30
Xenopus
1998), and subsequent work showed that Vegt controls
mesendoderm induction by directly activating a number of
nodal and nodal-related genes (nodal1, nodal2, nodal4 , and
the early expressed paralogs nodal5 and nodal6; Agius et
al., 2000; Clements et al., 1999; Hyde and Old, 2000; Kofron
et al., 1999; Lee et al., 2001; Takahashi et al., 2000). Vegt
likely functions mainly as a transcriptional activator, at least
maternally, although later or context-dependent repressive
roles have not been excluded. Consistent with this idea,
Vegt has been shown to mediate activating histone acetylation modifcations (Gao et al., 2016). In addition to these
transcriptional roles, depletion of the vegt transcript leads
to other localized mRNAs becoming delocalized ( Heasman
et al., 2001) and to disorganization of intermediate f laments
(Kloc, 2009), indicating that vegt has additional but still
unclear roles as a structural or regulatory RNA.
Several observations indicated that maternal regulation of nodal expression was especially relevant for mesoderm induction. First, a highly specifc Nodal antagonist
(CerS) blocked mesendoderm induction in vivo and in
Nieuwkoop assays (Agius et al., 2000). Only inhibition of
Nodal by CerS mimicked pan-Tgfβ inhibition (Agius et al.,
2000) and other Tgfβ molecules but not FGFs could rescue vegt-depletion (e.g. derrière/gdf3, Kofron et al., 1999).
Furthermore, an early/elevated aspect of nodal homologue
expression was dependent on maternal β -Catenin, which
could also synergize with Vegt to drive higher levels of
nodal expression and activity (Agius et al., 2000; Lee et al.,
2001; Rex et al., 2002). Last, this temporal aspect and the
absence of a unique (non-Nodal) dorsal signal was shown
in heterochronic, modifed-Nieuwkoop assays using conjugates of equatorial explants with β -Catenin-depleted vegetal masses, with activity in late-stage β -Catenin-depleted
vegetal explants equivalent to early-stage control explants
(Xanthos et al., 2002).
Vegt and Vegt-induced Nodal signals also regulate transcription factors important for endoderm, notably the mixrelated, gata4–6, and sox17a/b genes (Casey et al., 1999;
Clements and Woodland, 2003; Taverner et al., 2005; Xanthos
et al., 2001). It has been problematic to determine the exact
regulatory relationships among these genes, owing to possible overlapping roles of maternal and zygotic Vegt and
multiple feedback and cross-regulatory interactions. Recent
advances in genomic approaches implicated Vegt acting with
vegetally localized Otx1 and Foxh1 in the establishment and
function of pre-zygotic enhancer complexes at endodermal
loci (Paraiso et al., 2019; see Chapters 12 and 18).
3.4.2. MATERNAL SECRETED MOLECULES
IN GERM LAYER INDUCTION
The roles of maternal secreted molecules in germ layer induction remain relatively unclear. The identifcation of gdf1 as
encoding a Tgfβ family growth factor (Weeks and Melton,
1987) was suggestive, but initial antisense and dominantnegative loss of function experiments for gdf1 were equivocal
(Joseph and Melton, 1998; Kessler and Melton, 1995; Rebagliati
and Melton, 1987; Woolf et al., 1990). It also became problematic to envision a strong role for Gdf1 in mesendoderm induction given the absence of Tgfβ/Nodal activity in vegt- depleted
embryos. A revisitation of Gdf1 function using maternal
mRNA depletion revealed a requirement for this molecule
in the timing of Nodal versus BMP signaling and ultimately
in anterior patterning (Birsoy et al., 2006). One hypothesis is
that Gdf1 may affect Nodal signal propagation or sensitivity,
as was shown in studies of mouse embryos, Xenopus explants,
and embryonic stem cells (Fuerer et al., 2014; Tanaka et al.,
2007). Thus, endogenous Gdf1 may have no function in the
absence of properly regulated Nodal signals.
Tdgf1.3 (née Cripto/XCr-1/FRL1) is maternally supplied
but unlocalized and thought to function as a secreted Nodal
co-receptor and traffcking factor (Constam, 2009; Shen and
Schier, 2000). In Xenopus, maternal tdgf1.3 mRNA is translated primarily in the animal hemisphere ( Zhang et al., 2013)
but may also interact with maternal Wnt11b in dorsal signaling (Tao et al., 2005; see subsequently). A limited set of additional secreted signaling molecules are expressed maternally
in a non-localized fashion and are involved in modulating
Nodal, BMP, FGF, and other signaling pathways in the early
embryo (Ism1, Tsku, Ndp/Norrin, Grem1; Hsu et al., 1998;
Morris et al., 2007; Pera et al., 2002; Xu et al., 2012).
3.4.3. ECTODERM SPECIFICATION
In contrast to the detailed pathways known for mesendoderm
differentiation, much less is known about the initial specif -
cation of ectoderm in Xenopus. Ectoderm has been thought
of as a maternally programmed “default state” (Weinstein
and Hemmati-Brivanlou, 1999). Both inhibition of Tgfβ and
depletion of vegt allow vegetal cells to express ectodermal
genes and to adopt ectodermal cell adhesion and cell sorting
properties (Houston and Wylie, 2003; Zhang et al., 1998).
Because mesoderm forms out of prospective ectoderm there
does not appear to be an “animalizing” gradient as in sea
urchins (Dale et al., 1985; Nieuwkoop and Ubbels, 1972),
and thus maternal transcription factors must ultimately
specify ectoderm fate.
Two zygotic transcription factors were initially identifed as candidate targets of this maternal ectoderm specifying pathway, lhx5 and foxi1 (Houston and Wylie, 2003;
Mir et al., 2007; Suri et al., 2005). Lhx5 is necessary and
suffcient for normal ectoderm adhesion and cell sorting
properties but does not activate ectodermal gene expression or inhibit Tgfβ signaling, suggesting that Lhx5 primarily regulates cell behavior ( Houston and Wylie, 2003).
foxi1 is mosaically and dynamically expressed and tightly
regulated in the ectoderm, beginning dorsally and shifting
ventrally during gastrulation (Mir et al., 2008, 2007; Suri
et al., 2005). Foxi1 is required both for ectoderm adhesion
and for repression of Tgfβ signaling and mesoderm (Mir
et al., 2007; Suri et al., 2005). The mesoderm-inhibiting
function of Foxi1 is indirect, mediated through activation of Tbx2 (a transcriptional repressor) in the ectoderm
( Teegala et al., 2018).
Xenopus
1998), and subsequent work showed that Vegt controls
mesendoderm induction by directly activating a number of
nodal and nodal-related genes (nodal1, nodal2, nodal4 , and
the early expressed paralogs nodal5 and nodal6; Agius et
al., 2000; Clements et al., 1999; Hyde and Old, 2000; Kofron
et al., 1999; Lee et al., 2001; Takahashi et al., 2000). Vegt
likely functions mainly as a transcriptional activator, at least
maternally, although later or context-dependent repressive
roles have not been excluded. Consistent with this idea,
Vegt has been shown to mediate activating histone acetylation modifcations (Gao et al., 2016). In addition to these
transcriptional roles, depletion of the vegt transcript leads
to other localized mRNAs becoming delocalized ( Heasman
et al., 2001) and to disorganization of intermediate f laments
(Kloc, 2009), indicating that vegt has additional but still
unclear roles as a structural or regulatory RNA.
Several observations indicated that maternal regulation of nodal expression was especially relevant for mesoderm induction. First, a highly specifc Nodal antagonist
(CerS) blocked mesendoderm induction in vivo and in
Nieuwkoop assays (Agius et al., 2000). Only inhibition of
Nodal by CerS mimicked pan-Tgfβ inhibition (Agius et al.,
2000) and other Tgfβ molecules but not FGFs could rescue vegt-depletion (e.g. derrière/gdf3, Kofron et al., 1999).
Furthermore, an early/elevated aspect of nodal homologue
expression was dependent on maternal β -Catenin, which
could also synergize with Vegt to drive higher levels of
nodal expression and activity (Agius et al., 2000; Lee et al.,
2001; Rex et al., 2002). Last, this temporal aspect and the
absence of a unique (non-Nodal) dorsal signal was shown
in heterochronic, modifed-Nieuwkoop assays using conjugates of equatorial explants with β -Catenin-depleted vegetal masses, with activity in late-stage β -Catenin-depleted
vegetal explants equivalent to early-stage control explants
(Xanthos et al., 2002).
Vegt and Vegt-induced Nodal signals also regulate transcription factors important for endoderm, notably the mixrelated, gata4–6, and sox17a/b genes (Casey et al., 1999;
Clements and Woodland, 2003; Taverner et al., 2005; Xanthos
et al., 2001). It has been problematic to determine the exact
regulatory relationships among these genes, owing to possible overlapping roles of maternal and zygotic Vegt and
multiple feedback and cross-regulatory interactions. Recent
advances in genomic approaches implicated Vegt acting with
vegetally localized Otx1 and Foxh1 in the establishment and
function of pre-zygotic enhancer complexes at endodermal
loci (Paraiso et al., 2019; see Chapters 12 and 18).
3.4.2. MATERNAL SECRETED MOLECULES
IN GERM LAYER INDUCTION
The roles of maternal secreted molecules in germ layer induction remain relatively unclear. The identifcation of gdf1 as
encoding a Tgfβ family growth factor (Weeks and Melton,
1987) was suggestive, but initial antisense and dominantnegative loss of function experiments for gdf1 were equivocal
(Joseph and Melton, 1998; Kessler and Melton, 1995; Rebagliati
and Melton, 1987; Woolf et al., 1990). It also became problematic to envision a strong role for Gdf1 in mesendoderm induction given the absence of Tgfβ/Nodal activity in vegt- depleted
embryos. A revisitation of Gdf1 function using maternal
mRNA depletion revealed a requirement for this molecule
in the timing of Nodal versus BMP signaling and ultimately
in anterior patterning (Birsoy et al., 2006). One hypothesis is
that Gdf1 may affect Nodal signal propagation or sensitivity,
as was shown in studies of mouse embryos, Xenopus explants,
and embryonic stem cells (Fuerer et al., 2014; Tanaka et al.,
2007). Thus, endogenous Gdf1 may have no function in the
absence of properly regulated Nodal signals.
Tdgf1.3 (née Cripto/XCr-1/FRL1) is maternally supplied
but unlocalized and thought to function as a secreted Nodal
co-receptor and traffcking factor (Constam, 2009; Shen and
Schier, 2000). In Xenopus, maternal tdgf1.3 mRNA is translated primarily in the animal hemisphere ( Zhang et al., 2013)
but may also interact with maternal Wnt11b in dorsal signaling (Tao et al., 2005; see subsequently). A limited set of additional secreted signaling molecules are expressed maternally
in a non-localized fashion and are involved in modulating
Nodal, BMP, FGF, and other signaling pathways in the early
embryo (Ism1, Tsku, Ndp/Norrin, Grem1; Hsu et al., 1998;
Morris et al., 2007; Pera et al., 2002; Xu et al., 2012).
3.4.3. ECTODERM SPECIFICATION
In contrast to the detailed pathways known for mesendoderm
differentiation, much less is known about the initial specif -
cation of ectoderm in Xenopus. Ectoderm has been thought
of as a maternally programmed “default state” (Weinstein
and Hemmati-Brivanlou, 1999). Both inhibition of Tgfβ and
depletion of vegt allow vegetal cells to express ectodermal
genes and to adopt ectodermal cell adhesion and cell sorting
properties (Houston and Wylie, 2003; Zhang et al., 1998).
Because mesoderm forms out of prospective ectoderm there
does not appear to be an “animalizing” gradient as in sea
urchins (Dale et al., 1985; Nieuwkoop and Ubbels, 1972),
and thus maternal transcription factors must ultimately
specify ectoderm fate.
Two zygotic transcription factors were initially identifed as candidate targets of this maternal ectoderm specifying pathway, lhx5 and foxi1 (Houston and Wylie, 2003;
Mir et al., 2007; Suri et al., 2005). Lhx5 is necessary and
suffcient for normal ectoderm adhesion and cell sorting
properties but does not activate ectodermal gene expression or inhibit Tgfβ signaling, suggesting that Lhx5 primarily regulates cell behavior ( Houston and Wylie, 2003).
foxi1 is mosaically and dynamically expressed and tightly
regulated in the ectoderm, beginning dorsally and shifting
ventrally during gastrulation (Mir et al., 2008, 2007; Suri
et al., 2005). Foxi1 is required both for ectoderm adhesion
and for repression of Tgfβ signaling and mesoderm (Mir
et al., 2007; Suri et al., 2005). The mesoderm-inhibiting
function of Foxi1 is indirect, mediated through activation of Tbx2 (a transcriptional repressor) in the ectoderm
( Teegala et al., 2018).