Different thresholds of Wnt-Frizzled 7 signaling coordinate proliferation,
morphogenesis and fate of endoderm progenitor cells
Zheng Zhang, Scott A. Rankin, Aaron M. Zornn
Perinatal Institute, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center and the College of Medicine, University of Cincinnati, Cincinnati OH 45229, USA
a r t i c l e i n f o
Received 4 October 2012
Received in revised form
6 February 2013
Accepted 22 February 2013
Available online 3 April 2013
a b s t r a c t
Wnt signaling has multiple dynamic roles during development of the gastrointestinal and respiratory
systems. Differential Wnt signaling is thought to be a critical step in Xenopus endoderm patterning such
that during late gastrula and early somite stages of embryogenesis, Wnt activity must be suppressed
in the anterior to allow the specification of foregut progenitors. However, the foregut endoderm also
expresses the Wnt-receptor Frizzled 7 (Fzd7) as well as several Wnt ligands suggesting that the current
model may be too simple. In this study, we show that Fzd7 is required to transduce a low level of Wnt
signaling that is essential to maintain foregut progenitors. Foregut-specific Fzd7-depletion from the
Xenopus foregut resulted in liver and pancreas agenesis. Fzd7-depleted embryos failed to maintain the
foregut progenitor marker hhex and exhibited decreased proliferation; in addition the foregut cells were
enlarged with a randomized orientation. We show that in the foregut Fzd7 signals via both the Wnt/β-
catenin and Wnt/JNK pathways and that different thresholds of Wnt-Fzd7 activity coordinate progenitor
cell fate, proliferation and morphogenesis.
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The epithelial lining of the digestive and respiratory systems
and organs such as liver, pancreas, and lungs are derived from the
embryonic endoderm. The endoderm germ layer is specified
during gastrulation and is then patterned along the anterior–
posterior (A–P) axis into broad foregut and hindgut progenitor
domains, which become progressively subdivided into specific
organ lineages by a reiterative series of Wnt, FGF and BMP growth
factor signaling events (Zaret, 2008; Zorn and Wells, 2009). These
pathways are highly dynamic and in just a few hours of embry-
ogenesis, or at different ligand concentrations, the same signals
can have dramatically different effects on the same population of
endoderm cells (McLin et al., 2007; Serls et al., 2005; Wandzioch
and Zaret, 2009). The molecular mechanisms that regulate the
spatial-temporal activity of these pathways during endoderm
organogenesis are poorly understood. A detailed knowledge
of these complex signaling events will facilitate efforts to direct
the differentiation of human stem cells into different endoderm
lineages (Kroon et al., 2008; Si-Tayeb et al., 2010; Spence et al.,
2011; Zaret, 2008).
Wnt signaling is particularly dynamic during endoderm orga-
nogenesis. In Xenopus and zebrafish, maternal Wnt/β-catenin
signaling initially promotes gastrulation and anterior endoderm
fate during germ layer formation (Rankin et al., 2011; Schier and
Talbot, 2005; Zorn et al., 1999; Zorn and Wells, 2007). Only hours
later between mid-gastrula and early somite stages zygotic Wnt
signals have the opposite affect and repress foregut fate in the
anterior endoderm while promoting hindgut fate in the posterior
endoderm (Goessling et al., 2008; McLin et al., 2007). After
patterning into foregut and hindgut progenitors domains, distinct
Wnt signals then promote the specification, differentiation and/or
outgrowth of the lungs, liver, pancreas, stomach and intestine
(Lade and Monga, 2011; Murtaugh, 2008; Poulain and Ober, 2011;
Shin et al., 2011; Verzi and Shivdasani, 2008).
Our previous studies on the role of Wnt-signaling in Xenopus
endoderm patterning suggest that multiple Wnt ligands from the
lateral plate mesoderm including Wnt5a, 5b, 8 and 11 signal via
both the canonical Wnt/β-catenin and the non-canonical Wnt/JNK
pathways to promote hindgut fate and morphogenesis in the
posterior endoderm (Li et al., 2008; McLin et al., 2007). In the
canonical pathway binding of Wnt ligands (such as Wnt8 and
Wnt11) to Frizzled and LRP5/6 receptors causes the accumulation
of nuclear β-catenin, which interacts with TCF/LEF transcription
factors (Clevers, 2006; MacDonald et al., 2009) to activate target
genes that promote posterior endoderm fate including the homeo-
box genes vent1 and vent2 (collectively referred to here as vent1/2)
(McLin et al., 2007). There is an evidence suggesting that Wnt11
and/or Wnt5a/b also activate a β-catenin-independent Wnt/JNK
pathway in the endoderm, which signals via Rho-family GTPases
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Developmental Biology 378 (2013) 1–12
and Jun-N-terminal-kinase (JNK) (Kim and Han, 2005; Wallingford
and Habas, 2005) to regulate cytoskeleton dynamics, cell polarity
and cell shape changes during gut morphogenesis (Li et al., 2008;
Reed et al., 2009), although the precise cellular mechanisms are
In the anterior endoderm the Wnt-antagonist Sfrp5 suppresses
both the Wnt/β-catenin and Wnt/JNK pathways to promote fore-
gut development (Li et al., 2008). This has led to the model where
“Wnt-OFF” promotes foregut progenitors and “Wnt-ON” specifies
hindgut progenitors. However, this model may be too simplistic.
Sfrps have recently been shown to exhibit biphasic activity:
repressing Wnts at high concentrations but facilitating Wnt ligand
diffusion and signaling at low concentrations (Mii and Taira, 2009).
Moreover both Wnt11 and its putative receptor Frizzled 7 (Fzd7)
are expressed in the foregut endoderm (Djiane et al., 2000;
Li et al., 2008; Medina et al., 2000; Wheeler and Hoppler, 1999).
These observations led us to hypothesize that Fzd7 may mediate
a low level of Wnt signaling important for foregut progenitor
Although the role of Fzd7 in the foregut endoderm is unknown,
its function in Xenopus axis specification and gastrulation has been
well studied. In this context, gain-of-function and in vitro studies
have shown that Fzd7 can interact with various Wnt ligands,
(including Wnt5a, 8b and 11) and activate either canonical or non-
canonical Wnt pathways (Brown et al., 2000; Djiane et al., 2000;
Medina et al., 2000; Medina and Steinbeisser, 2000; Sumanas and
Ekker, 2001). Loss-of-function studies indicate that maternal Fzd7
signals via the Wnt/β-catenin pathway in dorsal axis specification
(Sumanas and Ekker, 2001; Sumanas et al., 2000), whereas zygotic
Fzd7 in the chordomesoderm regulates gastrulation cell move-
ments of via several non-canonical Wnt pathways. Specifically,
Fzd7 activation of a PKC pathway regulates tissue separation of the
mesoderm and ectoderm, whilst Fzd7/JNK regulates convergent
extension of the axial mesoderm (Kim et al., 2008; Medina et al.,
2004; Sumanas and Ekker, 2001; Winklbauer et al., 2001).
In this study we used targeted microinjection of fzd7 morpho-
linos (fzd7-MO) to specifically deplete Fzd7 from the foregut
endoderm. We demonstrate that Fzd7 is required to mediate a
low level of both Wnt/β-catenin and Wnt/JNK signaling that
coordinates foregut progenitor fate, proliferation and morphogen-
esis. Both Fzd7/β-catenin and Fzd7/JNK pathways contributed to
foregut fate and proliferation, whereas the JNK pathway (but not
β-catenin signaling) regulated cell morphology. Our data support a
revised model of endoderm patterning where Wnt signaling has
different thresholds along the A–P-axis such that high Wnt activity
promotes hindgut over foregut fate, but that a low essential
threshold of Wnt-Fzd7 activity is required to maintain foregut
Material and methods
Embryo manipulations and microinjections
Embryo manipulation and microinjections were performed as
described previously (McLin et al., 2007). To specifically target the
foregut endoderm and avoid the chordomedoserm we injected
fzd7-MOs and the various mRNAs used in this study (along with a
lineage tracer to confirm targeting) into the D1 cells of 32-cell stage
embryos, which give rise to the foregut (Moody, 1987). To knock-
down both Xenopus laevis Fzd7 homeologs we injected a mixture
of two characterized translation-inhibiting fzd7-MOs (25 ng each)
(Sumanas and Ekker, 2001): 5-CCGGCTCCAACAAGTGATCTCTGG-3
and 5-GCGGAGTGAGCAGAAATCGGCTGAT-3. The following mRNAs
were used: pCS107-Fzd7, pT7TS-Sfrp5, pCS107-Dkk1 (Li et al.,
2008), and GR-Lef-βCTA (Domingos et al., 2001). The following
plasmids were used: pCS2+c.a.JNK (Liao et al., 2006). Dexametha-
sone (1 μM; for GR constructs) and the following cell-soluble
inhibitors were dissolved in DMSO and added to the media at stage
11; JNK inhibitor SP600125 (50–100 μM), Rac1 inhibitor NSC23766
(100–200 μM), Cdc42 inhibitor Casin (50 μM), PKC inhibitor BIM
(40 μM), Ca2+-dependant PKC inhibitor Go6976 (40 μM), and Cam-
KII inhibitor, KN-93 (20 μM), Axin inhibitor XAV-939 (10–80 μM).
Inhibition of cell proliferation was achieved by addition of hydro-
xyurea (HU, 20 mM) to media at stage 9 and incubated until stages
12 and 19, as previously described (Ohnuma et al., 1999).
In situ hybridization and immunohistochemistry
In situ hybridization and immunohistochemistry were performed
as previously described (McLin et al., 2007; Sinner et al., 2004).
The following primary antibodies were used: rabbit anti-β-catenin
(1:250; H-102, Santa Cruz Biotechnologies), mouse anti-C-cadherin
(1:200; 6B6, DSHB), mouse anti-E-cadherin (1:200; 5D3, DSHB),
mouse anti-β1-integrin (1:500; 8C8, DSHB), rabbit anti-atypical-PKC
(1:100; sc-216 Santa Cruz Biotechnologies), rabbit anti-phospho-
histone H3 (1:250; Cell signaling), rabbit anti-Fzd7 (1:200; R&D
systems), rabbit anti-active-caspase-3 (1:250; BD Pharmigen). The
following secondary antibodies were used: goat anti-rabbit-cy5, goat
anti-rabbit-cy2 or goat anti-mouse-cy5 (1:300; Jackson Immunore-
search). Nuclei were counterstained with Topro-3. In all experiments
exactly the same confocal and camera settings were used for control
and manipulated sibling embryos.
TOP:flash and AP1:luciferase assay
Top-flash (150 pg), AP1:luciferase (150 pg; Stratagene), and
pRL-TK renilla (25 pg) (Li et al., 2008) plasmids were injected into
embryos as indicated in the text. Each experiment was performed
in triplicate using five embryos per replicate, and luciferase
activity was measured using a commercial kit (Promega). Lucifer-
ase activity was normalized to co-injected TK-renilla and the mean
relative activity of the triplicate samples was shown 7S.D. Each
experiment was repeated a minimum of 3 times and a represen-
tative result is shown.
Western blots were carried out as described (Cha et al., 2008).
Antibodies concentrations were rabbit anti-pJNK, (1:750; Cell
Signaling); rabbit anti-total JNK, (1:750; Cell Signaling); mouse
anti-C-cadherin (1:500; DSHB), mouse anti-E-cadherin (1:500;
DSHB); and mouse anti-tubulin (1:5000; Neomarker).
Graded reduction in Wnt signaling differentially impacts endoderm
The current model of endoderm patterning in Xenopus predicts
that “Wnt-ON” promotes hindgut fate in the posterior, whereas
“Wnt-OFF”, due to the Wnt-antagonist Sfrp5, promotes foregut
fate (Li et al., 2008; McLin et al., 2007). Although the posterior
expression of wnt8, wnt5a and wnt5b mRNAs are consistent with
this model (Li et al., 2008; McLin et al., 2007) close examination of
wnt11 and its putative receptor fzd7 indicate that they are
expressed in the foregut endoderm underlying the sfrp5 expres-
sion domain at stage 19 (Li et al., 2008; Supplementary Fig. S1).
This suggests that the current model may be too simplistic and led
us to hypothesize that a low level of Wnt-Fzd7 signaling might
have a positive role in foregut progenitor development.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
To test the hypothesis that a low level of Wnt signaling is
required for foregut development, we microinjected an increasing
doses of mRNA encoding Sfrp5 into the anterior endoderm and
assayed the expression of the foregut marker hhex and hindgut
markers vent1/2. A moderate dose of sfrp5 (500–800 pg mRNA)
expanded the hhex expression at the expense of vent1/2-expres-
sing hindgut domain (Fig. 1E–G, J–L), which is consistent with our
previous findings (Li et al., 2008). However, at higher doses of sfrp5
(2–3 ng), rather than expanded hhex we observed a loss of hhex
expression as well as reduced vent1/2 expression (Fig. 1I,N).
The non-cell autonomous effects on the hindgut endoderm were
expected as secreted Sfrp5 is predicted to readily diffuse (Mii and
Since Sfrps can sometimes (at low concentrations) potentiate
Wnt signaling we confirmed the Sfrp5 results by inhibiting Wnt
signaling using an alternative method: We treated embryos from
stages 11 to 19 with a does range of the cell-soluble small molecule
Wnt-inhibitor XAV-939; a tankyrase-inhibitor that stabilizes Axin
and thus promotes degradation of cytosolic β-catenin (Huang
et al., 2009). Recapitulating the dose-dependent effects of Sfrp5,
low concentration of XVA-939 (modest Wnt inhibition) expanded
hhex, whereas high concentrations of XVA-939 repressed both
hhex and vent1/2 (Supplementary Fig. S2).
These results support the hypothesis that a low level of
Wnt signaling is actually required for foregut development, with
hindgut progenitors requiring an even higher level of Wnt activity.
Fzd7 is required for foregut organogenesis
We next wanted to use a loss-of-function approach to test the
role of Wnt signaling in the foregut. Since multiple secreted Wnt
ligands are expressed in the ventral region of the embryo at this
time in development we focused on the role of the Wnt receptor
Fzd7. In addition to being expressed in the foregut endoderm, fzd7 is
also strongly expressed in the axial mesoderm (Supplementary
Fig. S1), and previous global knockdown approaches examining its
role in gastrulation (Djiane et al., 2000; Medina et al., 2000; Sumanas
and Ekker, 2001; Winklbauer et al., 2001) precluded analysis of later
digestive system development. To test the function of Fzd7 specifi-
cally in the foregut without disturbing its mesodermal role in
gastrulation, we injected a mixture of two well-characterized trans-
lation-blocking Fzd7antisense morpholinooligos (fzd7-MOs)
(Sumanas and Ekker, 2001) together with a red fluorescent tracer
into D1 cells of 32-cell stage embryos, which are fated to give rise
to the ventral foregut endoderm (Moody, 1987). Lineage analysis
confirmed that the fzd7-MOs were limited to the foregut (Fig. 2A).
Moreover these foregut-targeted embryos did not exhibit defects in
either convergent extension or mesoderm–ectoderm tissue separa-
tion, whereas control injections targeting the dorsal mesoderm
recapitulated the published gastrulation defects, confirming the
efficacy of the fzd7-MOs (Supplementary Fig. S3).
Depletion of Fzd7 protein from the membrane of foregut
endoderm cells was confirmed by immunostaining at stage 19
(Fig. 2B,C). When cultured until organ bud stages (42–45) approxi-
mately 75% (n¼35) of the fzd7-MO embryos exhibited dramatic
gut defects (Fig. 2N,O). Histology and examination of isolated gut
tubes revealed disrupted gut coiling, foregut edema, and severe
organ hypoplasia with little if any heart, liver or pancreas tissue
and a reduced stomach in most Fzd7 morphants (Fig. 2P–S).
To determine whether endoderm patterning and organ specifica-
tion was compromised, we examined various foregut markers
(Fig. 2) at multiple developmental stages. Initial expression of hhex
in the gastrula anterior endoderm was unaffected (data not show),
but by stage 19 hhex expression was dramatically down regulated
in the foregut progenitors of Fzd7-depleted embryos (Fig. 2E,F),
whereas expression of the pan-endodermal marker sox17β was not
changed (data not shown). At stage 35, when organ lineages are
specified, Fzd7 morphants failed to express liver (nr1h5; pre-
viously for1, Xenbase.org) and pancreas (pdx1) markers (Fig. 2H,I,
K,L). Expression of the cardiac differentiation marker tnni3 was not
significantly altered (data not shown), suggesting that the heart
defect in Fzd7 morphants at stage 42 was due to impaired cardiac
morphogenesis and not a failure of heart specification.
Xenbase.org) expression, which is expressed in both the liver
and the presumptive intestine, suggested that hindgut fate was
not compromised (Fig. 2R,S). The fact that a2m was not ectopically
expressed in the remnant foregut tissue of Fzd7 morphants argues
that the foregut progenitors did not adopt a hindgut fate as is the
case when Wnt/β-catenin is hyper-activated in the post-gastrula
anterior endoderm (McLin et al., 2007).
To confirm that the Fzd7 morphant phenotype was specifically
due to loss of Fzd7, we co-injected the fzd7-MOs along with a
synthetic fzd7 mRNA lacking MO-target sequence, which was
Fig. 1. Differential Wnt signaling patterns the Xenopus endoderm. Wnt signaling has different thresholds in the endoderm. Embryos were injected with a dose range of
mRNA encoding Sfrp5; 500 pg (B,G), 800 pg (C,H), 2 ng (D,I) and 3 ng (E,J). In situ hybridization for hhex (A–E) and vent1 and vent2, a mixture of both probes referred to as
vent1/2 (F–J) in stage 19 bisected embryos showed that low doses of Sfrp5 expanded the hhex expression domain (yellow dashed line) (B–D) at the expense of hindgut
markers vent1/2 (G–I). The highest dose of Sfrp5 resulted in a loss of hhex (E). The number of embryos with the illustrated phenotype is indicated in each panel.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
sufficient to rescue Fzd7 immunostaining in foregut cells and
restore foregut gene expression (Fig. 2D, G, J and M). We conclude
that Fzd7 is required to maintain foregut progenitors and for
subsequent foregut organogenesis.
Fzd7 is required for foregut cell morphology
We noticed from the residual Fzd7 immunostaining that Fzd7-
depleted foregut cells had abnormal morphology. Since Fzd7 can
Fig. 2. Fzd7-depletion disrupts foregut organogenesis. (A) Targeted injection of fzd7-MOs (50 ng) and red fluorescent tracer (RLDx) into the D1 cells of 32-cell stage Xenopus
embryos. Bight field (BF) and fluorescent view of representative bisected stage 20 embryo showing that the injection targeted the foregut (fg) and avoided the axial
mesoderm. (B-D) Confocal immunostaining confirmed that the fzd7-MOs resulted in a loss of Fzd7 protein from the foregut cell membrane (B,C), which was rescued by
injection of Fzd7 mRNA lacking MO-target sequence (D). (E–M) In situ hybridization showed that fzd7-MO embryos failed to maintain hhex at stage 20 and did not express
the liver (nr1h5) and pancreas/duodenum (pdx1) markers at stage 35, which could be partially rescued by injection of fzd7 mRNA. (N, O) Ventral view of stage 45 embryos
showing foregut edema and defective intestinal coiling with shortened gut in fzd7-MO injected embryos. (P,Q) H&E-stained section of a control (P) and a fzd7-MO-injected
embryo (Q) that lacks foregut organs including liver (lv) and stomach (st). (R, S) In situ hybridization of a2m in isolated stage 42 gut tubes showed loss of foregut organs such
as liver (lv), stomach (st) and pancreas (p) and shortened intestine (In). The number of embryos with the illustrated phenotype is indicated in each panel.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
activate non-canonical Wnt signaling to regulate cytoskeleton
dynamics and cell adhesion in other contexts (Djiane et al.,
2000; Medina et al., 2000), we examined this more carefully.
Removing the neural plate to observe the surface of the foregut
endoderm at stage 19, we found that the Fzd7-depleted foregut
cells were enlarged and loosely adherent in comparison to controls
(Fig. 3A,B). Cell adhesion and cell shape are regulated by interac-
tions between cell surface adhesion molecules such as Cadherins,
which in turn are linked to the actin cytoskeleton by Catenins
(Adams et al.,1996; Tao et al., 2007). Immunostaining showed that
while control foregut cells were arranged in an organized poly-
gonal array, Fzd7-depleted cells were larger, round and disorga-
nized, typical of reduced cell adhesion (Rozario et al., 2009; Witzel
et al., 2006). Many of the enlarged fzd7-MO foregut cells exhibited
reduced C-cadherin and β1-integrin at cell membrane as well as
reduced levels of cortical β-catenin and F-actin at the inner cell
surface (Fig. 3C–J). This effect was more mosaic for β-catenin and
C-cadherin and correlated with cells that received a high dose of
the fzd7-MO (based on co-injected lineage label; data not shown).
Western blotting analysis of dissected stage 19 foreguts demon-
strated that the total amount of C-cadherin and E-cadherin were
not significantly changed (Fig. 3N), suggesting that the loss of Fzd7
impacts cadherin localization rather than expression.
To quantify the changes in cell size and polarity we measured
the length, width and orientation of foregut cells in control
and Fzd7-depleted embryos. In mid-sagittal sections of control
embryos the long axis of foregut cells was predominantly vertical;
oriented parallel to the dorsal-ventral axis. In contrast, the Fzd7-
depleted foregut cells were significantly larger (although their
length-to-width ratio was unchanged) and the orientation of their
long axes was randomized (Fig. 3K–M). We also assayed spindle
orientation in foregut cells undergoing mitosis by α-tubulin
immunostaining. The mitotic spindles were similarly oriented
along the long axis in controls cells but randomized in Fzd7
morphants (data not shown). Together, these data show that
Fzd7 is required for foregut cell adhesion, size and orientation.
Fzd7 is required for foregut cell proliferation
During analyses of the mitotic spindles we observed fewer
dividing cells in the foregut of Fzd7 morphants. Given the well-
known role of Wnt signaling in regulating proliferation of multiple
cells types we examined this in more detail. Immunostaining
of phospho-histone H3 (PH3) to mark cells undergoing mitosis
revealed that Fzd7-depleted embryos indeed had significantly
fewer proliferating cells in the foregut at stage 19 (Fig. 4). Analysis
Fig. 3. Fzd7-depletion causes defects in foregut cell morphology. (A,B) Bight field view of the foregut surface at stage 20 showed that Fzd7 morphants exhibit larger loosely
adherent cells (B), compared to controls (A). (C–J) Confocal immunostaining of the foregut (anterior left, dorsal up) with β1-integrin (C,D), C-cadherin (E,F), β-catenin (G,H)
and phalloidin (F-actin) (I,J) showed decreased cell adhesion molecules and reduced cytoskeleton in the enlarged foregut cells of Fzd7 morphants. All of the images were
taken using same setting for control and fzd7-MO embryos. (K,L) Quantitation of cell size and orientation; foregut cell length, width and orientation in control (K) and fzd7-
MO injected embryos (L) were measured from β-catenin immunostaining (green) using Image-J. Nuclei shown in blue, fzd7-MO/RLDx in red. All images were oriented with
dorsal up. The yellow line marks the long axis of foregut cells, and quantification shows the frequency of orientations of the long axis of cells in Fzd7 morphants (L,L') and
control (K,K') n¼ the total number of foregut cells from 5 uninjected (K') and 5 fzd7-MO embryos (L' ). (M) The relative length, width, and length/width radio in control and
Fzd7-depleted foregut cells.**po0.01 relative to controls in Student's t-test. (N) Western blot analysis shows no significant changes of total E-cadherin and C-cadherin level
in the foregut explants.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
of earlier stage embryos indicated that the reduced proliferation
was evident as early as mid-gastrula (Fig. 4), prior to when we first
observed defects in gene expression or cell morphology.
To test whether other defects in Fzd7 morphants were primar-
ily due to the loss of proliferation, we treated blastula embryos
with hydroxyurea (HU), which inhibits cell proliferation (Ohnuma
et al., 1999). PH3 staining confirmed that HU treatment from the
blastula stage reduced proliferation at stages 12 and 19 compar-
able to that of fzd7-MO-injected embryos (Supplementary Fig. S4).
However the HU treated embryos did not exhibit any disruption in
foregut cell morphology nor was there a loss of foregut gene
expression. On the contrary HU treatment resulted in expanded
hhex in the liver at stage 35 (Supplementary Fig. S4). This suggests
that reduced proliferation alone cannot account for the loss of
foregut identify in Fzd7 morphants. However, we postulate that
the decreased proliferation may contribute to later foregut organ
bud hypoplasia. TUNEL assays and active caspase-3 staining
indicated that there was no significant cell death in either controls
or Fzd7-depleted embryo at stage 19 (Supplementary Fig. S5).
We conclude that Fzd7 has multiple roles in the foregut
including maintenance of cell proliferation, foregut gene expres-
sion and proper cell morphology. Moreover the data suggest that
the disrupted gene expression and altered cell morphology in Fzd7
morphants is unlikely to be due primarily to reduced cell
Fzd7 depletion results in reduced Wnt/β-catenin and Wnt/JNK activity
Fzd7 has been shown to stimulate canonical β-catenin, as well
as non-canonical Wnt pathways in different contexts (Medina
et al., 2000), although in most instances the activation of these
different downstream pathways is thought to be mutually exclu-
sive with the canonical and non-canonical pathways antagonizing
one another (Nemeth et al., 2007; Topol et al., 2003). To better
understand the molecular basis of Fzd7 function in the foregut we
assayed the status of the Wnt/β-catenin and Wnt/JNK intracellular
signaling pathways both of which are known to be active in the
Xenopus endoderm at this time (Li et al., 2008).
To measure endogenous β-catenin/Tcf transcriptional activity
downstream of canonical Wnt signaling we used a TOP:flash
reporter plasmid, which contains multiple TCF DNA-binding sites
driving luciferase expression. We injected the TOP:flash reporter,
with or without the fzd7-MOs, into D1 cells or D4 cells at 32-cell
stage, which will develop into future foregut and hindgut, respec-
tively (Moody, 1987) and measured luciferase activity at stage 19.
As expected, the hindgut had higher endogenous β-catenin/Tcf
activity than foregut. However control embryos did exhibit a
modest level of reporter activity in the foregut, which was
significantly reduced by fzd7-MO injection (Fig. 5A). We also
examined levels of the activated C-terminal dephosphorylated
form of β-catenin by western blot of foregut explants (Fig. 5B)
and by measuring intensity of nuclear β-catenin immunostaining
(Fig. 5C–E), both of which were detected at a low level in the
foregut and dramatically reduced by Fzd7 depletion.
To measure non-canonical Wnt/JNK activity, we used an AP1:
luciferase reporter plasmid (Cheyette et al., 2002), which contains
AP1 (c-Jun/c-Fos) DNA-binding sites driving expression of Lucifer-
ase. Activated JNK phosphorylates c-Jun and stimulates c-Jun/c-Fos
mediated transcription. Injecting the AP1:luciferase reporter into
either the presumptive hindgut and foregut revealed that JNK was
active in both regions, although slightly higher in the hindgut.
Moreover Fzd7 depletion from the foregut resulted in significantly
reduced AP1:luciferase activity (Fig. 5A), and western blot analysis
of foregut explants confirmed that phospho-JNK levels were
reduced in Fzd7 morphants (Fig. 5B).
These data demonstrate that in the foregut Fzd7 transduces
a low but detectable level of both Wnt/β-catenin and Wnt/JNK
Fzd/β-catenin and Fzd/JNK coordinate foregut progenitor
proliferation, gene expression and morphology
To determine whether different aspects of the Fzd7 morphant
phenotype were due to reduced β-catenin and/or JNK signaling, we
performed a series of loss of function and rescue experiments. First
we specifically inhibited either the Wnt/β-catenin pathway (by
microinjecting RNA encoding the canonical Wnt-antagonist Dkk1
in the anterior endoderm at 32-cell stage) or inhibited the Wnt/JNK
pathway (by adding the JNK-inhibitor SB600125; 100 μM to the
culturing medium at stage 11) and determined to what extent
either of these could recapitulate the fzd7-MO phenotype. TOP:
flash and AP1:luciferase assays confirmed that at stage 19 Dkk1
only inhibit β-catenin activity and did not impact JNK activity,
whereas the JNK-inhibitor only repressed the AP1:luciferase and did
not change TOP:flash activity (data not shown). PH3 immunostain-
ing revealed that JNK inhibition caused a significant reduction in
foregut cell proliferation at both stages 12 and 19, similar to Fzd7
morphants, whereas Dkk1 overexpression repressed foregut cell
proliferation predominantly at stage 12 (Fig. 6A). This indicates that
Fig. 4. Fzd7-depletion causes reduced cell proliferation. (A–D) Confocal immunos-
taining of phospho-Histone h3 (PH3; green), nuclei (blue) and fzd7-MO/RLDx (red)
at stage 12 (A,B) and stage 19 (C,D) show that Fzd7-depleted embryos exhibit
reduced foregut (outlined in dashed yellow line) proliferation. (E) Mean number of
PH3 positive cells in the foregut 7S.D. *po0.05 relative to sibling controls in
Student's t-test (n¼4 embryos/ condition).
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
both β-catenin and JNK activity are required for foregut cell
Next we examined foregut gene expression and found that the
JNK-inhibitor or high levels of Dkk1 mRNA (1500 pg) could both
suppress, but not totally eliminate, hhex expression (Fig. 6I, M, N).
Lower doses of Dkk1 (o500 pg) expanded hhex (data not shown)
similar to what we observed with Sfrp5 low dose over expression
(Fig. 1), which is consistent with the model that reducing, but not
completely eliminating β-catenin activity, expands the foregut. Inter-
estingly, different doses of JNK inhibition did not exhibit a similar
bimodal impact on hhex expression and we never observed increased
hhex expression at any dose of the JNK inhibitor (data not shown).
These data suggest that both β-catenin and JNK activity are required
for robust foregut gene expression, and that β-catenin regulates
foregut versus hindgut fate in a dose responsive manner.
We next examined cell morphology in the Dkk1-injected
and JNK-inhibited embryos by immunostaining of cytoskeletal
β-catenin. Dkk1 had no impact on cytoskeletal β-catenin even
though it caused a reduction of the nuclear β-catenin, confirming
the suppression of canonical Wnt signaling. In contrast, the JNK
inhibitor caused enlarged foregut cells with reduced cortical
β-catenin similar to Fzd7 loss of function (Fig. 6C,D,G,H). Prolonged
JNK inhibition also prevented elongation of the endoderm that
normally occurs between stages 15–30 (Supplementary Fig. S6).
This observation is similar to previous reports of Sfrp5 and
dominant negative Dsh overexpression (Li et al., 2008) consistent
with a role for Wnt/JNK-mediated gut morphogenesis.
We also tested a number of other inhibitors to different
intracellular effectors of non-canonical Wnt signaling including
inhibitors of: CamKII, receptor coupled G-proteins, PI3 kinase,
Cdc42, Rac1 and PKC. None of these had an obvious impact on
foregut cell proliferation (data not shown). Rac1 inhibition
partially phenocopied fzd7-MO by suppressing hhex expression,
whereas Cdc42 and PKC inhibition caused an increase in the size of
foregut cells, similar to the Fzd7 morphants (Supplementary
Fig. S7; data not shown). These findings suggest Rac1, Cdc42 and
PKC may also participate in non-canonical Wnt/Fzd7 signaling to
regulate gene expression and/or cell morphology in the foregut.
To further confirm that the Fzd7 morphant phenotype was due
to the loss of both the Wnt/β-catenin and Wnt/JNK pathways we
preformed rescue experiments co-injecting the fzd7-MOs with
RNA encoding either constitutively active JNK (caJNK) (Liao et al.,
2006) or a hormone inducible Lef1-β-catenin fusion construct (GR:
Lef-βCTA, which constitutively activates Tcf/Lef-β-catenin targets
in the presence of dexamethasone) (Domingos et al., 2001).
We targeted these injections to the presumptive foregut endo-
derm, which avoids the axial mesoderm and as expected all the
injected embryos gastrulated normally. Both caJNK (200 pg) and
GR:Lef-βCTA (200 pg, induced at stage 11) partially rescued foregut
proliferation (Fig. 6B) and hhex expression (Fig. 6K,L) in fzd7-MOs,
whereas only the caJNK rescued cell morphology (Fig. 6E,F).
Reporter assays demonstrated that caJNK only activated the AP1:
luc reporter and that GR:Lef-βCTA only activated the TOP:flash
reporter (data not shown).
In these rescue experiments we again observed a dose respon-
sive effect in the Wnt/β-cat pathway. The same dose of GR:Lef-
βCTA (200 pg) that rescued hhex in Fzd7-depleted embryos had
the opposite effect and repressed hhex when injected into control
Fig. 5. Fzd7 depletion results loss of both Wnt/β-catenin and Wnt/JNK activity in the foregut. (A) Fzd7-depletion resulted in a reduction of β-catenin/Tcf and JNK/AP1 activity
in the foregut. TOP:flash or AP1:Luciferase reporter plasmids were injected into either the D1 foregut endoderm cells or the D4 hindgut endoderm cells at the 32-cell stage,
with or without fzd7-MO as indicated. The TOP:Flash reporter is an indicator of β-catenin/Tcf activity, while the AP1:luciferase reporter is an indicator of JNK-mediated c-Jun/
c-Fos (AP1) activity. At stage 20 luciferase activity was measured, in triplicate. The average relative luciferase activity, normalized to co-injected pRTK:Renila, from three
biological replicates per condition is shown 7S.D. *po0.05 and ** po0.01 relative to control foregut in Student's t-test. (B) Western blot showed decreased phospho-JNK1/2
(p-JNK) and a loss of dephosphorylated active β-catenin and total cytosolic β-catenin in the foregut explants at stage 19. (C–D): Confocal immunostaining showed reduced
nuclear β-catenin levels in Fzd7 morphant foregut tissue (D) relative to controls (C), at stage 20. (E) Mean pixel intensity of nuclear β-catenin staining measured using Image-
J 7S.D (foregut cells were scored from 5 embryos/ condition).
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
embryos (89% n¼19; not shown). This is probably because con-
trols have endogenous Wnt/Fzd7 signaling and the injection
elevates β-catenin activity above the threshold for foregut identity.
Consistent with this, injection of a 3-fold higher dose of GR:Lef-
βCTA RNA (600 pg) into fzd7-MO embryos no longer rescued hhex
(90%, n¼20, not shown). Unlike GR:Lef-βCTA, caJNK did not have a
bimodal impact on gene expression and it never repress hhex at
any of the doses tested. However, we did observe a caJNK dose
effect on cell morphology with 200 pg of caJNK RNA rescuing the
large cell size in the fzd7-MO as described above, whereas 600 pg
of caJNK resulted in smaller than normal, disorganized foregut
cells (data not shown) similar to previous reports of elevated Wnt/
JNK activity caused by Sfrp5-depletion (Li et al., 2008).
We conclude from these experiments that Fzd7 signals via both
the β-catenin and JNK pathways in the foregut. Foregut progenitor
proliferation and gene expression require both Fzd7/β-catenin and
Fzd7/JNK signaling, whereas the JNK, but not the β-catenin, path-
way regulates foregut cell morphology. Moreover the data are
consistent with the hypothesis that a low level of Wnt/Fzd7
activity promotes foregut development whereas, high levels
Different thresholds of Fzd7/β-catenin regulate endoderm fate
The previous model of endoderm patterning predicted that
“Wnt-ON” promotes hindgut and represses foregut fate whereas a
“Wnt-OFF” state is required to specify foregut progenitors (McLin
et al., 2007). Our data here indicate that this is an over
simplification and suggests that endoderm progenitor develop-
ment is controlled by multiple thresholds of Wnt/β-catenin signal-
ing: (1) when β-catenin activity is reduced below a critical
threshold, as in the Fzd7 morphants progenitor development is
arrested; (2) in response to a low level of Wnt/β-catenin the
endoderm cells adopt a foregut fate at the expense of hindgut
endoderm fate; and (3) in response to a high level of Wnt/β-
catenin, endoderm cells adopt a mid/hindgut fate and repress
foregut fate. To more thoroughly test this hypothesis, we modu-
lated both Fzd7 levels and β-catenin signaling in a progressive
series of overlapping doses to determine if we could indeed
generate embryos with each of the three predicted endoderm
To stimulate a dose response of Wnt/β-catenin activity, we
treated control or Fzd7-depleted sibling embryos from stage 10 to
stage 20 with different concentrations of the small molecule BIO,
which inhibits GSK3 and thus stabilizes β-catenin (Sato et al.,
2004). In control un-manipulated stage 20 embryos, hhex and
vent1/2 are expressed in a reciprocal pattern, with hhex marking
the foregut and vent1/2 marking the mid/hindgut progenitors
(Fig. 7C,D). As the dose of BIO (and therefore β-catenin activity)
was increased, hhex was down regulated and vent1/2 was ectopi-
cally expanded into the foregut domain (Fig. 7A); this indicates
that the anterior endoderm has adopted a hindgut fate. We next
progressively reduced Fzd7 levels by injecting different doses of
the fzd7-MOs. Consistent with a multiple threshold model; partial
knockdown of Fzd7 (25 ng of the fzd7-MOs) resulted in a modest
expansion of hhex domain and modest down-regulation of vent1/2
Fig. 6. Fzd7 signals via both β-catenin and JNK coordinate foregut cell proliferation, gene expression and cell morphology. (A) Inhibition of either Wnt/β-catenin or JNK
pathways reduced foregut cell proliferation. Embryos were either injected with RNA encoding Dkk1 (500 pg) to block the Wnt/β-catenin pathway or treated with the cell
soluble JNK inhibitor SB600125 (JNKi; 100 μM). Mean number of PH3 positive cells in the foregut +/−S.D. *po0.05 and ** po0.01compared to controls (n¼4 embryos/
condition). (B) Activation of either β-catenin or JNK signaling rescued cell proliferation in Fzd7 morphants. Embryos were injected with fzd7-MOs (50 ng) with or without
RNA encoding a constitutively active JNK (c.a. JNK; 200 pg) or a hormone inducible GR:Lef-βCTA (β-cat; 200 pg) activated by 1 μM dexamethasone at stage 11. Mean number
of PH3 positive foregut cells at stage 12 7S.D. *po0.05 relative to control and **po0.05 relative to fzd7-MO alone in Student's t-test (n¼4 embryos/ condition). (C–H) Fzd7/
JNK signaling regulates cell shape. Confocal immunostaining of β-catenin at stage 20 showed that c.a.JNK injection (F) rescued the cell-size defects in Fzd7 morphants (D),
whereas activation of the GR:Lef-βCTA (β-cat) did not (E). The JNK inhibitor (JNKi) caused a reduction of cytoskeletal β-catenin and increased cell size (H), phenocopying fzd7-
MO (D), whereas Dkk1 (1.5 ng) had no effects on cell morphology (G). (I–N) Both Fzd7/β-catenin and Fzd7/JNK regulate gene expression. In situ hybridization to stage 20
embryos showed that co-injection of either GR:Lef-βCTA (β-cat) (K) or c.a.JNK (L) restored hhex expression in Fzd7 morphants (J), whereas the JNK inhibitor (N) or high levels
of Dkk1 (1.5 ng) (M) suppressed hhex. The number of embryos with the illustrated phenotype is indicated in each panel.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
(Fig. 7E,F). In contrast, injection of 50 ng of the fzd7-MOs, which
resulted in a more complete Fzd7 depletion caused a loss of hhex
(Fig. 7G) as we have already shown in Fig. 2. Most importantly this
loss of hhex in the complete Fzd7 knockdown was not accompa-
nied by an expansion of vent1/2 (Fig. 7H) as was seen when
β-catenin activity was elavated (Fig. 7B); this suggests that foregut
development was arrested rather than being re-specified to a
hindgut fate. When we progressively added back β-catenin signal-
ing to the 50 ng fzd7-MO injected embryos (via BIO treatment),
we found that a low dose of BIO restored hhex expression (Fig. 7I)
whereas a higher BIO dose once again repressed hhex and
expanded vent1/2 (Fig. 7K,L). We conclude from these experiments
that different thresholds of Wnt/Fzd7/β-catenin signaling control
endoderm progenitor fate in the Xenopus embryo.
Thresholds of Wnt/Fzd7 signaling coordinate endoderm progenitor
Previous studies suggested a model of Xenopus endoderm
patterning where “Wnt-OFF” promotes foregut development and
“Wnt-ON” specifies hindgut (Li et al., 2008; McLin et al., 2007).
However, our results here support a revised model where multiple
thresholds of Wnt/Fzd7/β-catenin and Wnt/Fzd7/JNK activity
coordinate cell fate, proliferation and morphogenesis (Fig. 8). Our
results shed light on the dynamic role of Wnt signaling during
endoderm development and may help to resolve a number of
disparate observations in the literature reporting differential
effects of Wnt signaling on endoderm lineages (Goessling et al.,
2008; Goss et al., 2009; Lade and Monga, 2011; Ober et al., 2006;
Poulain and Ober, 2011).
Our findings here together with previous reports suggest that
the high levels of Wnt/β-catenin signaling, which occur in the
posterior, cause endoderm cells to adopt a hindgut fate and
repress foregut identity. In the anterior endoderm the Wnt-
antagonist Sfrp5 (Li et al., 2008) maintains Wnt/Fzd7/β-catenin
activity at a low (but essential) threshold required to maintain
foregut progenitors and repress hindgut fate. However, if β-catenin
signaling is too low (as in Fzd7 morphants) endoderm progenitor
development is blocked and proliferation is dramatically reduced
With respect to Wnt/Fzd7/JNK signaling we propose that there
may be differential activity between the deep and surface endo-
derm cells. In Xenopus the early endoderm is not a single cell layer
sheet as in mouse but rather a mass of tissue approximately 15–20
cells thick. Our results together with previous studies suggested
that Wnt/JNK activity is required in the deep endoderm (foregut
and hindgut) for polarized cell movements and gut elongation
(Li et al., 2008 and Supplementary Fig. S6). We demonstrate that
JNK activity in the foregut endoderm requries Fzd7 and that when
the threshold of Wnt/JNK activity is too low (as in the Fzd
morphants) both the deep and surface endoderm cells exhibit an
enlarged size, reduced adhesion and have a random orientation.
On the other hand when JNK activity is too high, such as when
caJNK is over expressed or when Sfrp5 is depleted (Li et al., 2008)
cell morphology and adhesion is also disrupted. The observation
that too much Wnt/JNK or too little Wnt/JNK can cause similar
phenotypes has also been reported in other contexts (Kim and
Han, 2005; Wallingford and Habas, 2005). Recent evidence sug-
gests that Sfrps can exert biphasic concentration dependent
activities; inhibiting Wnts at high concentration and facilitating
Wnt signaling at low concentrations (Mii and Taira, 2009). We
postulate that in foregut surface cells, which specifically express
Sfrp5, Wnt/Fzd7/JNK activity is maintained at a low but essential
threshold necessary to form an epithelium, and that diffusion of
low levels of Sfrp5 protein into the deep foregut tissue may
facilitate Wnt/Fzd7/JNK activity to promoting morphogenesis as
well as maintain hhex expression and proliferation (Fig. 8C).
Fzd7 stimulates both Wnt/β-catenin and Wnt/JNK pathways to
coordinate foregut cell identity, morphogenesis and proliferation
Fzd7 and its putative ligands in the foregut Wnt11 and Wnt5a,
can stimulate either canonical Wnt or non-canonical Wnt trans-
duction pathways depending on the cellular context (Cha et al.,
2008; Medina et al., 2000; Mikels and Nusse, 2006; Sumanas and
Ekker, 2001; Tao et al., 2005). However, there is little evidence that
Wnt/Fzd signaling can activate both pathway simultaneoulsy in
the same tissue; indeed in most instances the canonical and non-
canonical branches appear to be mutually antagonistic (Grumolato
Fig. 7. Multiple thresholds of Wnt/Fzd7/β-catenin activity pattern the endoderm.
(A–L) Fzd7/β-catenin signaling levels were modulated in a dose response with
different combination of fzd7-MOs and/or treatment with BIO, a GSK3 inhibitor
that stabilizes β-catenin. In situ hybridization of the foregut marker hhex and the
mid/hindgut markers vent1/2 in stage 20 embryos showed that increasing dose of
BIO and therefore increasing β-catenin activity decreased hhex while expanding
vent1/2 (A, B) relative to untreated controls (C, D). A partial knockdown of Fzd7
(25 ng of fzd7-MOs) resulted in a modest increase in hhex and reduction of vent1/2
(E, F), whereas a complete Fzd7 knockdown by 50 ng of the fzd7-MO caused a loss
of hhex, which was not accompanied by an expansion of vent1/2 (G, H). A low dose
of BIO (5 uM in I,J) rescued hhex expression in fzd7-depleted embryos (I, J), whereas
a higher BIO dose (10 uM) resulted in the foregut adopting a hindgut fate and
expressing ectopic vent1/2 (K, L). The number of embryos with the illustrated
phenotype is indicated in each panel.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
et al., 2010; Topol et al., 2003). Our data indicate that in the
Xenopus foregut Fzd7 activates both Wnt/β-catenin and Wnt/JNK
pathways, which cooperate rather than antagonize each other to
coordinate foregut progenitor proliferation and gene expression.
Although we cannot rule out the possibility that different cells
in the foregut activate β-catenin or JNK, our data suggest that these
two pathways act in parallel rather that in a linear fashion since
manipulation of one pathway did not appear to impact the activity
of the other.
The fact that the Fzd7 morphant phenotype is distinct from
the previous reported Wnt11 foregut-knockdown (Li et al., 2008)
suggests that Fzd7 may interact with multiple, redundant Wnt
ligands including Wnt5a, Wnt5b, Wnt8 and Wnt11. Interestingly
maternal Wnt5a and Wnt11 are able to form heteromeric com-
plexes to activate canonical signaling in the Xenopus blastula
(Cha et al., 2009). Future studies will test whether different
combinations of Wnt ligands signal through Fzd7 to elicit distinct
Fzd7/JNK regulation of cadherin and the cytoskeleton in the foregut
Wnt/JNK can regulate cell polarity, motility and the cytoskele-
ton in many contexts (Bovolenta et al., 2006; Kim and Han, 2005;
Seifert and Mlodzik, 2007). Previous work suggests that Wnt/JNK
activity is required in the deep endoderm for early gut elongation
(Li et al., 2008) and consistent with this Fzd7 morphants exhibit
a short gut. We postulate several possible mechanisms by which
Fzd7/JNK-mediated signaling might influence foregut cell adhe-
sion and the cytoskeleton:
1. Fzd7-mediated JNK activity might directly regulate the inter-
action between the actin cytoskeleton and cell adhesion com-
plexes. For example in human primary keratinocytes JNK
activity is required for the association of β-catenin to β-catenin/
E-cadherin at adhesion junctions (Lee et al., 2011). If JNK were
playing a similar role in the Xenopus foregut this might account
for the altered cadherin localization and loss of cortical actin in
2. Alternatively the actin cytoskeleton could be the primary target
of Fzd7 regulation. Non-canonical Wnt/Fzd signaling regulates
small GTPases including Cdc42, Rho and Rac (Schlessinger et al.,
2009), which can modulate JNK and regulate the formation of
actin stress fibers, and these can in turn influence the localiza-
tion of cadherins to nascent adhesion junction (Chu et al.,
2004; Vaezi et al., 2002; Vasioukhin et al., 2000).
3. Fzd7 may also regulate the cadherin cycling to the membrane.
In the zebrafish gastrula Wnt11/Fzd7 can influence cell cohe-
sion by regulating E-cadherin endocytosis via GTPase Rab5c
(Ulrich et al., 2005). In addition Wnt11−/−mouse cardiomyo-
cytes exhibit abnormal localization of N-cadherin, β-catenin
and actin (Nagy et al., 2010), similar to Fzd7 morphants.
4. Finally it is possible that Fzd7 regulates the activity of other
adhesion molecules such as proto-cadherins (Schambony and
Wedlich, 2007) or Flamingo the apical cadherin Wnt/PCP
co-receptor (Usui et al., 1999).
Using a foregut specific loss-of-function we demonstrate that
Fzd7 mediates a low, but essential level of Wnt/β-catenin and
Wnt/JNK signaling that is required for foregut development.
Together with previous results our data support a model where
Sfrp5-Wnt-Fzd7 interactions spatially regulate different thresholds
of Wnt/β-catenin and Wnt/JNK signaling that coordinate endo-
derm progenitor fate, proliferation and morphogenesis.
Fig. 8. A model of how Wnt/Fzd7/Sfrp5 regulate β-catenin and JNK signaling to coordinate endoderm fate, morphogenesis. (A) Schematic of a neurula embryo (anterior left)
showing expression of the fzd7 (green), wnt11, wnt5 and wnt8 in the ventral mesoderm and endoderm (blue) and the Wnt-antagonist sfrp5 in the surface of the foregut
endoderm (red). The spatial expression pattern of receptors, ligands and antagonists is postulated to establish differential Wnt activity in the endoderm. (B) Different
thresholds of β-catenin/TCF activity (red line) pattern endoderm with high activity promoting hindgut progenitor fate (hg), where as a low but essential level of β-catenin/
TCF is required to maintain foregut (fg) fate. (C) Differential Fzd7/JNK activity might regulate cell shape, adhesion and morphogenesis in the foregut (green). Fzd7/JNK
signaling in the deep endoderm promotes cell adhesion and the oriented cell shape required for gut elongation. Sfrp5 in the surface layer reduces JNK activity to a low but
essential level to establish an epithelium. If JNK activity is too low (as in Fzd7 morphants) cells become enlarged, loosely adherent with a random orientation.
Z. Zhang et al. / Developmental Biology 378 (2013) 1–12
We are grateful to Dr. Heisenberg and Dr. Kuan for reagents and
to members of the Zorn and Wells labs for helpful suggestions.
This work was supported by NIH grant DK070858 to AMZ.
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