78
Xenopus
7.2.1. CANONICAL NOTCH SIGNALING
Excellent reviews describe the typical mechanism by
which Notch signals, which is summarized in Figure 7.1A
(Davis and Turner, 2001; Lai, 2004; Fortini, 2009; Kopan
and Ilagan, 2009; Jorissen and De Strooper, 2010; Kovall
and Blacklow, 2010; Tanigaki and Honjo, 2010; Groot and
Vooijs, 2012; Bray, 2016). In the absence of signaling, a
complex containing the DNA-binding protein RBPJ and
co-repressors occupies the enhancers of Notch targets to
silence them by recruiting histone deacetylases (HDACs)
or other chromatin-modifying enzymes. When the mature
Notch receptor is bound by a ligand, Delta (Dll), or Jagged
(Jag), presented by the sending cell undergoes a conformational change that exposes a cleavage site in its extracellular domain, which is then cleaved by a membrane-tethered
ADAM metalloprotease. This renders a membrane-tethered Notch intermediate (Notch extracellular truncation,
NEXT), which is cleaved at the transmembrane domain
by a γ-Secretase enzyme complex, whose active subunit is
Presenilin (Psen). This releases the NICD, which enters the
cell nucleus and forms a complex with RBPJ that recruits
the co-activator Mastermind-like (MAML), displacing
the RBPJ repressor complex and activating Notch-targets.
Typically, Notch targets are members of the hes/hey gene
families encoding bHLH-Orange (bHLH-O) transcriptional repressors.
Hes/Hey bHLH-O transcription factors (TFs) bind to
their target DNA sequence through the basic domain,
and also achieve transcriptional repression via: (1) a
C-terminal tetrapeptide motif WRPW/Y that recruits
transcriptional co-repressors of the TLE/Groucho family and (2) the Orange domain, located just C-terminal
to the bHLH domain, that controls selection of the bHLH
partner for heterodimerization. Hes proteins form homoor heterodimers with Hey proteins and repress transcription actively or passively. Active repression involves
DNA binding to the N box (CACNAG) or the class C site
[CACG(C/A)G] and recruitment of TLE/Groucho corepressors. Passive repression involves heterodimerization with other bHLH factors like E47. The WRPW motif
is also necessary for polyubiquitylation, which confers
short half-lives to Hes proteins by proteosome degradation, a key feature for their oscillatory expression during
somitogenesis (see Section 3.6) (Davis and Turner, 2001;
Bertrand et al., 2002; Huang et al., 2014; Kageyama et
al., 2007; Imayoshi and Kageyama, 2014). Notably, Hey1
lacks the WRPW motif and does not bind TLE/Groucho
(Pichon et al., 2004).
The traditional description of how the Notch pathway is used during development includes the following
( Figure 7.1B ). (1) In lateral inhibition, Notch prompts
binary cell fate choices in cell populations of equal developmental potential. The ligand-sending cell signals to its
neighbors, which in response repress ligand expression,
“ligand-sending cell fate,” and acquire an alternative fate
or remain as uncommitted precursors. This usually results
in salt-and-pepper patterns of cells of different fates, with
roughly regular spacing between specifc cell types. (2) In
lateral induction, the ligand-sending cell induces ligand
expression in its neighbors and instructs them to adopt the
same fate. This propagates a cascade of Notch activation
through a feld of adjacent cells. Also, some cases of boundary formation between two cell populations involve lateral
induction ( Lewis, 1998 ; Gazave et al., 2009 ; Sjöqvist and
Andersson, 2019 ). These models sometimes are insuff cient
to explain the complex mechanisms controlled by Notch
( Favarolo and López, 2018 ).
7.2.2. NON-CANONICAL NOTCH SIGNALING
Several core components of the canonical Notch pathway
also function in what are collectively known as non-canonical pathways; these are likely part of ancestral mechanisms for regulating cell differentiation in metazoans since
the canonical pathway did not appear until the bilaterian
lineage (Layden and Martindale, 2014). Non-canonical
pathways have been described in different cell contexts
and a variety of animal models, including (1) activation of
Notch targets through NICD without RBPJ participation;
(2) activation of Notch targets without NICD participation,
with or without RBPJ mediation (Sanalkumar et al., 2010;
Tanigaki and Honjo, 2010); (3) interaction with atypical
ligands, atypical nuclear cofactors, and other signaling
pathways (D’Souza et al., 2010; Heitzler, 2010); and (4)
non-nuclear Notch activities, independent of typical ligand
interaction and RBPJ-mediated transcription, involving
the cytoplasmic tyrosine kinase Abl (Heitzler, 2010) or
β-Catenin destabilization (Hayward et al., 2005; Hayward
et al., 2008; Sanders et al., 2009; Muñoz-Descalzo et al.,
2010; Acosta et al., 2011; Kwon et al., 2011). The latter was
frst described in Xenopus in the context of axis formation
(see Section 3.1).
7.3. NOTCH SIGNALING DURING
XENOPUS EMBRYOGENESIS
The Xenopus laevis and tropicalis genomes contain four
Notch receptor genes (notch1–4), three Delta-like ligand
genes (dll1, dlc, dll4), and two Jagged ligand genes (jag1,
jag2) (Michiue et al., 2017; Karimi et al., 2018) (Table
7.1) (Figure 7.1). Members of two groups of hes/hey genes
(hey1/hey2/hey-L and hes1–7) (Figure 7.2) are regulated
by the canonical Notch pathway (Davis and Turner, 2001;
Zhou et al., 2012) (Table 7.2). Most of them are up-regulated by Notch, but atypical responses were described for
a few hes1–7 genes (Tables 7.2, 7.3). In addition, crossregulation between Xenopus hes/hey genes was described
( Table 7.4 ).
Xenopus
7.2.1. CANONICAL NOTCH SIGNALING
Excellent reviews describe the typical mechanism by
which Notch signals, which is summarized in Figure 7.1A
(Davis and Turner, 2001; Lai, 2004; Fortini, 2009; Kopan
and Ilagan, 2009; Jorissen and De Strooper, 2010; Kovall
and Blacklow, 2010; Tanigaki and Honjo, 2010; Groot and
Vooijs, 2012; Bray, 2016). In the absence of signaling, a
complex containing the DNA-binding protein RBPJ and
co-repressors occupies the enhancers of Notch targets to
silence them by recruiting histone deacetylases (HDACs)
or other chromatin-modifying enzymes. When the mature
Notch receptor is bound by a ligand, Delta (Dll), or Jagged
(Jag), presented by the sending cell undergoes a conformational change that exposes a cleavage site in its extracellular domain, which is then cleaved by a membrane-tethered
ADAM metalloprotease. This renders a membrane-tethered Notch intermediate (Notch extracellular truncation,
NEXT), which is cleaved at the transmembrane domain
by a γ-Secretase enzyme complex, whose active subunit is
Presenilin (Psen). This releases the NICD, which enters the
cell nucleus and forms a complex with RBPJ that recruits
the co-activator Mastermind-like (MAML), displacing
the RBPJ repressor complex and activating Notch-targets.
Typically, Notch targets are members of the hes/hey gene
families encoding bHLH-Orange (bHLH-O) transcriptional repressors.
Hes/Hey bHLH-O transcription factors (TFs) bind to
their target DNA sequence through the basic domain,
and also achieve transcriptional repression via: (1) a
C-terminal tetrapeptide motif WRPW/Y that recruits
transcriptional co-repressors of the TLE/Groucho family and (2) the Orange domain, located just C-terminal
to the bHLH domain, that controls selection of the bHLH
partner for heterodimerization. Hes proteins form homoor heterodimers with Hey proteins and repress transcription actively or passively. Active repression involves
DNA binding to the N box (CACNAG) or the class C site
[CACG(C/A)G] and recruitment of TLE/Groucho corepressors. Passive repression involves heterodimerization with other bHLH factors like E47. The WRPW motif
is also necessary for polyubiquitylation, which confers
short half-lives to Hes proteins by proteosome degradation, a key feature for their oscillatory expression during
somitogenesis (see Section 3.6) (Davis and Turner, 2001;
Bertrand et al., 2002; Huang et al., 2014; Kageyama et
al., 2007; Imayoshi and Kageyama, 2014). Notably, Hey1
lacks the WRPW motif and does not bind TLE/Groucho
(Pichon et al., 2004).
The traditional description of how the Notch pathway is used during development includes the following
( Figure 7.1B ). (1) In lateral inhibition, Notch prompts
binary cell fate choices in cell populations of equal developmental potential. The ligand-sending cell signals to its
neighbors, which in response repress ligand expression,
“ligand-sending cell fate,” and acquire an alternative fate
or remain as uncommitted precursors. This usually results
in salt-and-pepper patterns of cells of different fates, with
roughly regular spacing between specifc cell types. (2) In
lateral induction, the ligand-sending cell induces ligand
expression in its neighbors and instructs them to adopt the
same fate. This propagates a cascade of Notch activation
through a feld of adjacent cells. Also, some cases of boundary formation between two cell populations involve lateral
induction ( Lewis, 1998 ; Gazave et al., 2009 ; Sjöqvist and
Andersson, 2019 ). These models sometimes are insuff cient
to explain the complex mechanisms controlled by Notch
( Favarolo and López, 2018 ).
7.2.2. NON-CANONICAL NOTCH SIGNALING
Several core components of the canonical Notch pathway
also function in what are collectively known as non-canonical pathways; these are likely part of ancestral mechanisms for regulating cell differentiation in metazoans since
the canonical pathway did not appear until the bilaterian
lineage (Layden and Martindale, 2014). Non-canonical
pathways have been described in different cell contexts
and a variety of animal models, including (1) activation of
Notch targets through NICD without RBPJ participation;
(2) activation of Notch targets without NICD participation,
with or without RBPJ mediation (Sanalkumar et al., 2010;
Tanigaki and Honjo, 2010); (3) interaction with atypical
ligands, atypical nuclear cofactors, and other signaling
pathways (D’Souza et al., 2010; Heitzler, 2010); and (4)
non-nuclear Notch activities, independent of typical ligand
interaction and RBPJ-mediated transcription, involving
the cytoplasmic tyrosine kinase Abl (Heitzler, 2010) or
β-Catenin destabilization (Hayward et al., 2005; Hayward
et al., 2008; Sanders et al., 2009; Muñoz-Descalzo et al.,
2010; Acosta et al., 2011; Kwon et al., 2011). The latter was
frst described in Xenopus in the context of axis formation
(see Section 3.1).
7.3. NOTCH SIGNALING DURING
XENOPUS EMBRYOGENESIS
The Xenopus laevis and tropicalis genomes contain four
Notch receptor genes (notch1–4), three Delta-like ligand
genes (dll1, dlc, dll4), and two Jagged ligand genes (jag1,
jag2) (Michiue et al., 2017; Karimi et al., 2018) (Table
7.1) (Figure 7.1). Members of two groups of hes/hey genes
(hey1/hey2/hey-L and hes1–7) (Figure 7.2) are regulated
by the canonical Notch pathway (Davis and Turner, 2001;
Zhou et al., 2012) (Table 7.2). Most of them are up-regulated by Notch, but atypical responses were described for
a few hes1–7 genes (Tables 7.2, 7.3). In addition, crossregulation between Xenopus hes/hey genes was described
( Table 7.4 ).