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Wnt Signaling in Tissue Differentiation
6.4. FUTURE DIRECTIONS AND IMPORTANT
QUESTIONS
6.4.1. WNT INTERACTIONS WITH OTHER SIGNALING
PATHWAYS
Historically, the canonical Wnt signaling pathway was
described, and later β-Catenin-independent pathways were
added that were thought to act independently of each other.
As described previously, multiple pieces of evidence indicated that these different branches of Wnt signaling are in
fact highly connected and that Wnt signaling should be represented as a signaling network. Recent evidence suggests
that, in addition, Wnt signaling interacts with other signaling pathways. First, YAP and TAZ—two components of the
Hippo signaling pathway—were found to associate with the
destruction complex (Azzolin et al., 2014), as well as other
Wnt signaling components (reviewed in Piccolo et al., 2014).
Second, Disheveled recently was shown to interact with
Ephrin signaling, specifcally with Sipa1L3, an interactor of
Epha4 (Rothe et al., 2017). This interaction was shown to
be crucial for proper eye development in Xenopus embryos
by balancing Wnt signaling. This study also raised the
possibility that Disheveled may interact with other signaling pathways such as Notch, a notion supported by earlier
Xenopus experiments that demonstrated an inhibitory crossregulation between Wnt and Notch signaling (Collu et al.,
2012). Taken together, these examples illustrate that in-depth
analyses of the molecular mechanisms underlying the crosstalk of Wnt signaling with other signal pathways, such as
Hippo, Ephrin, and Notch, warrant further investigation.
6.4.2. REGULATION OF GENE EXPRESSION BY
NON-CANONICAL WNT SIGNALING
Non-canonical Wnt signaling is narrowly considered by
many researchers to be the Wnt/PCP pathway regulating cell
polarity and cell migration by modulating the cytoskeleton
or the polarized sub-cellular distribution of its components.
However, since some components of the non-canonical Wnt
signaling pathway, such as JNK, CamKII, or Calcineurin,
are also known to be involved in gene regulation, does noncanonical Wnt signaling directly regulate gene expression?
The frst genes thought to be regulated at the transcriptional level by non-canonical Wnt, via JNK, were PAPC
(Feike et al., 2010; Schambony and Wedlich, 2007) and
EAF 2 (Maurus et al., 2005) in Xenopus and TGFβ2 ( Zhou
et al., 2007) in mice. Since JNK regulates phosphorylation of
the transcription factor ATF2, an ATF2-luciferase reporter
was used to map non-canonical Wnt signaling in Xenopus
embryos (Ohkawara and Niehrs, 2011). Further, using dorsal
marginal zone explants defcient of either Wnt5a or Wnt11,
Gradl and colleagues recently identif ed pbk as a Wnt5a
target gene, whereas rab11f p5 was shown to be a specif c
Wnt11b target gene (Wallkamm et al., 2016).
The likely best-described target gene of non-canonical Wnt signaling is given by the cell adhesion molecule
ALCAM. First identifed in a screen for non-canonical Wnt
target genes (Prieve and Moon, 2003), it was later found to
be regulated by Wnt11a during cardiogenesis in Xenopus
(Gessert et al., 2008). This fnding was subsequently confrmed in the zebrafsh (Choudhry and Trede, 2013). In follow-up studies, the promotor of alcam was isolated in the
Xenopus system. A Frizzled3 responsive element was identifed by the use of reporter gene assays. This element is regulated through ATF2 and Pax2 in the Xenopus pronephros
and chromatin IP experiments conf rmed in vivo binding in a
non-canonical Wnt-dependent manner (Cizelsky et al., 2014).
In the Xenopus eye, this gene also was found to be regulated
through non-canonical Wnt (Seigfried et al., 2017).
These studies raise the important question: Are there specifc non-canonical Wnt cis-regulatory responsive elements
on DNA level? It will be an important and fundamental
research question for the future to determine to what extent
non-canonical Wnt signaling regulates gene expression and
to identify unifying underlying mechanisms. Recent loss-offunction experiments in Xenopus have shown that Wnt5a or
Wnt11b regulate fewer genes than canonical Wnt signaling
(Wallkamm et al., 2016), demonstrating in principle that
Xenopus is a very suitable model in which to answer these
questions.
6.4.3. INTEGRATION OF WNT SIGNALING
INTO THE EMBRYONIC SIGNALING AND
REGULATORY ENVIRONMENT
The recent identifcation of direct Wnt target genes (e.g.
Afouda et al., 2020; Nakamura et al., 2016) highlighted the
importance of the co-regulatory environment in the embryo
to ensure that Wnt target genes are expressed at the right time
and in the right place. Recent articles have demonstrated
that Xenopus is a leading experimental model for investigating the embryonic function and molecular mechanisms
of such combinatorial regulation of Wnt target gene expression, such as with FGF signaling (Kjolby et al., 2019) or with
BMP signaling (Polevoy et al., 2019) but also to dissect the
role of chromatin modifcation (Hontelez et al., 2015) and,
most likely related to that, chromatin accessibility (Esmaeili
et al., 2020).
6.4.4. XENOPUS AS A MODEL FOR HUMAN DISEASE
CRISP/R-mediated gene editing technology has clearly
transformed the potential for Xenopus research to model
human disease, including some involving Wnt pathway
mechanisms. This approach was pioneered with TALENmediated gene editing of the apc gene in Xenopus tropicalis to create an animal model that successfully phenocopied
Familial Adenomatous Polyposis (Van Nieuwenhuysen et
al., 2015). Xenopus experiments discovered the molecular
connection between EMC1 variants in patients and Wnt
signaling and neural crest development consistent with the
observed diverse birth defects (Marquez et al., 2020). Several
heterotaxy-correlated birth-defect candidate genes have also
Wnt Signaling in Tissue Differentiation
6.4. FUTURE DIRECTIONS AND IMPORTANT
QUESTIONS
6.4.1. WNT INTERACTIONS WITH OTHER SIGNALING
PATHWAYS
Historically, the canonical Wnt signaling pathway was
described, and later β-Catenin-independent pathways were
added that were thought to act independently of each other.
As described previously, multiple pieces of evidence indicated that these different branches of Wnt signaling are in
fact highly connected and that Wnt signaling should be represented as a signaling network. Recent evidence suggests
that, in addition, Wnt signaling interacts with other signaling pathways. First, YAP and TAZ—two components of the
Hippo signaling pathway—were found to associate with the
destruction complex (Azzolin et al., 2014), as well as other
Wnt signaling components (reviewed in Piccolo et al., 2014).
Second, Disheveled recently was shown to interact with
Ephrin signaling, specifcally with Sipa1L3, an interactor of
Epha4 (Rothe et al., 2017). This interaction was shown to
be crucial for proper eye development in Xenopus embryos
by balancing Wnt signaling. This study also raised the
possibility that Disheveled may interact with other signaling pathways such as Notch, a notion supported by earlier
Xenopus experiments that demonstrated an inhibitory crossregulation between Wnt and Notch signaling (Collu et al.,
2012). Taken together, these examples illustrate that in-depth
analyses of the molecular mechanisms underlying the crosstalk of Wnt signaling with other signal pathways, such as
Hippo, Ephrin, and Notch, warrant further investigation.
6.4.2. REGULATION OF GENE EXPRESSION BY
NON-CANONICAL WNT SIGNALING
Non-canonical Wnt signaling is narrowly considered by
many researchers to be the Wnt/PCP pathway regulating cell
polarity and cell migration by modulating the cytoskeleton
or the polarized sub-cellular distribution of its components.
However, since some components of the non-canonical Wnt
signaling pathway, such as JNK, CamKII, or Calcineurin,
are also known to be involved in gene regulation, does noncanonical Wnt signaling directly regulate gene expression?
The frst genes thought to be regulated at the transcriptional level by non-canonical Wnt, via JNK, were PAPC
(Feike et al., 2010; Schambony and Wedlich, 2007) and
EAF 2 (Maurus et al., 2005) in Xenopus and TGFβ2 ( Zhou
et al., 2007) in mice. Since JNK regulates phosphorylation of
the transcription factor ATF2, an ATF2-luciferase reporter
was used to map non-canonical Wnt signaling in Xenopus
embryos (Ohkawara and Niehrs, 2011). Further, using dorsal
marginal zone explants defcient of either Wnt5a or Wnt11,
Gradl and colleagues recently identif ed pbk as a Wnt5a
target gene, whereas rab11f p5 was shown to be a specif c
Wnt11b target gene (Wallkamm et al., 2016).
The likely best-described target gene of non-canonical Wnt signaling is given by the cell adhesion molecule
ALCAM. First identifed in a screen for non-canonical Wnt
target genes (Prieve and Moon, 2003), it was later found to
be regulated by Wnt11a during cardiogenesis in Xenopus
(Gessert et al., 2008). This fnding was subsequently confrmed in the zebrafsh (Choudhry and Trede, 2013). In follow-up studies, the promotor of alcam was isolated in the
Xenopus system. A Frizzled3 responsive element was identifed by the use of reporter gene assays. This element is regulated through ATF2 and Pax2 in the Xenopus pronephros
and chromatin IP experiments conf rmed in vivo binding in a
non-canonical Wnt-dependent manner (Cizelsky et al., 2014).
In the Xenopus eye, this gene also was found to be regulated
through non-canonical Wnt (Seigfried et al., 2017).
These studies raise the important question: Are there specifc non-canonical Wnt cis-regulatory responsive elements
on DNA level? It will be an important and fundamental
research question for the future to determine to what extent
non-canonical Wnt signaling regulates gene expression and
to identify unifying underlying mechanisms. Recent loss-offunction experiments in Xenopus have shown that Wnt5a or
Wnt11b regulate fewer genes than canonical Wnt signaling
(Wallkamm et al., 2016), demonstrating in principle that
Xenopus is a very suitable model in which to answer these
questions.
6.4.3. INTEGRATION OF WNT SIGNALING
INTO THE EMBRYONIC SIGNALING AND
REGULATORY ENVIRONMENT
The recent identifcation of direct Wnt target genes (e.g.
Afouda et al., 2020; Nakamura et al., 2016) highlighted the
importance of the co-regulatory environment in the embryo
to ensure that Wnt target genes are expressed at the right time
and in the right place. Recent articles have demonstrated
that Xenopus is a leading experimental model for investigating the embryonic function and molecular mechanisms
of such combinatorial regulation of Wnt target gene expression, such as with FGF signaling (Kjolby et al., 2019) or with
BMP signaling (Polevoy et al., 2019) but also to dissect the
role of chromatin modifcation (Hontelez et al., 2015) and,
most likely related to that, chromatin accessibility (Esmaeili
et al., 2020).
6.4.4. XENOPUS AS A MODEL FOR HUMAN DISEASE
CRISP/R-mediated gene editing technology has clearly
transformed the potential for Xenopus research to model
human disease, including some involving Wnt pathway
mechanisms. This approach was pioneered with TALENmediated gene editing of the apc gene in Xenopus tropicalis to create an animal model that successfully phenocopied
Familial Adenomatous Polyposis (Van Nieuwenhuysen et
al., 2015). Xenopus experiments discovered the molecular
connection between EMC1 variants in patients and Wnt
signaling and neural crest development consistent with the
observed diverse birth defects (Marquez et al., 2020). Several
heterotaxy-correlated birth-defect candidate genes have also