188
Xenopus
importance of the maternal TF Otx1 in endoderm formation
(Paraiso et al., 2019). Like vegt, otx1 mRNA is localized
to the vegetal region, and Otx1 works with other maternal
TFs (e.g., Vegt and Foxh1) to promote the transcription of
endodermally expressed genes. Additionally, Otx1 is a dualfunction TF, as it also has a repressive role in endoderm by
suppressing fgf20 and fgf8 expression. This is important
because Fgf signaling promotes mesoderm development.
12.2.3. GRNS DURING GASTRULATION
While germ layer specifcation occurs along the animalvegetal axis, cortical rotation of the cytoplasm shortly after
fertilization transports Dishevelled, a component of Wnt
signaling, to the dorsal side. This results in activation of a
Wnt signaling cascade dorsally as revealed by enrichment of
Ctnnb1 (β-Catenin) to the nucleus, which causes transcriptional activation of homeobox genes sia1/2 at the blastula
stage in the mesendodermal region (Laurent et al., 1997;
Brannon et al., 1997). More specifcally, Ctnnb1 forms a
complex with Tcf/Lef members of the HMG box TF family on the regulatory regions (CRMs) of the sia1/2 genes
and the nodal5/6 genes prior to zygotic genome activation
(Figure 12.1A) (Laurent et al., 1997; Nishita et al., 2000; Rex
et al., 2002). This activation of sia1/2 and nodal5/6 provides
an essential spatial activating input that is needed to induce
the expression of the Spemann organizer gene goosecoid
(gsc) (Watabe et al., 1995; Laurent et al., 1997 ), which marks
this signaling center required for the formation of primary
body axis (Spemann and Mangold, 1924). In terms of gene
regulation, CRMs of gsc integrate multiple different inputs
and synergistically respond to these inputs (Figure 12.1B).
Sia1/2 directly bind to a CRM called the proximal element
located upstream of the gsc promoter at pregastrula stages
and activate its transcription (Laurent et al., 1997 ). In addition to the Sia1/2 input, a TF complex of Smad2/4-Foxh1
serves as an input for signaling pathway activators, including Gdf1/3 and a number of Nodal ligands, via another CRM
called the distal element. The combination of these different
inputs synergistically activates gsc transcription during late
blastula stages.
At the gastrula stage, shortly after gsc induction, additional transcription of genes encoding TFs including Mix1,
Otx2, and Lhx1 occurs in the organizer region (Mochizuki
et al., 2000). All of these zygotically expressed factors later
bind to the same gsc CRMs and maintain the expression
of gsc through a feedforward loop until the neurula stage
(Charney et al., 2017a; Paraiso et al., 2020). At about the
same time when gsc expression begins in the dorsal marginal
zone of late blastula-stage embryos, tbxt (brachyury) expression encompasses the entire marginal zone (Smith et al.,
1991). Thus, gsc and tbxt are briefy coexpressed within a
population of the Spemann’s organizer cells. However, the
gsc and the tbxt expression domains quickly segregate to
generate two non-overlapping expression domains, due to
Gsc’s ability to bind to a CRM of tbxt to suppress its expression in the organizer (Artinger et al., 1997; Figure 12.1B).
This results in generating spatially distinct gene expression
domains. Gsc-expressing cells will become the prechordal
plate mesoderm, and Tbxt-expressing cells will become the
chordamesoderm and the remaining ventral mesoderm.
Regarding tbxt regulation, its initial expression is
induced by intermediate levels of Nodal (Green and Smith,
1990), but its sustained expression is regulated by a positive feedback regulation via a CRM of tbxt that responds
to Foxh1/Smad2/4 input and later by Fgf signaling, which
helps sustain tbxt expression after gastrulation (Latinkić
et al., 1997 ). Gsc additionally represses the expression of
wnt8a and ventx2 genes in the organizer to promote head
formation by inhibiting the posteriorizing function of
these factors (Yao and Kessler, 2001; Latinkić et al., 1997;
Yasuoka et al., 2014). These repressive actions of Gsc illustrate how broadly overlapping expression of TFs can segregate into two spatially distinct domains (spatial exclusion)
with clear expression boundaries to ensure specif cation of
new cell states.
In Xenopus endoderm, the maternal TF Ctnnb1
(β-catenin) is required for high expression levels of several nodal genes at the onset of zygotic transcription. Both
inputs of Ctnnb1 and Foxh1/Smad2 promote the expression
of endodermal Sox17, Foxa, Gata, and Mix TF families,
(Afouda et al., 2020; Figure 12.1). A recent genomic study
shows that Sox17 and Ctnnb1 synergistically activate a subset of endodermal enhancers, and this activation is context
dependent, sometimes requiring Tcf/Lef, and sometimes
functioning independently of Tcf/Lef (Mukherjee et al.,
2020). In addition to promoting expression of mesendoderm
genes like gsc and hhex, Sox17 also represses ectodermal
and mesodermal gene transcription in endoderm and promotes an endodermal GRN program by activating endodermal target gene expression.
12.2.4. GRN SUBCIRCUITS IN XENOPUS EMBRYOS
Developmental genes, which control expression of large
gene batteries, are rapidly induced or repressed to control
the expression levels and duration of specifc gene expression in both space and time. Much of the complexity of the
transcriptional regulation involves the interplay of TFs on
CRMs that reside close to target genes. It is through these
mechanisms that feedback and feedforward loops are created. Here we examine common topological network subcircuits that are found during early Xenopus mesendoderm
development.
12.2.4.1. Autoregulation Subcircuits
The intrinsic ability of critical genes to self-regulate their
expression in development is important in maintaining cell
fate and differentiation. Autoregulatory mechanisms come
in a variety of forms. They can be positive or negative,
and they may be direct or indirect. Positive autoregulation occurs when a TF enhances its own rate of production
directly or indirectly. A common consequence of this type
of regulation is to sustain the expression of target genes
Xenopus
importance of the maternal TF Otx1 in endoderm formation
(Paraiso et al., 2019). Like vegt, otx1 mRNA is localized
to the vegetal region, and Otx1 works with other maternal
TFs (e.g., Vegt and Foxh1) to promote the transcription of
endodermally expressed genes. Additionally, Otx1 is a dualfunction TF, as it also has a repressive role in endoderm by
suppressing fgf20 and fgf8 expression. This is important
because Fgf signaling promotes mesoderm development.
12.2.3. GRNS DURING GASTRULATION
While germ layer specifcation occurs along the animalvegetal axis, cortical rotation of the cytoplasm shortly after
fertilization transports Dishevelled, a component of Wnt
signaling, to the dorsal side. This results in activation of a
Wnt signaling cascade dorsally as revealed by enrichment of
Ctnnb1 (β-Catenin) to the nucleus, which causes transcriptional activation of homeobox genes sia1/2 at the blastula
stage in the mesendodermal region (Laurent et al., 1997;
Brannon et al., 1997). More specifcally, Ctnnb1 forms a
complex with Tcf/Lef members of the HMG box TF family on the regulatory regions (CRMs) of the sia1/2 genes
and the nodal5/6 genes prior to zygotic genome activation
(Figure 12.1A) (Laurent et al., 1997; Nishita et al., 2000; Rex
et al., 2002). This activation of sia1/2 and nodal5/6 provides
an essential spatial activating input that is needed to induce
the expression of the Spemann organizer gene goosecoid
(gsc) (Watabe et al., 1995; Laurent et al., 1997 ), which marks
this signaling center required for the formation of primary
body axis (Spemann and Mangold, 1924). In terms of gene
regulation, CRMs of gsc integrate multiple different inputs
and synergistically respond to these inputs (Figure 12.1B).
Sia1/2 directly bind to a CRM called the proximal element
located upstream of the gsc promoter at pregastrula stages
and activate its transcription (Laurent et al., 1997 ). In addition to the Sia1/2 input, a TF complex of Smad2/4-Foxh1
serves as an input for signaling pathway activators, including Gdf1/3 and a number of Nodal ligands, via another CRM
called the distal element. The combination of these different
inputs synergistically activates gsc transcription during late
blastula stages.
At the gastrula stage, shortly after gsc induction, additional transcription of genes encoding TFs including Mix1,
Otx2, and Lhx1 occurs in the organizer region (Mochizuki
et al., 2000). All of these zygotically expressed factors later
bind to the same gsc CRMs and maintain the expression
of gsc through a feedforward loop until the neurula stage
(Charney et al., 2017a; Paraiso et al., 2020). At about the
same time when gsc expression begins in the dorsal marginal
zone of late blastula-stage embryos, tbxt (brachyury) expression encompasses the entire marginal zone (Smith et al.,
1991). Thus, gsc and tbxt are briefy coexpressed within a
population of the Spemann’s organizer cells. However, the
gsc and the tbxt expression domains quickly segregate to
generate two non-overlapping expression domains, due to
Gsc’s ability to bind to a CRM of tbxt to suppress its expression in the organizer (Artinger et al., 1997; Figure 12.1B).
This results in generating spatially distinct gene expression
domains. Gsc-expressing cells will become the prechordal
plate mesoderm, and Tbxt-expressing cells will become the
chordamesoderm and the remaining ventral mesoderm.
Regarding tbxt regulation, its initial expression is
induced by intermediate levels of Nodal (Green and Smith,
1990), but its sustained expression is regulated by a positive feedback regulation via a CRM of tbxt that responds
to Foxh1/Smad2/4 input and later by Fgf signaling, which
helps sustain tbxt expression after gastrulation (Latinkić
et al., 1997 ). Gsc additionally represses the expression of
wnt8a and ventx2 genes in the organizer to promote head
formation by inhibiting the posteriorizing function of
these factors (Yao and Kessler, 2001; Latinkić et al., 1997;
Yasuoka et al., 2014). These repressive actions of Gsc illustrate how broadly overlapping expression of TFs can segregate into two spatially distinct domains (spatial exclusion)
with clear expression boundaries to ensure specif cation of
new cell states.
In Xenopus endoderm, the maternal TF Ctnnb1
(β-catenin) is required for high expression levels of several nodal genes at the onset of zygotic transcription. Both
inputs of Ctnnb1 and Foxh1/Smad2 promote the expression
of endodermal Sox17, Foxa, Gata, and Mix TF families,
(Afouda et al., 2020; Figure 12.1). A recent genomic study
shows that Sox17 and Ctnnb1 synergistically activate a subset of endodermal enhancers, and this activation is context
dependent, sometimes requiring Tcf/Lef, and sometimes
functioning independently of Tcf/Lef (Mukherjee et al.,
2020). In addition to promoting expression of mesendoderm
genes like gsc and hhex, Sox17 also represses ectodermal
and mesodermal gene transcription in endoderm and promotes an endodermal GRN program by activating endodermal target gene expression.
12.2.4. GRN SUBCIRCUITS IN XENOPUS EMBRYOS
Developmental genes, which control expression of large
gene batteries, are rapidly induced or repressed to control
the expression levels and duration of specifc gene expression in both space and time. Much of the complexity of the
transcriptional regulation involves the interplay of TFs on
CRMs that reside close to target genes. It is through these
mechanisms that feedback and feedforward loops are created. Here we examine common topological network subcircuits that are found during early Xenopus mesendoderm
development.
12.2.4.1. Autoregulation Subcircuits
The intrinsic ability of critical genes to self-regulate their
expression in development is important in maintaining cell
fate and differentiation. Autoregulatory mechanisms come
in a variety of forms. They can be positive or negative,
and they may be direct or indirect. Positive autoregulation occurs when a TF enhances its own rate of production
directly or indirectly. A common consequence of this type
of regulation is to sustain the expression of target genes