102
Xenopus
neurogenesis through Notch-canonical trans-signaling,
involving hes5.1 activation in receiving cells, and through
Jag1-ICD cis-signaling, perhaps acting as a transcriptional
regulator not involving hes5.1 (Kiyota et al., 2001; Kiyota
and Kinoshita, 2004).
In the posterior neural plate, another member of the delta
gene family, dlc, which is only expressed by the medial
stripes (Figure 7.1C), appears to be necessary for terminal differentiation of primary neurons (Peres and Durston,
2006 ) ( Table 7.7 ).
7.3.4.2. Which hes1–7 Genes Are Involved
in Primary Neurogenesis?
There is a synexpression group transcribed in primary
neurogenesis domains, including hes6.1 and multiple hes5
genes (Figure 7.2). Of these, hes5.1, hes5.2, and hes5.4–5.7
are positively regulated by Notch signaling in some cases
through paired RBPJ binding sites (Tables 7.2, 7.3). hes5.5
needs direct additional input from proneural bHLH factors, whereas hes5.1 is indirectly up-regulated by them.
Therefore, Notch/RBPJ signaling is necessary for the
expression of hes5 genes in proneural domains in vivo, but
they also require regulation by additional inputs. In contrast, hes4, which is expressed in other domains and is
directly regulated by Notch through RBPJ binding sites,
was not up-regulated by neurog2 overexpression (Lamar
and Kintner, 2005).
So far, gain- and loss-of-function experiments show that
hes2, hes4, hes5.1, hes5.4, hes1, hes5.5, hes5.6, and hey1
are able to suppress primary neurogenesis but in different
domains of the neural plate (Table 7.8). hes1, hes2, and hes4
are involved in the development of the neural border and/or
its descendants (see subsequently) and, as well as hey1, they
are up-regulated by Notch (Tables 7.2, 7.3). hes4 and hey1
are expressed in the midline of the neural plate (future f oor
plate), while hes2 is expressed in the superfcial layer of the
intermediate and lateral primary neuron stripes (Figure 7.2),
where neural precursors continue to proliferate (Sölter et al.,
2006 ).
hes6.1 is expressed in scattered cells in the medial and lateral primary neuron domains (Figure 7.2). Interestingly,
it is repressed by Notch/RBPJ signaling and is required
for expression and activity of neurog2 and the neuronal
determination gene neurod1, thus relieving neuronal precursors from Notch-mediated lateral inhibition (KoyanoNakagawa et al., 2000; Murai et al., 2011) (Tables 7.2, 7.3,
7.8). Moreover, neurog2 and neurod1 induced hes6.1 in
the absence of protein synthesis in animal caps (Seo et al.,
2007), suggesting they directly activate hes6.1, establishing
a positive feedback loop. It was proposed that Hes6.1 promotes primary neurogenesis through direct protein-protein
antagonistic interactions with other Hes factors (e.g. Hes1,
Hes4) that inhibit neuronal differentiation and by sequestering TLE/Groucho co-repressors that antagonize bHLHO Hes proteins that directly repress proneural and neuronal
determination genes (Murai et al., 2011).
7.3.4.3. Regulation of the Cell-Cycle
There is evidence that Notch1 might inhibit the withdrawal
of neuroblasts from mitosis and prevent their differentiation through the negative regulation of p21-activated kinase
3 (pak3) (Souopgui et al., 2002). However, either blocking
Dll1 or excessive Notch1/RBPJ signaling inhibited mitosis in the neural plate (Vernon et al., 2006). Experiments
with mouse P19 cells and Xenopus embryos showed a differential sensitivity of the dll1 and neurod1 promoters to the
Cdk-dependent phosphorylation status of Neurog2: while
the dll1 promoter can be activated by hypo-phosphorylated
Neurog2 (in cells undergoing cycle lengthening) or by phospho-Neurog2 (in rapidly cycling progenitors), the neurod1
promoter can only be activated by hypo-phosphorylated
Neurog2. Hypo-phosphorylated Neurog2 was able and phospho-Neurog2 was unable to promote neuronal differentiation
in the presence of NICD1, indicating that the Cdk-dependent
Neurog2 phosphorylation status also determines its posttranscriptional sensitivity to Notch signaling (Hindley et al.,
2012). Therefore, it was proposed that hypo-phosphorylated
Neurog2 shifts the balance from progenitor maintenance to
neuronal differentiation. Similarly, studies employing the
proneural mouse Ascl1 in Xenopus mitotic and interphase
egg extracts and Xenopus embryos undergoing primary neurogenesis indicate that the Cdk-dependent phosphorylation
status of Ascl1 regulates its post-translational sensitivity
to Notch signaling. Hypo-phosphorylated Ascl1 probably
escapes Notch-mediated lateral inhibition through up-regulation of Myt1 (Ali et al., 2014). Hes1 is phosphorylated
by CyclinB/Cdk1 and CyclinA/Cdk2 in vitro, suggesting it
may be controlled by phosphorylation in the G2/M phase
(Hardwick and Philpott, 2015). Phosphorylation by prolinedirected kinases destabilizes Hes1 protein and decreases its
inhibitory activity on PN in vivo (Hardwick and Philpott,
2019 ).
7.3.5. NEURAL PLATE BORDER AND MIDBRAINHINDBRAIN BOUNDARY
Neural induction subdivides the embryonic ectoderm into
neural and non-neural regions, with an intervening transition zone known as the neural plate border (NB) zone. This
zone gives rise to neural crest cells (NCCs) and cranial placodes and is positioned by intermediate BMP levels as well
as local FGF and Wnt signaling that induce a number of NB
specifer genes (Stuhlmiller and García-Castro, 2012; Pla
and Monsoro-Burq, 2018; Grocott et al., 2012; Saint-Jeannet
and Moody, 2014; Steventon et al., 2014). Members of the
Notch pathway and hes1–7 genes are expressed throughout the development of the NB and its derivatives (Figure
7.2) ( Tables 7.9 , 7.10 ). dll1 is restricted to the NB by the
counterbalanced activities of a positive regulator, Irx1, and
a negative regulator, Snai1 (Glavic et al., 2004), and hes4
expression in the NB laterally restricts the neural plate
(Maharana and Schlosser, 2018). Interestingly, hes3 can promote neural plate fate at the expense of NCC and cranial
Xenopus
neurogenesis through Notch-canonical trans-signaling,
involving hes5.1 activation in receiving cells, and through
Jag1-ICD cis-signaling, perhaps acting as a transcriptional
regulator not involving hes5.1 (Kiyota et al., 2001; Kiyota
and Kinoshita, 2004).
In the posterior neural plate, another member of the delta
gene family, dlc, which is only expressed by the medial
stripes (Figure 7.1C), appears to be necessary for terminal differentiation of primary neurons (Peres and Durston,
2006 ) ( Table 7.7 ).
7.3.4.2. Which hes1–7 Genes Are Involved
in Primary Neurogenesis?
There is a synexpression group transcribed in primary
neurogenesis domains, including hes6.1 and multiple hes5
genes (Figure 7.2). Of these, hes5.1, hes5.2, and hes5.4–5.7
are positively regulated by Notch signaling in some cases
through paired RBPJ binding sites (Tables 7.2, 7.3). hes5.5
needs direct additional input from proneural bHLH factors, whereas hes5.1 is indirectly up-regulated by them.
Therefore, Notch/RBPJ signaling is necessary for the
expression of hes5 genes in proneural domains in vivo, but
they also require regulation by additional inputs. In contrast, hes4, which is expressed in other domains and is
directly regulated by Notch through RBPJ binding sites,
was not up-regulated by neurog2 overexpression (Lamar
and Kintner, 2005).
So far, gain- and loss-of-function experiments show that
hes2, hes4, hes5.1, hes5.4, hes1, hes5.5, hes5.6, and hey1
are able to suppress primary neurogenesis but in different
domains of the neural plate (Table 7.8). hes1, hes2, and hes4
are involved in the development of the neural border and/or
its descendants (see subsequently) and, as well as hey1, they
are up-regulated by Notch (Tables 7.2, 7.3). hes4 and hey1
are expressed in the midline of the neural plate (future f oor
plate), while hes2 is expressed in the superfcial layer of the
intermediate and lateral primary neuron stripes (Figure 7.2),
where neural precursors continue to proliferate (Sölter et al.,
2006 ).
hes6.1 is expressed in scattered cells in the medial and lateral primary neuron domains (Figure 7.2). Interestingly,
it is repressed by Notch/RBPJ signaling and is required
for expression and activity of neurog2 and the neuronal
determination gene neurod1, thus relieving neuronal precursors from Notch-mediated lateral inhibition (KoyanoNakagawa et al., 2000; Murai et al., 2011) (Tables 7.2, 7.3,
7.8). Moreover, neurog2 and neurod1 induced hes6.1 in
the absence of protein synthesis in animal caps (Seo et al.,
2007), suggesting they directly activate hes6.1, establishing
a positive feedback loop. It was proposed that Hes6.1 promotes primary neurogenesis through direct protein-protein
antagonistic interactions with other Hes factors (e.g. Hes1,
Hes4) that inhibit neuronal differentiation and by sequestering TLE/Groucho co-repressors that antagonize bHLHO Hes proteins that directly repress proneural and neuronal
determination genes (Murai et al., 2011).
7.3.4.3. Regulation of the Cell-Cycle
There is evidence that Notch1 might inhibit the withdrawal
of neuroblasts from mitosis and prevent their differentiation through the negative regulation of p21-activated kinase
3 (pak3) (Souopgui et al., 2002). However, either blocking
Dll1 or excessive Notch1/RBPJ signaling inhibited mitosis in the neural plate (Vernon et al., 2006). Experiments
with mouse P19 cells and Xenopus embryos showed a differential sensitivity of the dll1 and neurod1 promoters to the
Cdk-dependent phosphorylation status of Neurog2: while
the dll1 promoter can be activated by hypo-phosphorylated
Neurog2 (in cells undergoing cycle lengthening) or by phospho-Neurog2 (in rapidly cycling progenitors), the neurod1
promoter can only be activated by hypo-phosphorylated
Neurog2. Hypo-phosphorylated Neurog2 was able and phospho-Neurog2 was unable to promote neuronal differentiation
in the presence of NICD1, indicating that the Cdk-dependent
Neurog2 phosphorylation status also determines its posttranscriptional sensitivity to Notch signaling (Hindley et al.,
2012). Therefore, it was proposed that hypo-phosphorylated
Neurog2 shifts the balance from progenitor maintenance to
neuronal differentiation. Similarly, studies employing the
proneural mouse Ascl1 in Xenopus mitotic and interphase
egg extracts and Xenopus embryos undergoing primary neurogenesis indicate that the Cdk-dependent phosphorylation
status of Ascl1 regulates its post-translational sensitivity
to Notch signaling. Hypo-phosphorylated Ascl1 probably
escapes Notch-mediated lateral inhibition through up-regulation of Myt1 (Ali et al., 2014). Hes1 is phosphorylated
by CyclinB/Cdk1 and CyclinA/Cdk2 in vitro, suggesting it
may be controlled by phosphorylation in the G2/M phase
(Hardwick and Philpott, 2015). Phosphorylation by prolinedirected kinases destabilizes Hes1 protein and decreases its
inhibitory activity on PN in vivo (Hardwick and Philpott,
2019 ).
7.3.5. NEURAL PLATE BORDER AND MIDBRAINHINDBRAIN BOUNDARY
Neural induction subdivides the embryonic ectoderm into
neural and non-neural regions, with an intervening transition zone known as the neural plate border (NB) zone. This
zone gives rise to neural crest cells (NCCs) and cranial placodes and is positioned by intermediate BMP levels as well
as local FGF and Wnt signaling that induce a number of NB
specifer genes (Stuhlmiller and García-Castro, 2012; Pla
and Monsoro-Burq, 2018; Grocott et al., 2012; Saint-Jeannet
and Moody, 2014; Steventon et al., 2014). Members of the
Notch pathway and hes1–7 genes are expressed throughout the development of the NB and its derivatives (Figure
7.2) ( Tables 7.9 , 7.10 ). dll1 is restricted to the NB by the
counterbalanced activities of a positive regulator, Irx1, and
a negative regulator, Snai1 (Glavic et al., 2004), and hes4
expression in the NB laterally restricts the neural plate
(Maharana and Schlosser, 2018). Interestingly, hes3 can promote neural plate fate at the expense of NCC and cranial