68
Xenopus
signaling in more complex ways (discussed in Bovolenta et al.,
2014). An image emerges with potentially several kinds of
extracellular ligands (Wnt, Sfrp, Dkk, etc.) inf uencing Wnt
signaling at the membrane.
6.2.2. NON-CANONICAL WNT SIGNALING
Wnt proteins that failed to induce a secondary axis were
originally classifed as Wnt5a class members (Du et al., 1995;
Torres et al., 1996). So-called non-canonical Wnt signaling
was defned by its independence from β-Catenin (Figure 6.2).
Again, Xenopus served as a powerful model system to dissect
the function of those Wnt members and the molecular nature
of the activated signaling pathways. It turned out, however,
that non-canonical Wnt signaling involves more than just one
signaling pathway. According to their main molecular effectors or cellular effect, they were named Wnt/PCP (planar cell
polarity), Wnt/JNK (jun-N-terminal Kinase), Wnt/calcium,
or Wnt/Ror signaling pathways. For historical reasons, we
deal here with these pathways independently, but these originally described different pathways overlap heavily to represent a Wnt signaling network.
6.2.2.1. Wnt/PCP/JNK Signaling
Cell migration is thought to be regulated by Wnt/PCP
signaling, often also named Wnt/JNK signaling, such
as during vertebrate gastrulation (Djiane et al., 2000;
Wallingford et al., 2000) or migration of neural crest cells
(De Calisto et al., 2005). Xenopus assays including dorsal
marginal zone explants as well as Activin-induced animal
caps served to reveal the mechanisms and functions of this
non-canonical Wnt signaling pathway during gastrulation,
neural crest cell migration, and morphogenesis. This process is mediated by directed and regulated cell movements
called convergent extension. This involves polarization of
mesodermal cells and migration of cells toward the dorsal
midline, resulting in medio-lateral narrowing and anteriorposterior elongation of the embryo. Work in Xenopus showed
that Wnt11b is required for polarization of mesodermal cells
(Yamanaka and Nishida, 2007), whereas Wnt5a regulates
cell migration toward the dorsal midline (Schambony and
Wedlich, 2007). Use of such Xenopus explants helped to further characterize these pathways and to identify components
of this pathway, including Prickle (Veeman et al., 2003),
Ror2 and JNK (Schambony and Wedlich, 2007), PTK7 (Lu
et al., 2004; Podleschny et al., 2015), Cdc42 (Penzo-Mendez
et al., 2003), Rac and Rho (Habas et al., 2003), ROK (Kim
and Han, 2005), WGEF (Tanegashima et al., 2008), and
Daam1 (Habas et al., 2001).
Wnt/PCP signaling links to cilia formation (Wallingford
and Mitchell, 2011), which regulates left/right patterning. In
particular, Wnt11b is required for the polarization of the gastrocoel roof plate and subsequently for the cilia-driven leftward fow (Walentek et al., 2013). Explants of the Xenopus
animal cap were essential for these f ndings.
6.2.2.2. Wnt/Calcium Signaling
The unique feature of Xenopus also helped to elucidate Wnt/
calcium signaling (Figure 6.2). Groundbreaking work by
Randall Moon and colleagues indicated that certain Wnts trigger an intracellular calcium release (Slusarski et al., 1997a,
1997b). Work in other model systems later showed that this
calcium release occurs within seconds and thus is very likely
a direct response of Wnt signaling (Dejmek et al., 2006; Jenei
et al., 2009). The identifcation of calcium effectors in this
pathway again made use of the unique features of the Xenopus
system. Activation of PKC through calcium is accompanied
by a translocation of the enzyme toward the membrane, which
can be monitored in animal cap explants taking advantage of
fuorescence microscopy (Sheldahl et al., 1999). Activation of
Calcium-Calmodulin Dependent Kinase II (CaMKII) was
studied in cytoplasmic lysates of Xenopus embryos before the
onset of zygotic gene transcription at the midblastula transition
(Kuhl et al., 2000). Also, the calcium-dependent phosphatase,
Calcineurin, and its transcriptional regulator Nuclear Factor of
Activated T cells (NFAT) were shown to be regulated by Wnts
using Xenopus as a model system (Saneyoshi et al., 2002).
Wnt/calcium signaling was shown to be involved in dorsoventral patterning using Xenopus embryos (Kuhl et al., 2000).
6.2.3. INTEGRATION OF CANONICAL AND NONCANONICAL WNT SIGNALING
While the concept of “canonical” versus “non-canonical”
had been useful, cracks in this wall separating canonical
from non-canonical Wnt signaling appeared from the beginning; these have subsequently only grown larger and larger.
Canonical Wnt signaling may be justif ably defned in the
sense of involving components such as LRP co-receptors and,
of course, β-Catenin and its partners in the nucleus. Noncanonical Wnt signaling—it now appears—is really only
defned by not involving β-Catenin and is a collective term
for every other form of Wnt signaling. Cracks in the wall separating canonical from non-canonical Wnt signaling particularly appear where they share pathway components as shown
by work in Xenopus, such as Disheveled (Rothbacher et al.,
2000; Sokol, 1996; Wallingford et al., 2000) or Frizzled 7
functioning at the crossroads of different Wnt pathways
(Medina et al., 2000; Medina and Steinbeisser, 2000).
Furthermore, some components of one pathway inhibit
the activity of the other. NFAT, for example, is a calciumsensitive transcription factor activated by Wnt/calcium
signaling yet also inhibits canonical β-Catenin dependent
signaling in several models (Huang et al., 2011; Saneyoshi
et al., 2002; Wang et al., 2013). Nemo-like Kinase is activated by Wnt/calcium signaling involving CamKII, and
this fnally results in a downregulation of canonical Wnt/
β-Catenin signaling (Ishitani et al., 2003). Taken together,
these and other cross-regulatory effects between canonical
and non-canonical Wnt signaling resulted in the idea of a
Wnt signaling network (Kestler and Kuhl, 2008).
Xenopus
signaling in more complex ways (discussed in Bovolenta et al.,
2014). An image emerges with potentially several kinds of
extracellular ligands (Wnt, Sfrp, Dkk, etc.) inf uencing Wnt
signaling at the membrane.
6.2.2. NON-CANONICAL WNT SIGNALING
Wnt proteins that failed to induce a secondary axis were
originally classifed as Wnt5a class members (Du et al., 1995;
Torres et al., 1996). So-called non-canonical Wnt signaling
was defned by its independence from β-Catenin (Figure 6.2).
Again, Xenopus served as a powerful model system to dissect
the function of those Wnt members and the molecular nature
of the activated signaling pathways. It turned out, however,
that non-canonical Wnt signaling involves more than just one
signaling pathway. According to their main molecular effectors or cellular effect, they were named Wnt/PCP (planar cell
polarity), Wnt/JNK (jun-N-terminal Kinase), Wnt/calcium,
or Wnt/Ror signaling pathways. For historical reasons, we
deal here with these pathways independently, but these originally described different pathways overlap heavily to represent a Wnt signaling network.
6.2.2.1. Wnt/PCP/JNK Signaling
Cell migration is thought to be regulated by Wnt/PCP
signaling, often also named Wnt/JNK signaling, such
as during vertebrate gastrulation (Djiane et al., 2000;
Wallingford et al., 2000) or migration of neural crest cells
(De Calisto et al., 2005). Xenopus assays including dorsal
marginal zone explants as well as Activin-induced animal
caps served to reveal the mechanisms and functions of this
non-canonical Wnt signaling pathway during gastrulation,
neural crest cell migration, and morphogenesis. This process is mediated by directed and regulated cell movements
called convergent extension. This involves polarization of
mesodermal cells and migration of cells toward the dorsal
midline, resulting in medio-lateral narrowing and anteriorposterior elongation of the embryo. Work in Xenopus showed
that Wnt11b is required for polarization of mesodermal cells
(Yamanaka and Nishida, 2007), whereas Wnt5a regulates
cell migration toward the dorsal midline (Schambony and
Wedlich, 2007). Use of such Xenopus explants helped to further characterize these pathways and to identify components
of this pathway, including Prickle (Veeman et al., 2003),
Ror2 and JNK (Schambony and Wedlich, 2007), PTK7 (Lu
et al., 2004; Podleschny et al., 2015), Cdc42 (Penzo-Mendez
et al., 2003), Rac and Rho (Habas et al., 2003), ROK (Kim
and Han, 2005), WGEF (Tanegashima et al., 2008), and
Daam1 (Habas et al., 2001).
Wnt/PCP signaling links to cilia formation (Wallingford
and Mitchell, 2011), which regulates left/right patterning. In
particular, Wnt11b is required for the polarization of the gastrocoel roof plate and subsequently for the cilia-driven leftward fow (Walentek et al., 2013). Explants of the Xenopus
animal cap were essential for these f ndings.
6.2.2.2. Wnt/Calcium Signaling
The unique feature of Xenopus also helped to elucidate Wnt/
calcium signaling (Figure 6.2). Groundbreaking work by
Randall Moon and colleagues indicated that certain Wnts trigger an intracellular calcium release (Slusarski et al., 1997a,
1997b). Work in other model systems later showed that this
calcium release occurs within seconds and thus is very likely
a direct response of Wnt signaling (Dejmek et al., 2006; Jenei
et al., 2009). The identifcation of calcium effectors in this
pathway again made use of the unique features of the Xenopus
system. Activation of PKC through calcium is accompanied
by a translocation of the enzyme toward the membrane, which
can be monitored in animal cap explants taking advantage of
fuorescence microscopy (Sheldahl et al., 1999). Activation of
Calcium-Calmodulin Dependent Kinase II (CaMKII) was
studied in cytoplasmic lysates of Xenopus embryos before the
onset of zygotic gene transcription at the midblastula transition
(Kuhl et al., 2000). Also, the calcium-dependent phosphatase,
Calcineurin, and its transcriptional regulator Nuclear Factor of
Activated T cells (NFAT) were shown to be regulated by Wnts
using Xenopus as a model system (Saneyoshi et al., 2002).
Wnt/calcium signaling was shown to be involved in dorsoventral patterning using Xenopus embryos (Kuhl et al., 2000).
6.2.3. INTEGRATION OF CANONICAL AND NONCANONICAL WNT SIGNALING
While the concept of “canonical” versus “non-canonical”
had been useful, cracks in this wall separating canonical
from non-canonical Wnt signaling appeared from the beginning; these have subsequently only grown larger and larger.
Canonical Wnt signaling may be justif ably defned in the
sense of involving components such as LRP co-receptors and,
of course, β-Catenin and its partners in the nucleus. Noncanonical Wnt signaling—it now appears—is really only
defned by not involving β-Catenin and is a collective term
for every other form of Wnt signaling. Cracks in the wall separating canonical from non-canonical Wnt signaling particularly appear where they share pathway components as shown
by work in Xenopus, such as Disheveled (Rothbacher et al.,
2000; Sokol, 1996; Wallingford et al., 2000) or Frizzled 7
functioning at the crossroads of different Wnt pathways
(Medina et al., 2000; Medina and Steinbeisser, 2000).
Furthermore, some components of one pathway inhibit
the activity of the other. NFAT, for example, is a calciumsensitive transcription factor activated by Wnt/calcium
signaling yet also inhibits canonical β-Catenin dependent
signaling in several models (Huang et al., 2011; Saneyoshi
et al., 2002; Wang et al., 2013). Nemo-like Kinase is activated by Wnt/calcium signaling involving CamKII, and
this fnally results in a downregulation of canonical Wnt/
β-Catenin signaling (Ishitani et al., 2003). Taken together,
these and other cross-regulatory effects between canonical
and non-canonical Wnt signaling resulted in the idea of a
Wnt signaling network (Kestler and Kuhl, 2008).