66
Xenopus
epithelial cells and could later be linked to canonical Wnt
signaling. In contrast, members of the Wnt5a class failed
in the secondary axis induction assay but rather inhibited the activity of Wnt1 class members. They affected
Cadherin mediated cell adhesion (Torres et al., 1996), cell
migration (Tada and Smith, 2000), and tumor metastasis
(Dissanayake et al., 2007). Those members were linked to
non-canonical Wnt signaling.
6.2.1. CANONICAL WNT SIGNALING
6.2.1.1. Canonical Wnt Signaling in the Cytoplasm
Xenopus dorsal axis development served as a powerful
experimental assay to investigate mechanisms and functions of canonical Wnt signaling. Analysis of Xenopus
dorsal axis development served to confrm in vertebrates
important components of the canonical signal transduction
pathway in the cytoplasm, such as GSK3 (Yost et al., 1996 ),
APC (Vleminckx et al., 1997), Axin (Zeng et al., 1997), and
Casein Kinase I (CKI) (Peters et al., 1999). Crucially, the
hallmark of canonical Wnt pathway, β-Catenin was also
confrmed as the important downstream component causing
Xenopus axis induction (Funayama et al., 1995).
Xenopus research was particularly powerful at integrating the molecular functions at the core of the canonical
Wnt signal transduction pathway in the “β-Catenin destruction complex” (Hedgepeth et al., 1999; Itoh et al., 1998;
Xing et al., 2003), including the N-terminal phosphorylation of β-Catenin protein by GSK3 (Yost et al., 1996) and
CK1epsilon (Liu et al., 2002), which is further controlled by
Protein Phosphatase 2A (Li et al., 2001), leading to β-TrCPmediated degradation of the β-Catenin protein (Liu et al.,
1999; Marikawa and Elinson, 1998).
These discoveries led to the concept of the “futile
β-Catenin protein turnover cycle” (reviewed by Chen et al.,
2014). In the absence of canonical Wnt signaling, β-Catenin
protein is synthesized and then promptly degraded by the
β-Catenin destruction complex, whereas active upstream
Wnt signaling disrupts the β-Catenin destruction complex.
This results in stabilized β-Catenin protein that then functions to induce axis development and by extension all the
other functions of canonical Wnt signaling in other tissues
at other stages and of course in other organisms.
Xenopus researchers were able to re-constitute the
β-Catenin destruction complex in egg extracts (Hyde et al.,
2016; Salic et al., 2000) to study the biochemistry in detail,
which revealed that, in early Xenopus development, at least,
Axin protein is clearly the limiting component (Lee et al.,
2003) with likely implications for regulation of β-Catenin
destruction complex assembly (see subsection 2.1.3.).
6.2.1.2. Canonical Wnt Signaling in the Nucleus
Xenopus embryos clearly illustrate that activated canonical
Wnt signaling allows transport of β-Catenin protein into
the nucleus (Figure 6.1) (Schneider et al., 1996; Yost et al.,
1996 ), and again Xenopus dorsal axis development served
in the discovery of how β-Catenin regulates developmental change in the nucleus. β-Catenin does not make direct
contact with DNA. Xenopus experiments were crucial in
demonstrating functional interaction with the Lymphoid
Enhancer Factor/T-Cell Factor (LEF/TCF) family of DNAbinding proteins (Behrens et al., 1996; Molenaar et al., 1996 )
and the very f rst identifcation of some of the direct Wnt
target genes (sia, Brannon et al., 1997; nodal3, McKendry
et al., 1997; twin, a.k.a. sia2, Laurent et al., 1997 ; f bronectin, Gradl et al., 1999; engrailed2, McGrew et al., 1999; and
nodal5 and nodal6, Yang et al., 2002).
Xenopus experiments dissected what has subsequently been
named the “transcriptional switch” (reviewed by Ramakrishnan
et al., 2018). In the presence of nuclear β-Catenin, when the
canonical Wnt pathway is active, LEF/TCF proteins establish
a transcriptional activation complex together with other transcriptional co-activator proteins such as pCBP (Takemaru and
Moon, 2000). In the absence of nuclear β-Catenin, when the
canonical Wnt pathway is inactive, LEF/TCF proteins establish a transcriptional repression complex together with transcriptional co-repressors such as TLE/Groucho (Roose et al.,
1998). The structure of LEF/TCF proteins reveals functional
domains that mediate interaction with nuclear β-Catenin,
and thus transcriptional activation, with TLE/Groucho, and
thus transcriptional repression, and an HMG DNA binding
domain (reviewed by Hoppler and Kavanagh, 2007; Hoppler
and Waterman, 2014). The large protein complexes assembled
by LEF/TCF proteins on regulatory DNA sequences have
since been named the “Wnt enhanceosome” (Gammons and
Bienz, 2018) (Figure 6.2).
6.2.1.3. Canonical Wnt Signaling at the Membrane
Xenopus embryos were instrumental in confrming in vertebrates that Frizzled proteins function as Wnt receptors
(Yang-Snyder et al., 1996 ) and LRP5/6 (Tamai et al., 2000)
as important co-receptors in canonical Wnt signaling. The
interaction between the Xenopus Wnt8a ligand and Frizzled8
receptor has since been described in structural detail (Janda
et al., 2012). Xenopus experiments contributed critically to
the concept that in canonical signaling Wnt, Frizzled, and
LRP form a complex at the membrane (Holmen et al., 2002;
Tamai et al., 2000), with the intracellular domain of LRP
being important for the recruitment of Axin (Davidson et al.,
2005; Zeng et al., 2005) in a process that involves Disheveled
to form a mega-protein-complex that has subsequently been
named the “Wnt signalosome” (Bilic et al., 2007; Gammons
and Bienz, 2018).
Experiments with Xenopus embryos were particularly
fertile at identifying and studying extracellular partners
modifying Wnt signaling at the membrane. The Dickkopf
(DKK) family of proteins (Glinka et al., 1998) and Sclerostin
(SOST) (Semenov et al., 2005) bind to LRP5/6 co-receptors
(Mao et al., 2001). Secreted Frizzled related proteins (sFrp),
including Frzb (a.k.a. Sfrp3) (Leyns et al., 1997; Wang et al.,
1997 ) and at later stages Sfrp1 (Gibb et al., 2013), initially
were described as Wnt inhibitors, but they may modulate Wnt
Xenopus
epithelial cells and could later be linked to canonical Wnt
signaling. In contrast, members of the Wnt5a class failed
in the secondary axis induction assay but rather inhibited the activity of Wnt1 class members. They affected
Cadherin mediated cell adhesion (Torres et al., 1996), cell
migration (Tada and Smith, 2000), and tumor metastasis
(Dissanayake et al., 2007). Those members were linked to
non-canonical Wnt signaling.
6.2.1. CANONICAL WNT SIGNALING
6.2.1.1. Canonical Wnt Signaling in the Cytoplasm
Xenopus dorsal axis development served as a powerful
experimental assay to investigate mechanisms and functions of canonical Wnt signaling. Analysis of Xenopus
dorsal axis development served to confrm in vertebrates
important components of the canonical signal transduction
pathway in the cytoplasm, such as GSK3 (Yost et al., 1996 ),
APC (Vleminckx et al., 1997), Axin (Zeng et al., 1997), and
Casein Kinase I (CKI) (Peters et al., 1999). Crucially, the
hallmark of canonical Wnt pathway, β-Catenin was also
confrmed as the important downstream component causing
Xenopus axis induction (Funayama et al., 1995).
Xenopus research was particularly powerful at integrating the molecular functions at the core of the canonical
Wnt signal transduction pathway in the “β-Catenin destruction complex” (Hedgepeth et al., 1999; Itoh et al., 1998;
Xing et al., 2003), including the N-terminal phosphorylation of β-Catenin protein by GSK3 (Yost et al., 1996) and
CK1epsilon (Liu et al., 2002), which is further controlled by
Protein Phosphatase 2A (Li et al., 2001), leading to β-TrCPmediated degradation of the β-Catenin protein (Liu et al.,
1999; Marikawa and Elinson, 1998).
These discoveries led to the concept of the “futile
β-Catenin protein turnover cycle” (reviewed by Chen et al.,
2014). In the absence of canonical Wnt signaling, β-Catenin
protein is synthesized and then promptly degraded by the
β-Catenin destruction complex, whereas active upstream
Wnt signaling disrupts the β-Catenin destruction complex.
This results in stabilized β-Catenin protein that then functions to induce axis development and by extension all the
other functions of canonical Wnt signaling in other tissues
at other stages and of course in other organisms.
Xenopus researchers were able to re-constitute the
β-Catenin destruction complex in egg extracts (Hyde et al.,
2016; Salic et al., 2000) to study the biochemistry in detail,
which revealed that, in early Xenopus development, at least,
Axin protein is clearly the limiting component (Lee et al.,
2003) with likely implications for regulation of β-Catenin
destruction complex assembly (see subsection 2.1.3.).
6.2.1.2. Canonical Wnt Signaling in the Nucleus
Xenopus embryos clearly illustrate that activated canonical
Wnt signaling allows transport of β-Catenin protein into
the nucleus (Figure 6.1) (Schneider et al., 1996; Yost et al.,
1996 ), and again Xenopus dorsal axis development served
in the discovery of how β-Catenin regulates developmental change in the nucleus. β-Catenin does not make direct
contact with DNA. Xenopus experiments were crucial in
demonstrating functional interaction with the Lymphoid
Enhancer Factor/T-Cell Factor (LEF/TCF) family of DNAbinding proteins (Behrens et al., 1996; Molenaar et al., 1996 )
and the very f rst identifcation of some of the direct Wnt
target genes (sia, Brannon et al., 1997; nodal3, McKendry
et al., 1997; twin, a.k.a. sia2, Laurent et al., 1997 ; f bronectin, Gradl et al., 1999; engrailed2, McGrew et al., 1999; and
nodal5 and nodal6, Yang et al., 2002).
Xenopus experiments dissected what has subsequently been
named the “transcriptional switch” (reviewed by Ramakrishnan
et al., 2018). In the presence of nuclear β-Catenin, when the
canonical Wnt pathway is active, LEF/TCF proteins establish
a transcriptional activation complex together with other transcriptional co-activator proteins such as pCBP (Takemaru and
Moon, 2000). In the absence of nuclear β-Catenin, when the
canonical Wnt pathway is inactive, LEF/TCF proteins establish a transcriptional repression complex together with transcriptional co-repressors such as TLE/Groucho (Roose et al.,
1998). The structure of LEF/TCF proteins reveals functional
domains that mediate interaction with nuclear β-Catenin,
and thus transcriptional activation, with TLE/Groucho, and
thus transcriptional repression, and an HMG DNA binding
domain (reviewed by Hoppler and Kavanagh, 2007; Hoppler
and Waterman, 2014). The large protein complexes assembled
by LEF/TCF proteins on regulatory DNA sequences have
since been named the “Wnt enhanceosome” (Gammons and
Bienz, 2018) (Figure 6.2).
6.2.1.3. Canonical Wnt Signaling at the Membrane
Xenopus embryos were instrumental in confrming in vertebrates that Frizzled proteins function as Wnt receptors
(Yang-Snyder et al., 1996 ) and LRP5/6 (Tamai et al., 2000)
as important co-receptors in canonical Wnt signaling. The
interaction between the Xenopus Wnt8a ligand and Frizzled8
receptor has since been described in structural detail (Janda
et al., 2012). Xenopus experiments contributed critically to
the concept that in canonical signaling Wnt, Frizzled, and
LRP form a complex at the membrane (Holmen et al., 2002;
Tamai et al., 2000), with the intracellular domain of LRP
being important for the recruitment of Axin (Davidson et al.,
2005; Zeng et al., 2005) in a process that involves Disheveled
to form a mega-protein-complex that has subsequently been
named the “Wnt signalosome” (Bilic et al., 2007; Gammons
and Bienz, 2018).
Experiments with Xenopus embryos were particularly
fertile at identifying and studying extracellular partners
modifying Wnt signaling at the membrane. The Dickkopf
(DKK) family of proteins (Glinka et al., 1998) and Sclerostin
(SOST) (Semenov et al., 2005) bind to LRP5/6 co-receptors
(Mao et al., 2001). Secreted Frizzled related proteins (sFrp),
including Frzb (a.k.a. Sfrp3) (Leyns et al., 1997; Wang et al.,
1997 ) and at later stages Sfrp1 (Gibb et al., 2013), initially
were described as Wnt inhibitors, but they may modulate Wnt