268
Xenopus
section, we discuss how combinatorial mesenchymal signals, some of which are dependent on paracrine cross-talk
with the endoderm itself, induce organ lineages.
18.5.1. PHARYNGEAL ENDODERM AND THYROID
The pharyngeal endoderm contributes to the thymus, thyroid, and parathyroid. While the molecular markers of the
thymus and parathyroid are conserved between mammals
and Xenopus (Lee et al. 2013; Manley and Condie 2010),
the molecular mechanism of their development has not been
extensively investigated in Xenopus. We therefore focus on
conserved mechanisms of pharyngeal endoderm formation
and thyroid induction.
Pharyngeal endoderm cells express TFs such as Foxe1,
Tbx1, and Sox2 (Figure 18.5B), and FGF signaling during
NF20–NF34 promotes their expression (Rankin et al. 2012;
Shifey et al. 2012; Kurmann et al. 2015). BMP signaling
has temporally dynamic effects on pharyngeal endoderm
development. During neurula stages, high levels of BMP in
the foregut inhibit pharyngeal identity and directly repress
tbx1 (Stevens et al. 2017); however, subsequent BMP signaling, from NF20–NF34, is required for pharyngeal fate
and thyroid gene expression (Kurmann et al. 2015). Thyroid
progenitors, which co-express the TF genes nkx2–1, foxe1,
hhex, and pax2 (pax2 in Xenopus is analogous to pax8 in
mammals), are induced in response to combinatorial FGF
and BMP signals in a region of low RA activity (Wang et al.
2011; Kurmann et al. 2015). RA is an evolutionarily ancient
patterning signal that defnes the posterior boundary of the
pharynx in both invertebrates and vertebrates (Kelly and
Drysdale 2015). Studies in Xenopus have shown that in addition to suppressing foxe1+ pharyngeal fate (Rankin et al.
2018), RA acts as regulatory switch between thyroid and
lung: exogenous RA is suffcient to drive ectopic, lung-specifc surfactant gene expression in the thyroid/pharyngeal
domain, simultaneously suppressing thyroid and pharyngeal
markers (Wang et al. 2011; Rankin et al. 2018).
18.5.2. LUNG, TRACHEA, AND ESOPHAGUS
Reciprocal mesoderm-endoderm paracrine signaling
between the foregut lateral plate mesoderm and underlying
foregut endoderm is necessary for induction of Nkx2–1+
respiratory progenitors. Studies in Xenopus helped def ne
a conserved GRN regulating pulmonary induction that
involves RA, Hedgehog (HH), Wnt, and BMP signals, as
well as the TF Tbx5 ( Figure18.6A–F ). Mesoderm-produced
RA is required for HH ligand expression in the endoderm,
and HH ligands then signal back to the mesoderm via Gli
TFs, which act together with Tbx5 to promote expression of
wnt2/2b and bmp2/4/7 ligands in the ventral foregut mesoderm (Rankin et al. 2016; Steimle et al. 2018). Canonical
Wnt/β-Catenin signaling is necessary and suff cient for
nkx2–1 induction in the ventral foregut epithelium (Steimle
et al. 2018; Rankin et al. 2016). Ventral BMP signaling
restricts expression of the key esophageal TF Sox2 to the
dorsal foregut epithelium (Domyan et al. 2011; Rankin et al.
2015). In addition to its repressive effect on Sox2, BMP also
plays an additional, yet mechanistically unknown, function
in nkx2–1 induction or early maintenance, as the experimental removal of Sox2 cannot substitute for the role of BMP
during either Xenopus respiratory induction or during the
directed differentiation of human PSCs into respiratory progenitors in vitro (Trisno et al. 2018).
After D-V patterning, between NF37–NF44, the foregut
separates into a distinct ventral trachea and dorsal esophagus (Rankin et al. 2015; Nasr et al. 2019). In humans, disruptions in this process result in life-threatening congenital
defects such as esophageal atresia (a partial absence of
the esophagus), tracheoesophageal fstulas, or laryngotracheoesophageal clefts, which occur in about 1 out of
every 3500 live births (Brosens et al. 2014). Comparative
studies in Xenopus and mouse have identifed the conserved
cellular mechanism that control tracheal-esophageal (T-E)
morphogenesis (Nasr et al. 2019). These include the formation of a transient epithelial septum at the Sox2/Nkx2–1 dorsal/ventral boundary as well as the resolution of this septum,
a process involving endosome-mediated epithelial remodeling and localized extracellular matrix degradation (Figure
18.6G). Additional studies in Xenopus and mouse also found
a conserved requirement of the homeodomain TF Islet1 in
T-E separation (Kim et al. 2019), wherein Islet1 is thought
to help maintain nkx2–1 expression in boundary/midline
epithelial cells. Interestingly, in humans, chromosomal deletions at 5q11.2, which encompass the ISLET1 gene (and others), are found in patients with tracheal agenesis (de Jong
et al. 2010).
Experiments in Xenopus and mouse indicate that T-E
morphogenesis is regulated by HH, although the precise
HH/Gli-target genes are unclear. Indeed CRISPR-mediated
mutation of gli3 in Xenopus to mimic heterozygous truncating mutations observed in human GLI3 in Pallister-Hall
syndrome (OMIM #146510) resulted in defective epithelial
remodeling and laryngo-tracheoesophageal clefts, similar to
human Pallister-Hall syndrome patients (Nasr et al. 2019).
The genetic etiology of trachea-esophageal birth defects
is poorly understood, and although genome sequencing
of patients identifes many de novo variants, determining
which mutations are causative remains a major challenge;
groups such as the CLEARconsrtium.org are using Xenopus
CRISPR screens to validate candidate genes and place them
into developmental pathways involved in T-E development.
18.5.3. PANCREAS AND LIVER
Xenopus pancreas development has been extensively
reviewed, and important insights into evolutionarily conserved mechanisms governing pancreas induction have been
made in Xenopus (Kelly and Melton 2000; Pearl et al. 2009;
Kofent and Spagnoli 2016), including the initial cloning of
the master pancreatic TF gene pdx1 (Wright et al. 1989)
(Figure 18.5B). The use of Xenopus to study congenital pancreatic disorders and diseases such as diabetes, MODY, and
Xenopus
section, we discuss how combinatorial mesenchymal signals, some of which are dependent on paracrine cross-talk
with the endoderm itself, induce organ lineages.
18.5.1. PHARYNGEAL ENDODERM AND THYROID
The pharyngeal endoderm contributes to the thymus, thyroid, and parathyroid. While the molecular markers of the
thymus and parathyroid are conserved between mammals
and Xenopus (Lee et al. 2013; Manley and Condie 2010),
the molecular mechanism of their development has not been
extensively investigated in Xenopus. We therefore focus on
conserved mechanisms of pharyngeal endoderm formation
and thyroid induction.
Pharyngeal endoderm cells express TFs such as Foxe1,
Tbx1, and Sox2 (Figure 18.5B), and FGF signaling during
NF20–NF34 promotes their expression (Rankin et al. 2012;
Shifey et al. 2012; Kurmann et al. 2015). BMP signaling
has temporally dynamic effects on pharyngeal endoderm
development. During neurula stages, high levels of BMP in
the foregut inhibit pharyngeal identity and directly repress
tbx1 (Stevens et al. 2017); however, subsequent BMP signaling, from NF20–NF34, is required for pharyngeal fate
and thyroid gene expression (Kurmann et al. 2015). Thyroid
progenitors, which co-express the TF genes nkx2–1, foxe1,
hhex, and pax2 (pax2 in Xenopus is analogous to pax8 in
mammals), are induced in response to combinatorial FGF
and BMP signals in a region of low RA activity (Wang et al.
2011; Kurmann et al. 2015). RA is an evolutionarily ancient
patterning signal that defnes the posterior boundary of the
pharynx in both invertebrates and vertebrates (Kelly and
Drysdale 2015). Studies in Xenopus have shown that in addition to suppressing foxe1+ pharyngeal fate (Rankin et al.
2018), RA acts as regulatory switch between thyroid and
lung: exogenous RA is suffcient to drive ectopic, lung-specifc surfactant gene expression in the thyroid/pharyngeal
domain, simultaneously suppressing thyroid and pharyngeal
markers (Wang et al. 2011; Rankin et al. 2018).
18.5.2. LUNG, TRACHEA, AND ESOPHAGUS
Reciprocal mesoderm-endoderm paracrine signaling
between the foregut lateral plate mesoderm and underlying
foregut endoderm is necessary for induction of Nkx2–1+
respiratory progenitors. Studies in Xenopus helped def ne
a conserved GRN regulating pulmonary induction that
involves RA, Hedgehog (HH), Wnt, and BMP signals, as
well as the TF Tbx5 ( Figure18.6A–F ). Mesoderm-produced
RA is required for HH ligand expression in the endoderm,
and HH ligands then signal back to the mesoderm via Gli
TFs, which act together with Tbx5 to promote expression of
wnt2/2b and bmp2/4/7 ligands in the ventral foregut mesoderm (Rankin et al. 2016; Steimle et al. 2018). Canonical
Wnt/β-Catenin signaling is necessary and suff cient for
nkx2–1 induction in the ventral foregut epithelium (Steimle
et al. 2018; Rankin et al. 2016). Ventral BMP signaling
restricts expression of the key esophageal TF Sox2 to the
dorsal foregut epithelium (Domyan et al. 2011; Rankin et al.
2015). In addition to its repressive effect on Sox2, BMP also
plays an additional, yet mechanistically unknown, function
in nkx2–1 induction or early maintenance, as the experimental removal of Sox2 cannot substitute for the role of BMP
during either Xenopus respiratory induction or during the
directed differentiation of human PSCs into respiratory progenitors in vitro (Trisno et al. 2018).
After D-V patterning, between NF37–NF44, the foregut
separates into a distinct ventral trachea and dorsal esophagus (Rankin et al. 2015; Nasr et al. 2019). In humans, disruptions in this process result in life-threatening congenital
defects such as esophageal atresia (a partial absence of
the esophagus), tracheoesophageal fstulas, or laryngotracheoesophageal clefts, which occur in about 1 out of
every 3500 live births (Brosens et al. 2014). Comparative
studies in Xenopus and mouse have identifed the conserved
cellular mechanism that control tracheal-esophageal (T-E)
morphogenesis (Nasr et al. 2019). These include the formation of a transient epithelial septum at the Sox2/Nkx2–1 dorsal/ventral boundary as well as the resolution of this septum,
a process involving endosome-mediated epithelial remodeling and localized extracellular matrix degradation (Figure
18.6G). Additional studies in Xenopus and mouse also found
a conserved requirement of the homeodomain TF Islet1 in
T-E separation (Kim et al. 2019), wherein Islet1 is thought
to help maintain nkx2–1 expression in boundary/midline
epithelial cells. Interestingly, in humans, chromosomal deletions at 5q11.2, which encompass the ISLET1 gene (and others), are found in patients with tracheal agenesis (de Jong
et al. 2010).
Experiments in Xenopus and mouse indicate that T-E
morphogenesis is regulated by HH, although the precise
HH/Gli-target genes are unclear. Indeed CRISPR-mediated
mutation of gli3 in Xenopus to mimic heterozygous truncating mutations observed in human GLI3 in Pallister-Hall
syndrome (OMIM #146510) resulted in defective epithelial
remodeling and laryngo-tracheoesophageal clefts, similar to
human Pallister-Hall syndrome patients (Nasr et al. 2019).
The genetic etiology of trachea-esophageal birth defects
is poorly understood, and although genome sequencing
of patients identifes many de novo variants, determining
which mutations are causative remains a major challenge;
groups such as the CLEARconsrtium.org are using Xenopus
CRISPR screens to validate candidate genes and place them
into developmental pathways involved in T-E development.
18.5.3. PANCREAS AND LIVER
Xenopus pancreas development has been extensively
reviewed, and important insights into evolutionarily conserved mechanisms governing pancreas induction have been
made in Xenopus (Kelly and Melton 2000; Pearl et al. 2009;
Kofent and Spagnoli 2016), including the initial cloning of
the master pancreatic TF gene pdx1 (Wright et al. 1989)
(Figure 18.5B). The use of Xenopus to study congenital pancreatic disorders and diseases such as diabetes, MODY, and