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.
& 2013 Elsevier Inc. All rights reserved.
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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nCorresponding author. Fax: +1 513 636 4317.
E-mail address: email@example.com (A.M. Zorn).
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
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.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2013.02.024.
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