90
Xenopus
7.3.1. ESTABLISHING THE DORSAL-VENTRAL AXIS
The polarity of the initial dorsal-ventral (DV) axis is controlled by two antagonistic centers: the ventral center (VC)
secretes morphogens (BMP4, Wnt8a) that induce ventralposterior fates, and the dorsal center (DC) secretes antagonists and expresses repressors of ventral morphogens,
protecting the dorsal region from being ventralized and posteriorized thus promoting dorsal-anterior fates. These centers have been characterized in amphibians (De Robertis,
2009) and fsh (Thisse and Thisse, 2015). The DC is evident at the blastula stage and consists of: (1) the Nieuwkoop
center (NC) in vegetal cells and (2) the Blastula Chordinand Noggin-expressing (BCNE) center in marginal zone
and animal cells. The BCNE gives rise to most of the brain
and the organizer and secretes the neural inducers Noggin,
Chordin, and Nodal3, which trigger brain induction shortly
after mid-blastula transition (Wessely et al., 2001; Kuroda
et al., 2004).
While the molecular establishment of the DC has been
well documented (see Chapters 4 and 6 ), the early events
leading to the establishment of the VC were largely unknown;
our work found that Notch1 is involved (Acosta et al., 2011;
Castro Colabianchi et al., 2018). The frst clue was that
NICD1 down-regulated chordin and nodal3 in the BCNE.
Strikingly, RBPJ DBM did not affect their early expression, but notch1 knock-down in ventral cells expanded their
domains. This indicated that a ventral notch1 activity restricts
the BCNE to the dorsal side through an RBPJ -independent
pathway (Acosta et al., 2011). Indeed, NICD1 destabilized a
β-Catenin mutant lacking the GSK3 phosphorylation sites,
whereas notch1 knock-down increased its levels and ventrally expanded the domain in which β-Catenin was nuclear
in the blastula, indicating that maternal notch1 contributes
to conf ning nuclear β-Catenin to the dorsal side. Moreover,
when analyzed at tailbud or tadpole stages, NICD1 mRNA
injection resulted in a ventralized phenotype, whereas
notch1 knock-down, but not RBPJ DBM, favored dorsalanterior development. Notably, NICD1 blocked secondary
axis induction by ventral injection of ctnnb1 (β-catenin)
mRNA, indicating that Notch1 has ventralizing properties
because it interferes with the β-Catenin dorsalizing activity
(Acosta et al., 2011).
Although our functional experiments revealed a ventral,
non-canonical notch1 activity, it was not clear if this were
due to an asymmetric notch1 mRNA distribution or regulation of Notch1 activity. We found that both notch1 mRNA
and protein are enriched in the ventral region of Xenopus
embryos from fertilization to mid-blastula, with an opposite distribution of nuclear β-Catenin (Castro Colabianchi et
al., 2018), consistent with the proposed role for Notch1 in
destabilizing β-Catenin. This ventral enrichment of notch1
mRNA and protein is the earliest localized sign of ventral
development described so far in vertebrates, preceding the
ventral localization of wnt8a, bmp4, and ventx mRNAs
and dorsal localization of nuclear β-Catenin. Importantly,
we noticed nuclear Notch1 in ventral cells during cleavage
and mid-blastula stages, suggesting that besides the noncanonical role in destabilizing β-Catenin, Notch1 could be
poised to trigger transcriptional activity. Through a gene
reporter assay, we found that RBPJ -dependent transcriptional activity was higher on the ventral side at the onset of
gastrulation. Functional experiments involving NICD1, fulllength notch1, RBPJ DBM, and notch1 knock-down showed
that notch1 is necessary for the proper expression of VC
genes such as wnt8a, ventx, and bmp4. Canonical, RBPJdependent Notch1 activities are mainly involved in controlling their expression, but non-canonical Notch1 activities
might also contribute indirectly through β-Catenin destabilization and the known complex crosstalk between the DC
and the VC (Castro Colabianchi et al., 2018). Interestingly,
animal-dorsal expression of foxi1, which is necessary for
ectoderm development, is independent of Wnt/β-Catenin
signaling and is restricted to the dorsal region through a
Notch1/RBPJ-dependent mechanism (Mir et al., 2008).
Overall, this work supports the hypothesis that asymmetric
Notch1 activity, including both canonical and non-canonical
components, is involved in dorsal-ventral axis formation.
We proposed that Notch1 participates in forming the
initial DV axis via a dual, ventralizing role (Figure 7.3A):
(1) promoting the VC mainly through the canonical Notch/
RBPJ pathway and (2) restricting the DC by destabilizing
maternal β-Catenin independent of its phosphorylation by
GSK3 and RBPJ. Through this non-canonical pathway,
Notch1 ensures the elimination of β-Catenin from the ventral side that escapes from the GSK3-dependent degradation route. By inhibiting the early Wnt/β-Catenin pathway,
Notch1 contributes to preventing hyperdorsalization and
controls brain size by restricting the BCNE (Acosta et al.,
2011; Castro Colabianchi et al., 2018). Interestingly, in mammalian embryonic stem cells, membrane-bound Notch1
associates with hypophosphorylated β-Catenin, decreasing
its levels through the endocytic/lysosomal degradation pathway (Kwon et al., 2011). This fnding reinforces the conclusions of our work, the frst to study this non-canonical Notch
pathway in embryonic axis formation in vertebrates.
7.3.2. GERM LAYER FORMATION
In invertebrates, Notch signaling is a key pathway for the
induction of the germ layers, whereas in vertebrates, germ
layer induction and specifcation are controlled by several
TFs and signaling pathways (Favarolo and López, 2018).
In Xenopus, the presumptive array of germ layers can be
roughly predicted along the animal-vegetal axis of the egg
(Figure 7.3B). At cleavage stages, the animal cells approximate the ectoderm, the vegetal cells approximate the endoderm, and the intervening equator or marginal zone (MZ)
mostly contributes to the mesoderm (Dale and Slack, 1987;
Moody, 1987a ; Moody, 1987b). Thus, the MZ, which is composed of an involuting (IMZ) and a non-involuting (NIMZ)
region (Keller and Danilchik, 1988), constitutes a transition
area between germ layers whose limits need to be def ned
Xenopus
7.3.1. ESTABLISHING THE DORSAL-VENTRAL AXIS
The polarity of the initial dorsal-ventral (DV) axis is controlled by two antagonistic centers: the ventral center (VC)
secretes morphogens (BMP4, Wnt8a) that induce ventralposterior fates, and the dorsal center (DC) secretes antagonists and expresses repressors of ventral morphogens,
protecting the dorsal region from being ventralized and posteriorized thus promoting dorsal-anterior fates. These centers have been characterized in amphibians (De Robertis,
2009) and fsh (Thisse and Thisse, 2015). The DC is evident at the blastula stage and consists of: (1) the Nieuwkoop
center (NC) in vegetal cells and (2) the Blastula Chordinand Noggin-expressing (BCNE) center in marginal zone
and animal cells. The BCNE gives rise to most of the brain
and the organizer and secretes the neural inducers Noggin,
Chordin, and Nodal3, which trigger brain induction shortly
after mid-blastula transition (Wessely et al., 2001; Kuroda
et al., 2004).
While the molecular establishment of the DC has been
well documented (see Chapters 4 and 6 ), the early events
leading to the establishment of the VC were largely unknown;
our work found that Notch1 is involved (Acosta et al., 2011;
Castro Colabianchi et al., 2018). The frst clue was that
NICD1 down-regulated chordin and nodal3 in the BCNE.
Strikingly, RBPJ DBM did not affect their early expression, but notch1 knock-down in ventral cells expanded their
domains. This indicated that a ventral notch1 activity restricts
the BCNE to the dorsal side through an RBPJ -independent
pathway (Acosta et al., 2011). Indeed, NICD1 destabilized a
β-Catenin mutant lacking the GSK3 phosphorylation sites,
whereas notch1 knock-down increased its levels and ventrally expanded the domain in which β-Catenin was nuclear
in the blastula, indicating that maternal notch1 contributes
to conf ning nuclear β-Catenin to the dorsal side. Moreover,
when analyzed at tailbud or tadpole stages, NICD1 mRNA
injection resulted in a ventralized phenotype, whereas
notch1 knock-down, but not RBPJ DBM, favored dorsalanterior development. Notably, NICD1 blocked secondary
axis induction by ventral injection of ctnnb1 (β-catenin)
mRNA, indicating that Notch1 has ventralizing properties
because it interferes with the β-Catenin dorsalizing activity
(Acosta et al., 2011).
Although our functional experiments revealed a ventral,
non-canonical notch1 activity, it was not clear if this were
due to an asymmetric notch1 mRNA distribution or regulation of Notch1 activity. We found that both notch1 mRNA
and protein are enriched in the ventral region of Xenopus
embryos from fertilization to mid-blastula, with an opposite distribution of nuclear β-Catenin (Castro Colabianchi et
al., 2018), consistent with the proposed role for Notch1 in
destabilizing β-Catenin. This ventral enrichment of notch1
mRNA and protein is the earliest localized sign of ventral
development described so far in vertebrates, preceding the
ventral localization of wnt8a, bmp4, and ventx mRNAs
and dorsal localization of nuclear β-Catenin. Importantly,
we noticed nuclear Notch1 in ventral cells during cleavage
and mid-blastula stages, suggesting that besides the noncanonical role in destabilizing β-Catenin, Notch1 could be
poised to trigger transcriptional activity. Through a gene
reporter assay, we found that RBPJ -dependent transcriptional activity was higher on the ventral side at the onset of
gastrulation. Functional experiments involving NICD1, fulllength notch1, RBPJ DBM, and notch1 knock-down showed
that notch1 is necessary for the proper expression of VC
genes such as wnt8a, ventx, and bmp4. Canonical, RBPJdependent Notch1 activities are mainly involved in controlling their expression, but non-canonical Notch1 activities
might also contribute indirectly through β-Catenin destabilization and the known complex crosstalk between the DC
and the VC (Castro Colabianchi et al., 2018). Interestingly,
animal-dorsal expression of foxi1, which is necessary for
ectoderm development, is independent of Wnt/β-Catenin
signaling and is restricted to the dorsal region through a
Notch1/RBPJ-dependent mechanism (Mir et al., 2008).
Overall, this work supports the hypothesis that asymmetric
Notch1 activity, including both canonical and non-canonical
components, is involved in dorsal-ventral axis formation.
We proposed that Notch1 participates in forming the
initial DV axis via a dual, ventralizing role (Figure 7.3A):
(1) promoting the VC mainly through the canonical Notch/
RBPJ pathway and (2) restricting the DC by destabilizing
maternal β-Catenin independent of its phosphorylation by
GSK3 and RBPJ. Through this non-canonical pathway,
Notch1 ensures the elimination of β-Catenin from the ventral side that escapes from the GSK3-dependent degradation route. By inhibiting the early Wnt/β-Catenin pathway,
Notch1 contributes to preventing hyperdorsalization and
controls brain size by restricting the BCNE (Acosta et al.,
2011; Castro Colabianchi et al., 2018). Interestingly, in mammalian embryonic stem cells, membrane-bound Notch1
associates with hypophosphorylated β-Catenin, decreasing
its levels through the endocytic/lysosomal degradation pathway (Kwon et al., 2011). This fnding reinforces the conclusions of our work, the frst to study this non-canonical Notch
pathway in embryonic axis formation in vertebrates.
7.3.2. GERM LAYER FORMATION
In invertebrates, Notch signaling is a key pathway for the
induction of the germ layers, whereas in vertebrates, germ
layer induction and specifcation are controlled by several
TFs and signaling pathways (Favarolo and López, 2018).
In Xenopus, the presumptive array of germ layers can be
roughly predicted along the animal-vegetal axis of the egg
(Figure 7.3B). At cleavage stages, the animal cells approximate the ectoderm, the vegetal cells approximate the endoderm, and the intervening equator or marginal zone (MZ)
mostly contributes to the mesoderm (Dale and Slack, 1987;
Moody, 1987a ; Moody, 1987b). Thus, the MZ, which is composed of an involuting (IMZ) and a non-involuting (NIMZ)
region (Keller and Danilchik, 1988), constitutes a transition
area between germ layers whose limits need to be def ned