107
Notch Signaling in Early Embryogenesis
the NB through the positive regulation of the NB-specif er
hes4, which receives multiple regulatory inputs from other
pathways. Moreover, Notch1 can suppress nkx1–2 in presumptive NCC; this gene encodes a transcriptional repressor thought to inhibit neural fate to allow NCC induction
(Kurata and Ueno, 2003).
7.3.5.2. The Role of hes Genes in PPE Development
Notch signaling and genes of the hes1–7 group are also
involved in cranial placode development (Tables 7.9, 7.10).
Six1 and its co-activator Eya1 are crucial regulators of placode development (Brugmann et al., 2004; Riddiford and
Schlosser, 2016). hes4 is required for establishing the preplacodal ectoderm; the expression of notch2, six1, and eya1
in this tissue; and the development of the lens f eld (Murato
and Hashimoto, 2009; Maharana and Schlosser, 2018) (Table
7.10), placing hes4 upstream of the placodal program probably at the level of NB establishment. However, additional
gene cascades converge in setting this program, since other
PPE markers were not affected by hes4 knock-down (Murato
and Hashimoto, 2009). Recently, a NB gene regulatory network that cross-regulates with Six1/Eya1 was proposed for
controlling PPE and NCC specifcation. This GRN includes
Hes4 and other TFs expressed in the neural and non-neural
ectoderm (Maharana and Schlosser, 2018). Downstream of
this GRN are hes2, hes5.4, and hes5.6, which are expressed
in the PPE. They are presumptive direct targets of Six1/
Eya1, as they were up-regulated by them in the absence of
protein synthesis, and placodal hes5.4 and hes5.6 expression
require Six1/Eya1 function (Riddiford and Schlosser, 2016).
In neurogenic placodes, Six1/Eya1 control dll1 in a dosedependent manner. High Six1/Eya1 levels maintain proliferating placodal precursors, but as cells delaminate from the
placodes, Six/Eya1 levels are reduced and the neurogenesis
program is triggered, including the onset of dll1 expression (Schlosser et al., 2008). Notch1/RBPJ is required for
hes5.4 expression during placodal development (Tables 7.2,
7.3) (Riddiford and Schlosser, 2016). The requirement of
Notch signaling for hes2 and hes5.6 expression in this process is currently unknown, although hes5.6 is induced by
Notch1/RBPJ during gastrulation (Tables 7.2, 7.3). hes2 was
not induced by activation of the Notch pathway in naive or
neuralized animal cap explants but was moderately induced
in embryos, with ectopic expression restricted to the NB
(Sölter et al., 2006) (Tables 7.2, 7.3).
PPE hes5.4 expression requires two positive regulators:
(1) high Six1/Eya1 levels activate hes5.4 independently of
Notch/RBPJ and (2) Notch/RBPJ activates hes5.4 independently of Six1/Eya1, probably through lateral inhibition
( Tables 7.2 , 7.3 , 7.9 ). Subsequently, hes5.4 maintains placodal progenitors in an undifferentiated state, restricting primary neurogenesis upstream of proneural genes (Table 7.8).
As Six1/Eya1 activity declines, hes5.4 is required at low levels to promote neuronal differentiation. Intriguingly, hes5.4
is also required for neuronal differentiation downstream of
proneural genes, as terminal differentiation markers were
frequently decreased after hes5.4 knock-down (Table 7.8).
The details of the mechanism underlying these opposing
roles remain unresolved, but oscillation of hes5.4 expression
might be involved since hes5.4 represses its own transcription (Tables 7.4, 7.8) (Riddiford and Schlosser, 2017).
7.3.5.3. Other Roles for Notch Pathway
in Placode Development
Other evidence further supports Notch pathway involvement
in cranial placode development (Table 7.9). Potentiated by
Otx2, Notch can activate dmrta1/2 in the anterior ectoderm.
These genes encode TFs expressed in the presumptive olfactory placodes and are involved in olfactory neurogenesis
(Parlier et al., 2013). An RBPJ binding site was found in the
main enhancer of foxe3, a key TF required for lens placode
development (Ogino et al., 2008; Kenyon et al., 1999). dll1
and dlc are expressed in the adjacent presumptive retina,
from where they presumably induce foxe3 through Notch/
RBPJ. While an antimorphic dll1 produced severe head
defects, making the results diffcult to interpret, the antimorphic dlc produced a more restricted, lens-defective phenotype, indicating that dlc is involved in lens development
(Ogino et al., 2008).
7.3.5.4. The Midbrain/Hindbrain Boundary
The midbrain/hindbrain boundary (MHB) is considered
an organizing center because signals from this region
induce and pattern the adjacent mesencephalon and hindbrain (Anderson and Stern, 2016). hes7.1 is one of the f rst
genes to demarcate the presumptive MHB at early gastrula
stages. Notably, at neural plate stages, the hes7.1 domain
coincides with a hes5.1, hes5.2, and hes5.7 expression gap
(Shinga et al., 2001; Takada et al., 2005) (Figure 7.2) (Table
7.11). hes7.1 is necessary for MHB establishment through
repressing, probably directly, hes5.1, hes5.2, and hes5.7 in
this region, whereas these hes5 genes (which are positively
regulated by Dll/Notch signaling during primary neurogenesis) are thought to restrict hes7.1 to the MHB (Shinga
et al., 2001; Takada et al., 2005) (Tables 7.4, 7.11). Strikingly,
NICD1 abolished and RBPJ DBM did not affect MHB hes7.1
expression (Takada et al., 2005). It would be interesting to
address whether notch1 normally down-regulates hes7.1 by
a non-canonical pathway.
7.3.6. SOMITOGENESIS
The classic Clock and Wavefront hypothesis for vertebrate
somitogenesis, which involves Notch signaling, was originally postulated by experimental work based on Xenopus
(Cooke and Zeeman, 1976; Cooke, 1981). It explains the
sequential formation of vertebrate somites from the posterior presomitic mesoderm that is due to an oscillation
between permissive and non-permissive phases for segmentation, the so-called “segmentation clock” that is controlled
by hes genes. Their proteins act as pacemakers that cellautonomously cycle on and off through an autoregulatory
Notch Signaling in Early Embryogenesis
the NB through the positive regulation of the NB-specif er
hes4, which receives multiple regulatory inputs from other
pathways. Moreover, Notch1 can suppress nkx1–2 in presumptive NCC; this gene encodes a transcriptional repressor thought to inhibit neural fate to allow NCC induction
(Kurata and Ueno, 2003).
7.3.5.2. The Role of hes Genes in PPE Development
Notch signaling and genes of the hes1–7 group are also
involved in cranial placode development (Tables 7.9, 7.10).
Six1 and its co-activator Eya1 are crucial regulators of placode development (Brugmann et al., 2004; Riddiford and
Schlosser, 2016). hes4 is required for establishing the preplacodal ectoderm; the expression of notch2, six1, and eya1
in this tissue; and the development of the lens f eld (Murato
and Hashimoto, 2009; Maharana and Schlosser, 2018) (Table
7.10), placing hes4 upstream of the placodal program probably at the level of NB establishment. However, additional
gene cascades converge in setting this program, since other
PPE markers were not affected by hes4 knock-down (Murato
and Hashimoto, 2009). Recently, a NB gene regulatory network that cross-regulates with Six1/Eya1 was proposed for
controlling PPE and NCC specifcation. This GRN includes
Hes4 and other TFs expressed in the neural and non-neural
ectoderm (Maharana and Schlosser, 2018). Downstream of
this GRN are hes2, hes5.4, and hes5.6, which are expressed
in the PPE. They are presumptive direct targets of Six1/
Eya1, as they were up-regulated by them in the absence of
protein synthesis, and placodal hes5.4 and hes5.6 expression
require Six1/Eya1 function (Riddiford and Schlosser, 2016).
In neurogenic placodes, Six1/Eya1 control dll1 in a dosedependent manner. High Six1/Eya1 levels maintain proliferating placodal precursors, but as cells delaminate from the
placodes, Six/Eya1 levels are reduced and the neurogenesis
program is triggered, including the onset of dll1 expression (Schlosser et al., 2008). Notch1/RBPJ is required for
hes5.4 expression during placodal development (Tables 7.2,
7.3) (Riddiford and Schlosser, 2016). The requirement of
Notch signaling for hes2 and hes5.6 expression in this process is currently unknown, although hes5.6 is induced by
Notch1/RBPJ during gastrulation (Tables 7.2, 7.3). hes2 was
not induced by activation of the Notch pathway in naive or
neuralized animal cap explants but was moderately induced
in embryos, with ectopic expression restricted to the NB
(Sölter et al., 2006) (Tables 7.2, 7.3).
PPE hes5.4 expression requires two positive regulators:
(1) high Six1/Eya1 levels activate hes5.4 independently of
Notch/RBPJ and (2) Notch/RBPJ activates hes5.4 independently of Six1/Eya1, probably through lateral inhibition
( Tables 7.2 , 7.3 , 7.9 ). Subsequently, hes5.4 maintains placodal progenitors in an undifferentiated state, restricting primary neurogenesis upstream of proneural genes (Table 7.8).
As Six1/Eya1 activity declines, hes5.4 is required at low levels to promote neuronal differentiation. Intriguingly, hes5.4
is also required for neuronal differentiation downstream of
proneural genes, as terminal differentiation markers were
frequently decreased after hes5.4 knock-down (Table 7.8).
The details of the mechanism underlying these opposing
roles remain unresolved, but oscillation of hes5.4 expression
might be involved since hes5.4 represses its own transcription (Tables 7.4, 7.8) (Riddiford and Schlosser, 2017).
7.3.5.3. Other Roles for Notch Pathway
in Placode Development
Other evidence further supports Notch pathway involvement
in cranial placode development (Table 7.9). Potentiated by
Otx2, Notch can activate dmrta1/2 in the anterior ectoderm.
These genes encode TFs expressed in the presumptive olfactory placodes and are involved in olfactory neurogenesis
(Parlier et al., 2013). An RBPJ binding site was found in the
main enhancer of foxe3, a key TF required for lens placode
development (Ogino et al., 2008; Kenyon et al., 1999). dll1
and dlc are expressed in the adjacent presumptive retina,
from where they presumably induce foxe3 through Notch/
RBPJ. While an antimorphic dll1 produced severe head
defects, making the results diffcult to interpret, the antimorphic dlc produced a more restricted, lens-defective phenotype, indicating that dlc is involved in lens development
(Ogino et al., 2008).
7.3.5.4. The Midbrain/Hindbrain Boundary
The midbrain/hindbrain boundary (MHB) is considered
an organizing center because signals from this region
induce and pattern the adjacent mesencephalon and hindbrain (Anderson and Stern, 2016). hes7.1 is one of the f rst
genes to demarcate the presumptive MHB at early gastrula
stages. Notably, at neural plate stages, the hes7.1 domain
coincides with a hes5.1, hes5.2, and hes5.7 expression gap
(Shinga et al., 2001; Takada et al., 2005) (Figure 7.2) (Table
7.11). hes7.1 is necessary for MHB establishment through
repressing, probably directly, hes5.1, hes5.2, and hes5.7 in
this region, whereas these hes5 genes (which are positively
regulated by Dll/Notch signaling during primary neurogenesis) are thought to restrict hes7.1 to the MHB (Shinga
et al., 2001; Takada et al., 2005) (Tables 7.4, 7.11). Strikingly,
NICD1 abolished and RBPJ DBM did not affect MHB hes7.1
expression (Takada et al., 2005). It would be interesting to
address whether notch1 normally down-regulates hes7.1 by
a non-canonical pathway.
7.3.6. SOMITOGENESIS
The classic Clock and Wavefront hypothesis for vertebrate
somitogenesis, which involves Notch signaling, was originally postulated by experimental work based on Xenopus
(Cooke and Zeeman, 1976; Cooke, 1981). It explains the
sequential formation of vertebrate somites from the posterior presomitic mesoderm that is due to an oscillation
between permissive and non-permissive phases for segmentation, the so-called “segmentation clock” that is controlled
by hes genes. Their proteins act as pacemakers that cellautonomously cycle on and off through an autoregulatory