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First published online 23 May 2007
doi: 10.1242/dev.003855
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1 Department of Cell Biology, Duke University Medical Center, Durham, NC 27710,
USA.
2 Nashville VA Medical Center and the Departments of Surgery and Cell and
Developmental Biology, Vanderbilt University Medical Center, Nashville, TN
37232, USA.
3 Laboratory of Metabolism, NCI, NIH, Bethesda, MD 20892, USA.
4 Departments of Medicine and Cell and Developmental Biology, University of
Pennsylvania, PA 19104, USA.
5 Department of Genetics, University of North Carolina Neuroscience Center,
University North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Author for correspondence (e-mail:
b.hogan{at}cellbio.duke.edu)
Accepted 23 April 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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18% of
wild type there is no TEF. However, the esophagus is still abnormal, with
luminal mucus-producing cells, fewer p63+ cells, and ectopic
expression of genes normally expressed in glandular stomach and intestine. In
all hypomorphic embryos the forestomach has an abnormal phenotype, with
reduced keratinization, ectopic mucus cells and columnar epithelium. These
findings suggest that Sox2 plays a second role in establishing the boundary
between the keratinized, squamous esophagus/forestomach and glandular
hindstomach.
Key words: Sox2, Nkx2.1, p63, Mouse embryo, Mutant, Foregut development, Tracheoesophageal fistula, Metaplasia
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Another example is the congenital defect known as
tracheoesophageal fistula (TEF), in which the separation of the foregut into
trachea and esophagus is abnormal (for reviews, see
Brunner and van Bokhoven, 2005;
Que et al., 2006).
Insights into the mechanisms controlling the patterning, differentiation
and morphogenesis of foregut organs have come from a variety of experimental
and genetic manipulations (for reviews, see
Fukuda and Yasugi, 2005;
Grapin-Botton and Melton,
2000; Lemaigre and Zaret,
2004; Roberts,
2000). These studies highlight the importance of reciprocal
signaling between the endoderm and mesoderm in foregut patterning. For
example, tissue recombination studies in the chick embryo have shown that
during a window of early development culturing the epithelium of the small
intestine with stomach (gizzard) mesenchyme results in the epithelium
acquiring a stomach phenotype. By contrast, combining gizzard epithelium with
intestinal mesenchyme has the opposite effect
(Fukuda and Yasugi, 2005;
Ishii et al., 1998).
Typically, such epithelial-mesenchymal interactions involve conserved
intercellular signaling pathways and transcription factors, including those of
the hedgehog, fibroblast growth factor (FGF), activin, bone morphogenetic
protein (BMP), Wnt and retinoic acid pathways
(Calmont et al., 2006;
Kim, B. et al., 2005;
Kim, J. et al., 2005;
Kim et al., 2000;
Listyorini and Yasugi, 2006;
Matsuda et al., 2005;
Narita et al., 1998;
Narita et al., 2000;
Ramalho-Santos et al., 2000;
Shin et al., 2006;
Theodosiou and Tabin, 2003).
However, relatively little is known about the identity and function of the
downstream transcription factors (Aubin et
al., 2002; Jacobsen et al.,
2002; Kawazoe et al.,
2002; Kim, J. et al.,
2005).
Here, we address the role in anterior foregut development of Sox2, a member
of a family of evolutionarily conserved transcription factors containing an
Sry-related HMG box. Sox2 is already known to be critically important for
early vertebrate development. For example, in mouse and human it is essential
for maintaining the pluripotential phenotype of epiblast and embryonic stem
(ES) cells, in part by regulating the expression of nanog and repressing genes
that promote differentiation (Avilion et
al., 2003; Boyer et al.,
2005; Rodda et al.,
2005). In addition, Sox2 regulates the differentiation of neural
stem cells in the CNS and sensory cells in the neuroepithelium of the retina
and inner ear (Taranova et al.,
2006; Kiernan et al.,
2005). Sox2 is expressed at varying levels in the endoderm of
foregut-derived organs, including the tongue, esophagus, trachea and proximal
lung and stomach (Ishii et al.,
1998; Okubo et al.,
2005; Williamson et al.,
2006). Several lines of evidence indicate that this expression is
functionally significant. First, heterozygosity for SOX2 in humans is
associated with anophthalmia-esophageal-genital (AEG) syndrome (OMIM 600992).
In some affected infants the esophagus and trachea fail to separate normally
and the trachea is connected to the stomach by an abnormal distal esophagus
(Fantes et al., 2003;
Hagstrom et al., 2005;
Williamson et al., 2006).
Second, we recently established that Sox2 is required for the differentiation
of endoderm progenitor cells of the embryonic tongue into taste bud cells
versus keratinocytes (Okubo et al.,
2006). Finally, crucial processes in both the morphogenesis and
differentiation of the embryonic chick lung and proventriculus are associated
with dramatic changes in the level of Sox2. Significantly, chick Sox2 levels
are downregulated in the epithelium as it responds to morphogenetic signals
from the surrounding mesenchyme and gives rise to either distal buds (lung) or
secretory glands (proventriculus) (Ishii
et al., 1998). In the developing gizzard, chick Sox2 levels are
downregulated by combination with intestinal mesenchyme whereas CDXA, a key
regulator of intestinal development, is upregulated
(Fukuda and Yasugi, 2005;
Ishii et al., 1998).
Addressing the in vivo function of Sox2 in the mouse is hampered by the
fact that Sox2-null embryos die before gastrulation because of a
requirement in the inner cell mass of the blastocyst
(Avilion et al., 2003).
Recently, two hypomorphic alleles of Sox2, Sox2LP and
Sox2COND have been generated. Combining these alleles with
the Sox2EGFP-null allele gives compound mutant embryos in
which the level of Sox2 varies below 50%
(Taranova et al., 2006). Here,
we use these mutants to explore the dose-dependent role of Sox2 in the
differentiation and morphogenesis of several foregut-derived organs, including
the posterior pharynx, trachea, esophagus and stomach.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Sox2EGFP/+ has been backcrossed for multiple generations
to C57Bl/6 whereas the other two lines are maintained on the (129/SvxC57Bl/6)
background. A total of 42 Sox2EGFP/LP mutants were
generated and analyzed either postnatally or at E18.5. The number of
Sox2EGFP/COND mutants examined is shown in
Table 1. Embryos of the
Fgf10Tg(Myl3-lacZ)24Buck (Fgf10lacZ)
line (Kelly et al., 2001) were
generously provided by Saverio Bellusci (Children's Hospital Los Angeles).
Titf1tm1Shk heterozygotes are maintained on the Black
Swiss background. Homozygous null embryos were examined at E15.5
(n=2) and E16.5 (n=1), together with wild-type
littermates.
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) and
Chemicon AB5603 affinity-purified rabbit polyclonal] and anti-Nkx2.1 (mouse
monoclonal clone 8G7G3/1; Lab Vision, Fremont, CA). For wholemount
immunostaining with phosphorylated ERK1/2 antibody (rabbit polyclonal; Cell
Signaling Technology, Danvers, MA) and X-gal staining followed by paraffin
embedding and sectioning, see Que et al.
(Que et al., 2006). Anterior
stomach samples were taken proximally, whereas posterior samples were from the
corpus and/or antrum, well away from the junction with the forestomach.
PAS and Alcian blue staining
Sections were treated with or without amylase (alpha-amylase from
Aspergillus oryzae, Fluka #10065: 0.5% in distilled water at 37°C
for 15 minutes), then stained in Periodic Acid Schiff (PAS) solution. For
Alcian blue staining, sections were treated with 3% acetic acid solution for 3
minutes, stained in Alcian blue solution for 30 minutes, and counterstained
with eosin.
Organ culture
Explants of E10.0 (32 somite) foregut tissues (from posterior pharynx to
the stomach) or E11.0 isolated esophagi from Sox2EGFP/+
embryos were cultured in suspension in groups of 2-3 in a 500-700 µl drop
of Dulbecco's modified Eagle's medium (DMEM)/F-12(1:1) medium (Gibco) on a
glass coverslip in a humidified chamber. The medium contained 0.5% bovine
serum albumin (BSA) Fraction V (Roche), 5 µg/ml each of transferrin and
insulin and 5 ng/ml selenous acid (ITS, Mediatech), 100 U/ml penicillin G, 100
µg/ml streptomycin, 0.1 mM non-essential amino acid (Gibco) and 1:100
chemically defined lipid concentrate (Gibco, 11905). They were cultured for 36
hours at 37°C in 5% CO2, with or without 50 ng/ml Fgf10
(R&D Systems), and the optimal dose was found to inhibit the separation of
foregut into trachea and esophagus. The cultures were repeated three times.
After culture, foregut explants were either photographed, fixed in 4% PFA,
dehydrated, embedded in paraffin and sectioned at 7 µm, or harvested for
RNA extraction.
BrdU labeling
BrdU (Amersham Bioscience, Little Chalfont, UK) was injected
intraperitoneally into pregnant females at a dose of 10 µl per gram
bodyweight. After 1 hour, embryonic tissues were collected and fixed in 4% PFA
in PBS (pH 7.4). For immunohistochemistry, BrdU monoclonal antibody (clone
BU33; Sigma) was used, in combination with MOM kit (Vector Laboratories,
Burlingame, CA). For quantifying proliferation index at least 2000 cells were
counted from at least seven sections of three different embryos of the
following phenotype: wild type, Sox2GFP/COND with TEF, and
Sox2GFP/COND without TEF.
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RT-PCR
Total RNA was extracted by RNeasy (Qiagen), and cDNA was synthesized from
500 ng total RNA by SuperScript (Invitrogen). PCR was performed using the
following primer sets:
Tff1: 5'-GCACAAGGTGATCTGTGTCCTC-3',
5'-CCCGGACACTGTCATCAAAAC-3'; Tff2:
5'-GTGAGCAGTGCTTTGATCTTGG-3',
5'-GGCACTTCAAAGATCAGGTTGG-3'; Muc5B:
5'-CGGTACAGCCCAATGAAACC-3',
5'-GGAAGCAATCATGGAAGTCC-3'; Agr2:
5'-TCCAAGACAAGCAACAGACC-3',
5'-GTCTTTAGCAGCTTGAGAGC-3'; Clca3:
5'-TCACTGACCTGAAGGCCAGC-3',
5'-CAGTGCAAACCTAGTGTCAC-3'; Reg3g:
5'-AGATGCTTCCCCGTATAACC-3',
5'-ACTCTAGGCCTTGAATTTGC-3'; Cdx1:
5'-GGACGCCCTACGAATGGATG-3',
5'-AACTCCTCCTTGACGGGCAC-3'; Sox2:
5'-AACGGCTCGCCCACCTACAGC-3',
5'-CAGGGGCAGTGTGCCGTATTTGG-3'; Nkx2.1:
5'-CGCCATGTCTTGTTCTACCTT-3',
5'-AGCGTCTGTCCTCCCTTCT-3'; TAp63:
5'-GCATATCTGGGATTTTCTGGAAC-3',
5'-GGAGCCCCAGGTTCGTGTA-3'; deltaNp63:
5'-GCAGAAAAGAGGAGAGCAGC-3', 5'-GGAGCCCCAGGTTCGTGTA-3';
ß-actin: see Okubo et al. (Okubo et
al., 2005).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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).
Sox2EGFP is also expressed in developing lingual and palatine
glands (see Fig. S1 in the supplementary material). We detail here the
temporal and spatial pattern of Sox2 expression in the foregut giving rise to
the esophagus, trachea and stomach (Fig.
1A-F,M-Q). At E9.5, Sox2 is localized in all endoderm cells of the
undivided foregut. Protein levels are somewhat higher dorsally, in the future
esophagus, and lower ventrally, in the future trachea
(Fig. 1D). This dorsal-ventral
difference is very clear one day later
(Fig. 1E), and is even more
marked after separation of the two tubes
(Fig. 1F,N). Subsequently, the
esophageal endoderm becomes multilayered and develops into a stratified
squamous epithelium. Sox2 levels are always highest in the cells adjacent to
the basal lamina (Fig. 1F,
Fig. 2H), with lower levels
suprabasally. Significantly, the Sox2hi cells also express high
levels of p63 (Fig. 1H,I,
Fig. 2I), a transcription
factor required for the development of many stratified epithelia, including
the esophagus, and the basal cells of the trachea
(Daniely et al., 2004). RT-PCR
shows that the E11.5-18.5 esophagus expresses a higher level of the TAp63
isoform than of the 
Np63 isoform (data not shown).
The high expression of Sox2 in the dorsal foregut is clearly reciprocal to
that of Nkx2.1 (also known as thyroid transcription factor 1, Titf1 - Mouse
Genome Informatics). This homeodomain protein plays a key role in regulating
trachea and lung development, and promotes the transcription of genes
expressed in mature airway epithelial cells, including Scgb1a1 (also
known as CC10, CCSP) (Ray et al.,
1996). As shown in Fig.
1J-L, levels of Nkx2.1 in the undivided foregut are highest
ventrally, in the future trachea and primary lung buds.
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Hypomorphic mutants die postnatally and have defects in the posterior pharynx
No Sox2EGFP/+, Sox2LP/+ or
Sox2COND/+ heterozygous embryos examined at any stage had
obvious defects in foregut development. However, at P0 all
Sox2EGFP/COND compound mutants showed labored breathing
and died with air in the stomach. In approximately 60% of these mutants this
phenotype might have resulted from the presence of TEF (see below). However,
all the Sox2EGFP/LP mutants also died soon after birth,
some with labored breathing and air in the stomach, and none of these mutants
had even a small TEF, after serial sectioning of the trachea and esophagus.
This raises the possibility that defects in the dynamic interaction between
the epiglottis and the soft palate are responsible for the misrouting of air
into the esophagus. Histological analysis did reveal abnormalities in the
posterior pharyngeal region of P0 Sox2EGFP/LP mutants, in
addition to the absence of taste buds previously described
(Okubo et al., 2006). Although
the circumvallate papilla forms, there are only a few, undeveloped Ebner's
glands arising from it. In addition, there is a complete absence of serous-
and mucous-secreting glands in the posterior tongue and soft palate (see Fig.
S1D-G in the supplementary material). It is possible that the abnormalities in
the soft palate disturb its interactions with the epiglottis, but whether this
is the only cause of death is not presently known.
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). As discussed below, analysis of
Sox2EGFP/COND mutants revealed a clear correlation between
levels of Sox2 in the esophagus and the TEF phenotype.
Sox2EGFP/COND compound mutants with EA/TEF have a
dorsally located fistula connecting the undivided foregut/trachea with a tube
leading to the stomach (Fig.
2B,E,F). This tube is known as the distal esophagus
(Brunner and van Bokhoven,
2005) but for simplicity will be called hereafter the fistula. In
wild-type littermates at E18.5 the epithelium of the esophagus is stratified
(but not yet keratinized) and most of the cells adjacent to the basal lamina
are Sox2hi and p63hi, and negative for Nkx2.1
(Fig. 2G-J). In striking
contrast, the Sox2EGFP/COND fistula is mostly composed of
a simple columnar epithelium, although multilayered regions are present in
some sections (Fig. 2Q and see
Fig. S2B,C in the supplementary material). Significantly, nuclear staining for
Sox2 is absent or barely above background by immunohistochemistry and only a
few p63+ cells are present (Fig.
2R,S). However, the nuclei of most of the columnar cells are
strongly positive for Nkx2.1 (Fig.
2T). Scattered cells score positively for Scgb1a1, a secreted
glycoprotein characteristic of Clara cells of the trachea and proximal airways
of the lung (Fig. 2U). Analysis
also shows the presence of ciliated cells, and mucus-producing cells staining
positively with Alcian blue (see Fig. S2B,C in the supplementary material).
The epithelium therefore resembles that of the normal E18.5 trachea, with
Clara, ciliated and mucus-secreting cells (the mucus cells later
disappear).
It is possible that the ectopic expression of Nkx2.1 in the fistula of Sox2EGFP/COND mutants is a late event, occurring well after the failure of the foregut to separate. We therefore examined Sox2EGFP/COND mutants at E12.5, shortly after the separation normally takes place. This revealed ectopic Nkx2.1 expression and reduced Sox2 in the dorsal undivided foregut even at this time (data not shown).
In the Sox2EGFP/COND mutants without TEF, and in all Sox2EGFP/LP mutants, the esophageal phenotype is variable. There are columnar regions where the epithelium is mostly multilayered, although not as thick as wild type and not squamous (Fig. 2L, note scale difference). There are fewer p63+ and K14+ basal cells than normal (Fig. 2N and Fig. 3F). In addition, many cells in the uppermost (luminal) layer are positive for markers of mucus production, namely Alcian blue, amylase-resistant PAS and Muc5AC (Fig. 3G,H and data not shown). These markers are never seen in wild-type esophagus at any stage of development. Significantly, none of the esophageal cells in these hypomorphic mutants ectopically express Nkx2.1 (Fig. 2O).
The immunohistochemical studies described above suggest that there is a correlation between the levels of Sox2 expression in Sox2EGFP/COND mutants and the presence/absence of TEF. To quantify this finding we performed western blot analysis of protein extracted from either the esophagus or the fistula of E15.5 embryos of various genotypes and phenotypes (Fig. 4). As expected, the level of Sox2 protein in the Sox2EGFP/+ esophagus is approximately 50% of that in wild-type (+/+) embryos. The level of Sox2 protein is further reduced, to approximately 18% of wild type, in the esophagus of Sox2EGFP/COND embryos without TEF. In agreement with the immunohistochemical findings, Sox2 protein cannot be detected above background in the fistula of compound mutants with TEF.
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7% in the fistula of
mutants with TEF (n=3) (P<0.05). However, this value is
not significantly different from that seen in the wild-type trachea (5%)
(P>0.05). It therefore appears that the reduced epithelial
proliferation of the mutant fistula is secondary to its acquisition of a
tracheal-like phenotype.
In Nkx2.1-null mutants the undivided foregut resembles the esophagus
The ectopic expression of Nkx2.1 in the fistula of
Sox2EGFP/COND mutants with TEF raised the possibility that
Sox2 and Nkx2.1 normally negatively regulate each other's
expression during the dorsal/ventral (D/V) patterning of the foregut. We
therefore examined the undivided foregut of embryos homozygous for a null
mutation of Nkx2.1 at E15.5-E16.5 (n=3). In
Nkx2.1-null mutants the foregut does not separate into trachea and
esophagus and the primary lung buds fail to branch but form sacs that do not
express genes specific to mature pulmonary phenotypes
(Minoo et al., 1999). As
previously reported, the undivided foregut anterior to the lung sacs has an
abnormal morphology and the epithelium is multilayered. Immunohistochemistry
clearly shows upregulation of Sox2 in the mutant foregut, so that expression
in the cells adjacent to the basal lamina resembles that in the esophagus of
wild-type littermate controls (Fig.
5A,D and insets). In addition, the basal epithelial cells stain
strongly for p63, again as in the normal esophagus and unlike the trachea
(Fig. 5B,E). Finally, staining
with antibody to smooth muscle actin shows a continuous ring of smooth muscle
around the mutant foregut anterior to the lung sacs. This abnormal muscle
pattern (and the absence of tracheal cartilage) has been described before
(Minoo et al., 1999) and is
characteristic of that seen in the esophagus
(Fig. 5C,F). Upregulation of
Sox2 and p63 was less dramatic in the most anterior undivided foregut (data
not shown), although cartilage development is much reduced. Taken together,
these results support a model in which absence of Nkx2.1 can result
in the dorsalization of the ventral foregut endoderm and its acquisition of an
esophagus-like phenotype.
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), whereas
overexpression of Fgf10 in the stomach downregulates Sox2
(Nyeng et al., 2007). In the
chick embryo, chick Sox2 levels decrease in distal lung primordia undergoing
budding morphogenesis in response to the distal mesenchyme
(Ishii et al., 1998) that
specifically expresses Fgf10 (Cardoso and
Lu, 2006). We therefore hypothesized that FGFs, including Fgf10,
regulate Sox2 expression in the foregut endoderm. In support of this idea,
analysis of Fgf10lacZ embryos at E10.5 shows that
Fgf10 is expressed in the mesoderm surrounding the ventral undivided
foregut (Fig. 6A,C). Moreover,
immunohistochemistry shows phosphoERK1/2-positive cells in the adjacent
ventral endoderm (Fig. 6B). To
test the functional significance of these findings we exploited the fact that
the foregut at this stage continues to separate into trachea and esophagus
when cultured in vitro (Fig.
6E,F). Addition of 50 ng/ml Fgf10 to the medium inhibits this
continued separation (Fig. 6G).
Moreover, analysis of the samples showed that Fgf10 induces a significant
decrease in both Sox2 and p63 in the esophagus, which does not become
multilayered (Fig. 6H-O). We
also tested the effect of exogenous Fgf10 on Sox2 expression in the
isolated esophagus. As shown in Fig.
6D, Fgf10 downregulates Sox2 and p63 and
promotes Nkx2.1 expression in the E11.0 esophagus in culture. Taken
together, these results support a model in which FGF signaling from the
ventral mesenchyme regulates the reciprocal expression patterns of
Sox2 and Nkx2.1 in the early foregut endoderm.
The esophagus of Sox2EGFP/LP and Sox2EGFP/COND mutants without TEF ectopically expresses genes characteristic of glandular stomach and intestine
Based on the abnormal histology of the esophagus of compound mutants
without TEF described above, we performed microarray analysis to compare
global gene expression in the E18.5 esophagus of
Sox2EGFP/LP and Sox2EGP/+ embryos
(data not shown). This revealed the misexpression in the mutant esophagus of
genes such as Tff1 (trefoil factor 1), Tff2
(trefoil factor 2), Muc5B (mucin 5 subtype B),
Agr2 (anterior gradient 2), Clca3 (chloride
channel calcium activated 3) and Reg3g (regenerating
islet-derived 3 gamma). All these genes are normally expressed in the
posterior glandular stomach or intestine. However, there was no significant
change in the level of transcripts for the homeodomain genes, Cdx1
and Cdx2, crucial regulators of the intestinal lineage
(Roberts, 2000). RT-PCR
analysis of cDNA derived from the esophagus of Sox2EGFP/LP
mutants confirmed these results (Fig.
3I). They also showed ectopic expression of all of the genes,
again except Cdx1, in the esophagus of
Sox2EGFP/COND mutants without TEF.
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Anterior stomach of all hypomorphic Sox2 mutants has characteristics of posterior stomach
Sox2 is initially expressed throughout the embryonic stomach but becomes
restricted to the anterior before birth
(Fig. 1O,P). In the mouse this
region is stratified and keratinized and Sox2 expression is highest in the
basal cells that are also K14+ and p63hi
(Fig. 1Q-S). In all classes of
compound mutant, the anterior stomach is highly dysmorphic and variable in
histology (Fig. 7F-J and see
Fig. S4 in the supplementary material). Some regions resemble the simple
secretory epithelium of the posterior glandular stomach in which the cells are
negative for K14 and p63 (Fig.
7F,G and see Fig. S4 in the supplementary material). Other regions
are multilayered but do not stain for involucrin, a marker for keratinization
(Fig. 7H). Like the esophagus
of Sox2EGFP/LP mutants, and
Sox2EGFP/COND mutants without TEF, the forestomach also
contains numerous ectopic luminal cells that stain positively with Alcian
blue, PAS (amylase resistant) and antibodies to Muc5AC and Tff2
(Fig. 7I,J and data not shown).
These mucin-producing cells are present both in the columnar regions and in
the upper layers of the multilayered epithelium.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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D/V patterning of the foregut and the etiology of tracheoesophageal fistula
One model to explain our findings is that Sox2 and Nkx2.1 play reciprocal
roles in the D/V patterning of the undivided foregut and the specification of
the future dorsal esophagus and ventral trachea, respectively. In particular,
we suggest that Sox2 normally directly or indirectly represses Nkx2.1, and
vice versa. According to this model, if the level of Sox2 in the undivided
foregut falls below a certain threshold early in development Nkx2.1 is
ectopically expressed dorsally and there is a cell-autonomous ventralization
of the foregut. This, in turn, disrupts the normal separation of the tube,
which is mediated by cellular mechanisms as yet largely unknown
(Que et al., 2006). In
addition, the endodermal tube connected to the stomach expresses Nkx2.1 and is
specified as a trachea and develops a tracheal rather than esophageal
phenotype. By contrast, when Nkx2.1 is absent, Sox2 is strongly upregulated
ventrally, at least in the region of the foregut anterior to the abnormal lung
sacs, and this region now acquires characteristics of the esophagus rather
than trachea. The fact that this dorsalization was less evident in the most
anterior foregut suggests that here other genes, for example Nkx2.5,
may compensate for the absence of Nkx2.1. Future studies must
determine whether the reciprocal regulation of Sox2 and Nkx2.1 is direct or
indirect. Significantly, it has been reported that Sox2 represses Nkx2.1 in
human ES cells (Boyer et al.,
2005), but the precise mechanism by which this is achieved is not
known. In addition, it will be important to develop genetic tools to
conditionally inactivate or overexpress Sox2, Nkx2.1 and other potential key
regulatory genes in different regions of the early foregut. We also need to
identify more genes that function as specific markers for early esophagus and
trachea in order to follow changes in cell fate.
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; Que et al.,
2006). Here, we provide strong evidence that Fgf10 made in the
ventral mesenchyme is one such factor. We show that exogenous Fgf10
upregulates Nkx2.1 and downregulates Sox2 and interferes with normal
separation of the trachea and esophagus. However, Fgf10 cannot be the only FGF
mediating normal patterning in vivo because Fgf10-null mutants have a
separate trachea and esophagus (Min et
al., 1998). Other FGFs expressed in the ventral foregut mesenchyme
(Serls et al., 2005) may
compensate for the absence of Fgf10 in these mutants. Our findings raise the
possibility that mutations in genes acting in the FGF signaling pathway
function as modifiers of TEF with variable penetrance, as seen in
Sox2EGFP/COND hypomorphic mutants. This could be tested in
the future by introducing specific mutations into the genetic background of
Sox2EGFP/COND embryos.
Role of Sox2 in regulating epithelial morphogenesis
Our data suggest that in addition to regulating the D/V patterning of the
anterior foregut Sox2 also plays a crucial role in the two other processes,
which may be tightly interconnected: the epithelial organization of the
endoderm and the patterning and differentiation of the esophagus and
stomach.
In terms of epithelial organization we argue that high levels of Sox2
promote the morphogenesis of the endoderm into a stratified, squamous
epithelium versus a pseudostratified, columnar epithelium. Thus, in the normal
esophagus and forestomach of the mouse, where Sox2 levels are high in the
basal cells, the epithelium develops into a thick stratified and keratinized
layer to protect the organs from damage by food. Such stratified epithelia,
including those in the esophagus, are characteristically renewed from a
population of p63+, K14+ basal cells
(Seery and Watt, 2000). Both
loss- and gain-of-function studies suggest that p63 isoforms play crucial
roles in regulating the development of stratified epithelia, including that of
the esophagus (for a review, see McKeon,
2004). Instead of being stratified and keratinized the epithelium
of the p63-null mutant esophagus consists of a simple columnar
ciliated epithelium, and both the esophagus and the trachea lack basal cells
(Daniely et al., 2004). By
contrast, misexpression of p63 in the epithelium of the distal lung leads to
the development of a multilayered epithelium that is Nkx2.1+
(Koster et al., 2004). Recent
evidence suggests that p63 isoforms regulate the expression of multiple genes
associated with epithelial cell adhesion and shape, as well as proliferation
and survival (Carroll et al.,
2006; Vigano et al.,
2006). Here, we find that Sox2 and p63 are coexpressed in basal
cells of the esophagus and forestomach and that a reduction in the level of
Sox2 in hypomorphic mutants is associated with a reduction in the number of
p63+ and K14+ cells. This raises the possibility that
Sox2 directly regulates the transcription of p63 and/or K14 in epithelial
cells of developing foregut organs.
Role of Sox2 in patterning of the esophagus and stomach
A striking feature of the phenotype of all Sox2 hypomorphic
embryos is that the esophagus and forestomach ectopically express genes
normally restricted to the posterior glandular stomach, including mucin genes
and genes encoding trefoil factor peptides. Moreover, mucin-producing cells
are present on the luminal surface of the epithelium, even when it is
multilayered. This finding has several implications. First, it suggests that
Sox2 normally plays a role in establishing the boundary between the
forestomach and glandular stomach by directly or indirectly repressing genes
expressed in the posterior epithelium. Second, regions of multilayered
epithelium with luminal goblet-like cells have been seen in the dysmorphic
lesions of patients with Barrett's esophagus. It has been suggested that this
phenotype is a transition phase in the complex pathogenic process by which the
epithelium of the esophagus is replaced by epithelium resembling stomach or
intestine (Glickman et al.,
2001; Shields et al.,
2001). Our results therefore raise the possibility that
downregulation of Sox2 plays a role in the etiology of columnar mucous cell
metaplasia in the esophagus.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2521/DC1
|
ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Center for Integrative Bioscience, National Institutes of
Natural Sciences, Okazaki, Aichi 444-8787, Japan
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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