Download Endoderm development in vertebrates: fate mapping

Develop. Growth Differ. (2005) 47, 343– 355
Review
Blackwell Publishing, Ltd.
Endoderm development in vertebrates: fate mapping,
induction and regional specification
Kimiko Fukuda1 and Yutaka Kikuchi2,*
1
Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minamiohsawa, Hachioji, Tokyo 192-0397,
Japan and 2Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya 464-8602, Japan
The formation of the vertebrate body plan begins with the differentiation of cells into three germ layers:
ectoderm, mesoderm and endoderm. Cells in the endoderm give rise to the epithelial lining of the digestive
tract, associated glands and respiratory system. One of the fundamental problems in developmental biology
is to elucidate how these three primary germ layers are established from the homologous population of cells
in the early blastomere. To address this question, ectoderm and mesoderm development have been
extensively analyzed, but study of endoderm development has only begun relatively recently. In this review,
we focus on the ‘where’, ‘when’ and ‘how’ of endoderm development in four vertebrate model organisms: the
zebrafish, Xenopus, chick and mouse. We discuss the classical fate mapping of the endoderm and the more
recent progress in characterizing its induction, segregation and regional specification.
Key words: endoderm, fate mapping, induction, regional specification.
Introduction
Gastrulating vertebrate embryos generate the three
germ layers, known as ectoderm, mesoderm and
endoderm, during early development. The induction
and differentiation of endodermal cells and the formation of organs derived from the gut tube have been
poorly analyzed in comparison to ectoderm or mesoderm. However, in the past few years, zebrafish
mutants and knockout mice which have disrupted
endoderm formation, and the molecular functions of
Xenopus genes involved in endoderm formation,
have been extensively analyzed. As a result of these
studies, our understanding of endoderm development
is now fairly advanced in early vertebrate embryos.
In particular, these genetic and molecular biological
approaches have led to a more detailed understanding of the molecular regulation of the endoderm.
In spite of these efforts, however, many mechanisms, such as the fate choice of endodermal cells,
the migration of endodermal cells during gastrulation
and the regional specification of the endoderm along
the anterior–posterior axis, which are essential to fully
understanding its differentiation, are not well understood. Here, we attempt to summarize both classical
and more recent findings in the study of endoderm
development including fate mapping, the molecular
pathways involved in its generation leading to the
endoderm, its segregation from the ectoderm and
mesoderm and its regional specification.
Fate maps and the timing of endoderm
commitment
To analyze ‘how’ the endoderm differentiates, it is
essential to know ‘where’ the endoderm is derived
and ‘when’ differentiation begins. There have been a
number of reports describing fate maps of the early
vertebrate embryo that show the origin of the endoderm in various species and in various ways. In addition, by using mainly implantation techniques, many
of these studies have revealed the timing of endoderm fate.
Xenopus
*Author to whom all correspondence should be addressed.
Email: [email protected]
Received 15 June 2005; accepted 16 June 2005.
Many fate maps have been compiled for amphibian
embryos over a number of years. Vogt (1929) generated
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K. Fukuda and Y. Kikuchi
a famous fate map of the uropod embryo using vital
dyes at the early gastrulation stage. Subsequently, a
fate map of the Xenopus embryo at the gastrula
stage using a vital dye was described by Keller
(1975; 1976). More recently, many researchers have
constructed amphibian fate maps using fluorescent
dyes (Dale & Slack 1987; Chalmers & Slack 2000).
In the amphibian embryo, there is quite a high
degree of topographic projection from the very early
stages. However, there is also an element of indeterminacy, as endoderm fate is segregated from both
the mesodermal and ectodermal fate at a relatively
early stage compared to other vertebrates. At the
32-cell stage, the vegetal-most blastomeres (Fig. 1D1–
4 Xenopus) have been shown to contribute to the
endoderm (Nakamura & Kishiyama 1971; Nakamura
et al. 1978; Dale & Slack 1987) and significant endodermal contributions have also been observed from
the dorsal marginal blastomeres (Fig. 1C1–2 Xenopus)
(Nakamura et al. 1978; Dale & Slack 1987). Moreover,
vegetal blastomeres contribute specifically to endoderm, whereas dorsal marginal blastomeres also
contribute to dorsal mesoderm, including the notochord and somites. These observations indicate that
whilst there is a general tendency for vegetal blastomeres to differentiate into endoderm, the mesodermal and endoderm fates do overlap partially and fate
determination is not complete at the 32-cell stage.
Another study of the vegetal pole stage 6 cells (the
morula) of Xenopus laevis has shown that they contribute progeny to all three germ layers (Heasman
et al. 1984). However, by the midblastula stage
(stage 8), the cells are smaller and more confined to
the vegetal pole and are observed to contribute
exclusively to the endoderm. At early gastrula
(stage 10), the prospective lining of the archenteron
(the prospective endoderm), the prospective neural
area and the prospective epidermal area are represented on the surface, whereas the prospective mesoderm is in the deep layer of the marginal zone
(Fig. 1 Xenopus) (Nieuwkoop & Florshtz 1950; Keller
1975; Keller 1976). A thin superficial layer around
the vegetal pole, extending relatively further upward
from the blastopore pigment line and covering the
notochordal, somatic, and lateral plate mesoderm,
also contributes to the endoderm (Fig. 1 Xenopus).
These superficial cells invaginate as a continuous
layer to form the lining of the archenteron during gastrulation (Nieuwkoop & Florshtz 1950; Keller 1975).
At the onset of gastrulation, prospective pharyngeal
endoderm leads to the involution of the mesoderm
through the blastopore (Keller 1976). However, cells
in the deep layer of the vegetal pole don’t contribute
to the archenteron roof or floor at the gastrula or neurula but will become intestinal endoderm in tadpoles
after the elongation of the gut (Chalmers & Slack
2000). These findings reveal that in Xenopus, endoderm fate is segregated prior to gastrulation.
After fate mapping was established, some reports
investigated whether the restriction of cell fates at
midblastula reflected cell determination by implanting labeled vegetal pole cells into the blastocoels of
the host embryos and analyzing the tissues, including the labeled progeny. When a single vegetal pole
cell from the morula (stage 6) and midblastula was
isolated and transplanted into the blastocoel of the
late blastula, their progeny were found in all three
germ layers (Heasman et al. 1984). When vegetal
pole cells from early gastrula were assayed in this
way, however, their progeny contributed only to gut
endoderm (Heasman et al. 1984). These results suggest that vegetal pole cells became committed by
the beginning of gastrulation and further analysis
of their progenies in more detail showed that this
Fig. 1. Comparison of endoderm
fate maps of vertebrates. Presumptive endoderm cells develop from
common progenitor cells of both
the mesoderm and endoderm in
zebrafish, chicken and mouse
embryos. In Xenopus embryos,
some endodermal cells arise from
marginal cells which give rise to
both endoderm and mesoderm.
Endoderm cells are located very
close to the node or blastopore
(*) at the onset of gastrulation and
invaginate into the deep layer.
Endoderm development in vertebrates
commitment was gradual (Wylie et al. 1987). In addition, single vegetal cells cultured with other vegetal
cells became more committed than single cells alone
or single cells cultured with other cell types (Wylie
et al. 1987). It seems therefore that endoderm
commitment is a cell autonomous process, but that
cell–cell interaction among vegetal pole cells also
contributes to this pathway.
Zebrafish
Zebrafish fate map studies began in the 1990s and
these fate maps showed that both endoderm and
mesoderm also originate from a common progenitor,
but more extensively than in Xenopus. Both germ
layers derive from cells near the blastoderm margin
(Kimmel et al. 1990) and both involute into the
forming hypoblast (Warga & Kimmel 1990). In 40%
epyboly, just prior to gastrulation, endoderm progenitor cells are located in a narrow field of cells
along the margin (Fig. 1 zebrafish) (Kimmel et al.
1990; Warga & Nüsslein-Volhard 1999). Furthermore,
there is asymmetric distribution of the endoderm progenitor in the margin, and more endoderm progenitor
is found dorsally than ventrally (Warga & NüssleinVolhard 1999). In addition, most endoderm progenitors
are located within a 2-cell diameter of the blastoderm
margin and no progenitors are found more than a 4cell diameter from this margin (Warga & NüssleinVolhard 1999). At the onset of gastrulation, the majority of the endoderm progenitor cells are the earliest
deep involuting cells from the blastoderm margin in
the newly formed hypoblast (Warga & NüssleinVolhard 1999). Hence, there are mere endoderm
progenitors at the marginal zone of the shield stage
(gastrulation stage) embryo (Melby et al. 1996; Warga
& Nüsslein-Volhard 1999).
The fate commitment of the endoderm also occurs
just after the onset of gastrulation (Ho & Kimmel 1993;
David & Rosa 2001). When marginal cells from late
blastulae (30 –40% epiboly) are transplanted into
animal blastomeres, they contribute mostly to neuroectodermal tissue, consistent with the fate maps of
animal blastomeres, and only a small proportion of
transplanted cells contribute to the endoderm (David
& Rosa 2001). This contribution to the endoderm dramatically increases, however, when marginal cells
from embryos are transplanted at the onset of the
gastrulation (50% epiboly) (David & Rosa 2001).
Chick
Among the higher vertebrates, fate mapping has
been most extensively characterized in the chick
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embryo (Bellairs 1953; Bellairs 1957; Rosenquist 1966;
Rosenquist 1971; Fontaine & Le Douarin 1977; Kirby
et al. 2003; Lawson & Schoenwolf 2003). In amniotes
such as chick, all of the embryonic tissues arise from
the epiblast in the early embryo. HNK-1 antibodypositive cells, which are common progenitors of both
endoderm and mesoderm (Stern & Canning 1990),
are randomly distributed within the epiblast before
streak formation occurs (stage XII–XIII) (Canning &
Stern 1988). At stage XII−2 (early primitive streak
stage), these stained cells are distributed in the more
posterior and medial regions. Finally, the primitive
streak contains more HNK-positive cells than more
remote regions. The chick embryo fate maps have
revealed that endoderm progenitors are distributed
at the posterior region and form a winged shape,
hinged at the posterior end of the midline at stage X
(early blastula). This region also gives rise to mesoderm, especially notochord (Hatada & Stern 1994).
This prospective endoderm region moves towards
the midline and progresses anteriorly during the
subsequent stages (Selleck & Stern 1991; Hatada &
Stern 1994). After the formation of the primitive
streak, endoderm progenitors are located in the
deep layer of the anterior primitive streak and in the
Hensen’s node (Fig. 1, chick) (Selleck & Stern 1991;
Garcia-Martinez & Schoenwolf 1993). These endoderm progenitors are also mesodermal progenitors
and disappear from the node immediately following
the onset of gastrulation (stage 4) (Selleck & Stern
1991). During gastrulation, endoderm cells invaginate into the lower layer (ventral-most layer) either
directly or indirectly after lateral migration in the middle layer (Kimura et al. unpubl. data). When endoderm progenitors in the anterior primitive streak are
transplanted into the posterior streak, which contains
only mesodermal progenitors, almost all of the grafted
cells contribute to the lateral plate mesoderm. After
the onset of gastrulation (stage 4), when cells in the
middle layer lateral to the Hensen’s node are grafted
into the middle layer lateral to the posterior primitive
streak, where cells normally contribute to the mesoderm, almost all of the transplanted cells contribute
to endoderm. These results show that the fate determination of endoderm cells occurs during gastrulation (Kimura et al. unpubl. data).
Mouse
In the mouse embryo, the distribution of endoderm
progenitor cells is essentially the same as in the
chick embryo. In the epiblast, which is the sole
source of all embryonic tissue, presumptive endoderm becomes available in the posterior region of
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K. Fukuda and Y. Kikuchi
the prestreak embryo. This region, including the
region of presumptive head processes, partially overlaps with the presumptive mesodermal region. After
streak formation, the presumptive endoderm area
extends anteriorly and this anterior area continues to
overlap with the presumptive head process/notochord (Fig. 1, mouse) (Lawson et al. 1986; Lawson
& Pedersen 1987).
In summary, vertebrate endoderm cells arise from
common progenitor cells of both endoderm and mesoderm, at which time fate segregation occurs, following endoderm fate determination. At gastrulation, at
least a portion of the endoderm progenitor cells involute from the surface to a deep layer in front of the
mesodermal cells.
Molecular pathways leading to endoderm
induction
Zebrafish
Our understanding of the molecular mechanisms
underlying endoderm induction in the zebrafish originates from the genetic and molecular biological studies
of five mutants: one-eyed-pinhead (oep), bonnie and
clyde (bon), faust (fau), casanova (cas) and spielohen-grenzen (spg) (Zhang et al. 1998b; Kikuchi
et al. 2000; Dickmeis et al. 2001; Kikuchi et al. 2001;
Reiter et al. 2001; Sakaguchi et al. 2001; Lunde et al.
2004; Reim et al. 2004). oep encodes a member of
the EGF-CFC family of proteins and its protein product acts as cofactor of Nodal (a TGF-β super family
protein). The maternal-zygotic oep (MZoep) mutant,
which lacks both maternal and zygotic Oep, fails to
form any endoderm cells and most mesoderm cells
(Zhang et al. 1998b; Gritsman et al. 1999). In the
zebrafish blastula embryo, two Nodal genes, cyclops
(cyc) and squint (sqt), are expressed throughout the
entire marginal domain and the double mutant (cyc;
sqt ) of these genes is very similar to the MZoep
mutant, in that it is entirely deficient of endoderm
cells (Feldman et al. 1998). These results implicate
Nodal as an essential signaling molecule for endoderm induction in the zebrafish embryo.
bon and fau encode a Mix-type homeodomain transcription factor and the transcription factor Gata5,
respectively. bon is expressed in both the mesendoderm and mesoderm domains where the panmesoderm marker no tail (ntl; homologue of Xenopus
brachyury) is also expressed. In contrast, fau/gata5
expression is restricted to the mesendoderm domain
along the animal–vegetal axis in late blastula and
early gastrula embryos (Alexander et al. 1999). Both
bon and fau/gata5 expression is completely lost in
MZoep and cyc; sqt double mutants, demonstrating
that their expression is regulated by Nodal signaling
(Alexander & Stainier 1999; Rodaway et al. 1999;
Reiter et al. 2001). bon and fau/gata5 mutants contain approximately 10% and approximately 60% of
the normal number of endoderm cells, respectively,
as assessed by the early endoderm marker genes
sox17, a high-mobility-group (HMG) transcription
factor gene (Alexander & Stainier 1999) and foxA2,
a winged helix/forkhead transcription factor gene
(formerly known as axial (Strähle et al. 1993)) (Kikuchi
et al. 2000; Reiter et al. 2001). These genetic findings
indicate that both factors are crucial downstream
effectors of Nodal signaling during endoderm induction in the zebrafish.
Although the endoderm phenotype in bon or fau/
gata5 disrupted embryos is severe, endoderm cells
still remain in bon or fau/gata5 single and double
mutants (Kikuchi et al. 2000; Reiter et al. 2001). The
partial endoderm induction phenotype in these mutants
is likely to be a reflection of the redundant activity
of other genes such as Mezzo, which is another Mixtype transcription factor whose expression domain
and stage profile overlaps with Bon. In addition, both
gain-of-function and loss-of-function experiments
using antisense morpholino oligonucleotide (MO) further suggest that Mezzo is a redundant factor of Bon
(Poulain & Lepage 2002). In fact, based on overexpression experiments and gene expression analyses
using mutant embryos, Bon, Fau/Gata5 and Mezzo
are in almost parallel position with each other (Reiter
et al. 2001; Poulain & Lepage 2002)(Fig. 2).
The cas mutant completely lacks all endodermal
cells and organs derived from gut tube. However,
mesoderm induction appears to be normal in cas
mutants (Alexander et al. 1999). cas encodes a
Sox-type transcription factor, and its expression is
restricted to endodermal progenitors in the marginal
region in the late blastula embryo (Dickmeis et al.
2001; Kikuchi et al. 2001; Sakaguchi et al. 2001). The
expression of cas around the marginal domain is
absent in MZoep and cyc; sqt mutants and the
number of cas-expressing cells is reduced in bon and
fau/gata5 mutants, suggesting that cas is a downstream target of Nodal, Bon and Fau/Gata5 (Kikuchi
et al. 2001). Cas can also transform a mesodermal
fate to an endodermal fate when it is overexpressed,
whereas the fate of ectodermal cells in the animal
pole region cannot be changed to an endodermal
fate by this overexpression (Kikuchi et al. 2001).
These data indicate that cas is an essential gene,
but not master gene, for endoderm induction and
additional factors are necessary for converting ectodermal into endodermal fates.
Endoderm development in vertebrates
347
Fig. 2. Molecular pathway leading
to endoderm in the zebrafish and
Xenopus. In the zebrafish, all hierarchical cascades are based on
genetic studies. In contrast, most
of the regulatory pathways in
Xenopus have been elucidated
using animal cap assays and knockdown experiments. (1) Maternal
factor (X) upstream of Nodal
(Cyc, Sqt) is unknown. (2) squint
is a maternal transcript. (3) Tar;
Taram-a, type-I TGFβ receptor
(Renucci et al. 1996) (4) Xnrs;
Xnr1, Xnr2, Xnr4, Xnr5 and Xnr6
(5) Mix-type homeobox proteins;
Mixer, Mix.1, Mix.2, Bix.1, Bix.2
(Milk), Bix.3 and Bix.4.
Recently, the spg mutant, that was originally identified as a brain mutant, was also shown to have
defects in endoderm induction (Lunde et al. 2004;
Reim et al. 2004). spg, encoding the transcription
factor Pou2/Oct4, is a maternal gene and zygotic
spg is expressed ubiquitously prior to gastrulation
(Belting et al. 2001; Burgess et al. 2002). To elucidate the function of Spg in endoderm induction,
maternal-zygotic spg mutants (MZspg) were generated by injection of spg mRNA into homozygous
eggs. In MZspg, expression of the early endoderm
markers, sox17 and foxA2, is never initiated and
although induction of cas mRNA is detected at the
blastula stage, cas expression is not maintained during the gastrula stage (Lunde et al. 2004; Reim et al.
2004). Thus, spg is necessary for the induction of
sox17 and foxA2, and for the maintenance of cas
expression. Additionally, sox17 promoter–luciferase
reporter assays have shown that Cas and Spg synergistically regulate sox17 expression to regulate
endoderm induction (Reim et al. 2004).
In summary, Figure 2 illustrates the molecular regulatory cascade leading to endoderm induction in
the zebrafish. Whereas Gata4, Gata5 and Gata6 are
thought to be involved in heart and endoderm development in various model organisms (Charron &
Nemer 1999; Molkentin 2000), zebrafish Gata4 and
Gata6 function in endoderm induction is not well
understood. The maternal factor (X in Fig. 2)
upstream of Nodal has not yet been identified and
although foxA1 and foxA3 are known to be
expressed in endodermal cells and act downstream
of Cas, the position of these factors in the regulatory
cascade remains unclear. Analyses of five zebrafish
mutants have revealed many aspects of the molecular cascade downstream of Nodal signaling. It will
be interesting to investigate whether signaling other
than Nodal regulates endoderm induction or if other
signaling pathways cross-talk with Nodal signaling.
Xenopus
The Xenopus endoderm originates from the vegetal
region, where the endodermal cells are intermingled
with yolk cells, in blastula stage embryos. In the Xenopus
embryo, the maternal transcript VegT, encoding a
T-box transcription factor, is localized in the vegetal
hemisphere of the egg and early embryo, and the
zygotic VegT transcript is restricted to the equatorial
region, where mesoderm cells are formed (Lustig
et al. 1996; Stennard et al. 1996; Zhang & King 1996;
Horb & Thomsen 1997). Endoderm is never induced,
however, and mesoderm is mainly formed from the
vegetal region, in VegT-depleted embryos (Zhang
et al. 1998a). Moreover, VegT overexpression can ectopically induce the expression of some early endoderm
maker genes, indicating that maternal VegT is necessary for endoderm formation and mesoderm patterning in the Xenopus embryo (Zhang et al. 1998a).
A recent study using animal cap assays has reported
that another maternal factor, Sox7, is also a crucial
regulator of endoderm induction and that the ability
of VegT to induce endoderm genes, except for sox17
and Xenopus Nodal-related genes (Xnrs; Xnr1, Xnr2
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K. Fukuda and Y. Kikuchi
and Xnr4), appeared to depend upon Sox7 activity
(Zhang et al. 2005).
Five Nodal-related genes (Xnr1, Xnr2, Xnr4, Xnr5
and Xnr6) are reported to be involved in mesendoderm induction in Xenopus (reviewed by Schier
2003). The Xnr5 and Xnr6 in the vegetal hemisphere
are induced by VegT in a cell autonomous manner
and are inducers of Xnr1, Xnr2 and Xnr4 (Takahashi
et al. 2000). The cleavage mutant, Xnr2, suppresses
some endoderm marker expression (Osada & Wright
1999). Additionally, the Nodal-related genes Xnr1,
Xnr2 and Xnr4 and the Veg1-like TGF-β family member derrière can restore endoderm gene expression
in VegT-depleted embryos (Xanthos et al. 2001). Furthermore, the ability of Sox7 to induce endodermal
genes is inhibited by Cerberus, which is an antagonist
of Nodal (Zhang et al. 2005). These data demonstrate that Nodal-related genes function downstream
of Sox7 and VegT and that the Xnrs play important
roles in endoderm induction. However, the function
of individual Xnrs in endoderm induction remains to
be elucidated.
The seven Mix-type homeodomain factors (Mixer,
Mix.1, Mix.2, Bix1, Bix2/Milk, Bix3 and Bix4) and
three Gata factors (Gata4, Gata5 and Gata6) function
downstream of VegT and Nodal-related factors (Xanthos et al. 2001) and are thought to be involved in
endoderm induction (Weber et al. 2000; Afouda et al.
2005). The Mix-type genes are expressed in the
vegetal region and some are also expressed in the
marginal domain which gives rise to mesoderm cells.
When overexpressed in animal caps, these Mix-type
genes exhibit different abilities to activate endoderm
gene expression: Mix.1 can activate endoderm gene
expression only when co-expressed with the homeobox gene siamois (Lemaire et al. 1998); Bix1 overexpression at low and high levels induces mesoderm
and endoderm gene expression, respectively (Tada
et al. 1998); Bix2/Milk appears to promote endoderm
gene expression at the expense of mesoderm gene
expression (Ecochard et al. 1998) and Bix4 can
rescue endoderm, but not mesoderm, gene expression in VegT-depleted embryos (Casey et al. 1999).
In addition, Mixer is a strong endoderm inducer
when overexpressed (Henry & Melton 1998) and MOmediated knockdown data shows that Mixer plays
an essential role in controlling the amount of mesoderm induction by the vegetal cells (Kofron et al.
2004).
The three zygotic Gata genes (gata4, gata5 and
gata6) initiate their expression in vegetal cells fated
to form endoderm at the onset of gastrulation. The
functional analyses of three these genes demonstrate
that Gata5 and Gata6 function as activators of endo-
derm genes such as sox17 and HNF1β, whereas the
function of Gata4 to induce endoderm genes is
dependent upon gata6 induction (Afouda et al. 2005).
Additionally, Gata5 enhances the ability of Mixer to
induce sox17α expression and acts upstream of
Xhex and gata4 in cooperation with Mixer (Xanthos
et al. 2001).
In summary, Figure 2 illustrates the molecular
regulatory cascade leading to endoderm formation
in Xenopus embryos. The hierarchical relationship,
however, between individual factors in this cascade
such as the Mix-type homeobox proteins, the Xnrs
and the Gata proteins is still not yet fully clear due
to their functional redundancies.
Mouse
Our understanding of endoderm formation in vertebrates originates mainly from studies of the zebrafish
and Xenopus. Although there are several differences
between the molecular regulatory cascades in these
species, as shown in Figure 2, it seems that the
factors involved in endoderm induction and its associated regulatory pathways are conserved between
these two vertebrates (Fig. 2). In addition to these
species, recent studies have shown that analyses of
knockout mice have also contributed to our understanding of endoderm development. In mouse, the
hypomorphic allele of Nodal is associated with a
complete lack of a definitive endoderm, indicating
that Nodal is also an essential factor in the murine
embryo, as has been observed in zebrafish and
Xenopus (Lowe et al. 2001). Additionally, the Mixtype homeodomain protein, Mixl1, and the Sox-type
HMG domain protein, Sox17, are also necessary for
definitive endoderm formation in the mouse embryo,
as is the case for the zebrafish and Xenopus (Hart
et al. 2002; Kanai-Azuma et al. 2002).
In contrast to these factors, it is not yet clear whether the Gata family of transcription factors (Gata4,
Gata5 and Gata6) function in endoderm induction in
the mouse (Molkentin 2000). The knockout mice for
each individual Gata factor do not show any defects
in endoderm induction, presumably because of the
functional redundancy among family members. However, these Gata genes are expressed in extraembryonic tissues, heart and definitive endoderm and
transfection of Gata genes into non-endodermal cells
can induce specific endoderm gene expression such
as IFABP, gastric H+/K+-ATPase and HNF4 (Maeda
et al. 1996; Gao et al. 1998; Morrisey et al. 1998).
These data suggest that Gata transcription factors
are involved in endoderm differentiation in mouse. In
addition, although the molecular regulatory pathways
Endoderm development in vertebrates
leading to endoderm induction in the mouse have
not yet been analyzed, it is interesting to note
that Nodal, Mix-type, Sox and Gata factors regulate
endoderm development in three vertebrates: the
zebrafish, Xenopus and mouse.
Fate choice between endoderm and
mesoderm/ectoderm
In the zebrafish embryo, endodermal and mesodermal cells are intermingled along the margin of the
blastoderm and in the Xenopus embryo, endodermal
and mesodermal cells are generated from the C-tier
domain. Furthermore, as observed for zebrafish and
Xenopus embryos, mesodermal and definitive
endodermal cells arise from the epiblast and migrate
through the primitive streak in the mouse. It is
unclear, however, how individual mesendoderm cells
decide their own fate in vertebrates. On the other
hand, specification between endodermal and secondary mesenchymal cells (SMC), which give rise to
the majority of the mesodermal cells in the sea urchin
embryo, is regulated by Notch signaling (Sherwood
& McClay 1999). Notch signaling functions within the
presumptive SMC and plays a crucial role in the
differential specification of SMC and endoderm in
the sea urchin embryo. In the zebrafish, endoderm
progenitors arise in the marginal domain in a scattered, not clustered, arrangement, as assessed by
cas expression (Kikuchi et al. 2004). Based on the
endoderm induction pattern, it seems that Notch signaling may regulate the fate choice of endoderm and
mesoderm in the zebrafish. notch1a, deltaC and deltaD are expressed in the marginal domain, which
gives rise to cells of either the endoderm or mesoderm (Kikuchi et al. 2004). Moreover, the number of
endoderm cells is reduced by the activation of Notch
signaling, but is not increased by inhibition of Notch
signaling (Kikuchi et al. 2004). These data suggest
that an additional signaling mechanism other than
Notch is necessary for the segregation of endoderm
and mesoderm in the zebrafish, and that the segregation mechanisms in vertebrates are more complicated than in the sea urchin.
Recent studies have also shown that maternal B1type Sox transcription factors, which are expressed
in animal pole region, regulate germ layer formation
in both Xenopus and zebrafish. In Xenopus embryos
injected with antibodies against Sox3, the expression
levels of endodermal and mesodermal markers are
increased and the normal animal–vegetal patterning
of mesoderm and endoderm is disrupted (Zhang
et al. 2004). Maternal Sox3 can regulate Xnr5 expression and the reduction of Xnr5 generates an abnor-
349
mal gene expression profile and disrupted patterning
of the mesendoderm (Zhang et al. 2004) (Fig. 2).
These data demonstrate that the maternal B1-type
Sox proteins are involved in the fate choices of ectoderm and mesendoderm via the regulation of Nodalrelated gene expression.
Regionalization
After the endoderm layer is established during
gastrulation, it gradually becomes regionalized into
anteroposteriorly and dorsolaterally divided organs.
Compared with the study of early endoderm differentiation and late organogenesis, however, the molecular pathways that control this process remained
largely unknown.
Movement of the endoderm
In Xenopus, the ventral and margin superficial layer
of the blastula, which is presumptive endoderm,
becomes invaginated to form the lining of the archenteron. This invagination begins first, and is at its
strongest level, in a restricted area just dorsal to the
blastopore which becomes an anterior-most lining
(Fig. 3a,b). The suprablastopore region then becomes
the archenteron roof, whereas the sub-blastopore
region contributes to the archenteron floor (Fig. 3a–c)
(Keller 1975). These archenteron cells lining both roof
and floor eventually intermingle and contribute to the
dorsal gut wall after cell arrangement (Chalmers &
Slack 2000). On the other hand, cells in the vegetal
pole and the deep layer of the vegetal hemisphere,
contribute to the ventral cell layer of the gut wall
(Chalmers & Slack 2000).
In amniotes, the endoderm first forms a sheet
structure which is situated in the most ventral layer
of the embryo (Fig. 3d), then folds from the anterior
and generates the foregut at the early somite stages
(Fig. 3f,g). Later, the endodermal sheet also folds
from the posterior to make the hindgut. These two
folding events then join together to complete the formation of the gut tube.
In the chick, each region of the endoderm along
the antroposterior or dorsoventral axis appears as
the most ventral layer at different times. At stage 2,
the first population of the presumptive endoderm
appears as a very limited region in the ventral layer
under the rostral primitive streak (Rosenquist 1966;
Rosenquist 1971; Rosenquist 1972). These cells contribute only to the mid/hindgut and until gastrulation,
the presumptive mid/hindgut region expands laterally
and caudally (Kimura et al. unpubl. data). The presumptive dorsal foregut region invaginates into the
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K. Fukuda and Y. Kikuchi
Fig. 3. Cell movement of the endoderm in Xenopus and chicken embryo. (a–c) Xenopus embryo. (a) lateral view of stage 10+.
(b) ventral view of stage 10+ and c; medial section of stage 18. (d–g) Ventral view of the chicken embryo. (d) stage 4, (e) stage 5,
(f) stage 7, and (g) stage 11.
ventral layer just prior to gastrulation and expands
only laterally (Fig. 3d). Meanwhile, the presumptive
foregut region emerges from the epiblast into the
ventral layer through the middle layer (Kimura et al.
unpubl. data) (Fig. 3e). The shape of the ventral
foregut region at stage 5 is arch-like and the outside
part of the arch contributes to hepatogenetic cells
(Rosenquist 1971; Fukuda-Taira 1981; Tremblay &
Zaret 2005), whereas the inside part contributes to
the trachea and thyroid (Rosenquist 1971). Additionally, reports using mouse embryos have shown that
the fate map of the murine endoderm is very similar
to that of the chick (Lawson et al. 1986; Lawson &
Pedersen 1987; Tremblay & Zaret 2005).
Regional specification
Transplantation experiments have shown that in
Xenopus, endoderm cells have no regionality until
gastrulation when endoderm fate commitment occurs
(Heasman et al. 1984). Additionally, in the chick the
presumptive foregut endoderm can differentiate into
hindgut endoderm when transplanted into the posterior region of the embryo (Kimura et al. unpubl.
data). On the other hand, at the somite stage, presumptive small intestine endoderm differentiates into
intestinal epithelium even when cultured with stomach mesenchyme (Yasugi et al. 1991; Hiramatsu &
Yasugi 2004). Endoderm fragments from each region
of somite stage embryos can be cultured alone and
can partially differentiate according to their fate map
(Sumiya 1976a; Sumiya 1976b; Matsushita 1999).
These results demonstrate that regional commitment
within the endoderm occurs during the somite stage.
In fact, there have been several genes identified that
are expressed in a regional-specific manner during
the somite stages. The earliest of these regional specific markers to be characterized are the paired-type
homeobox-containing proteins, Pax1 and 9. They
begin to be expressed in the presumptive lateral
foregut endoderm just before somitogenesis in the
chick (Muller et al. 1996) and mouse (Deutsch et al.
1988; Timmons et al. 1994; Neubuser et al. 1995).
Pax-9-deficient mice lack derivatives of the third and
Endoderm development in vertebrates
fourth pharyngeal pouches (Peters et al. 1998). Furthermore, the finding that heterotopical transplanted
endoderm fragments from the presumptive lateral
foregut endoderm express Pax1 and 9 shows that
the expression of these genes in the pharyngeal
pouch endoderm is intrinsic (Muller et al. 1996).
In the zebrafish, mutants which are deficient the
tbx1 gene show defects in the pharyngeal pouches
and associated structures (Piotrowski et al. 1996;
Piotrowski & Nusslein-Volhard 2000; Piotrowski et al.
2003). In addition, tbx1 is also important for development of the pharyngeal arch in Xenopus (Ataliotis
et al. 2005). Taken together with reports that the haploinsufficiency of Tbx1 may be a major determinant
of cardiac and craniofacial birth defects associated
with DiGeorge syndrome in humans (Lindsay et al.
2001; Yamagishi et al. 2003), it is possible that Tbx1
is important to pharyngeal pouch endoderm differentiation. In the mouse embryo, however, tbx1 is
expressed only in the mesoderm in the early stages
and expands its expression in the endoderm at a
later stage (Chapman et al. 1996; Garg et al. 2001;
Yamagishi et al. 2003).
The vertebrate Caudal homologue, CdxA and the
HMG domain containing the Sox2 gene begin to be
expressed in caudal and rostral endoderm at the 8–
10 somite stages (Duprey et al. 1988; Frumkin et al.
1993; Suh et al. 1994; Ishii et al. 1997; Freund et al. 1998;
Ishii et al. 1998; Wood & Episkopou 1999; Beck et al.
2003). These genes continue to be expressed in the
endoderm and this expression never overlaps. At a
later stage, Sox2 is expressed in the endoderm of the
esophagus and stomach, whereas CdxA is detected
in the endoderm of the small and large intestine.
A CdxA-deficient mouse shows malformation of the
intestinal epithelium (Suh et al. 1994; Tamai et al.
1999), indicating that this factor is important for intestinal development. The functions of these genes during the somite stages and the pathways that regulate
their expression are still unknown however. At the
somite stage, there are additional regional marker
genes expressed, the paraHox transcription factor gene,
Pdx1 and the homeobox gene, Hex1. These genes
become active in the presumptive pancreas (Slack
1995; Offield et al. 1996; Kim & Melton 1998; Wang
et al. 2001) and liver regions (Yatskievych et al. 1999;
Bogue et al. 2000; Zorn & Mason 2001), respectively
at the somite stage. The Pdx1-deficient mouse shows
significant defects in pancreas development (Slack
1995; Offield et al. 1996) and the Hex1-deficient
mouse shows hepatocyte defects (Keng et al. 2000;
Martinez Barbera et al. 2000).
In summary, the endoderm is regionalized with
some potency by each organ in at least the somite
351
stages and each region expresses different transcription factors which may be important to further
development. In Drosophila, the regionalization of the
endoderm is affected by the mesoderm (Bienz 1994;
Bienz 1997). In mouse, the mesoderm is adjacent to
the endoderm, which is very important during development. Moreover, when endoderm fragments from
gastrulation embryos are cultured with various adjacent tissues, the region specific genes that are
expressed are dependent on the adjacent tissues
(Wells & Melton 2000). In addition, FGF4, which is
expressed in the adjacent mesoderm, can induce
the differentiation of endoderm in a concentrationdependent manner. In the zebrafish, mutants which
affect bmp signaling show no influence upon the
induction of endoderm precursors but produce
abnormal gut phenotypes (Tiso et al. 2002). The swirl
(bmp2b) mutant shows expansion of the pharyngeal
region and reduction of the pancreas and posterior
gut region, whereas the chordino (chordin) mutant
shows a reduction in the pharyngeal region and expansion of the pancreatic and posterior gut regions (Tiso
et al. 2002). When the activity of the enzyme responsible for early embryonic retinoic acid synthesis,
retinaldehyde dehydrogenase 2 (RALDH2/ALDH1a2)
is inhibited in mouse embryos, the pharyngeal
endoderm develops rudimentary and pouch-derived
organs which fail to establish. Raldh2 expression is
restricted to the posterior-most pharyngeal mesoderm and RA target genes (Hoxa1, Hoxb1) are
downregulated in both the pharyngeal endoderm
and mesoderm of these mutant embryos. Thus, RA
is one of the diffusible mesodermal signals that patterns the pharyngeal endoderm (Niederreither et al.
2003). In summary, endoderm regionalization is controlled by soluble factors which are provided by the
adjacent germ layers.
Conclusion
We describe the recent advances made in our
understanding of endoderm specification and differentiation during early vertebrate development. The
genetic, biological and molecular approaches using
vertebrate model organisms such as the zebrafish,
Xenopus, chick and mouse have greatly contributed
to the elucidation of endoderm formation. However,
many questions still remain and a great number of
experiments will be necessary in the future to address these. We believe that the elucidation of the
molecular mechanisms underlying endoderm development improves not only our knowledge of basic
developmental biology, but also our understanding
of the regeneration of endodermal organs such as
352
K. Fukuda and Y. Kikuchi
the lung, liver, pancreas and intestine. In particular,
we contend that the elucidation of the pathways that
control the segregation of the three germ layers and
regulate the regional specification of endoderm will
be useful in future studies that seek to understand
the differentiation of embryonic stem cells to various
types of endodermal organs.
Acknowledgements
Y. K. is supported by the Ministry of Education, Culture, Sports, Science and Technology and the
Mitsubishi Foundation and K. F. is supported by the
Ministry of Education, Culture, Sports, Science and
Technology.
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