1 Cell and Developmental Biology Program, Fox Chase Cancer Center, 7701 Burholme
Aveue, Philadelphia, PA 19111, USA
2 Neural Development Unit, Institute of Child Health, 30 Guilford Street, London
WC1N 1EH, UK
3 Division of Mammalian Development, National Institute for Medical Research,
The Ridgeway, London NW7 1AA, UK
Author for correspondence (e-mail:
zaret{at}fccc.edu)
Accepted 3 November 2003
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SUMMARY |
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Key words: Homeobox, Differentiation, Endoderm, Pancreas, Morphogenesis
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Introduction |
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In vertebrates, the pancreas emerges from ventral and dorsal domains of the
gut endoderm epithelium (Slack,
1995). The early pancreatic transcription factor Pdx1 (also known
as Ipf1) (Ohlsson et al.,
1993
; Offield et al.,
1996
) is first detected in ventral-lateral domains of endoderm in
mouse embryos at the 7 somite pair stage (7S), approximately day 8.5 of
gestation (E8.5) (Gannon and Wright,
1999
), and marks the ventral pancreatic progenitor cells
(Gu et al., 2002
). This is
coincident with the first appearance of hepatic gene expression in an adjacent
domain of ventral foregut endoderm cells
(Gualdi et al., 1996
;
Deutsch et al., 2001
). During
this period, the ventral foregut endoderm cells constitute a leading edge of
epithelium that grows ventrally, causing the curved foregut sheet to form a
tube. At the 6-7S stage, the cardiogenic mesoderm elaborates fibroblast growth
factors that can induce the liver program in explants of adjacent endoderm and
simultaneously inhibit the pancreas program
(Jung et al., 1999
;
Deutsch et al., 2001
). Ventral
endoderm cells that become positioned past the cardiogenic mesoderm, prior to
this point, can execute the pancreas program. Presently there is no
understanding of whether a specific morphogenetic control mechanism allows the
prospective ventral pancreatic domain to be positioned appropriately during
specification.
Mouse genetic studies have shown that ventral and dorsal pancreatic
patterning requires different sets of transcription factors. The bHLH
transcription factor gene Ptf1ap48 is necessary
for the specification of all ventral but not all dorsal pancreatic cells
(Kawaguchi et al., 2002;
Krapp et al., 1998
); the
homeobox genes Isl1 and Hlxb9 are necessary for the
specification of all dorsal but not ventral pancreatic cells
(Ahlgren et al., 1997
;
Harrison et al., 1999
;
Li et al., 1999
); and the
cut-homeodomain factor gene Hnf6 (Onecut1) controls the
timing of appearance of both ventral and dorsal pancreatic cells
(Jacquemin et al., 2003
), as
well as aspects of early liver development
(Clotman et al., 2002
). Ectopic
expression of the homeobox gene Pdx1 expands the domain of dorsal
pancreatic progenitors (Ferber et al.,
2000
; Grapin-Botton et al.,
2001
; Horb et al.,
2003
). The particular cell functions that are regulated by these
pancreatic factors are unknown, and it is unclear whether they directly or
indirectly activate the pancreatic gene program.
While homeobox transcription factors can directly control the expression of
genes that confer cell-specific differentiation
(Garcia-Bellido, 1975), they
also control genes that affect the growth, movement, or death of aggregates of
cells (Graba et al., 1997
),
and thus are hypothesized to control differentiation at the level of tissue
morphology (Weatherbee et al.,
1998
). Hex is a homeobox-containing gene in the
Antennapedia/Ftz class (Crompton
et al., 1992
) that is expressed in anterior endoderm cells at
embryonic day 7.0 of mouse gestation (E7.0) and subsequently in the
ventral-lateral foregut (Thomas et al.,
1998
) that gives rise to the ventral pancreas and the liver.
Hex is expressed in the liver bud
(Thomas et al., 1998
;
Bogue et al., 2000
), but
whether Hex is expressed in the nascent ventral pancreas has been
unknown. In Hex-null mouse embryos at E7.5, the definitive endoderm
appears compromised in its anterior displacement of the visceral endoderm
(yolk sac), as seen by the expression pattern of Foxa2
(Martinez-Barbera et al.,
2000
). Chimeric embryo experiments have demonstrated that these
morphological defects are intrinsic to the definitive endoderm and were not
due to defective signaling from the visceral endoderm
(Martinez-Barbera et al.,
2000
). Hex-null embryos grow to the E11.5 stage and
undergo normal turning and gut tube closure, but they lack a liver, a thyroid,
and parts of the forebrain (Keng et al.,
2000
; Martinez-Barbera et al.,
2000
). Thus, while Hex may be transiently required for
morphogenesis of the anterior definitive endoderm, the basis for this control
is not understood and it appears not to grossly affect the morphogenesis of
the embryo; nor is it known how Hex may control gut tissue
development.
While studying the basis for the liver defect in Hex-null embryos, we discovered that Hex controls the growth, rather than the specification, of the liver bud. We further discovered that Hex controls the proliferation of ventral foregut endoderm cells prior to liver induction and that, unexpectedly, Hex controls the specification of the ventral pancreas. However, embryo tissue explant assays show that Hex operates as a pancreatic specification factor by a heretofore-unappreciated mechanism; by allowing a subset of endoderm cells to grow past a mesodermal signaling center in the embryo. The approaches taken here may be used to determine whether other developmental transcription factors act at a morphogenetic level during endoderm patterning, rather than as direct regulators of tissue-specific genes.
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Materials and methods |
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HexlacZ expression and histology
Embryos were fixed in 4% paraformaldehyde in PBS at 4°C for 30-60
minutes, washed with PBS on ice twice for 10 minutes, then stained in X-gal
solution in PBS: 0.2% X-gal, 2 mM MgCl2, 5 mM potassium
ferricyanide, 5 mM potassium ferrocyanide and 0.02% NP-40, overnight at
37°C. Embryos were rinsed twice with PBS for 10 minutes, post-fixed in 4%
paraformaldehyde in PBS, and photographed digitally. For immunostaining,
embryos were dehydrated through a PBT (PBS +0.1% Tween 20)-methanol series and
stored in methanol at -20°C. Embryos were embedded in paraffin wax and
sectioned at 4-7 µm. Slides were immunostained with peroxidase or alkaline
phosphatase using the avidin-biotin-HRP method. Boiling in 10 mM sodium
citrate buffer pH 6.0 for 10 minutes was used for unmasking; secondary
antibody was used at 1:2000 dilution. The bound peroxidase was visualized by
reaction with a Vector-SG substrate, while the alkaline phosphatase was
visualized using BM-Purple (Roche); sections were counterstained with Eosin
and mounted with Permount. Primary antibodies to the following antigens (made
in rabbit unless otherwise indicated) were used at the indicated dilutions:
Pdx1 (a generous gift from C. Wright, Vanderbilt University, TN), 1:5000;
Hlxb9 (a generous gift from J. Kehrl), 1:8000; glucagon (Maine
Biotechnologies) 1:180; Isl1 (40.2D6 from the Hybridoma Bank at the University
of Iowa) 1:2000; Foxa2 (Santa Cruz; made in goat) 1:150 and Gata4 (Santa Cruz;
made in goat) 1:150.
In situ hybridization
Whole-mount in situ hybridization on embryos
(Wilkinson, 1992) and explants
(Rossi et al., 2001
) has been
described previously. Probes included Mrg1
(Dunwoodie et al., 1998
),
Hex (Thomas et al.,
1998
), Foxa2 (Ang et
al., 1993
; Dufort et al.,
1998
), albumin (Alb)
(Cascio and Zaret, 1991
) and
-fetoprotein (Cascio and Zaret,
1991
). A Pdx1 cDNA probe from nt 132-863 of the coding
region was cloned from embryonic RNA by RT-PCR into the pCR-Script plasmid.
After whole-mount in situ hybridization, embryos were post-fixed in 4% PFA,
photographed with a Pixera Pro150ES camera mounted on a Nikon SMZ-U
stereomicroscope, dehydrated in ethanol, cleared in xylenes, embedded in
paraffin wax, and sectioned at 6 µm.
RNA isolation and RT-PCR cycle step analysis
RNA was isolated as described (Gualdi
et al., 1996) and resuspended in 10 µl of nuclease free water
(Ambion). One µl of the total RNA was quantified using RiboGreen® RNA
Quantitation Kit (Molecular Probes). RNA (5-10 ng for cultured explants and
50-60 ng for primary embryonic tissue) was reverse transcribed using
Superscript II (Invitrogen) and oligo(dT15). Reverse transcribed
cDNA (1 µl) was amplified in 40 µl of 20 mM Tris-HCl (pH 8.4) containing
50 mM KCl, 1.5 mM MgCl2, 50 µM of each deoxynucleotide
triphosphate, 1 U Amplitaq DNA polymerase (Roche), 8 pmol of each specific
oligonucleotide, and 0.3 pmol of oligonucleotide phosphorylated with
[
-32P]ATP. Multiple PCR cycle steps were analyzed by gel
electrophoresis, to be sure that the reactions were in the exponential range
of PCR (Gualdi et al., 1996
).
Cycle number for each sample was normalized with actin levels as follows. A
first round of PCR reactions for actin was done and the cycle range with an
exponential increase of PCR product was assigned to each sample (approx. 28-32
cycles). Next, the other genes were assayed adjusting the cycle number in each
sample to the inter-sample variability found with actin (total range used was
30-42 cycles). When actin and albumin were assayed in the same PCR reaction,
actin primers were added after 6-8 cycles with albumin primers
(Jung et al., 1999
). Other
primers were described by Deutsch et al.
(Deutsch et al., 2001
) except:
lacZ #825 (bottom strand, for RT)
5'-ACTCCAACGCAGCACCATCAC-3' and #824 (top strand)
5'-TACTGTCGTCGTCCCCTCAAA-3' (331 bp product) and Prox1
#829 (bottom strand, for RT) 5'-CAGAGATGAGCAGGAACCAACAG-3' and
#824 (top strand) 5'-GCACTACAACAAAGCAAATGACT-3' (361 bp
product).
Embryo tissue isolation and culture
After mating HexlacZ mice, noon of the day of
the appearance of a vaginal plug was considered as E0.5. For endoderm explant
culture experiments, embryo tissues were harvested at E8.5 and staged by
somite number; only tissue from embryos younger than 7S were used. Embryo
tissues were dissected at 37°C in PBS under a dissecting microscope
(60x magnification) with electrolytically etched tungsten needles, and
cultured as described previously (Gualdi
et al., 1996). In some experiments, endoderm alone was isolated by
incubating the anterior portion of the embryo in 0.125% pancreatin (Sigma),
0.025% polyvinylpyrrolidone-40, and 20 mM Hepes (pH 7.5) in PBS at 4°C for
2 minutes, prior to dissection. Explants were cultured in 8-well glass
microwell slides (LabTek) coated with type I rat tail collagen (Collaborative
Biomedical Products) in DMEM (Invitrogen) containing 10% calf serum (Hyclone)
(Gualdi et al., 1996
). Beating
cardiac mesoderm cells in live explants, previously shown to be
-actin
positive (Gualdi et al.,
1996
), were evident by microscopy and annotated on photographs of
explants. Cultures were maintained at 37°C and 5% CO2 in
air.
Cell proliferation and apoptosis analyses
BrdU incorporation was detected using the BrdU Labeling and Detection Kit
II (Roche). Pregnant animals were injected with BrdU reagent and sacrificed
after 2 or 3.5 hours, and embryos were processed following standard
histological procedures (see above). BrdU immunostaining was done using the
manufacturer's protocol. Immunostaining of phospho-histone H3 (Cell Signaling)
was performed as described above at a 1:50 dilution. BrdU and Phospho-H3 were
quantified by alternately taking digital images of a section with bright-field
(to determine the positive-stained cells) and phase contrast (to determine the
total number of cells) illumination. The limits of the ventral endoderm domain
selected for quantification at 5-8S were established caudally by the
visceral/definitive endoderm border (Foxa2-negative/Foxa2-positive transition)
in neighboring sections and rostrally by mapping the Hex-positive
domain observed in the wild-type embryos. At least three
Hex-/- embryos were analyzed for each stage and assay.
Probability value was determined by the homoscedastic one tailed t-test.
Apoptotic cells were detected using the In Situ Cell Death Detection Kit
(Roche).
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Results |
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|
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Defect in ventral-lateral endoderm proliferation and positioning in Hex-/- embryos
Considering that Hex is expressed in the ventral-lateral endoderm
(Fig. 2A) (Thomas et al., 1998), we
wished to determine whether there is a growth defect in
Hex-/- endoderm prior to and during organogenic
patterning. We first used Foxa2/Hnf3b expression as a marker of
ventral definitive endoderm cells (Ang et
al., 1993
), as Foxa2 is intrinsically necessary for
ventral foregut endoderm development
(Dufort et al., 1998
). At the
8S stage (E8.5), the domain of ventral-lateral foregut cells expressing
Foxa2 was clearly reduced in Hex-/- embryos,
compared to wild type, but other Foxa2-positive domains, such as the
notochordal plate, were normal (Fig.
2B,D; ventral foregut, red arrows; notochordal plate, arrowheads).
By contrast, there was no change in Mrg1 mRNA expression, either
spatially or quantitatively, which is a marker of the adjacent septum
transversum mesenchyme in foregut mesoderm cells
(Fig. 2C,E; red arrows)
(Dunwoodie et al., 1998
), nor
in the position of the cardiogenic mesoderm
(Martinez-Barbera et al.,
2000
) (also see below).
|
Hex control of ventral endoderm morphogenesis could be at the
level of changes in cell shape, size, migration or proliferation. We found
that the leading edge of ventral endoderm cells in Hex-/-
embryos appeared identical in shape and size to those in wild type
(Fig. 2G,H,P,Q). We therefore
investigated the proliferative rate of the endoderm cells. A total of 80
sections of wild-type and Hex-null embryos were scored for
phosphorylated histone H3 (P-H3), a mitotic marker
(Schmiesing et al., 2000), in
the ventral-lateral endoderm cells that normally express Hex. As seen
in Fig. 2I,J (red boxed
regions) and quantitated in Fig.
2M, the leading ventral-lateral edge of definitive endoderm cells
exhibited a statistically significant decrease in P-H3 labeling in
Hex-/- embryos, compared with wild type, at the 5-6S stage
(P<0.05). The same domain of cells also exhibited a statistically
significant decrease in BrdU incorporation in Hex-/-
embryos (Fig. 2K,L, red boxed
regions; N, `ventral-lateral', red bars; P<0.01). BrdU
incorporation in cells located in dorsal-rostral or ventral-rostral endodermal
domains, where Hex is not normally expressed
(Fig. 2F), did not have a
decreased proliferation rate (Fig.
2K,L; orange and blue boxed regions;
Fig. 2N, orange and blue bars).
Thus, the reduced ventral-lateral endoderm domain in Hex-null embryos
and the excess of visceral endoderm cells in the gut is due to a localized
defect in cell proliferation of the definitive endoderm. These results
emphasize how Hex controls the growth of a discrete domain of
endoderm.
Failure of ventral pancreatic specification and morphogenesis in Hex-/- embryos
The Hex-/- deficiency in ventral foregut endoderm that
is normally caudal to the cardiogenic mesoderm led us to investigate the
consequences for pancreas development. As expected
(Ohlsson et al., 1993;
Offield et al., 1996
), we
found that in wild-type E8.5 embryos (9S), Pdx1 mRNA is expressed in
the ventral-most cells in the anterior intestinal portal
(Fig. 3A; `ventral'); the same
cells that normally express Hex
(Fig. 3B) and fail to become
positioned there in Hex-null embryos
(Fig. 2Q). E9.5 embryos,
heterozygous for the Hex-null allele with lacZ under the
control of the Hex promoter, exhibited cytoplasmic staining for
ß-galactosidase in a caudal extension of the hepatic bud
(Fig. 3C; blue labeled cells).
Nuclear Pdx1 protein was also present in the cells of the caudal extension
(Fig. 3D,E), but not the liver
bud (data not shown). By contrast, the dorsal pancreatic endoderm was Pdx1
positive, but HexlacZ negative
(Fig. 3C; data not shown).
These results were confirmed by in situ hybridization for endogenous
Hex mRNA and immunostaining for Pdx1 (data not shown). Thus,
Hex is expressed in ventral, but not dorsal, pancreatic progenitor
cells.
|
Glucagon-positive cells first appear in the ventral pancreas at E10.5-11.0 of wild-type embryos (data not shown), but they were not found ventrally in Hex-/- embryos, even in a rare embryo surviving to E12.5 (Fig. 3S,T, dashed arrow points to absence of glucagon cells and of any pancreatic mass). Significantly, after analyzing all fore- and midgut sections from multiple embryos stained for the above markers, we conclude that there was no budding or morphogenesis of the prospective ventral pancreas domain in any Hex-/- embryo (n=40; Fig. 3J,L,N,P,T; data not shown). By contrast, the budding and branching of the dorsal pancreas and the expression of dorsal pancreatic factors, such as Isl1 at these stages, was normal (Fig. 3Q,R), indicating that the midgestation lethality of the Hex mutation does not block pancreatic development per se. We conclude that Hex is required for the initial specification of the ventral pancreatic endoderm, and in the absence of such, there is a complete failure in pancreatic morphogenesis and differentiation.
Hex is not necessary to activate the ventral pancreatic program
Although the pancreatic specification defect in Hex-null embryos
superficially might resemble that seen with other transcription factor mutants
that block dorsal or ventral pancreatic development
(Edlund, 2002;
Wilson et al., 2003
), we
wished to rigorously assess whether Hex is directly required to
activate early pancreas genes, or if it controls pancreatic fate by an
indirect, morphogenetic mechanism. We therefore used an embryonic tissue
explant system (Gualdi et al.,
1996
; Deutsch et al.,
2001
). Ventral-lateral, definitive endoderm was isolated from 4-6S
embryos, prior to the time of pancreatic and hepatic induction, and was
cultured for 48 hours in the absence of cardiogenic mesoderm. We previously
showed that such Foxa2-positive explants contain trace amounts of
septum transversum mesenchyme cells (Rossi
et al., 2001
) and default to a pancreatic fate
(Deutsch et al., 2001
). The
Hex-/- endoderm explants showed normal viability and, like
wild-type endoderm explants, exhibited little if any growth
(Fig. 4C)
(Jung et al., 1999
;
Deutsch et al., 2001
). The
ability of the dissected ventral-lateral endoderm to initiate hepatic gene
expression, if cultured with cardiogenic mesoderm
(Fig. 1B;
Fig. 4D, lanes 16-19), proves
that we were isolating prospective ventral foregut endoderm from the
Hex-null embryos. We performed RT-PCR analysis on ventral endoderm
explants without cardiogenic mesoderm and compared the gene expression
patterns with those of ventral and dorsal midgut domains of intact E9.5
embryos, which represent a comparable developmental stage
(Deutsch et al., 2001
). We
note that the Hex-null endoderm explants without cardiogenic mesoderm
remained Alb negative, like wild-type endoderm explants
(Fig. 4B, lanes 3-12)
(Jung et al., 1999
). The
wild-type, E9.5 ventral midguts (uncultured) expressed Alb, as
expected, whereas the comparable tissue from Hex-null embryos at E9.5
did not, as previously reported (Fig.
4A, lanes 6-13) (Keng et al.,
2000
). Also, the Hex-null explants expressed the
HexlacZ marker
(Fig. 4B, lanes 9-12), further
emphasizing that we isolated the correct cell population (see
Fig. 2A).
Strikingly, RT-PCR analysis of explant RNAs showed that the
Hex-null ventral endoderm appeared fully competent to activate early
pancreas genes, including Pdx1 and the proendocrine genes Isl1,
Ngn3, and ß2/NeuroD
(Ahlgren et al., 1997;
Gradwohl et al., 2000
;
Naya et al., 1995
;
Schwitzgebel et al., 2000
)
(Fig. 4B, lanes 9-12). This was
in sharp contrast to the in vivo situation, where, by E9.5, the
Hex-/- ventral endoderm failed to activate these genes
(Fig. 4A, lanes 10-13). The low
levels of Pdx1 and Isl1 expression seen in the ventral
midgut RNAs of E9.5 Hex-/- embryos in
Fig. 4A can be accounted for by
the normal expression of these genes in prospective duodenal
(Fig. 3K) and lateral
mesenchymal cells (Fig. 3Q),
respectively (Ahlgren et al.,
1997
; Offield et al.,
1996
). In addition, the induction of high levels of Isl1
expression in the Hex-/- ventral endoderm explants was
suppressed when cardiogenic mesoderm was included
(Fig. 4D, compare lanes 3, 4
with 9-12), as in control embryos (Fig.
4D, lanes 1, 2, 5-8) (Deutsch
et al., 2001
).
These data show that despite the complete absence of ventral pancreas development in Hex-null embryos, Hex is not necessary for the ventral foregut endoderm to activate early pancreatic genes. We conclude that in Hex-null embryos, the failure of an endodermal domain to be positioned beyond the cardiogenic mesoderm, and escape its hepatic-inducing signaling, results in the absence of the pancreatic program. Thus, Hex normally controls the growth and positioning of a specific endodermal domain to allow ventral pancreas organogenesis.
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Discussion |
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|
Although Hex, like other homeobox factors, was originally considered to
regulate differentiation genes (Brickman et
al., 2000; Denson et al.,
2000
), there are examples of Hex controlling cell proliferation in
other developmental contexts. Newman et al.
(Newman et al., 1997
) showed
that in Xenopus embyros, Hex is expressed in vascular
endothelial cells and that overexpression increases endothelial cell numbers.
Hex is expressed in basal cells of the developing chick dermis
(Obinata et al., 2002
) and
overexpression in those cells in culture increases both their proliferative
rate and the epidermal area. We found that when Hex is deleted, the
proliferation of endodermal epithelium cells decreases and there is a decrease
in the area spanned by those cells. Thus, Hex-controlled
proliferation, at the cellular level, dictates the rate at which the leading
edge of the endodermal epithelium is positioned relative to the cardiogenic
mesoderm. This apparently becomes a timing mechanism so that a segment of the
endoderm can avoid the onset of hepatogenic signaling by the cardiac mesoderm
at the 6-7 S stage (Jung et al.,
1999
) and thereby initiate pancreatic development.
The ability of Hex-null ventral endoderm to activate liver but not
pancreas genes in vivo, and pancreas but not liver genes in vitro (in the
absence of cardiac mesoderm), provides further evidence that this endoderm
cell population is bipotent for hepatic and pancreatic fates
(Deutsch et al., 2001).
Lineage tracing studies of Kawaguchi et al.
(Kawaguchi et al., 2002
) show
that in Ptf1a-null embryos, which fail to specify a ventral pancreas
but have normal amounts of ventral endoderm, and thus are unlike
Hex-null embryos, the ventral endoderm cells adopt a
duodenal-intestinal fate. Taking all of these studies together, the data
suggest that prospective ventral-lateral endoderm cells may be competent to
execute at least three different fates. Hepatogenic signaling induces the
liver fate, an as yet unidentified signal induces or permits the pancreatic
fate (Lammert et al., 2001
;
Kumar et al., 2003
), and in
the absence of both cardiac signaling and the expression of Ptf1a,
the cells execute an intestinal-duodenal fate.
In summary, the Hex homeobox gene controls the ventral pancreatic
program indirectly, by maintaining the proliferation rate and consequently the
positioning of ventral foregut endoderm cells relative to the mesoderm. It
remains to be seen whether other endodermal transcription factors besides
Hex control the position of the endoderm relative to other mesodermal
and ectodermal signaling centers. An HMG box-containing gene that controls
endoderm development, Sox17
(Kanai-Azuma et al., 2002),
also affects the amount of definitive endoderm cells and the activation of
Pdx1. Sox17 also affects endodermal apoptosis, though how this may be
connected to patterning is unknown. We suggest that Sox17 and perhaps
other early endodermal transcription factors
(Wells and Melton, 1999
) could
be more critical for the morphogenetic control of tissue interactions than for
the direct regulation of cell type-specific genes during patterning.
Continuing with this theme, at later stages in development or in adults, such
transcription factors could be more important for the growth of different
cells within an emerging or regenerating tissue, thereby affecting the timing
and extent of cell interactions that are required for further differentiation
events.
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ACKNOWLEDGMENTS |
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Footnotes |
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