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1005Development 125, 1005-1015 (1998)Printed in Great Britain ©
The Company of Biologists Limited 1998DEV6328
Integrated FGF and BMP signaling controls the progression of
progenitor cell
differentiation and the emergence of pattern in the embryonic
anterior
pituitary
Johan Ericson 1,2, Stefan Norlin 1, Thomas M. Jessell 2 and
Thomas Edlund 1,*1Department of Microbiology, Umeå University,
S-901 87 Umeå, Sweden2Howard Hughes Medical Institute, Department
of Biochemistry and Molecular Biophysics, Center for Neurobiology
and Behavior,Columbia University, New York, NY 10032, USA *Author
for correspondence (e-mail: [email protected])
Accepted 7 January 1998: published on WWW 17 February 1998
The mechanisms by which inductive signals control theidentity,
proliferation and timing of differentiation ofprogenitor cells in
establishing spatial pattern indeveloping vertebrate tissues remain
poorly understood.We have addressed this issue in the embryonic
anteriorpituitary, an organ in which distinct hormone cell types
aregenerated in a precise temporal and spatial order from
anapparently homogenous ectodermal primordium. Weprovide evidence
that in this tissue the coordinate controlof progenitor cell
identity, proliferation and differentiationis imposed by spatial
and temporal restrictions in FGF- andBMP-mediated signals. These
signals derive from adjacentneural and mesenchymal signaling
centers: theinfundibulum and ventral juxtapituitary mesenchyme.
The
infundibulum appears to have a dual signaling function,serving
initially as a source of BMP4 and subsequently ofFGF8. The ventral
juxtapituitary mesenchyme appears toserve as a later source of BMP2
and BMP7. In vitro, FGFspromote the proliferation of progenitor
cells, prevent theirexit from the cell cycle and contribute to the
specificationof progenitor cell identity. BMPs, in contrast, have
noapparent effect on cell proliferation but instead appear toact
with FGFs to control the initial selection of thyrotrophand
corticotroph progenitor identity.
Key words: Progenitor cell, Cortocotroph, Pituitary, BMP,
FGF,Spatial patterning
SUMMARY
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INTRODUCTION
In vertebrate embryos, insight into the mechanisms by whsecreted
factors control tissue development has emerged fseveral different
systems. Studies of mesodermal and netube patterning have provided
information on the role inductive factors in controlling the
spatial pattern of progenitcell identity and fates (Tanabe and
Jessell, 1996; Bumcrot McMahon, 1995; Smith, 1995) but have not
resolved how ttiming of terminal cell differentiation is regulated.
In otheembryonic tissues, notably the limb bud, the time at which
celeave the cell cycle appears to be an important determinantheir
eventual identity (Tabin, 1995) but here it is less clear
henvironmental signaling is coordinated with temporal controon
progenitor cell identity. Thus, the mechanisms by whitemporal and
spatial controls on progenitor cell differentiatiare coordinated to
establish vertebrate tissue pattern rempoorly understood.
We have attempted to address this issue through an anaof the
development of a specialized endocrine organ, pituitary gland. We
selected the pituitary for analysis becaueach of its component
hormone cell types have been defi(reviewed in Treier and Rosenfeld,
1996) and are known to
ichromuraloforandherllst ofowls
chon
ain
lysisthesened be
generated in a precise temporal and spatial order from
aapparently homogeneous ectodermal primordium (Simmons al., 1990;
Japón et al., 1994; reviewed in Voss and Rosenfe1992). The
pituitary has a dual embryonic origin. The posteriopituitary
derives from the infundibulum (INF), an evaginationof the ventral
diencephalion, whereas the anterior pituitar(AP) derives from
Rathke’s pouch (RP), a specialized regioof the oral roof ectoderm.
In the mouse, AP development initiated as the oral roof ectoderm
involutes towards the ventrdiencephalon to form RP. Subsequently,
RP detaches from tadjacent ectoderm to form the AP. Soon after
detachment the AP, an early TSH-secreting population of thyrotrophs
igenerated in its ventral-most domain whereas ACTH
secretincorticotrophs differentiate in an adjacent,
intermediatedomain. At this stage, cells in the dorsal domain,
close to thINF, continue to proliferate (Drolet et al., 1991; Ikeda
andYoshimoto 1991). These dorsal cells differentiate only at
latestages, generating a second population of thyrotrophs, as was
somatotrophs, gonadotrophs and lactotrophs. The generatof most of
these later-born hormone cell populations appeato be controlled by
the activity of the POU-domain protein Pit1(Li et al., 1990; Lin et
al., 1994; Simmons et al., 1990; Sornsoet al., 1996; Rhodes et al.,
1994).
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J. Ericson and others
Several transcription factors in addition to Pit1 have beshown
to regulate the commitment, proliferation andifferentiation of
pituitary cell types (Sheng et al., 199Sornson et al., 1996; Treier
and Rosenfeld, 1996). Amonthese, members of the LIM homeobox gene
family aexpressed by cells in the AP prior to the onset of hormone
gexpression. The LIM homeobox gene Lhx3(Lim3/PLim) isexpressed over
a prolonged period of AP development (Bet al., 1995; Zhadanov et
al., 1995). Lhx3null mice exhibit anarrest of AP development and
most hormone-producing ctypes are absent (Sheng et al., 1996).
Another LIM homeogene, Isl1, is expressed in a variety of hormone
secreting ctypes, including cells in the AP (Thor et al., 1991).
The ealethality of Isl1 null mice (Pfaff et al., 1996) has so
faprecluded an analysis of its function in pituitary developmebut
Isl1 is known to be required for the generation of pancreaendocrine
cell types (Ahlgren et al., 1997).
The apposition of the INF and RP has led to suggestions
signaling between these two cell groups influences the groof cells
in the AP (Schwind, 1928; Diakoku, 1982). Consistewith this, mice
in which Nkx2.1, a transcription factoexpressed in the neural tube
has been inactivated by gtargeting show defects in the development
of the vendiencephalon and the differentiation of the AP is also
impai(Kimura et al., 1996). However, the source, identity
amechanism of action of the inductive signals that control tearly
development of the AP remain to be defined. In tpaper, we examine
the origin and identity of secreted factthat control the
differentiation of progenitor cells in the Ausing LIM homeobox
genes and polypeptide hormonesmolecular markers in combination with
in vitro assays pituitary cell differentiation.
MATERIALS AND METHODS
AnimalsEmbryos were collected from CBa/B6 mice. The day of
tappearance of the vaginal plug was considered embryonic day(E0.5).
Somite number was used to determine developmental stup to E11.
Isolation and culture of Rathke’s pouchRP and associated ventral
diencephalic tissue was dissected at 4L15 medium (GibcoBRL) and
incubated with Dispase (Yamada et 1993) for 7 minutes. RP, the
ventral hypothalamus and the INF wthen separated from surrounding
mesenchyme. The venjuxtapituitary mesenchyme (VJM) was isolated
from E11.5 embryRP explants were isolated from 28-31 somite embryos
prior to Idown-regulation. In experiments involving COS cells, RP
tissue wderived from 35-37 somite (E10.5) and E11.5 embryos. The
INF wisolated from E10.5-E12.5 embryos. Explants were cultured
collagen gels (Yamada et al., 1993) in OPTI-MEM
(GibcoBRLsupplemented with N2 (GibcoBRL) for 24-96 hours. DEAE
Affi-geblue beads (Bio-Rad) or heparin beads (Pharmacia) were
soakePBS or in PBS containing recombinant FGF8 or FGF2 (GibcoBR
cDNA clonesLhx3 and FGF8 cDNAs were cloned from E11 AP and
INFrespectively, using RT-PCR. The human BMP2 expression
constructwas provided by P. Brickell, human BMP7cDNA by K. Liem and
themouse BMP4expression construct by R. Derynck.
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Expression and purification of FGF8 The FGF8 cDNA was used to
isolate a cDNA corresponding to thFGF8(3) splice variant (Crossley
and Martin, 1995). For bacteriexpression, the pET32 expression
system (Novagen) was used. His tag was fused to the N terminus of
FGF8(3)and the fusion proteinwas purified on a Ni-NTA column
(Qiagen). The eluted protein wrefolded in 5 mM glutathione by
successive removal of the ureadialysis. The purity (90%) and
concentration (approx. 500 µg/ml) ofthe FGF8 protein was estimated
from a coomassie-stained proteinusing BSA as a reference
standard.
Expression in COS cells and preparation of
conditionedmediaTransfection and aggregation of COS cells were
performed described by Roelink et al. (1994). Control vectors were
without insor contained ShhcDNA in antisense orientation.
Conditioned mediumfrom BMP2or mock-transfected COS cells were
concentrated 30 fousing Centricon spin filters. 10-25 µl of
concentrated conditionedmedium was added to 0.5 ml culture
medium.
Immunohistochemistry and in situ
hybridizationImmunohistochemistry was performed as described by
Yamada e(1991). Isl1 was detected by using rabbit anti-Isl1
antibodies (Thoal., 1991; Ericson et al., 1992). Antisera to αGSU,
ACTH and TSHβwere obtained from the National Hormone and Pituitary
PrograNIDDK, USA. PCR fragments and cDNAs (see above) were
usedtemplates to prepare digoxigenin-labeled RNA probes. In
shybridization was performed essentially as described by
SchaeWiemers and Gerfin Moser (1993).
BrdU labelingAfter 40 hours in culture, RP explants were
incubated with BrdU (µM, Sigma) for 75 minutes and the fixed in 4%
paraformaldehydBrdU+ cells were detected using an anti-BrdU
monoclonal antibo(Becton-Dickinson).
RESULTS
Progression of cell differentiation in Rathke’s pouch The
pattern of cell differentiation in the embryonic mouse Acan be
monitored by the spatially restricted expressionhormone genes
(Rhodes et al., 1994). The pattern of cell tyin the AP is first
evident around E12.5. Thyrotrophs, definby expression of the
α-glycoprotein (αGSU) and thyroidstimulating hormone β(TSHβ)
subunits, differentiate in aventral domain (Fig. 1A′,B′) and
corticotrophs, defined byexpression of ACTH, differentiate in the
intermediate doma(Fig. 1C′). At this stage cells in the dorsal
domain, close to tINF, remain proliferative and do not express
terminal markeof hormone cell differentiation (Fig. 1D′; see also
Ikeda andYoshimoto, 1991).
To investigate how this early pattern of cell types generated
from cells in RP we first examined the tempoexpression, in situ, of
the LIM homeobox genes Isl1and Lhx3.At the 10 somite stage (E8.5),
expression of Isl1 is detecteoral roof ectoderm cells that underlie
the ventral diencephaand give rise to RP (Fig. 1B). At E9.5, all RP
cells express I(Fig. 1H and data not shown) but at later times
(E10.5-E1Isl1 expression is gradually extinguished from RP cells
locaclosest to the INF (Fig. 1N). The expression of Isl1,
howevpersists in cells in the ventral domain of RP and by E11.5,
thcells have begun to express αGSU (Fig. 1T and data not
-
1007Patterning of the anterior pituitary
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shown). We refer to these ventral Isl1+/αGSU+ cells (Fig.1X,A′
,B′) as prospective thyrotrophs since only at E12.5, they initiate
expression of TSHβ, a definitive thyrotrophmarker (Fig. 1B′ and
data not shown). Expression of Lhx3(Bach et al., 1995; Zhadanov et
al., 1995) is initiated at thesomite stage (E9.5) in the dorsal
domain of RP (Fig. 1A,G) afrom E10.5-E11.5 Lhx3appears to be
expressed at high leveby all cells in RP (Fig. 1M,S; Bach et al.,
1995; Zhadanoval., 1995). By E12.5, however, expression of Lhx3 is
no longeruniform: there is a high level in the dorsal domain of the
Aa moderate level in the intermediate domain and a very level in
the ventral domain (Fig. 1Y). Thus, Isl1 and Lhx3appear initially
to be coexpressed by cells in RP but thexpression subsequently
segregates into distinct domwithin the AP (Fig. 1Y,Z).
The early potential of Rathke’s pouch cellsTo test if the
regulation of transcription factor expression athe early pattern of
hormone cell differentiation within RP controlled intrinsically or
by signals from surrounding tissuewe isolated RP explants from E10
embryos. At this stavirtually all cells still express Isl1 and Lhx3
(Fig. 1G,H anddata not shown). Explants were cultured in vitro for
65 houand the expression of Lhx3, Isl1, αGSU and TSHβwasmonitored.
In these explants, ~90% of cells maintained Iexpression (Fig. 2B)
and cells expressed only low levelsLhx3 (Fig. 2A). Most Isl1+ cells
expressed αGSU (Fig. 2C),but fewer than 1% of cells expressed TSHβ
(data not shown).The down-regulation of Lhx3and the maintenance of
Isl1expression in RP explants suggests that signals
providedsurrounding cell types are required to establish the
norpattern of LIM homeobox gene expression and the venrestriction
of thyrotroph differentiation in RP. In addition, thmaintenance of
Isl1 expression and the onset of αGSUexpression in vitro shows that
most RP progenitor cells acqmolecular markers characteristic of
prospective thyrotrophsthe absence of additional signals. The
absence of expressioTSHβ in vitro, however, indicates that these
cells are not ato progress to a definitive thyrotroph state.
Patterning of Rathke’s pouch by the infundibulumThe INF abuts
the dorsal region of Rathke’s pouch aconstitutes one potential
source of factors that might reguthe early expression of LIM
homeobox genes and the patof hormone cell differentiation. To test
this possibility, E10 Rexplants were grown in vitro, together with
INF tissue isolatfrom E10.5 embryos. After 65 hours in coculture,
Isl1 aαGSU expression was absent from most (80-90%) cells the
residual Isl1+ and αGSU+ cells were confined to the regionof the
explant distant from the junction with the INF (Fig2E,F). Lhx3
expression was maintained by most cells withthese explants, with
those cells located close to INF tisexpressing Lhx3at a higher
level than more distant cells (Fi2D). Complete extinction of Isl1
and αGSU expression wasachieved when two INF explants were placed
at opposite sof RP (Fig. 2H,I) and now Lhx3expression was
maintained aa high level by most cells (Fig. 2G). Similar results
weobserved when RP explants were grown with E11.5-E12.5 I(data not
shown). Basal hypothalamic tissue isolated at E1did not mimic the
ability of the INF to restrict the expressioof Isl1 in RP explants
(data not shown). Thus, within t
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relevant region of the embryonic ventral diencephalon, ability
to pattern RP and control the position of prospectthyrotroph
differentiation appears to be restricted to the IN
FGFs mimic the patterning activity of theinfundibulumTwo
secreted factors, BMP4 and FGF8, are expressed inembryonic INF
(Jones et al., 1991; Heikinheimo et al., 19Crossley and Martin,
1995; MacArthur et al., 1995), raising possibility that one or both
contribute to the patterning activof the INF. To begin to assess
the potential contributionsthese two factors, we analyzed their
profiles of expressionthe INF over the period that the early
pattern of RP cellsestablished. The expression of BMP4 in the
presumptive INFis initiated at E8.5 and a high level of BMP4is
detected byE9.5 (Fig. 1C,I; Jones et al., 1991), a stage when
Isl1expressed by all RP cells (Fig. 1H). By E11.5, howevexpression
of BMP4has been extinguished (Fig. 1U; Jonesal.,1991). FGF8
expression is initiated in the presumptive Iat E9.25 (Fig. 1D,J)
and persists until at least E14.5 (Fig. 1and data not shown;
Crossley and Martin, 1995). The onseFGF8 expression in the INF
coincides with that of Lhxexpression (Fig. 1A,G) and precedes the
extinction of Isl1RP cells (Fig. 1N). The ability of the INF over
the period E10to E12.5 to extinguish Isl1 and promote
Lhx3expression thus,corresponds more closely to the temporal
expression of FGF8than of BMP4.
To investigate whether FGF8 can mimic the ability of tINF to
repress Isl1 and maintain Lhx3 expression, E10 RPexplants were
grown for 65 hours in contact with beaadsorbed with FGF8 protein.
In these explants Lhx3expressionwas maintained at a high level and
Isl1+, αGSU+ cells wererestricted to the region of the explant
distant from the be(Fig. 3D-F). In contrast, with control beads
most cells ceato express Lhx3(Fig. 3A) and Isl1 and αGSU expression
weredetected in many cells (Fig. 3B,C). Complete repression of was
not observed in presence of FGF8 beads, possibly becof the low
activity of our bacterially expressed FGF8 prote(Fig. 3E). Since
FGF8 and FGF2 show similar inductiactivities in many tissues
(Fallon et al., 1994; Cohn et al., 19Crossley et al., 1996b;
Mahmood et al., 1995; Neubuser et1997) we tested whether FGF2 also
repressed Isl1 expresin RP explants. Explants grown with FGF2 beads
did nexpress Isl1 and αGSU (Fig. 3H,I) and Lhx3 was
expresseduniformly at a high level (Fig. 3G). Thus, both FGF2 and
FGmimic the ability of the INF to repress Isl1,
maintainLhx3andcontrol the pattern of prospective thyrotroph
differentiatioSince the INF does not express FGF2, its signaling
activitlikely to be mediated by FGF8.
We next examined whether cells in the ventral domain of that
have progressed to an Isl1+/αGSU+ state remain sensitiveto signals
from the INF and to FGFs. Explants isolated frthe ventral domain of
E11.5 AP were cultured for 40 houeither alone, with E11.5 INF or
with FGF beads and texpression of Isl1, αGSU and TSHβ examined.
Under all threeconditions, >90% of cells within these explants
maintained I(Fig. 3K,N and data not shown) and αGSU (data not
shown)expression despite exposure to INF or FGF2. These cells
expressed the definitive thyrotroph marker TSHβ (Fig.
3L,O).Conversely, few if any cells maintained expression of Lh(Fig.
3J,M). These results show that by E11.5, Isl1+/αGSU+
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1008
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J. Ericson and others
Fig. 1.Gene expression during early stages of pituitary
development. (A-F) E8.5, Lhx3 is not expressed (A) whereas Isl1
expression (B) can bedetected in ectodermal cells that underlie the
presumptive diencephalon. BMP4 is expressed in diencephalic cells
overlying the future RP (C)whereas no expression of FGF8 (D) or
BMP2(E) is present in the vicinity of the presumptive RP at this
stage. (G-K) E9.5,Lhx3 is initiated andis expressed by most cells
(G) and Isl1 by all cells (H) in Rathkes pouch (RP), BMP4is
expressed at high levels in the presumptiveinfundibulum (INF) (I),
FGF8expression is initiated in the INF (J) whereas no
BMP2expression is detected in the vicinity of RP at this stage(K).
(M-Q) E10.5, Lhx3is expressed by all or most cells in RP (M),
expression of Isl1is extinguished in dorsal RP cells (N), BMP4
isexpressed in the INF (O), FGF8expression is maintained by the INF
(P) and BMP2expression is detected at low levels in ventral
juxtapituitarymesenchymal (VJM) cells (Q). (S-W) E11.5,
Lhx3expression is maintained in most AP cells (S), Isl1 expression
is confined to the ventral-mostdomain of the AP (T), BMP4 is no
longer expressed in the INF (U), FGF8expression is maintined in the
INF (V) and BMP2-expressing VJMcells abuts the ventral domain of
the AP (W). (Y-C′) E12.5, Lhx3 is expressed in a non-uniform manner
in the AP: high levels in the dorsaldomain adjacent to the INF,
moderate levels in the intermediate domain and negligible levels in
the ventral domain (Y); Ventral expression ofIsl1 (Z) coincides
with thyrotroph differentiation shown by the expression of αGSU
(A′) and TSHβ (B′) in the same ventral domain.Corticotrophs,
defined by expression of ACTH, differentiate in an adjacent dorsal
position to the thyrotrophs (C′). (F,L,R,X,D′)
Schematicrepresentation of gene expression and cell differentiation
at different stages of pituitary development: gray/red
checkered,Isl1+/Lhx3+ cells(except in F); gray, Lhx3+ cells; light
red, Isl1+/αGSU+ presumtive thyrotrophs; red, Isl1+/αGSU+/TSHβ+
definitive thyrotrophs; green, ACTH+definitive corticotrophs. di,
presumptive diencephalon; oe, oral roof ectoderm; pm, pharyngeal
membrane; U, undifferentiated cells; H,presumptive hypothalamus.
AP, anterior pituitary.
cells are no longer sensitive to inhibition by FGFs and thappear
to be committed to a definitive thyrotroph fate.
Late FGF8 signaling controls corticotrophdifferentiationThe
activity of the INF and the expression of FGF8 aremaintained at
stages after the commitment of thyrotrophs (F1V), raising the
possibility that the differentiation of othehormone cell types may
be regulated by FGF8-mediat
us
ig.red
signals from the INF. To test this possibility we first
analyzedthe fate of the Isl1− progenitor cells present in the
intermediateand dorsal domains of the AP. By E12.5-E13, Isl1−
progenitorcells in the intermediate domain have begun to
differentiatinto ACTH+ corticotrophs (Fig. 1C′; Fig. 4A), whereas
Isl1−cells in the dorsal domain continue to proliferate and fail
toexpress hormone markers. We examined whether threstriction of
corticotroph differentiation to the intermediatedomain observed in
vivo is recapitulated when E11.5 AP i
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1009Patterning of the anterior pituitary
,iateomis
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Fig. 2. Patterning of Rathke’s pouch by the infundibulum and
FGF8.(A-C) E10 RP explants grown alone for 65 hours express low
levelsof Lhx3(A) whereas most cells maintain expression of Isl1 (B)
(88%of cells, n=25 explants) and many of these initiate expression
ofαGSU (C). (D-F)Lhx3expression is maintained in RP cells
whencocultured with E10.5 INF tissue (D). Lhx3expression is higher
inRP cells adjacent to INF compared to more distant cells. Isl1
isdownregulated in RP by INF tissue and cells that maintain
Isl1expression (20% of cells, n=10 explants) are located at a
distancefrom the INF (E). αGSU expression is detected in RP cells
located ata distance from the INF tissue (F). (G-I) Most cells
within RPexpress high levels of Lhx3(G) when cocultured with two
INFwhereas Isl1 (H) and αGSU (I) are not expressed in RP
whencultured with two INF (
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J. Ericson and others
Fig. 5. BMP2 promotes prospective thyrotroph and
suppressescorticotroph differentiation. (A) Few cells (95%, n=8
explants) RP cells grown with COS cellsexpressing BMP2 express
Isl1. (C) BMP2 fails to induce Isl1 in Ell.5APD explants (n=6
explants). (D) Few cells in E10.5 RP cells grownwith
mock-transfected COS cells express αGSU. (E) Most E10.5 RPcells
grown with BMP2 expressing COS cells express αGSU.(F) BMP2 fails to
induce αGSU expression in Ell.5 APD explants(n=6 explants). (G)
ACTH+ cells are detected in El0.5 RP explantsgrown with
mock-transfected COS cells, (n=8 explants). (H) ACTHexpression is
suppressed by exposure of El0.5 RP explants to BMP2-expressing COS
cells (n=8 explants). (I) ACTH+ cells are detected inell.5 APD
explants exposed to BMP2 (n=6 explants).
Fig. 4. Infundibulum and FGFs control corticotroph
differentiationand maintain Lhx3+ progenitor cells. (A) At E13,
corticotrophs,defined by expression of ACTH, differentiate in the
intermediatedomain of the AP. (B) Numerous ACTH+ cells are detected
in theintermediate and dorsal domains of E11.5 AP explants cultured
alonefor 40 hours. No ACTH+ cells are detected in the ventral
domain ofAP (region adjacent to dotted line) that expresses TSHβ
(data notshown). (C) E11.5 AP explant grown in contact with INF.
Only cellsat a distance from the INF tissue initiate expression of
ACTH.(D) Numerous ACTH− cells (17%, n=8) are detected in E11.5
dorsalAP (APD) explants grown alone or with PBS soaked beads (data
notshown) for 40 hours. (E) Expression of ACTH is suppressed at
highlevels in APD explants grown in contact with E11.5 INF.(F)
Expression of ACTH is suppressed at high levels in APD
explantsgrown in contact with FGF2 soaked beads. b, FGF2 bead. (G)
Veryfew Lhx3+ cells are detected in Ell.5 dorsal AP (APD)
explantsgrown alone or with PBS-soaked beads for 40 hours. (H)
Lhx3expression is maintained at high levels in APD explants grown
incontact with Ell.5 INF. (I) Lhx3expression is maintained at
highlevels in APD explants grown in contact with FGF2-soaked
beads.
restrict corticotroph differentiation to the intermediate domaof
the AP.
BMPs promote prospective thyrotroph and suppresscorticotroph
differentiationWe next considered whether the early patterning of
cells in AP can be accounted for exclusively by FGF-mediatsignaling
from the INF. The ventral domain of RP contaiIsl1+/αGSU+
presumptive thyrotrophs and is flanked by VJMcells that express
BMP2 and BMP7 (Fig. 1Q,W and data shown). Moreover, the presumptive
INF itself expresses BMat a stage Isl1 is expressed uniformly
within RP (Fig. 1C,I adata not shown). These observations prompted
us to examwhether BMPs expressed by the VJM might promote
Isexpression in RP cells.
To test this possibility, RP and dorsal AP explants weisolated
at E10.5 and Ell.5 respectively and were grown forhours in contact
with COS cells expressing BMP2 or wmock-transfected COS cells. At
E10.5, Isl1 expression h
in
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notP4ndinel1
re 60ithad
ceased in approx. 50% of cells (Fig. 1N) and in dorsal AP ell.5
no cells expressed Isl1 (Fig. 1T). When E10.5 RP explanwere grown
on control COS cells, 95% and of αGSU in approx. 80% ofRP cells
(Fig. 5B,E) and conversely ACTH expression wainhibited (Fig. 5H).
Similar results were obtained with VJMBMP4 and BMP7 (data not
shown). These results providevidence that BMPs can reinitiate Isl1
expression in E10.5 Rcells and promote the generation of
prospective thyrotrophsthe expense of corticotrophs. When E11.5
dorsal AP explawere cultured in the presence or absence of BMP2 no
inductof Isl1 or αGSU was detected (Fig. 5C,F) and ACTHexpression
was not repressed (Fig. 5I). Thus by E11corticotroph progenitors
have lost the capacity to switch tothyrotroph progenitor state in
response to BMPs. This resultcomplementary to the refractory
behavior of Ell.5 prospectivthyrotrophs to the inhibitory actions
of FGFs.
Integration of FGF and BMP signaling by anteriorpituitary
cellsThe opponent activities of FGFs and BMPs on thestablishment of
progenitor cell identity raised the issue of t
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1011Patterning of the anterior pituitary
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Fig. 6.Regulation of anterior pituitary differentiation by
integrationof BMP and FGF signaling. (A) Isl1 is expressed by most
E10 RPcells cultured with BMP2 containing medium (c.m.) for 65
hours(>95% Isl1+ cells/explant, n=6 explants). (B) Isl
expression isextinguished in E10 RP explants when cultured in
contact with FGbeads and c.m. from mock-transfected COS cells (
-
1012
olete
sisF
s,e
tnt
g of
eng
cts
ls,ofult
s
gsheel
hefg.to,e
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J. Ericson and others
INF appears to have a dual signaling function, serving initiaas
a source of BMP4 and subsequently of FGF8. The Vappears to serve as
a later source of BMP2 and BMP7. In vFGFs promote the proliferation
of progenitor cells, prevetheir exit from the cell cycle and
contribute to the specificatiof progenitor cell identity. BMPs, in
contrast, have no appareffect on cell proliferation but instead
appear to act with FGto control the initial selection of thyrotroph
and corticotropprogenitor identity. Our results suggest a model in
which coordinated actions of BMPs and FGFs regulate the tempand
spatial progression of progenitor cell differentiation thunderlies
the emergence of pattern in the developing AP.
The dual roles of FGF8 and BMPs in controlling Adevelopment that
are suggested from these studies hparallels with proposed
mechanisms of cell patterning in otvertebrate tissues. In the
developing limb, FGF8 is expresin the apical ectodermal ridge (AER)
and appears to maintunderlying mesenchymal cells in a proliferative
state (reviewby Tabin, 1995; Tickle, 1995). The patterning of
tissues thderive from the limb mesenchyme appears, however,
todependent upon the actions of other secreted signals, SHedgehog
and possibly BMPs (Niswander and Martin 199Francis et al., 1994;
Laufer et al., 1994; Yang and Niswan1995; Zou and Niswander 1996).
There is also evidence the coordinate activities of BMPs and FGF8
control tposition of initiation of tooth development (Neubuser et
a1997). Thus, the integration of FGF and BMP signalinappears to
coordinate the timing and pattern of cdifferentiation in many
vertebrate tissues.
Coordinated FGF8 and BMP signaling controls theprogression of
progenitor cell differentiationWe discuss our results in the
context of a model which mprovide insight into how the integration
of FGF and BMsignaling coordinates the spatial and temporal control
of cdifferentiation in the embryonic AP. We focus first othyrotroph
differentiation, which we consider in thresuccessive stages: the
establishment of an early I+
thyrotroph progenitor state, the progression to an
Isl1+/αGSU+prospective thyrotroph state and the conversion
Isl1+/αGSU+/TSHβ+ definitive thyrotrophs (Fig. 7A).
At early stages of AP development in vivo, all cells in Rexpress
Isl1 and when grown in vitro cells in RP explanmaintain Isl1
expression but stop proliferating and initiaexpression of αGSU.
These observations suggest that RPinitially composed of cells with
the properties of thyrotropprogenitors and that these cells have
the potential to progto an αGSU+ state characteristic of
prospective thyrotrophThey also raise the question of how the early
thyrotroprogenitor state is established. BMP4 is expressed in the
at early stages, prior to the onset of FGF8 expression
andexpression persists over the period that Isl1 is expresuniformly
within RP. Moreover, in vitro, BMPs can reinitiateIsl1 expression
in RP cells (Fig. 7). These findings raise possibility that an
early phase of BMP4 signaling from the INestablishes the thyrotroph
progenitor state.
Once the thyrotroph progenitor state is established,appears that
the maintenance of this state can ocindependently of BMP signaling,
but only in the absence FGFs. Exposure of RP progenitors to FGFs
switches cells fra prospective thyrotroph state to a corticotroph
progenitor st
llyJMitro,ntonentFsh
theoralat
Pave
hersedainedat
beonic3;
derthathel.,gell
ayPellnesl1
to
Ptste ishresss.phINF its
sed
theF
itcurofomate.
Thus BMPs and FGFs have opponent activities in the controf
progenitor cell identity. In the presence of both factors, thaction
of BMPs is dominant and the thyrotroph progenitor stais maintained
(Fig. 7B). The progression of thyrotrophprogenitors to cells with
markers of prospective thyrotrophcan also occur independently of
BMP signaling but again blocked in the presence of FGFs. At this
step, however, FGsignaling is dominant (Fig. 7A). Thus, in the
presence of FGFthyrotroph progenitors do not exit from the cell
cycle and thonset of expression of αGSU is inhibited. The hierarchy
ofFGF and BMP signaling appears therefore to differ asuccessive
stages of thyrotroph differentiation. The opponeactivities of FGFs
and BMPs on Isl1 expression in APdevelopment has parallels with
studies of tooth patterninwhich have shown that BMP4 signaling
opposes the actionsFGFs in the regulation of Pax9 expression
(Neubuser et al.,1997).
What controls the conversion of prospective into
definitivthyrotrophs? In contrast to the two earlier steps, FGF
signalifails to inhibit this third step in thyrotroph
differentiation.Moreover, this step appears to require a signal
that is distinfrom BMPs, since E10 RP cells grown in the presence
of BMPdo not convert into definitive thyrotrophs. However,
ventraIsl1+ cells isolated at E11.5 can generate definitive
thyrotrophsuggesting that this signal is supplied around the time
establishment of the presumptive thyrotroph state. This resargues
that the early Isl1+/αGSU+ cells detected in RP explantsin vitro
are likely to represent the precursors of thyrotrophrather than of
gonadotrophs, a late born αGSU+ cell populationwhich expresses the
βsubunits of LH and FSH rather than ofTSH (Simmons et al., 1990;
Japon et al., 1994). Our findintherefore support a model in which
three distinct steps in tdifferentiation of RP progenitors into
thyrotrophs can bdelineated (Fig. 7A), with each step exhibiting a
differentiasensitivity to BMP and FGF signaling.
Our results further suggest that the establishment of
tcorticotroph progenitor state initially requires the extinction
oIsl1 expression by RP cells in response to FGF signalinHowever, as
with thyrotroph progenitors, the progression definitive
corticotrophs is blocked by FGF signaling. Thuscorticotroph
differentiation may exhibit a biphasic dependencon FGF signaling;
an early phase which requires FGF signaliand a late phase which
requires the evasion of FGF signaThe late phase appears not to
require the extrinsic facimplicated in the establishment of the
definitive thyrotropstate (Fig. 7A), since early corticotroph
progenitors convert definitive corticotrophs when grown alone in
vitro.
Temporal and spatial constraints on FGF and BMPsignaling
underlie the patterning of the anteriorpituitaryOur findings
suggest also that the early patterning of the Adepends on temporal
and spatial constraints on FGF and BMsignaling. We discuss below
how such constraints migregulate the pattern of thyrotroph and
corticotroph generatioand maintain a pool of undifferentiated
progenitor cells (Fig7B).
Cells in RP appear initially to represent an uniformpopulation
of thyrotroph progenitors. The subsequenrestriction of thyrotroph
progenitors to the ventral domain othe AP may be imposed by the
gradual switch in the signalin
-
1013Patterning of the anterior pituitary
terndr
is
trol of pituitary cell differentiation and pattern by FGF8 and
BMPs.howing the progression of thyrotroph and corticotroph
differentiation and BMP signaling at different steps in this
program. The existence of anired for the conversion of prospective
to definitive thyrotrophs is alsong the source and range of action
of inductive signals that control the of progenitor cells in
Rathke’s pouch (RP) and the emergent pattern ofembryonic AP.
(E9-10) BMP4 and FGF8 signaling from thelishes a uniform population
of proliferative thyrotroph progenitors withinst from the INF but
FGF8 signaling is maintained and as a consequenceuished, thus
establishing a proliferative Isl− corticotroph progenitoriation of
these cells into corticotrophs is, however, prevented by theirFGF8.
BMP2/7 signaling, provided at this stage by the ventral
e (VJM) maintains Isl1 expression in adjacent ventral RP cells,
despite and thus establishes a ventral thyrotroph progenitor
population. from the INF maintains the proliferation and prevents
the differentiationrs in the dorsal and intermediate domains of the
anterior pituitary. Thef progenitor cells results in the
progressive ventral displacement ofuch that they come to be located
beyond the range of FGF8 signaling. As aress to the prospective
thyrotroph state and shortly after becomeE12-13) Maintained FGF8
signaling from the INF expands still furtherrogenitor population
and as a consequence the most ventral of theseted beyond range of
FGF8 signaling. Since these progenitors are also
his time refractory to BMP2/7 signaling, they progress to an
ACTH+
te. Cells in the ventral domain express TSHβ by this time and
arey to gene expression: gray/red check, Isl1+, Lhx3+; gray, Lhx3+;
pink,d, Isl1+, αGSU+, TSHβ+, Lhx3−; Green, ACTH+. For further
details see
properties of the INF, from BMP4 to FGF8. Once FGF8expressed,
however, cells in the ventral domain of RP, maexposed to this
factor since a high level of Lhx3expression canbe detected
ventrally. If this is the case, how then is thyrotroph progenitor
statemaintained by ventral AP cells? Ourresults raise the
possibility that theexpression of BMP2 and BMP7 bythe VJM provides
a local source ofBMPs that maintains the thyrotrophprogenitor state
despite exposure ofcells to FGFs. Between E11.5 and12.5, however,
Lhx3expression isdown-regulated and αGSUexpression is initiated by
ventralIsl1+ cells, suggesting that by thistime cells have escaped
the influenceof FGF signaling and thus, may nolonger depend on BMPs
provided bythe VJM (Fig. 7B).
Corticotrophs are generated in adomain immediately dorsal to
that ofthyrotrophs and derive fromprogenitors that have
extinguishedexpression of Isl1, presumably inresponse to FGF8
signaling from theINF. However, corticotrophprogenitors are
initially evenlydistributed in the dorsal andintermediate domains
of the AP. Therestriction in the differentiation ofcorticotrophs to
the intermediatedomain of the AP is likely to reflectthe subsequent
escape of moreventrally located corticotrophprogenitors from a
dorsal source ofFGF signaling. In addition, sinceBMPs appear to
induce thyrotrophprogenitors at the expense ofcorticotroph
progenitors, theestablishment of a corticotrophprogenitor
population may requireevasion of BMP signaling. At earlystages this
could to be achieved bythe loss of BMP4 from the INF andby a
limitation in the range of actionof BMPs derived from the VJM
andlater, by a loss of competence ofcorticotroph progenitors to
respondto BMPs by reinitiating expressionof Isl1.
Cell proliferation, tissuegrowth and pituitary patternAt early
stages, cells in the mostdorsal region of the AP
remainproliferative and hormone cell typesdo not differentiate.
Themaintenance of a pool of dorsalprogenitors is likely to reflect
thepersistence of FGF signaling. Our
Fig. 7. Model for the con(A) Schematic diagram sthe influence of
FGF andadditional factor (x) requshown. (B) Model showiidentity and
proliferationcell differentiation in the infundibulum (INF)
estabRP. (E10-11) BMP4 is loIsl1 expression is extingpopulation.
The differentmaintained exposure to juxtapituitary mesenchymtheir
exposure to FGF8,(E11-12) FGF8 signalingof corticotroph
progenitocontinued proliferation othyrotroph progenitors
sconsequence, cells progcommitted thyrotrophs. (the dorsal
corticotroph pprogenitors become locabeyond range of, or by
tdefinitive corticotroph stadefinitive thyrotrophs. KeIsl1+, αGSU+,
Lhx3+/−; Retext.
isy be
the
studies do not address the mechanisms by which lagenerated
hormone cell types, lactotrophs, somatotrophs agonadotrophs, arise
from the dorsal proliferative progenitocell population. However,
the perpetuation of FGF signaling
-
1014
es
ox
ls
eem.
a
e
d
id,
ndd.
1.
t-1
use
b
for
n
te
and
J. Ericson and others
likely to expand the population of dorsal progenitors furthesuch
that more distantly located progenitor cells progressivescape FGF
signaling, and initiate terminal differentiatioTranscription
factors such as Prop1 and Pit1 are requiredthe generation of most
hormone cells that eventuadifferentiate within this dorsal domain
(Ingraham et al., 198Li et al., 1990; Lin et al., 1994; Sornson et
al., 1996; Treand Rosenfeld, 1996). However, the identity of
factors thmight control the subtype identity of these cells
remaiunclear.
One final implication of our findings is that the
FGF-drivepromotion of cell proliferation is itself an important
element AP development. On the assumption that the range of
FGsignaling is restricted spatially, any expansion in the size of
progenitor population will lead, in a self-limiting manner, tthe
escape of more distant cells from the influence of FGsignals. Thus,
the FGF-driven expansion in the progenitor cpopulation in the AP
may contribute both to the timing oterminal differentiation and to
the resulting pattern of hormocell types. A similar FGF-driven
promotion of cell proliferatiohas been suggested to control the
timing of mesenchymal differentiation along the proximodistal axis
of the developinlimb (reviewed by Tabin, 1995) and the temporal
progressof neural differentiation in the mesencephalon (Crossley et
1996a; Lee et al., 1997). FGF signaling may therefore havgeneral
role in coordinating temporal and spatial aspectstissue patterning
in vertebrates.
J.E. and S.N. contributed equally to this work. We thank P.
BrickeC. Dickson, R. Derynck, K. Liem and C. MacArthur for reagents
aU. Nordstrom, H. Westphal and H. Sheng for helpful discussions.
thank the National Hormone and Pituitary Program, NIDDK, USA
fproviding antisera. This work was supported by grants to T. E.
frothe Swedish MRC and Strategic Research Foundation, to J. E. fthe
Swedish MRC and the Swedish Institute, and to T. M. J. from NIH. J.
E. is a Research Associate and T. M. J. is an Investigatothe Howard
Hughes Medical Institute.
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