265
Digestive and Respiratory System
blastula and gastrula stages (Paraiso et al. 2019; Gentsch
et al. 2019; Afouda et al. 2020). Additional ChIP-seq studies
suggest unique combinations of HD TFs can either promote
or repress transcription: for example, Oxt2/Lim1 complexes
stimulate transcription of dorsal-anterior endoderm genes
like hhex, whereas Otx2/Goosecoid complexes repress ventral-posterior genes such as ventx1/2/3 and wnt8 ( Yasuoka
et al. 2014; Yasuoka et al. 2019).
During gastrulation, there is a dramatic switch in the region
of active Wnt signaling in the embryo, which can be visualized with transgenic Wnt reporter embryos Tg(WRE:dGFP),
wherein canonical Tcf/Lef sites drive destabilized GFP
expression (Figure 18.3D; Tran et al. 2010). Maternal Wnt
signaling is high in the dorsal mesendoderm and low in the
ventral side of the blastula and early gastrula, but as gastrulation proceeds, the pattern is switched as zygotic wnt8
becomes expressed in ventral posterior mesendoderm and
Wnt-antagonists are expressed in the organizer.
In the ventral-posterior GRN, zygotic Wnt8/β-Catenin
and Bmp4/Smad1 act in a feedforward loop (Hoppler and
Moon 1998; Fuentealba et al. 2007; Kjolby et al. 2019)
and directly activate expression of the Ventx family of
HD transcriptional repressors (Ventx1/2/3) (Figure 18.3C;
Onichtchouk et al. 1996; Karaulanov et al. 2004; Hikasa
et al. 2010). Ventx factors repress transcription of dorsalanterior genes, including goosecoid and hhex ( Sander et al.
2007; Rankin et al. 2011); thus, a mutually antagonistic
cross-repressive loop exists between the ventral-posterior
and dorsal-anterior GRNs in the early gastrula embryo
( Figure 18.3E ).
18.4.2. NEURULA-STAGE ENDODERM PATTERNING
During gastrulation, the hhex-expressing anterior endoderm migrates to the future ventral foregut adjacent to the
heart as the blastopore closes and the archenteron opens.
Immediately behind the migrating anterior endoderm, a thin
layer of dorsal endoderm forms the roof of the archenteron,
whereas the ventx-expressing endodermal mass makes up
the bulk of the ventral gut tissue. During the neurula and
early somitogenesis stages (NF12–NF25), the endoderm
continues to be patterned along its D-V and A-P axes by
combinatorial BMP, Wnt/β-Catenin, RA, and FGF signaling
(Figure 18.4A), and distinct domains of regional endoderm
TF expression are obvious by NF20 (Figure 18.4B). This
post-gastrula patterning in Xenopus is comparable to events
that occur during E7-E9 in mouse gestation and 3–8 days of
many human PSC differentiation protocols.
During this post-gastrula period, bmp2/4/7 are expressed
in the ventral splanchnic mesoderm, while BMP antagonists noggin and chordin are produced dorsally from the
notochord, generating a D-V gradient of phosphorylated
Smad1/5/9 (pSMAD1) activity in the embryo (Figure
18.4A,B). High BMP promotes ventral gene expression in
both the foregut and hindgut, whereas low BMP permits a
dorsal endoderm identity (Stevens et al. 2017). BMP from
the ventral mesoderm induces expression of a number of
secreted Wnt antagonists in the foregut including sfrp1/2/5
(Kenny et al. 2012; Stevens et al. 2017), which are required
to maintain a low level of Wnt11-Fzd7 signaling essential for
proliferation, morphogenesis, and identity of hhex+ progenitors (McLin et al. 2007; Li et al. 2008; Zhang et al. 2013a).
In the posterior domain, BMP and Wnt ligands secreted
from the mesoderm cooperate to maintain ventx+ hindgut
fate and induce the expression of cdx2, a HD TF essential
for intestinal development in all vertebrates (Stevens et al.
2017 ).
RNA-seq and ChIP-seq analyses of stage NF20 embryos
have uncovered how foregut and hindgut progenitor cells
transcriptomes are regulated by BMP/pSmad1 and Wnt/βCatenin at a genomic level (Stevens et al. 2017). These studies identif ed hundreds of enhancers co-occupied by Smad1
and β-Catenin and revealed the unexpected observation that
their chromatin occupancy was associated with both transcriptional activation and repression (Stevens et al. 2017).
Moreover, Wnt-inhibited enhancers often lacking canonical TCF DNA-binding motifs, suggesting a novel mode of
repression, which remains to be resolved.
RA signaling regulates both D-V and A-P pattern, with
RA production being spatially controlled by restricted expression of the RA-synthesizing enzyme Aldh1a2 (Raldh2) in
anterior lateral plate mesoderm and RA-degrading Cyp26
enzymes in pharyngeal and hindgut territories (Figure
18.4A,B). This is thought to generate a gradient of RA
activity highest in dorsal-posterior foregut and lower in
the pharynx and hindgut. Genomic studies have uncovered RA-target genes and identifed a number of feedback
mechanisms between BMP, Wnt, RA, and FGF. RA promotes expression of Wnt-antagonists such as ndrg1a and
sfrps ( Zhang et al. 2013b; Damianitsch et al. 2009) and high
levels of ventral BMP restrict aldh1a2 so that it is more
robustly expressed dorsally (Stevens et al. 2017). FGFs from
the posterior mesoderm co-operate with Wnt to promote
the caudal expression of both cdx2 and cyp26, which limits
RA activity in the hindgut (Shiotsugu et al. 2004; Deimling
and Drysdale 2011). RA in turn restricts expression of fgf8
and fgf4 to the pharynx and hindgut (Shiotsugu et al. 2004;
Arima et al. 2005).
The result of this combinatorial BMP, Wnt, RA, and FGF
signaling is the restricted expression of lineage-promoting
TFs, a number of which are shown in Figure 18.4B. Mutations
in many of these TFs are associated with human congenital
syndromes affecting the digestive or respiratory systems:
for example, tbx1 is expressed in the pharyngeal endoderm
and is lost in 22q11.2 Deletion syndrome (DiGeorge syndrome, OMIM# 188400); rfx6 marks posterior foregut and
mutations cause Mitchell-Riley syndrome (OMIM # 615710;
Smith et al. 2010); mnx1 is expressed in dorsal endoderm
lining the archenteron roof, and mutations are associated
with Currarino syndrome (OMIM #176450; Han et al. 2020);
Digestive and Respiratory System
blastula and gastrula stages (Paraiso et al. 2019; Gentsch
et al. 2019; Afouda et al. 2020). Additional ChIP-seq studies
suggest unique combinations of HD TFs can either promote
or repress transcription: for example, Oxt2/Lim1 complexes
stimulate transcription of dorsal-anterior endoderm genes
like hhex, whereas Otx2/Goosecoid complexes repress ventral-posterior genes such as ventx1/2/3 and wnt8 ( Yasuoka
et al. 2014; Yasuoka et al. 2019).
During gastrulation, there is a dramatic switch in the region
of active Wnt signaling in the embryo, which can be visualized with transgenic Wnt reporter embryos Tg(WRE:dGFP),
wherein canonical Tcf/Lef sites drive destabilized GFP
expression (Figure 18.3D; Tran et al. 2010). Maternal Wnt
signaling is high in the dorsal mesendoderm and low in the
ventral side of the blastula and early gastrula, but as gastrulation proceeds, the pattern is switched as zygotic wnt8
becomes expressed in ventral posterior mesendoderm and
Wnt-antagonists are expressed in the organizer.
In the ventral-posterior GRN, zygotic Wnt8/β-Catenin
and Bmp4/Smad1 act in a feedforward loop (Hoppler and
Moon 1998; Fuentealba et al. 2007; Kjolby et al. 2019)
and directly activate expression of the Ventx family of
HD transcriptional repressors (Ventx1/2/3) (Figure 18.3C;
Onichtchouk et al. 1996; Karaulanov et al. 2004; Hikasa
et al. 2010). Ventx factors repress transcription of dorsalanterior genes, including goosecoid and hhex ( Sander et al.
2007; Rankin et al. 2011); thus, a mutually antagonistic
cross-repressive loop exists between the ventral-posterior
and dorsal-anterior GRNs in the early gastrula embryo
( Figure 18.3E ).
18.4.2. NEURULA-STAGE ENDODERM PATTERNING
During gastrulation, the hhex-expressing anterior endoderm migrates to the future ventral foregut adjacent to the
heart as the blastopore closes and the archenteron opens.
Immediately behind the migrating anterior endoderm, a thin
layer of dorsal endoderm forms the roof of the archenteron,
whereas the ventx-expressing endodermal mass makes up
the bulk of the ventral gut tissue. During the neurula and
early somitogenesis stages (NF12–NF25), the endoderm
continues to be patterned along its D-V and A-P axes by
combinatorial BMP, Wnt/β-Catenin, RA, and FGF signaling
(Figure 18.4A), and distinct domains of regional endoderm
TF expression are obvious by NF20 (Figure 18.4B). This
post-gastrula patterning in Xenopus is comparable to events
that occur during E7-E9 in mouse gestation and 3–8 days of
many human PSC differentiation protocols.
During this post-gastrula period, bmp2/4/7 are expressed
in the ventral splanchnic mesoderm, while BMP antagonists noggin and chordin are produced dorsally from the
notochord, generating a D-V gradient of phosphorylated
Smad1/5/9 (pSMAD1) activity in the embryo (Figure
18.4A,B). High BMP promotes ventral gene expression in
both the foregut and hindgut, whereas low BMP permits a
dorsal endoderm identity (Stevens et al. 2017). BMP from
the ventral mesoderm induces expression of a number of
secreted Wnt antagonists in the foregut including sfrp1/2/5
(Kenny et al. 2012; Stevens et al. 2017), which are required
to maintain a low level of Wnt11-Fzd7 signaling essential for
proliferation, morphogenesis, and identity of hhex+ progenitors (McLin et al. 2007; Li et al. 2008; Zhang et al. 2013a).
In the posterior domain, BMP and Wnt ligands secreted
from the mesoderm cooperate to maintain ventx+ hindgut
fate and induce the expression of cdx2, a HD TF essential
for intestinal development in all vertebrates (Stevens et al.
2017 ).
RNA-seq and ChIP-seq analyses of stage NF20 embryos
have uncovered how foregut and hindgut progenitor cells
transcriptomes are regulated by BMP/pSmad1 and Wnt/βCatenin at a genomic level (Stevens et al. 2017). These studies identif ed hundreds of enhancers co-occupied by Smad1
and β-Catenin and revealed the unexpected observation that
their chromatin occupancy was associated with both transcriptional activation and repression (Stevens et al. 2017).
Moreover, Wnt-inhibited enhancers often lacking canonical TCF DNA-binding motifs, suggesting a novel mode of
repression, which remains to be resolved.
RA signaling regulates both D-V and A-P pattern, with
RA production being spatially controlled by restricted expression of the RA-synthesizing enzyme Aldh1a2 (Raldh2) in
anterior lateral plate mesoderm and RA-degrading Cyp26
enzymes in pharyngeal and hindgut territories (Figure
18.4A,B). This is thought to generate a gradient of RA
activity highest in dorsal-posterior foregut and lower in
the pharynx and hindgut. Genomic studies have uncovered RA-target genes and identifed a number of feedback
mechanisms between BMP, Wnt, RA, and FGF. RA promotes expression of Wnt-antagonists such as ndrg1a and
sfrps ( Zhang et al. 2013b; Damianitsch et al. 2009) and high
levels of ventral BMP restrict aldh1a2 so that it is more
robustly expressed dorsally (Stevens et al. 2017). FGFs from
the posterior mesoderm co-operate with Wnt to promote
the caudal expression of both cdx2 and cyp26, which limits
RA activity in the hindgut (Shiotsugu et al. 2004; Deimling
and Drysdale 2011). RA in turn restricts expression of fgf8
and fgf4 to the pharynx and hindgut (Shiotsugu et al. 2004;
Arima et al. 2005).
The result of this combinatorial BMP, Wnt, RA, and FGF
signaling is the restricted expression of lineage-promoting
TFs, a number of which are shown in Figure 18.4B. Mutations
in many of these TFs are associated with human congenital
syndromes affecting the digestive or respiratory systems:
for example, tbx1 is expressed in the pharyngeal endoderm
and is lost in 22q11.2 Deletion syndrome (DiGeorge syndrome, OMIM# 188400); rfx6 marks posterior foregut and
mutations cause Mitchell-Riley syndrome (OMIM # 615710;
Smith et al. 2010); mnx1 is expressed in dorsal endoderm
lining the archenteron roof, and mutations are associated
with Currarino syndrome (OMIM #176450; Han et al. 2020);