260
Xenopus
maturity onset diabetes of the young (MODY), trachealesophageal fstula/esophageal atresia, and intestinal malrotation (Pearl et al. 2011; Nasr et al. 2019; Grzymkowski et al.
2020; Edwards and Zorn 2021). It is estimated that about
80% of human disease-associated genes have Xenopus
orthologs. The online biomedical knowledgebase Xenbase.
org (Nenni et al. 2019) facilitates human disease modeling
by curating Xenopus genomic, expression, genotype, and
phenotype data from the published literature and integrating this with orthologous human gene, anatomy, and disease
with links to National Center for Biotechnology Information
(NCBI), Online Mendelian Iheritance in Man (OMIM), and
human disease ontology (DO) resources.
In this chapter, we summarize the current understanding of Xenopus endoderm organogenesis. We organize our
discussion in four major sections: (1) a temporal overview of
Xenopus endoderm organogenesis; (2) endoderm germ layer
specifcation, (3) progressive pattering of the endoderm gut
tube, and (4) organ induction. In each section, we highlight
conserved molecular mechanisms, pointing out both historical and recent contributions of Xenopus research. We also
emphasize how the use of emerging technologies in Xenopus
continues to inform our molecular understanding of human
congenital birth defects and disease.
18.2. TEMPORAL OVERVIEW OF XENOPUS
ENDODERM ORGANOGENESIS
The molecular mechanisms of endoderm formation, patterning, organ induction, and early morphogenesis are
largely conserved amongst vertebrate species ( Zorn and
Wells 2009) and are largely indistinguishable between the
two commonly used species Xenopus laevis and Xenopus
tropicalis. Figure 18.1 summarizes key phases in Xenopus
endoderm development, showing a developmental fate map
(Figure 18.1A), example gene expression (Figure 18.1B), and
a schematic of cell lineages (Figure 18.1C ); in addition, we
present the approximate timing of similar developmental
transitions in mouse embryos and during the directed differentiation of human PSCs (Figure 18.1D).
Endoderm organogenesis begins with the segregation
of pluripotent embryonic cells into ectoderm, mesoderm,
and endoderm germ layers during the blastula and gastrula
stages. Pioneering fate-mapping and transplantation studies in the 1980s demonstrated that the yolk-rich vegetal pole
cells of the early cleavage stage embryo give rise to endoderm (Figure 18.1A; Moody 1987; Dale and Slack 1987), and
by early gastrula stage, these vegetal cells are committed to
their endoderm fate (Heasman et al. 1984; Wylie et al. 1987).
A critical milestone in our understanding of endoderm germ
layer specif cation was the discovery that the TGFβ/Activin/
Nodal family of growth factors could induce mesoderm and
endoderm fate in pluripotent blastula ectoderm animal cap
explants in a concentration-dependent manner (Smith et al.
1990; Asashima et al. 1990). Based on these studies, the use
of Activin has since become the standard approach to differentiate mouse and human PSCs into endoderm (Kubo et
al. 2004; D’Amour et al. 2005; Loh et al. 2014). This, along
with the discovery of the transcription factor (TF) Sox17
in Xenopus (Hudson et al. 1997), the f rst unambiguous
molecular marker of vertebrate defnitive endoderm, opened
the door to studying the molecular mechanisms controlling
endoderm formation. As detailed in the subsequent section,
TGFβ/Nodal signaling activates the expression of sox17 and
other key TF encoding genes such as foxa1 in the endoderm
lineage as early as the blastula stage (approximately f ve
hours post-fertilization [hpf], Nieuwkoop and Faber [NF]
stage 8 [NF8]; Nieuwkoop and Faber 1994) (Figure 18.1A, B).
During blastula and neurula stages of development (NF9–
NF20), the endoderm is progressively patterned along the
anterior and posterior (A-P) and dorsal-ventral (D-V) axes
into progenitor domains characterized by restricted expression of different lineage promoting TFs. Initial endoderm
patterning is coincident with endoderm formation (NF9–11)
and is coordinated with global D-V axial patterning of all
three germ layers by Wnt and BMP signals. This initial
regionalization is further elaborated during neurula and
early somite stages (NF12–25) by posteriorizing WNT,
BMP, FGF, and retinoic acid (RA) signals from the surrounding splanchnic mesoderm, which further subdivide
the endoderm into foregut, midgut, and hindgut domains
that have different developmental potential (Rankin et al.
2018): foregut-derived organs include the pharynx, thyroid,
thymus, parathyroid, esophagus, trachea, lungs, liver, pancreas, and stomach; midgut endoderm gives rise to the small
intestine; and hindgut endoderm gives rise to the large intestine and cloaca (Figure 18.1C ) (Chalmers and Slack 2000).
Between NF15 and NF35 (approximately two days), the
broad progenitor domains are progressively restricted, and
organ lineages are induced by continued combinatorial signaling between the splanchnic mesoderm and endoderm
epithelium. This process involves many of the same mesoderm-derived WNT, BMP, RA, and FGF signals that control
early patterning, which act reiteratively in various combinations and with additional signals such as endodermal-derived
Hedgehog (Shifey et al. 2012; Rankin et al. 2016; Rankin
et al. 2018). The bidirectional nature of this signaling coordinates organ identity in the germ layers so that the correct
mesoderm develops in contact with the appropriate endoderm. Embryological experiments show that by NF35, organ
fates are mostly specifed and a number of lineage-specif c
markers are expressed (Horb and Slack 2001; Shifey et al.
2012; Rankin et al. 2018).
Between NF35-NF43 early endoderm organ morphogenesis occurs, which include the emergence of lung, liver,
and pancreatic organ buds that evaginate from the gut tube
and intermingle with the adjacent mesoderm. The anterior
foregut tube separates into a distinct trachea and esophagus,
while the intestine elongates and undergoes a stereotypical
coiling process similar to mammals (Rankin et al. 2015;
Chalmers and Slack 2000; Grzymkowski et al. 2020). By
NF43 (3.5 dpf) the entire gut tube can be readily microdissected, and distinct organ buds are obvious under the
stereomicroscope ( Figure 18.1A , B ). Xenopus tadpoles begin
Xenopus
maturity onset diabetes of the young (MODY), trachealesophageal fstula/esophageal atresia, and intestinal malrotation (Pearl et al. 2011; Nasr et al. 2019; Grzymkowski et al.
2020; Edwards and Zorn 2021). It is estimated that about
80% of human disease-associated genes have Xenopus
orthologs. The online biomedical knowledgebase Xenbase.
org (Nenni et al. 2019) facilitates human disease modeling
by curating Xenopus genomic, expression, genotype, and
phenotype data from the published literature and integrating this with orthologous human gene, anatomy, and disease
with links to National Center for Biotechnology Information
(NCBI), Online Mendelian Iheritance in Man (OMIM), and
human disease ontology (DO) resources.
In this chapter, we summarize the current understanding of Xenopus endoderm organogenesis. We organize our
discussion in four major sections: (1) a temporal overview of
Xenopus endoderm organogenesis; (2) endoderm germ layer
specifcation, (3) progressive pattering of the endoderm gut
tube, and (4) organ induction. In each section, we highlight
conserved molecular mechanisms, pointing out both historical and recent contributions of Xenopus research. We also
emphasize how the use of emerging technologies in Xenopus
continues to inform our molecular understanding of human
congenital birth defects and disease.
18.2. TEMPORAL OVERVIEW OF XENOPUS
ENDODERM ORGANOGENESIS
The molecular mechanisms of endoderm formation, patterning, organ induction, and early morphogenesis are
largely conserved amongst vertebrate species ( Zorn and
Wells 2009) and are largely indistinguishable between the
two commonly used species Xenopus laevis and Xenopus
tropicalis. Figure 18.1 summarizes key phases in Xenopus
endoderm development, showing a developmental fate map
(Figure 18.1A), example gene expression (Figure 18.1B), and
a schematic of cell lineages (Figure 18.1C ); in addition, we
present the approximate timing of similar developmental
transitions in mouse embryos and during the directed differentiation of human PSCs (Figure 18.1D).
Endoderm organogenesis begins with the segregation
of pluripotent embryonic cells into ectoderm, mesoderm,
and endoderm germ layers during the blastula and gastrula
stages. Pioneering fate-mapping and transplantation studies in the 1980s demonstrated that the yolk-rich vegetal pole
cells of the early cleavage stage embryo give rise to endoderm (Figure 18.1A; Moody 1987; Dale and Slack 1987), and
by early gastrula stage, these vegetal cells are committed to
their endoderm fate (Heasman et al. 1984; Wylie et al. 1987).
A critical milestone in our understanding of endoderm germ
layer specif cation was the discovery that the TGFβ/Activin/
Nodal family of growth factors could induce mesoderm and
endoderm fate in pluripotent blastula ectoderm animal cap
explants in a concentration-dependent manner (Smith et al.
1990; Asashima et al. 1990). Based on these studies, the use
of Activin has since become the standard approach to differentiate mouse and human PSCs into endoderm (Kubo et
al. 2004; D’Amour et al. 2005; Loh et al. 2014). This, along
with the discovery of the transcription factor (TF) Sox17
in Xenopus (Hudson et al. 1997), the f rst unambiguous
molecular marker of vertebrate defnitive endoderm, opened
the door to studying the molecular mechanisms controlling
endoderm formation. As detailed in the subsequent section,
TGFβ/Nodal signaling activates the expression of sox17 and
other key TF encoding genes such as foxa1 in the endoderm
lineage as early as the blastula stage (approximately f ve
hours post-fertilization [hpf], Nieuwkoop and Faber [NF]
stage 8 [NF8]; Nieuwkoop and Faber 1994) (Figure 18.1A, B).
During blastula and neurula stages of development (NF9–
NF20), the endoderm is progressively patterned along the
anterior and posterior (A-P) and dorsal-ventral (D-V) axes
into progenitor domains characterized by restricted expression of different lineage promoting TFs. Initial endoderm
patterning is coincident with endoderm formation (NF9–11)
and is coordinated with global D-V axial patterning of all
three germ layers by Wnt and BMP signals. This initial
regionalization is further elaborated during neurula and
early somite stages (NF12–25) by posteriorizing WNT,
BMP, FGF, and retinoic acid (RA) signals from the surrounding splanchnic mesoderm, which further subdivide
the endoderm into foregut, midgut, and hindgut domains
that have different developmental potential (Rankin et al.
2018): foregut-derived organs include the pharynx, thyroid,
thymus, parathyroid, esophagus, trachea, lungs, liver, pancreas, and stomach; midgut endoderm gives rise to the small
intestine; and hindgut endoderm gives rise to the large intestine and cloaca (Figure 18.1C ) (Chalmers and Slack 2000).
Between NF15 and NF35 (approximately two days), the
broad progenitor domains are progressively restricted, and
organ lineages are induced by continued combinatorial signaling between the splanchnic mesoderm and endoderm
epithelium. This process involves many of the same mesoderm-derived WNT, BMP, RA, and FGF signals that control
early patterning, which act reiteratively in various combinations and with additional signals such as endodermal-derived
Hedgehog (Shifey et al. 2012; Rankin et al. 2016; Rankin
et al. 2018). The bidirectional nature of this signaling coordinates organ identity in the germ layers so that the correct
mesoderm develops in contact with the appropriate endoderm. Embryological experiments show that by NF35, organ
fates are mostly specifed and a number of lineage-specif c
markers are expressed (Horb and Slack 2001; Shifey et al.
2012; Rankin et al. 2018).
Between NF35-NF43 early endoderm organ morphogenesis occurs, which include the emergence of lung, liver,
and pancreatic organ buds that evaginate from the gut tube
and intermingle with the adjacent mesoderm. The anterior
foregut tube separates into a distinct trachea and esophagus,
while the intestine elongates and undergoes a stereotypical
coiling process similar to mammals (Rankin et al. 2015;
Chalmers and Slack 2000; Grzymkowski et al. 2020). By
NF43 (3.5 dpf) the entire gut tube can be readily microdissected, and distinct organ buds are obvious under the
stereomicroscope ( Figure 18.1A , B ). Xenopus tadpoles begin