx brains is altered in all Tyro3
expressing regions. In the cortex, Tyro3 expression is markedly reduced, and appears
virtually absent in layer 2. In the hippocampus, the thickness of the band expressing
Tyro3 in CA1 is reduced, and the neurons of the dentate gyrus are often marked with
internal regions of dark staining. The hypothalamus and cerebellum have a complete
absence of Tyro3 in the arcuate and ventromedial nuclei, and of the Purkinje cells,
respectively. In summary, Tyro3 is not only expressed in regions and neuronal
populations affected in anx mutant homozygotes, but in these animals, Tyro3
71
expression is markedly reduced. To date, no other gene with known or postulated roles
in neurodevelopment has been found to have such widespread change in its level of
pattern or expression in anx/anx animals. Thus Tyro3 expression in normal and mutant
animals is consistent with its possible role as the anx causative gene.
4.3 Partial rescue in transgenic mice
The presence of either GFP-tagged mouse or full-length human Tyro3 transgene
in anx/anx mice imparted partial rescue of the anx homozygous phenotype, reducing
the phenotype severity and increasing the overall health and bodyweight of these
animals at P21, and nearly doubling the lifespan of anx/anx homozygotes. The
question of whether this increase in weight is due to the reduced hyperactivity seen in
these animals, leading to less energy expenditure, or to somewhat corrected energy
and appetite regulation, is in part answered by NPY immunohistochemistry performed
by Dr. Sabine Cordes. In the hypothalamus of normal mice, axons from the arcuate
nucleus project anteriorly and dorsally to target areas including the paraventricular
nucleus and lateral hypothalamus, among other regions. Neuropeptide Y, which is
produced in the cell bodies of arcuate nucleus neurons, can be identified by the diffuse
staining ventral to this region where it is being transported in axons leading to the
presynaptic terminal. In anx/anx animals, Neuropeptide Y is found in the cell bodies of
arcuate nucleus neurons, with very little detectable staining in ventral regions (Cordes,
unpublished). Although Neuropeptide Y production does not appear affected, the
sequestering of the protein within arcuate nucleus neuron is the result of a failure of
proper cell body and possibly axon migration, thereby stunting the growth of these
72
projections (Cordes, unpublished). Interestingly, the hypothalamus of the transgenic
anx/anx mouse shows a phenotype intermediate between that of normal and anx/anx
mice. There appears to be a greater amount of NPY staining in the region ventral to the
arcuate nucleus, though not as great as in the normal brain, and there is less apparent
NPY staining in the arcuate nucleus neurons themselves compared to the anx/anx
hypothalamus. In P21 anx/anx mice, NPY-positive neurons are present in small
numbers in the arcuate nucleus, but some reside in the median eminence, a region
normally devoid of NPY-positive neurons. The transgenic anx/anx hypothalamus shows
NPY-positive neurons in mid-migration moving out of the median eminence towards the
arcuate nucleus. This intermediate phenotype at the neurobiological level appears to
reflect the intermediate effect of improved body weight in transgenic anx/anx animals.
The question as to the effect of the anx mutation on the absence of Tyro3 in key
areas of the hypothalamus involved in energy balance and appetite regulation gives rise
to a number of considerations. First, the failure of arcuate nucleus neurons to project
from the cell body suggests that Tyro3 is involved in axonal outgrowth and/or axonal
stability. Second, Tyro3 may also be responsible for stabilizing synapses between
incoming axons and target locations via cell adhesion or signaling at the terminal fields.
The inclusion of both Ig and FNIII domains in the extracellular region suggests that
Tyro3 expression in these cells may induce multiple consequences in order for axons to
project in the proper orientation and to secure proper synapses. Furthermore, Tyro3 is
known to mediate its downstream effects via homophilic interactions with other Tyro3
molecules. A disruption of the interactions of Tyro3 and the reduced expression within
the hypothalamus may ultimately result in the alterations observed in the hypothalamus.
73
The question remains as to why there is only partial rescue of the anx phenotype
in these transgenic mice. Initially, we had hypothesized that the C-terminal GFP tag
might disrupt the ability of Tyro3 receptors to signal normally by interfering with Tyro3
dimerization, or that GFP disallowed phosphorylation of tyrosine kinase targets.
Alternatively, GFP-mediated interactions between expressed transgenes may lead to
constitutive kinase activity causing additional disruptions in downstream activity.
Previous finding have shown that overexpression of Tyro3 causes ligand-independent
activation. However, full-length Tyro3 failed to fully rescue the anx phenotype and
these animals exhibited the same extent of partial rescue. We are currently
investigating several other possibilities. First, the transgenes may not contain all the
regulatory regions upstream of the 9.5kb genomic region cloned into the transgene or
perhaps within intronic regions which were removed between exons 3 to 18. Due to
this, normal Tyro3 may not be expressed in all the endogenous brain regions and/or at
physiologically relevant levels. Second, the anx mutation may cause dominant aversive
defects which cannot be completely overcome by the presence of a transgene. In this
case, the anx mutation might be a gain-of-function mutation that leads to a disruption of
normal brain development. Our most recent evidence from the cerebellum of mice
transgenic for the R7W-Tyro3 construct supports this hypothesis. As performed by Dr.
Sabine Cordes, immunohistochemical staining of Purkinje cells using an antibody
against calbindin showed that in the anx/anx cerebellum, Purkinje cells are present
though rounder and less uniformly-spaced than normal Purkinje cells, however, a large
number of Purkinje cells are missing in anx/anx cerebellum expressing R7W-Tyro3. In
the case that the anx mutation was a loss-of-function mutation, we would expect that
74
the presence of R7W-Tyro3 would have no effect on Purkinje cells in anx/anx mice.
These data suggest that the R7W-Tyro3 allele indeed has gain of function activity that is
deleterious to Purkinje cells and possibly, other neurons. These observations may
reflect a functional deficit in signaling through Tyro3-mediated pathways considering the
vast dendritic arborizations and inputs received by these cells, and thus neuronal
stimulation may also be a requirement for neuron survival.
4.4 Evidence suggestive that Tyro3 is causative of the anx mutation
In the anx/anx animal, we have identified a point mutation in Tyro3, a receptor
tyrosine kinase that was initially implicated for having roles in oncogenesis. The onset,
level, and pattern of Tyro3 expression within the nervous system are strongly correlated
to serotonergic innervation and targeting, and its role in neural development. Firstly,
Tyro3 expression in serotonergic target areas, namely the cortex, the hippocampus, the
hypothalamus, and the cerebellum, is upregulated just after birth during a period of time
when axonal targeting and synaptogenesis of serotonergic projections is maximal. It is
also these target areas that have extensive innervation due to aberrant hypersprouting
of serotonergic projections in the anx/anx animal. Furthermore, Tyro3 is expressed in
the brainstem similar to regions where the raphe nuclei reside. Secondly, mice that are
homozygous for this point mutation are the only animals that exhibit anx phenotype
characteristics, which has been shown in nearly 200 affected animals in 3 different
background strains. No other animal that is either heterozygous or lacking the mutation
altogether shows any abnormal behaviours associated with the anx phenotype. Thirdly,
Tyro3 expression in anx/anx brains is greatly affected in its level and pattern of
75
expression. In some areas, such as the cortex and hippocampus, there appears to be a
reduction of detectable Tyro3 mRNA as assayed by RNA in situ hybridization. In other
areas, such as the arcuate nucleus and ventromedial nucleus of the hypothalamus, and
of the Purkinje cells of the cerebellum, there is no detectable Tyro3 expression which is
clearly evident in normal brains. Fourthly, anx/anx animals carrying a normal Tyro3
transgene show partial rescue in bodyweight at P21 and of the failure to thrive
phenotype. These transgenic animals show attenuated anxiogenic behaviours, appear
healthier, and live almost twice as long as their non-transgenic anx/anx littermates.
Taken together, these data strongly suggest that the point mutation in Tyro3 is the
causative agent of the anorexia mutation and its associated phenotypes and
behaviours.
4.5 Possible roles of Tyro3
In anx/anx homozygotes, the reduction in Tyro3 mRNA may result from several
possibilities. The point mutation itself may lead to instability of the RNA transcript
thereby deteriorating prior to translation on its own or it may also be directed towards an
mRNA decay pathway. Tyro3 mRNA trafficking might also be disrupted, disallowing
transcripts to reach target areas prior to translation. However, if the anx mutation is
indeed a gain of function mutation in Tyro3, retrograde signaling from overactive Tyro3
may somehow downregulate Tyro3 mRNA transcription. In non-neural cells, the
intracellular kinase domain has been shown to immunoprecipitate with src, RanBPM,
and, in particular, the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI3K) and
activation of Tyro3 leads to Akt phosphorylation. Akt phosphorylation has been shown
76
to affect neuronal survival, axon specification, axon outgrowth, neurite outgrowth, cell
polarity, neuroprotective effects due to injury and neurotoxicity. Interestingly, Akt
overexpression has been shown to inhibit cell differentiation in adult hippocampal neural
progenitor cells (Peltier et al., 2007). We are currently investigating whether Akt
phosphorylation is affected in anx/anx brains via Western Blot analysis and in R7W-
Tyro3 expressing cell lines.
At this point, our observations point to mislocalization of Tyro3 RNA and protein
as the likely primary cause of the anx phenotype, and, from preliminary Western
analyses, Akt phosphorylation and thus, Tyro3 signaling is only mildly impacted.
77
CONCLUSION
Partial rescue of the anx/anx phenotype by the presence of a normal Tyro3
transgene combined with disruptions in the mRNA expression in mutant brains and
100% linkage to a point mutation within the signal sequence lead us to believe that
Tyro3 is the anx causative gene. Our most recent data suggest that this is a gain-of-
function mutation and we are exploring whether RNA localization abnormalities
particularly evident in the cerebellum are a primary or secondary consequence of the
R7W-Tyro3 mutation and/or this mutation disrupts Tyro3 signaling. Continued
biochemical analysis to assess changes in the brain and in downstream signaling
pathways will elucidate the mechanisms underlying the neurobiological phenotypes
observed in the anx/anx mouse, and may ultimately provide greater insight into the
neurological pathologies of human affective disorders at the genetic, molecular, and
cellular level.
78
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