263
Digestive and Respiratory System
Charney et al. 2017a, 2017b; Gentsch et al. 2019), where they
appear to act as pioneering factors to decondense the chromatin prior to genome activation and inductive signaling.
The vegetally localized TFs cooperate with a maternal Wnt pathway active on the dorsal side of the embryo
(Tao et al. 2005; Cha et al. 2008a). In dorsal vegetal cells,
β-Catenin translocates to the nucleus, interacts with Tcf/Lef
family TFs, and directly regulates expression of genes such
as siamois ( Figure 18.2A , D ). β-Catenin also contributes to
epigenetic priming of enhancers, recruiting the arginine
methyltransferase Prmt2 to modify histones and establish
a poised chromatin architecture (Blythe et al. 2010). The
key result of this pre-pattern by maternal Wnt and maternal
TFs is the initiation of zygotic nodal1–6 gene expression at
the mid-blastula transition in vegetal cells fated to become
endoderm (Xanthos et al. 2001, 2002; Agius et al. 2000; Rex
et al. 2002).
18.3.2. ENDODERM INDUCTION PHASE
During the “induction phase” (NF8–NF10), high levels of
Nodal signaling promote endoderm fate, while the overlying equatorial cells experience lower Nodal concentrations
and are induced to adopt a tbxt-expressing mesoderm fate
( Figure 18.2B , E ) ( Cha et al. 2004 , 2008b ; Gentsch et al.
2013; Charney et al. 2017b). Nodal-induced Smad2/Foxh1
complexes co-bind enhancers along with maternal TFs and
βCatenin/TCF complexes to cooperatively stimulate the
expression of an evolutionarily conserved group of zygotic
endodermal TFs including Sox17, Gata4–6, Foxa1–4, and
Mix/Bix family members, which collectively promote endoderm fate (Figure 18.2E) ( Zorn and Wells 2009; Charney
et al. 2017b). Recent genomic data suggest Smad2/Foxh1
act on super enhancers bound by maternal VegT/Oxt1/Sox7,
thus linking pre-patterning to the onset of zygotic gene
expression (Paraiso et al. 2019; Gentsch et al. 2019).
18.3.3. ENDODERM COMMITMENT PHASE
During the “commitment” phase (NF10–NF12), the zygotic
endoderm TFs function collectively with Nodal and Wnt
signaling in a series of feed-forward loops to promote each
other’s expression and establish endoderm identity (Figure
18.2C; Sinner et al. 2006; Charney et al. 2017b). Interactions
between the mutually repressive endoderm GRN and a TbxtFGF mesodermal GRN refne the boundary between the
endoderm and mesoderm cells (Figure 18.2G). Genomic analysis recently demonstrated that Sox17 promotes endoderm
commitment via multiple mechanisms: (1) Sox17 activates
the expression of many key endodermal genes such as hnf1b;
(2) Sox17 and β-Catenin co-bind about 30% of all endoderm
enhancers to synergistically activate endoderm genes, illustrating a cooperation with the Wnt pathway; (3) Sox17 directly
binds and suppresses mesoderm/ectoderm genes such as
tfap2a; and (4) Sox17 acts as a negative feedback regulator to
restrain nodal ligand expression ( Figure 18.2F , G ; Mukherjee
et al. 2020). These studies provide molecular insights into
the pioneering studies of Chris Wylie, Janet Heasman, Jim
Smith, and colleagues in the 1980s that demonstrated vegetal
cells become committed to endoderm fate during gastrulation
(Heasman et al. 1984; Wylie et al. 1987). A spatial-temporal
catalog of TFs expressed in the gastrula endoderm has been
generated (Blitz et al. 2017), and many TFs remain to be
incorporated into the endoderm GRN. Additional details on
the GRNs of germ layer formation can be found in Cho and
Blitz (Chapter 12 of this book).
18.4. ENDODERM PATTERNING
In the following section, we discuss the molecular control of
endoderm patterning during gastrulation (Figure 18.3) and
during neurula/somitogenesis stages (Figure 18.4), which
defne the foregut, midgut, and hindgut progenitor domains.
18.4.1. GASTRULA-STAGE ENDODERM PATTERNING
Gastrula endoderm pattern is intimately linked to its initial
formation. In the 1990s and early 2000s, pioneering work
by Eddy De Robertis, Richard Harland, Christof Niehrs,
and colleagues defned the molecular nature of the vertebrate organizer wherein secreted Wnt/BMP-antagonists,
expressed in the dorsal-anterior organizer mesendoderm,
regulate pattern of all three germ layers during gastrulation;
this work in Xenopus identifed principles that turned out
to be conserved in all vertebrates (De Robertis and Kuroda
2004; see also Chapter 4 of this book).
In Figure 18.3, we highlight molecular players driving
distinct dorsal-anterior and ventral-posterior GRNs, which
result in anterior and posterior endoderm domains identif -
able by expression of the TFs hhex and ventx1/2/3, respectively ( Zorn et al. 1999; Rankin et al. 2011). Interestingly,
these gastrula endoderm domains have distinct organforming competence due to BMP and Wnt-dependent early
patterning (Rankin et al. 2018).
In the dorsal-anterior GRN, maternal dorsal Wnt signaling co-operates with maternal vegetal TFs to drive
high levels of zygotic Nodal signaling and induce a number of organizer-specifc homedomain (HD) TFs, including
Siamois (Figure 18.2D), Twin/Sia2, Otx2, Lim1/Lhx1, and
Goosecoid (Figure 18.3E; Zorn et al. 1999; Rankin et al.
2011). These HD TFs in turn activate expression of BMP/
Wnt-antagonists, including Chordin, Noggin, Cerberus,
Dkk1, and Sfrps (secreted frizzled related proteins, Sfrp1,
Sfrp2, and Frzb/Sfrp3) (Figure 18.3C,E); the HD TFs also
promote transcription of hhex. We have performed extensive
cis-regulatory analyses of the hhex locus, characterizing
Wnt, Nodal, and HD TF responsive promoter and enhancer
elements as well as Ventx-mediated repressive elements
(Figure 18.3E; Rankin et al. 2011). Recent genome-wide
analyses have expanded these fndings and def ned superenhancers that are thought to act as transcriptional hubs
that integrate combinations of the HD TFs as well as Foxh1/
Smad2 and Tcf/β-Catenin complexes to control transcription of regionally expressed endoderm genes throughout
Digestive and Respiratory System
Charney et al. 2017a, 2017b; Gentsch et al. 2019), where they
appear to act as pioneering factors to decondense the chromatin prior to genome activation and inductive signaling.
The vegetally localized TFs cooperate with a maternal Wnt pathway active on the dorsal side of the embryo
(Tao et al. 2005; Cha et al. 2008a). In dorsal vegetal cells,
β-Catenin translocates to the nucleus, interacts with Tcf/Lef
family TFs, and directly regulates expression of genes such
as siamois ( Figure 18.2A , D ). β-Catenin also contributes to
epigenetic priming of enhancers, recruiting the arginine
methyltransferase Prmt2 to modify histones and establish
a poised chromatin architecture (Blythe et al. 2010). The
key result of this pre-pattern by maternal Wnt and maternal
TFs is the initiation of zygotic nodal1–6 gene expression at
the mid-blastula transition in vegetal cells fated to become
endoderm (Xanthos et al. 2001, 2002; Agius et al. 2000; Rex
et al. 2002).
18.3.2. ENDODERM INDUCTION PHASE
During the “induction phase” (NF8–NF10), high levels of
Nodal signaling promote endoderm fate, while the overlying equatorial cells experience lower Nodal concentrations
and are induced to adopt a tbxt-expressing mesoderm fate
( Figure 18.2B , E ) ( Cha et al. 2004 , 2008b ; Gentsch et al.
2013; Charney et al. 2017b). Nodal-induced Smad2/Foxh1
complexes co-bind enhancers along with maternal TFs and
βCatenin/TCF complexes to cooperatively stimulate the
expression of an evolutionarily conserved group of zygotic
endodermal TFs including Sox17, Gata4–6, Foxa1–4, and
Mix/Bix family members, which collectively promote endoderm fate (Figure 18.2E) ( Zorn and Wells 2009; Charney
et al. 2017b). Recent genomic data suggest Smad2/Foxh1
act on super enhancers bound by maternal VegT/Oxt1/Sox7,
thus linking pre-patterning to the onset of zygotic gene
expression (Paraiso et al. 2019; Gentsch et al. 2019).
18.3.3. ENDODERM COMMITMENT PHASE
During the “commitment” phase (NF10–NF12), the zygotic
endoderm TFs function collectively with Nodal and Wnt
signaling in a series of feed-forward loops to promote each
other’s expression and establish endoderm identity (Figure
18.2C; Sinner et al. 2006; Charney et al. 2017b). Interactions
between the mutually repressive endoderm GRN and a TbxtFGF mesodermal GRN refne the boundary between the
endoderm and mesoderm cells (Figure 18.2G). Genomic analysis recently demonstrated that Sox17 promotes endoderm
commitment via multiple mechanisms: (1) Sox17 activates
the expression of many key endodermal genes such as hnf1b;
(2) Sox17 and β-Catenin co-bind about 30% of all endoderm
enhancers to synergistically activate endoderm genes, illustrating a cooperation with the Wnt pathway; (3) Sox17 directly
binds and suppresses mesoderm/ectoderm genes such as
tfap2a; and (4) Sox17 acts as a negative feedback regulator to
restrain nodal ligand expression ( Figure 18.2F , G ; Mukherjee
et al. 2020). These studies provide molecular insights into
the pioneering studies of Chris Wylie, Janet Heasman, Jim
Smith, and colleagues in the 1980s that demonstrated vegetal
cells become committed to endoderm fate during gastrulation
(Heasman et al. 1984; Wylie et al. 1987). A spatial-temporal
catalog of TFs expressed in the gastrula endoderm has been
generated (Blitz et al. 2017), and many TFs remain to be
incorporated into the endoderm GRN. Additional details on
the GRNs of germ layer formation can be found in Cho and
Blitz (Chapter 12 of this book).
18.4. ENDODERM PATTERNING
In the following section, we discuss the molecular control of
endoderm patterning during gastrulation (Figure 18.3) and
during neurula/somitogenesis stages (Figure 18.4), which
defne the foregut, midgut, and hindgut progenitor domains.
18.4.1. GASTRULA-STAGE ENDODERM PATTERNING
Gastrula endoderm pattern is intimately linked to its initial
formation. In the 1990s and early 2000s, pioneering work
by Eddy De Robertis, Richard Harland, Christof Niehrs,
and colleagues defned the molecular nature of the vertebrate organizer wherein secreted Wnt/BMP-antagonists,
expressed in the dorsal-anterior organizer mesendoderm,
regulate pattern of all three germ layers during gastrulation;
this work in Xenopus identifed principles that turned out
to be conserved in all vertebrates (De Robertis and Kuroda
2004; see also Chapter 4 of this book).
In Figure 18.3, we highlight molecular players driving
distinct dorsal-anterior and ventral-posterior GRNs, which
result in anterior and posterior endoderm domains identif -
able by expression of the TFs hhex and ventx1/2/3, respectively ( Zorn et al. 1999; Rankin et al. 2011). Interestingly,
these gastrula endoderm domains have distinct organforming competence due to BMP and Wnt-dependent early
patterning (Rankin et al. 2018).
In the dorsal-anterior GRN, maternal dorsal Wnt signaling co-operates with maternal vegetal TFs to drive
high levels of zygotic Nodal signaling and induce a number of organizer-specifc homedomain (HD) TFs, including
Siamois (Figure 18.2D), Twin/Sia2, Otx2, Lim1/Lhx1, and
Goosecoid (Figure 18.3E; Zorn et al. 1999; Rankin et al.
2011). These HD TFs in turn activate expression of BMP/
Wnt-antagonists, including Chordin, Noggin, Cerberus,
Dkk1, and Sfrps (secreted frizzled related proteins, Sfrp1,
Sfrp2, and Frzb/Sfrp3) (Figure 18.3C,E); the HD TFs also
promote transcription of hhex. We have performed extensive
cis-regulatory analyses of the hhex locus, characterizing
Wnt, Nodal, and HD TF responsive promoter and enhancer
elements as well as Ventx-mediated repressive elements
(Figure 18.3E; Rankin et al. 2011). Recent genome-wide
analyses have expanded these fndings and def ned superenhancers that are thought to act as transcriptional hubs
that integrate combinations of the HD TFs as well as Foxh1/
Smad2 and Tcf/β-Catenin complexes to control transcription of regionally expressed endoderm genes throughout