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Wnt Signaling in Tissue Differentiation
6.2.4. INTEGRATED UNDERSTANDING OF WNT SIGNALING
IN VERTEBRATE EMBRYONIC DEVELOPMENT
While axis establishment and gastrulation assays have
served to study fundamental pathway mechanisms in gene
regulation and control of morphogenesis, at the same time,
Xenopus experiments have contributed to our general understanding of wider functional roles for Wnt signaling in vertebrate embryonic development, such as: (1) mesoderm
induction (e.g. Liu et al., 2005; Schohl and Fagotto, 2003)
and patterning (e.g. Hoppler et al., 1996 ); (2) patterning of the
neural plate ( Domingos et al., 2001; McGrew et al., 1997),
including neural crest induction (LaBonne and BronnerFraser, 1998) and migration (De Calisto et al., 2005), as well
as midbrain development (Kunz et al., 2004); and (3) organogenesis of the eye (Maurus et al., 2005; Rasmussen et al.,
2001), kidney (Lyons et al., 2004; Saulnier et al., 2002), and
heart (Marvin et al., 2001; Schneider and Mercola, 2001)
(see section IIIA). These investigations have led to a comprehensive and integrated understanding of Wnt signaling in
embryonic development of a typical model vertebrate.
6.3. RECENT ADVANCES
Xenopus research continues to make contributions at the
very forefront of new and important discoveries about
Wnt signaling. For example, it has been a mystery how
Wnt proteins with hydrophobic protein structures are able
to establish a signaling gradient in the extracellular space
through tissues. The importance of proteoglycans was demonstrated with Xenopus embryos, particularly the role of
extracellular sulfate and acetylate modifcations in regulating dispersal through tissues of Wnt signals, as well as Sfrp
proteins (recently reviewed by Mii and Takada, 2020). At
the membrane, the regulation of receptor turnover and the
intricate role of R-spondins in Wnt signaling has become
better understood (Chang et al., 2020; Chen et al., 2020;
Ding et al., 2018; Szenker-Ravi et al., 2018). Recent genomics studies (e.g. Nakamura et al., 2016) suggest functions for
β-Catenin beyond its association with LEF/TCF proteins.
For example, a functional interplay between Wnt pathwayregulated β-Catenin and Sox17 was recently carefully dissected in Xenopus endoderm patterning (Mukherjee et al.,
2020 ). Xenopus further contributes toward a more detailed
understanding of non-canonical Wnt signaling pathways,
particularly in regulating morphogenetic movements during
gastrulation and neurulation (e.g. Butler and Wallingford,
2018; Shindo et al., 2019). Recent fundamental discoveries
about Wnt signaling in neural crest induction and patterning
are groundbreaking far beyond the Xenopus model system,
including pluripotency of neural crest (Buitrago-Delgado
et al., 2015), surprising functions of Dkk2 (Devotta et al.,
2018), and a role for non-canonical Wnt signaling in neural
crest cells (Ossipova et al., 2018). Space constraints unfortunately prevent listing all of the important and excellent
research advances that have been facilitated by the Xenopus
model; in the following sections, three areas are explored.
6.3.1. WNT SIGNALING IN CARDIAC ORGANOGENESIS
Work in Xenopus was instrumental for deciphering roles
for Wnt signaling during heart development (reviewed by
Hoppler and Conlon, 2020; Hoppler et al., 2014). Whereas
initial work focused on the role of inhibitors of canonical signaling (Marvin et al., 2001; Schneider and Mercola, 2001), our
two laboratories were the frst to identify and confrm a role
for non-canonical Wnt signaling during this process (Afouda
et al., 2008; Pandur et al., 2002). Wnt11b as an extracellular
ligand together with JNK and PKC as intracellular signaling
mediators were shown to be involved in this process (Pandur
et al., 2002). Our work also showed how Wnt11b is tied into
a network of GATA transcription factors during this early
phase of cardiac development (Afouda and Hoppler, 2011;
Afouda et al., 2008). This initial fnding in Xenopus was later
confrmed in other biological models including murine and
human embryonic stem cells (Mazzotta et al., 2016).
Later during cardiac development, Wnt11a is important
for terminal differentiation (Gessert et al., 2008; Hempel et
al., 2017), ventricular trabeculation, and outfow tract formation both in Xenopus (Hempel et al., 2017) and mice (Nagy
et al., 2010; Zhou et al., 2007). Of interest is the f nding that
the cell adhesion molecule Alcam turned out to be critically
involved in this process (Gessert et al., 2008; Hempel et al.,
2017). During the normal cardiac differentiation program,
Wnt11a is regulated by the canonical Wnt signaling inhibitor
Dkk1 (Guo et al., 2019). It remains unclear at the moment
how inhibition of canonical Wnt signaling regulates Wnt11a
expression on a molecular level.
The precise regulation of both canonical and noncanonical Wnt signaling seems to be crucial for cardiogenesis. Inhibitors of canonical Wnt signaling, such as
Dkk1, Crescent, FrzB, and Sizzled, were shown to trigger
cardiogenesis in Xenopus in overexpression experiments
(Schneider and Mercola, 2001). In contrast, loss of Dkk1
experiments indicated its role during cardiac differentiation
(Guo et al., 2019). Also, the canonical Wnt inhibitor Sfrp1
turned out to be important to regulate the size of the heart
muscle during development via regulation of Wnt6 signaling
(Gibb et al., 2013), as explained in the next section.
6.3.2. MODELING WNT SIGNALING
Work in Xenopus was also instrumental for work on mathematical modeling of Wnt signaling. The group of Marc
Kirschner (Lee et al., 2003) built a mathematical model of
the pathway, focused on the main intracellular components
of canonical Wnt signaling with a set of ordinary differential equations (ODEs). This f rst model supported a rigorous
analysis of this part of Wnt signaling in silico allowing the
prediction of embryology experiments. Based on experiments
Wnt Signaling in Tissue Differentiation
6.2.4. INTEGRATED UNDERSTANDING OF WNT SIGNALING
IN VERTEBRATE EMBRYONIC DEVELOPMENT
While axis establishment and gastrulation assays have
served to study fundamental pathway mechanisms in gene
regulation and control of morphogenesis, at the same time,
Xenopus experiments have contributed to our general understanding of wider functional roles for Wnt signaling in vertebrate embryonic development, such as: (1) mesoderm
induction (e.g. Liu et al., 2005; Schohl and Fagotto, 2003)
and patterning (e.g. Hoppler et al., 1996 ); (2) patterning of the
neural plate ( Domingos et al., 2001; McGrew et al., 1997),
including neural crest induction (LaBonne and BronnerFraser, 1998) and migration (De Calisto et al., 2005), as well
as midbrain development (Kunz et al., 2004); and (3) organogenesis of the eye (Maurus et al., 2005; Rasmussen et al.,
2001), kidney (Lyons et al., 2004; Saulnier et al., 2002), and
heart (Marvin et al., 2001; Schneider and Mercola, 2001)
(see section IIIA). These investigations have led to a comprehensive and integrated understanding of Wnt signaling in
embryonic development of a typical model vertebrate.
6.3. RECENT ADVANCES
Xenopus research continues to make contributions at the
very forefront of new and important discoveries about
Wnt signaling. For example, it has been a mystery how
Wnt proteins with hydrophobic protein structures are able
to establish a signaling gradient in the extracellular space
through tissues. The importance of proteoglycans was demonstrated with Xenopus embryos, particularly the role of
extracellular sulfate and acetylate modifcations in regulating dispersal through tissues of Wnt signals, as well as Sfrp
proteins (recently reviewed by Mii and Takada, 2020). At
the membrane, the regulation of receptor turnover and the
intricate role of R-spondins in Wnt signaling has become
better understood (Chang et al., 2020; Chen et al., 2020;
Ding et al., 2018; Szenker-Ravi et al., 2018). Recent genomics studies (e.g. Nakamura et al., 2016) suggest functions for
β-Catenin beyond its association with LEF/TCF proteins.
For example, a functional interplay between Wnt pathwayregulated β-Catenin and Sox17 was recently carefully dissected in Xenopus endoderm patterning (Mukherjee et al.,
2020 ). Xenopus further contributes toward a more detailed
understanding of non-canonical Wnt signaling pathways,
particularly in regulating morphogenetic movements during
gastrulation and neurulation (e.g. Butler and Wallingford,
2018; Shindo et al., 2019). Recent fundamental discoveries
about Wnt signaling in neural crest induction and patterning
are groundbreaking far beyond the Xenopus model system,
including pluripotency of neural crest (Buitrago-Delgado
et al., 2015), surprising functions of Dkk2 (Devotta et al.,
2018), and a role for non-canonical Wnt signaling in neural
crest cells (Ossipova et al., 2018). Space constraints unfortunately prevent listing all of the important and excellent
research advances that have been facilitated by the Xenopus
model; in the following sections, three areas are explored.
6.3.1. WNT SIGNALING IN CARDIAC ORGANOGENESIS
Work in Xenopus was instrumental for deciphering roles
for Wnt signaling during heart development (reviewed by
Hoppler and Conlon, 2020; Hoppler et al., 2014). Whereas
initial work focused on the role of inhibitors of canonical signaling (Marvin et al., 2001; Schneider and Mercola, 2001), our
two laboratories were the frst to identify and confrm a role
for non-canonical Wnt signaling during this process (Afouda
et al., 2008; Pandur et al., 2002). Wnt11b as an extracellular
ligand together with JNK and PKC as intracellular signaling
mediators were shown to be involved in this process (Pandur
et al., 2002). Our work also showed how Wnt11b is tied into
a network of GATA transcription factors during this early
phase of cardiac development (Afouda and Hoppler, 2011;
Afouda et al., 2008). This initial fnding in Xenopus was later
confrmed in other biological models including murine and
human embryonic stem cells (Mazzotta et al., 2016).
Later during cardiac development, Wnt11a is important
for terminal differentiation (Gessert et al., 2008; Hempel et
al., 2017), ventricular trabeculation, and outfow tract formation both in Xenopus (Hempel et al., 2017) and mice (Nagy
et al., 2010; Zhou et al., 2007). Of interest is the f nding that
the cell adhesion molecule Alcam turned out to be critically
involved in this process (Gessert et al., 2008; Hempel et al.,
2017). During the normal cardiac differentiation program,
Wnt11a is regulated by the canonical Wnt signaling inhibitor
Dkk1 (Guo et al., 2019). It remains unclear at the moment
how inhibition of canonical Wnt signaling regulates Wnt11a
expression on a molecular level.
The precise regulation of both canonical and noncanonical Wnt signaling seems to be crucial for cardiogenesis. Inhibitors of canonical Wnt signaling, such as
Dkk1, Crescent, FrzB, and Sizzled, were shown to trigger
cardiogenesis in Xenopus in overexpression experiments
(Schneider and Mercola, 2001). In contrast, loss of Dkk1
experiments indicated its role during cardiac differentiation
(Guo et al., 2019). Also, the canonical Wnt inhibitor Sfrp1
turned out to be important to regulate the size of the heart
muscle during development via regulation of Wnt6 signaling
(Gibb et al., 2013), as explained in the next section.
6.3.2. MODELING WNT SIGNALING
Work in Xenopus was also instrumental for work on mathematical modeling of Wnt signaling. The group of Marc
Kirschner (Lee et al., 2003) built a mathematical model of
the pathway, focused on the main intracellular components
of canonical Wnt signaling with a set of ordinary differential equations (ODEs). This f rst model supported a rigorous
analysis of this part of Wnt signaling in silico allowing the
prediction of embryology experiments. Based on experiments