105
Notch Signaling in Early Embryogenesis
placodes, suggesting it also participates in setting the neural
plate/NB boundary, although this could not be conf rmed by
hes3 knock-down, perhaps due to compensation by other hes
genes (Hong and Saint-Jeannet, 2018).
7.3.5.1. The Role of hes4 in NB and NCC Development
hes4 is expressed in prospective NCC territories, along with
notch1 (Glavic et al., 2004; Vega-López et al., 2015), and
appears to act through partially counteracting pathways to
promote and restrict foxd3 expression to NCC (Maharana
and Schlosser, 2018).
Neural induction subdivides the ectoderm into neural
and non-neural regions. The transition zone is the neural
border, from which neural crest cells, placodes, the bordering non-neural ectoderm, and dorsal neural tube segregate
(Stuhlmiller and García-Castro, 2012; Pla and MonsoroBurq, 2018). The pre-placodal ectoderm (PPE) forms a
horseshoe-shaped domain surrounding the neural plate at
its anterior end and later segregates into individual placodes
(Stuhlmiller and García-Castro, 2012; Grocott et al., 2012;
Saint-Jeannet and Moody, 2014; Steventon et al., 2014; Pla
and Monsoro-Burq, 2018). NCC development begins during
gastrulation, when the NB is induced and stabilized and progresses through sequential steps, with the NB-TFs controlling the onset of NCC induction within the NB at the end of
gastrulation and during the neural plate stage. This is followed by NCC specif cation, which occurs through a multistep process during neurulation and involving the activation
of a new set of genes encoding specifc NCC transcription
factors (NCC-TFs), which are not shared with contiguous
populations. These NCC-TFs are activated in a stereotyped
sequence, with an early step (activation of sox9 since NF11;
snai2 and foxd3 since NF12–12.5) and a maturation step,
with the activation of late NCC-TFs (sox10 from NF13–14,
twist1). NCC migration begins at the end of neurulation
(once the neural folds fuse at the midline, transforming
the neural plate into the neural tube) and continues during
organogenesis, during which post-migratory NCCs colonize
target tissues and organs, where they differentiate into multiple cell types (Pegoraro and Monsoro-Burq, 2013; Pla and
Monsoro-Burq, 2018).
There are conficting interpretations concerning the
role of hes4 in NB/NCC development and its regulation by
Notch signaling in these tissues (Tables 7.9, 7.10). Although
hes4 is already expressed in the NB at mid-gastrula (Tsuji
et al., 2003) and is considered an NB specif er in vivo, this
TF alone can not initiate NCC specifcation in animal cap
explants (Milet et al., 2013). Analysis at advanced neurula stages indicated that Dll1 signals to presumptive NCC
through Notch1/RBPJ inducing hes4, which represses bmp4
to ensure optimal BMP signaling levels for NCC specif cation (Glavic et al., 2004). Others showed that during NCC
specif cation, hes4 restricts dll1 in NCC for the survival
and maintenance of precursors in a mitotic, undifferentiated state (Nagatomo and Hashimoto, 2007). Strikingly,
another group showed that the NCC hes4 domain was unaffected in neurulae following activation or blockade of the
RBPJ-dependent pathway at mid-gastrula and noticed that
hes4 and dll1 domains partially overlap in the anterior,
lateral neural plate. They proposed that Dll1 favors NCC
precursor proliferation rather than controlling the balance between primary neurons and NCC fates. Through a
DNA-binding independent mechanism, hes4 transiently and
indirectly induces dll1 expression, leading to NCC proliferation and differentiation. Through a cell-autonomous, DNAbinding-dependent mechanism, hes4 up-regulates early NB
genes and is required for NCC survival and maintenance in
an undifferentiated state (Nichane et al., 2008a ; Nichane
et al., 2008b). More recently, other authors showed that in the
pre-migratory NCC territory, hes1 and hes4 are positively
regulated by Dll1/Notch/RBPJ whereas BMP down-regulates hes4 (Nagatomo and Hashimoto, 2007; Vega-López
et al., 2015). These authors propose that hes1 and hes4 are
required for several processes during NCC development.
First, both promote NCC specifcation at the expense of
neural plate and epidermis independent of cell proliferation;
then Hes4 acts as a transcriptional repressor during NCC
specifcation and is later required for their survival during
neurulation; f nally, hes4 is required cell-autonomously to
initiate NCC migration and their differentiation into the cranial skeleton.
Wnt and FGF signaling are necessary for hes4 expression,
whereas BMP down-regulates it in the presumptive NCC at
neural plate stages (Nichane et al., 2008a ; Vega-López et al.,
2015). Others identifed three hes4 expression phases during NB/NCC development. First, during NB induction (early
gastrula), hes4 expression is insensitive to BMP signaling
but requires down-regulation of the Wnt pathway. Then,
during NCC induction (mid-gastrula), it requires Wnt and
down-regulation of BMP signaling. Finally, both pathways
are required for hes4 expression during NCC maintenance
in early neurula (Steventon and Mayor, 2012). FGF signaling
was proposed to regulate hes4 and dll1 in the NB through
Stat3.1, which is phosphorylated by FGF/FGFR4. Whereas
low Stat3.1 activity up-regulates hes4, high Stat3.1 activity
promotes dll1 expression and Dll1/Notch signaling (Nichane
et al., 2010). Overall, it is clear that hes4 is required for NCC
development in Xenopus, but controversies still exist about
the underlying mechanisms (for discussion, see Vega-López
et al., 2015). Time-dependent opposite responses to the same
experimental Notch perturbation, which were observed in
other contexts in Xenopus (Contakos et al., 2005; Revinski
et al., 2010), might underlie the conficting results between
different studies. Some reviews regard Notch signaling as
an important source for NCC maintenance rather than as a
key player in NB induction during gastrulation (Stuhlmiller
and García-Castro, 2012; Pla and Monsoro-Burq, 2018).
Curiously, most studies analyzed the effects of perturbing
Notch signaling on hes4 too late to address if this pathway
plays an early role in establishing the NB hes4 domain
(Table 7.9). However, we observed a clear expansion of the
NB hes4 domain at mid-gastrula after constitutive Notch1
activation beginning at cleavage stages (López et al., 2005),
suggesting that Notch participates in the establishment of
Notch Signaling in Early Embryogenesis
placodes, suggesting it also participates in setting the neural
plate/NB boundary, although this could not be conf rmed by
hes3 knock-down, perhaps due to compensation by other hes
genes (Hong and Saint-Jeannet, 2018).
7.3.5.1. The Role of hes4 in NB and NCC Development
hes4 is expressed in prospective NCC territories, along with
notch1 (Glavic et al., 2004; Vega-López et al., 2015), and
appears to act through partially counteracting pathways to
promote and restrict foxd3 expression to NCC (Maharana
and Schlosser, 2018).
Neural induction subdivides the ectoderm into neural
and non-neural regions. The transition zone is the neural
border, from which neural crest cells, placodes, the bordering non-neural ectoderm, and dorsal neural tube segregate
(Stuhlmiller and García-Castro, 2012; Pla and MonsoroBurq, 2018). The pre-placodal ectoderm (PPE) forms a
horseshoe-shaped domain surrounding the neural plate at
its anterior end and later segregates into individual placodes
(Stuhlmiller and García-Castro, 2012; Grocott et al., 2012;
Saint-Jeannet and Moody, 2014; Steventon et al., 2014; Pla
and Monsoro-Burq, 2018). NCC development begins during
gastrulation, when the NB is induced and stabilized and progresses through sequential steps, with the NB-TFs controlling the onset of NCC induction within the NB at the end of
gastrulation and during the neural plate stage. This is followed by NCC specif cation, which occurs through a multistep process during neurulation and involving the activation
of a new set of genes encoding specifc NCC transcription
factors (NCC-TFs), which are not shared with contiguous
populations. These NCC-TFs are activated in a stereotyped
sequence, with an early step (activation of sox9 since NF11;
snai2 and foxd3 since NF12–12.5) and a maturation step,
with the activation of late NCC-TFs (sox10 from NF13–14,
twist1). NCC migration begins at the end of neurulation
(once the neural folds fuse at the midline, transforming
the neural plate into the neural tube) and continues during
organogenesis, during which post-migratory NCCs colonize
target tissues and organs, where they differentiate into multiple cell types (Pegoraro and Monsoro-Burq, 2013; Pla and
Monsoro-Burq, 2018).
There are conficting interpretations concerning the
role of hes4 in NB/NCC development and its regulation by
Notch signaling in these tissues (Tables 7.9, 7.10). Although
hes4 is already expressed in the NB at mid-gastrula (Tsuji
et al., 2003) and is considered an NB specif er in vivo, this
TF alone can not initiate NCC specifcation in animal cap
explants (Milet et al., 2013). Analysis at advanced neurula stages indicated that Dll1 signals to presumptive NCC
through Notch1/RBPJ inducing hes4, which represses bmp4
to ensure optimal BMP signaling levels for NCC specif cation (Glavic et al., 2004). Others showed that during NCC
specif cation, hes4 restricts dll1 in NCC for the survival
and maintenance of precursors in a mitotic, undifferentiated state (Nagatomo and Hashimoto, 2007). Strikingly,
another group showed that the NCC hes4 domain was unaffected in neurulae following activation or blockade of the
RBPJ-dependent pathway at mid-gastrula and noticed that
hes4 and dll1 domains partially overlap in the anterior,
lateral neural plate. They proposed that Dll1 favors NCC
precursor proliferation rather than controlling the balance between primary neurons and NCC fates. Through a
DNA-binding independent mechanism, hes4 transiently and
indirectly induces dll1 expression, leading to NCC proliferation and differentiation. Through a cell-autonomous, DNAbinding-dependent mechanism, hes4 up-regulates early NB
genes and is required for NCC survival and maintenance in
an undifferentiated state (Nichane et al., 2008a ; Nichane
et al., 2008b). More recently, other authors showed that in the
pre-migratory NCC territory, hes1 and hes4 are positively
regulated by Dll1/Notch/RBPJ whereas BMP down-regulates hes4 (Nagatomo and Hashimoto, 2007; Vega-López
et al., 2015). These authors propose that hes1 and hes4 are
required for several processes during NCC development.
First, both promote NCC specifcation at the expense of
neural plate and epidermis independent of cell proliferation;
then Hes4 acts as a transcriptional repressor during NCC
specifcation and is later required for their survival during
neurulation; f nally, hes4 is required cell-autonomously to
initiate NCC migration and their differentiation into the cranial skeleton.
Wnt and FGF signaling are necessary for hes4 expression,
whereas BMP down-regulates it in the presumptive NCC at
neural plate stages (Nichane et al., 2008a ; Vega-López et al.,
2015). Others identifed three hes4 expression phases during NB/NCC development. First, during NB induction (early
gastrula), hes4 expression is insensitive to BMP signaling
but requires down-regulation of the Wnt pathway. Then,
during NCC induction (mid-gastrula), it requires Wnt and
down-regulation of BMP signaling. Finally, both pathways
are required for hes4 expression during NCC maintenance
in early neurula (Steventon and Mayor, 2012). FGF signaling
was proposed to regulate hes4 and dll1 in the NB through
Stat3.1, which is phosphorylated by FGF/FGFR4. Whereas
low Stat3.1 activity up-regulates hes4, high Stat3.1 activity
promotes dll1 expression and Dll1/Notch signaling (Nichane
et al., 2010). Overall, it is clear that hes4 is required for NCC
development in Xenopus, but controversies still exist about
the underlying mechanisms (for discussion, see Vega-López
et al., 2015). Time-dependent opposite responses to the same
experimental Notch perturbation, which were observed in
other contexts in Xenopus (Contakos et al., 2005; Revinski
et al., 2010), might underlie the conficting results between
different studies. Some reviews regard Notch signaling as
an important source for NCC maintenance rather than as a
key player in NB induction during gastrulation (Stuhlmiller
and García-Castro, 2012; Pla and Monsoro-Burq, 2018).
Curiously, most studies analyzed the effects of perturbing
Notch signaling on hes4 too late to address if this pathway
plays an early role in establishing the NB hes4 domain
(Table 7.9). However, we observed a clear expansion of the
NB hes4 domain at mid-gastrula after constitutive Notch1
activation beginning at cleavage stages (López et al., 2005),
suggesting that Notch participates in the establishment of