DEVELOPMENTAL DYNAMICS 226:118 –127, 2003
BRIEF COMMUNICATION
Molecular Components of the Endoderm
Specification Pathway in Xenopus tropicalis
Anjali D’Souza,1 Monica Lee,1 Nicola Taverner,2 Julia Mason,2 Samantha Carruthers,2 James C. Smith,2
Enrique Amaya,2 Nancy Papalopulu,2 and Aaron M. Zorn1,2*
Xenopus laevis has been instrumental in elucidating a conserved molecular pathway that regulates vertebrate
endoderm specification. However, loss-of-function analysis is required to resolve the precise function of the genes
involved. For such analysis, antisense oligos and possibly forward genetics are likely to be more effective in the
diploid species Xenopus tropicalis than in the pseudotetraploid Xenopus laevis. Here we have isolated most of the
tropicalis genes in the endoderm specification pathway, specifically, tVegT, tMixer, tMix, tBix, tGata6, tSox17␣,
tSox17␤, tFoxA1, tHex, and tCerberus, which lack the redundant copies that are found in laevis. In situ hybridization
analysis has revealed identical expression patterns between the orthologous tropicalis and laevis endoderm genes,
thus suggesting conserved genetic functions. Furthermore, we noted that the smaller tropicalis embryos gave better
probe penetration than in laevis whole-mount in situ hybridizations—allowing us to visualize transcripts in the deep
endoderm in tropicalis, which is difficult in laevis. This study illustrates how an entire genetic pathway can be quickly
transferred from laevis to tropicalis due to high sequence conservation between the sister species and the large
number of tropicalis-expressed sequence tags that are now available. Developmental Dynamics 226:118 –127, 2003.
© 2002 Wiley-Liss, Inc.
Key words: Xenopus tropicalis; endoderm; development; VegT; Mixer; Mix; Bix; Sox17; Gata; FoxA1; Hex; Cerberus
Received 1 August 2002; Accepted 23 August 2002
INTRODUCTION
In the vertebrate blastula, a small
group of several hundred endoderm
cells gives rise to the epithelial lining
of the respiratory and gastrointestinal tract as well as to the liver,
lungs, pancreas, thyroid, and thymus (Wells and Melton, 1999). Recently, work in Xenopus laevis has
elucidated the framework of a conserved molecular pathway that initiates vertebrate endoderm development (reviewed in Stainier, 2002).
In the current model, the maternal
T-box transcription factor VegT,
which is localized to the vegetal re-
1
gion of the Xenopus embryo (Zhang
and King, 1996; Stennard et al., 1996;
Lustig et al., 1996; Horb and Thomsen, 1997), initiates endoderm development. VegT activity results in the
transcription of zygotic endodermal
genes (Zhang et al., 1998; Clements
et al., 1999; Xanthos et al., 2001),
which include the following: nodalrelated proteins (Xnr1,2,4,5,6) and
derriere, which are members of the
transforming growth factor-beta
(TGF␤) growth factor family (Jones
et al., 1995; Joseph and Melton,
1997; Sun et al., 1999; Takahashi et
al., 2000); homeodomain proteins of
the Mixer/Mix/Bix family (Rosa, 1989;
Vize, 1996; Henry and Melton, 1998;
Tada et al., 1998; Casey et al., 1999);
the zinc finger factors Gata 4, 5, and
6 (Jiang and Evans, 1996; Weber et
al., 2000; Xanthos et al., 2001); and
two closely related HMG domain
transcription factors, Sox17␣ and
Sox17␤ (Husdon et al., 1997).
VegT appears to function primarily
by means of TGF␤ signaling (Kofron
et al., 1999; Xanthos et al., 2001),
which is required for activating
and/or maintaining the expression
of the Mix-like genes, Gata4/5/6,
and Sox17␣/␤ (Henry et al., 1996;
Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology, Cincinnati, Ohio
Wellcome Trust/Cancer Research UK Institute, Cambridge, United Kingdom
Grant sponsor: NIH; Grant number: HD42572.
*Correspondence to: Aaron M. Zorn, Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet
Avenue Room: TCHRF 2564, Cincinnati, OH, 45229-3039. E-mail: aaron.zorn@chmcc.org
2
DOI 10.1002/dvdy.10201
© 2002 Wiley-Liss, Inc.
ENDODERM GENES IN Xenopus tropicalis 119
TABLE 1. Xenopus tropicalis Genes Isolateda
Gene
Name
EST Accession No.
Sanger Centre
Clone I.D.
AL648546
AL643926
AL628918
AL629418
AL648100
AL648004
AL654910
AL649368
AL678593
AL773836
tGas 032n19
LIG5d04
tGas 011n03
tGas 017o02
tGas 036b21
tGas 038a07
tGas 043b04
tGas 049c03
tGas 059c07
tGas 071m08
AL594754
AL595122
AL595475
AL630110
AL650996
AL679936
tGas
tGas
tGas
tGas
tGas
tGas
AL683701
AL797310
AL785917
BQ395911
BQ396239
tGas 068m21
tNeu 122b04
tNeu 137p21
n.a.
n.a.
n.a.
185Cb
tNeu072i06
tGas107m23
n.a.
n.a.
n.a.
tFoxA1
tHex
tVegT
tMixer
tMix
tBix
tGata6
tSox17␣
tSox 17␤
tCerberus
Accession No. of Full-Length
Clone Used in this Study
Restriction Enzyme Used
for In Situ Probe
AF180352
001e10
002g10
005d23
009g11
048o19
052f22
Pending
HindIII
EcoR1
AY157638
EcoR1
AY157639
EcoR1
EcoR1
Pending
HindIII
HindIII
HindIII
AY157635
HindIII
tGas004n06
tGas029g13
tGas121m01
AY157636
HindIII
n.a.
tNeu062i17
AY157637
HindIII
n.a.
n.a.
tGas023c10
tGas075h01
Pending
BamHI
AL638969
AL631153
AL650019
AL648180
AL672540
LG10f05
tGas 016c17
tGas 030p22
tGas 038b01
tGas 54i20
AY157640
BamH1
HindIII
a
b
n.a., not applicable, clones were isolated by filter hybridization.
This clone was isolated before the library was arrayed.
Clements et al., 1999; Yasuo and Lemaire, 1999; Xanthos et al., 2001).
These transcription factors promote
endodermal cell fate and the expression of endoderm marker genes
such as the hepatic nuclear factors
(HNF1␤ and FoxA1/HNF3; Husdon et
al., 1997; Henry and Melton, 1998;
Alexander and Stainier, 1999; Weber
et al., 2000; Xanthos et al., 2001;
Stainier, 2002). As a result, vegetal
cells are committed to form
endodermal tissues by the midgastrula stage (Wylie et al., 1987).
Early endodermal patterning occurs simultaneously with general
endoderm specification, as evident
by the asymmetric expression of the
homeodomain transcription factor
Hex (Newman et al., 1997) and the
secreted growth factor antagonist
Cerberus (Bouwmeester et al., 1996)
in the anterior endoderm. The available evidence indicates that the
overlapping activities of endoderminducing nodal signals and the maternal Wnt/␤-catenin signals induce
the expression of Hex and Cerberus
in the anterior endoderm (Zorn et al.,
1999).
Although the genes described
above have been implicated in
endoderm specification, their precise roles are unclear. The majority of
functional studies to date have relied on overexpression experiments
120 D'SOUZA ET AL.
Figure 1.
Figure 2.
in Xenopus laevis. To rigorously define the function of these genes and
to resolve the epistatic relationships
between them requires “loss-offunction” analysis. Antisense morpholino oligos, used to block the
translation of specific target RNAs,
should provide a rapid and efficient
way to achieve “loss-of-function”
analysis in Xenopus (Heasman et al.,
2000; Heasman, 2002). The related
diploid species Xenopus (Silurana)
tropicalis may be better for antisense studies because, unlike the
pseudotetraploid Xenopus laevis, it
does not have redundant copies of
each gene (Amaya et al., 1998; Nutt
et al., 2001). In laevis, the different
mRNA copies often have divergent
sequences in the 5⬘ untranslated regions (UTRs) where the antisense oligos bind; for this reason, depleting
one copy with a morpholino leaves
the other untouched. For example,
in laevis there are two copies of
Figure 3.
ENDODERM GENES IN Xenopus tropicalis 121
Sox17␣ (Sox17␣1 and Sox17␣2) (Hasegawa et al., 2002) and two copies
of Gata6 (Jiang and Evans, 1996;
Gove et al., 1997) both with divergent 5' UTR sequences. When examining genes in laevis, one must target
both copies of a gene to ensure efficient depletion by the morpholino
oligo. In addition, the diploid tropicalis system has the promise of forward genetic analysis while retaining the advantages of easy
embryologic manipulation available
in laevis (Amaya et al., 1998; Khokha
et al., 2003).
As a prelude to examining
endoderm specification by “loss-offunction,” we have isolated fulllength X. tropicalis homologues of
the transcription factors involved.
We found the endoderm genes are
conserved in tropicalis and consistent with its diploid genome, there is
only one copy of each gene, which
should simplify functional analysis. By
using in situ hybridization, we present
a detailed expression analysis in
tropicalis embryos, with an emphasis
on gastrula stages when endoderm
specification occurs. The expression
patterns of the genes we examined
were almost identical to those described in laevis, suggesting tropicalis experiments are likely to be relevant to laevis and vice versa. We
found that detection of gene expression in the deep endoderm was
more efficient in tropicalis than laevis. As a result, we observed some
previously unreported expression
domains. Thus, we have assembled
most of the molecular components
of the endoderm specification pathway in X. tropicalis.
RESULTS AND DISCUSSION
To isolate X. tropicalis orthologues of
X. laevis endoderm genes, we generated two arrayed cDNA libraries
from gastrula and neurula tropicalis
embryos. We isolated X. tropicalis
clones from these cDNA libraries, either by high stringency screening of
arrayed filters with laevis probes or
by searching the publicly available
Sanger Centre tropicalis expressed
sequence tag (EST) and National
Center for Biotechnology Information (NCBI) databases. We only
examined cDNAs and ESTs that
appeared to encode full-length
clones, that is, they had the putative
start of translation based on the laevis sequence. Table 1 summarizes
the ESTs and full-length clones that
we have identified in this study.
To examine gene expression in
tropicalis embryos, we used the
same whole-mount in situ hybridization protocol we routinely use for
endodermal genes in laevis (Sive et
al., 2000, with modifications detailed
in the Experimental Procedures section), producing excellent results
(Fig. 1A). In fact, we observed superior penetration of probes in tropicalis embryos, allowing us to visualize
gene expression in the deep
endoderm tissue by sectioning after
the whole-mount hybridization procedure (Fig. 1D). In contrast, with
laevis, probes never penetrated into
the deep endoderm tissue by using
whole-mount in situ hybridization
(compare Fig. 1B with D). In laevis,
hybridization to paraffin-sectioned
embryos is required to fully detect
gene expression in the deep endoderm (Fig. 1C; Butler et al., 2001). The
smaller size of the tropicalis embryo
most likely accounts for the more efficient penetration. As a result, for
experimental analysis of endoderm
formation by in situ hybridization,
tropicalis embryos are easier to work
with than laevis.
VegT
The vegetally localized, maternal Tbox transcription factor VegT initiates endoderm formation in Xenopus laevis (Zhang et al., 1998;
Xanthos et al., 2001). Vempati and
King had previously deposited the
full-length sequence of tropicalis
VegT (tVegT) in Genbank, which is
93% identical to laevis VegT at the
amino acid level. To examine the expression of tVegT which, has not
been reported, we searched the
Sanger Centre tropicalis expressed
sequence tag (EST) databases and
identified a full-length tVegT clone
(Table 1), which we used for in situ
hybridizations. As expected, tVegT is
vegetally localized in tropicalis oocytes and is expressed in the presumptive endoderm and mesoderm
at early gastrula (Fig. 2). As in laevis,
tVegT expression is down-regulated
in the endoderm by the end of gastrulation but maintained in the lateral and ventral mesoderm of the
blastopore lip (data not shown).
Fig. 1. In situ hybridization. A comparison of the whole-mount in situ hybridization procedure between Xenopus tropicalis and Xenopus
laevis embryos. A: Whole-mount in situ hybridization with species-specific Sox17␣ probes to laevis (left) and tropicalis (right) gastrula. A
ventral view with dorsal up is shown. Probes penetrate more efficiently in tropicalis whole-mount hybridizations than they do in laevis
embryos. Compare a midsagittal, Vibratome section of a laevis gastrula previously hybridized by whole-mount with a Sox17␣ probe (B)
with a laevis embryo for which the Sox17␣ in situ hybridization was preformed on a paraffin section (C). Notice the lack of staining in the
central deep endoderm of the sectioned laevis whole-mount. D: In contrast, a midsagittal, Vibratome section of a tropicalis gastrula,
previously stained by tSox17␣, whole-mount shows complete penetration of the probe into the deep endoderm. E: A schematic of a
midsagittal gastrula embryo. As in all the sections above, dorsal is to the left. Yellow indicates the deep and superficial endoderm, orange
the mesoderm, and white the ectoderm.
Fig. 2. Xenopus tropicalis VegT. Whole-mount in situ hybridization analysis of tVegT mRNA expression in tropicalis oocyte and embryos.
A: tVegT mRNA is localized to the vegetal half of a stage VI oocyte. The bleached animal pole is up. A lateral view of a cleared
whole-mount in situ hybridization of early gastrula embryo (B) and a midsagittal section of the same whole-mount embryo(C) shows tVegT
expression throughout the endoderm with stronger expression in the mesoderm. Dorsal is to the left. The brown color in the animal cap
is pigment. st., embryonic stage.
Fig. 3. Xenopus tropicalis Mix-like genes. Whole-mount in situ hybridization analysis of tropicalis gastrula embryos with tMixer (A–C), tMix
(D–F), and tBix (G–I) antisense probes. The top row (A,D,G) shows stage (st.) 10.5 early gastrula embryos. The middle row (B,E,H) shows later
stage 11 gastrula. In each case, a vegetal view is shown with dorsal up. The bottom row (C,F,I) shows midsagittal sections of whole-mount,
stage 10.5 embryos. Dorsal is to the left.
122 D'SOUZA ET AL.
Fig. 4. Xenopus tropicalis Gata6. Whole-mount in situ hybridization analysis of tGata6
expression in tropicalis embryos. A vegetal view of stage (st.) 10 (A) and stage 11 (B)
gastrula embryos show tGata6 transcripts localized to the deep endoderm but absent from
the superficial endoderm (red arrow). C: A midsagittal hemisection of a stage 10.5 gastrula
previously stained by whole-mount (dorsal lip to the left) shows tGata6 transcripts abundant in the leading edge endoderm and confirms the lack of expression in the superficial
endoderm (red arrow). A tGata6 whole-mount of a stage 22, early tail bud embryo(D) and
a midsagittal section of the same embryo (E) is shown with anterior to the right. ld, liver
diverticulum; cm, cardiac mesoderm; lpm, lateral plate mesoderm. F: A section of a stage
26, whole-mount embryos shows tGata6 expression in the endoderm and the cardiac
mesoderm. G: By stage 32, tGata6 is strongly expressed in the mid-gut endoderm as well
as the cardiac and lateral plate mesoderm.
Mix-like
In Xenopus laevis, the “Mix-like” homeodomain transcription factors
Mixer, Mix1/2, and Bix1-4 have been
implicated in endoderm differentiation (Henry and Melton, 1998; Ecochard et al., 1998; Casey et al.,
1999). By using all of the different laevis Mix-like sequences, we searched
the EST database to identify cDNAs
encoding full-length Mix-like homeodomain proteins in tropicalis. From
approximately 100,000 gastrula and
neurula stage ESTs, we only found
three different tropicalis Mix-like sequences which appear to correspond to one tMixer gene, one tMix
gene, and only one tBix gene. In total, we identified one tMixer EST,
eight nearly identical tMix ESTs, and
six nearly identical tBix ESTs, all of
which correspond to full-length
cDNAs (Table 1). In contrast, the
pseudotetraploid laevis has a more
complex set of genes, with one
Mixer (Henry and Melton, 1998), two
Mix genes (Rosa, 1989; Vize, 1996),
and four Bix genes (Tada et al., 1998;
Ecochard et al., 1998). The slight variation in the EST sequences within the
tMix and tBix clusters (on average
98% identical) was in some cases
due to sequencing errors. The minor
sequence differences may also be
due to allelic variation as the cDNA
libraries used for the ESTs were generated from several different outbred animals (A.Z. and E.A. unpublished observations). By comparison,
the different laevis copies of the
genes, arising from its pseudotetraploidy, vary considerably more: X.
laevis Mix1 and Mix2 are only 85%
identical, whereas Bix1– 4 are on average only 80% identical to each
other at the nucleotide level.
We isolated one of each of the
full-length tMixer, tMix, and tBix
clones for further analysis (Table 1).
At the amino acid level, tMixer was
78% identical to laevis Mixer, tMix
was 74% and 75% identical to laevis
Mix1 and Mix2, and tBix was 78%,
74%, 75%, and 73% identical to laevis
Bix1– 4, respectively. In situ hybridization with tMixer, tMix, and tBix probes
to gastrula stage tropicalis embryos
(Fig. 3) revealed the expression
pattern predicted from laevis.
tMixer is expressed throughout the
deep and superficial endoderm of
the gastrula, with expression strongest across the endoderm/mesoderm boundary. By comparison,
tMix and tBix transcripts are predominantly localized to the mesoderm,
with relatively lower levels in the
deep endodermal mass at early
gastrula. By late gastrula, their
endodermal expression has declined to undetectable levels, but
the mesodermal expression persists.
In summary, we have found that
tropicalis has only three “Mix-like”
genes: tMixer, tMix, and tBix; and
their expression patterns are identical to their laevis counterparts. In
contrast, laevis has at least seven
different “Mix-like” genes, suggesting that functional analysis of this
gene family will be more straightforward in tropicalis.
Gata6
We searched the ESTs databases for
clones encoding tropicalis members
of the Gata4/5/6 family of zinc finger
transcription factors, which have
been implicated in vertebrate
endoderm development. We found
five nearly identical ESTs encoding
full-length tGata6 (Table 1). We did
not find any Gata5-like ESTs and only
three tGata4 ESTs, all of which encoded partial clones. Focussing on
one of the tGata6 clones, we found
that tGata6 uses an alternative upstream ATG like mouse and human
GATA6, which has not been reported in the laevis Gata6 cDNAs
(Brewer et al., 1999). In laevis, a
downstream, internal ATG is assumed to be used; therefore, the
tGata6 has a longer open reading
frame than the laevis Gata6 cDNAs.
Over the common regions, tGata6
ENDODERM GENES IN Xenopus tropicalis 123
umented. The tGata6 expression
pattern that we observe is reminiscent of that described for laevis
Gata5 (Jiang and Evans, 1996; Weber et al., 2000). Gain-of-function experiments suggest that Gata4 and
Gata5 are more important than
Gata6 in laevis endoderm specification; however, we have not found
any tropicalis Gata5 sequences in
the gastrula and neurula ESTs. Perhaps tGata6, rather than tGata5,
plays a more important role in tropicalis endoderm development. It will
be important to test this, as well as to
isolate full-length tGata4 to examine
the functional importance of each
of these genes in endoderm development.
Sox17
Fig. 5. Xenopus tropicalis Sox17␣/␤. In situ hybridization analysis of tropicalis embryos
with Sox17␣ (A–E) and Sox17␤ (F–J) antisense probes. Whole-mount (A,C,F,H) and sectioned (B,G) gastrula show that tSox17␣ and tSox17␤ are identically expressed in throughout the deep and superficial endoderm (red arrows). D: At early tail bud stage (anterior to
the left), tSox17␣ is strongly expressed in the posterior endoderm but absent in the anterior
endoderm, except behind the cement gland. I: By comparison at stage 22 (anterior to the
left), tSox17␤ transcripts are almost absent from the entire endoderm, except behind the
cement gland. E: At stage 32, tSox17␣ expression persists in the extreme posterior
endoderm, the gall bladder (g) precursors and in endothelial cells. J: In late tail bud
stages, tSox17␤ is only expressed in a small patch of cells in the head. st, embryonic stage.
was 94% identical to laevis Gata6 at
the amino acid level. By in situ hybridization, we found that tGata6
was expressed in the presumptive
deep endoderm of the gastrula (Fig.
4A–C) but not in the superficial epithelial endoderm of the blastopore
lip (Fig. 4B,C red arrows). At tail bud
stages (Fig. 4D–G), tGata6 mRNA
was localized to the deep midgut
endoderm, with levels highest near
the liver diverticulum and weaker in
the head and posterior endoderm.
The dorsal midgut endoderm underlying the axial mesoderm also expressed high levels of tGata6 (Fig.
4E,F). In addition, tGata6 mRNA was
detected in the cardiac and lateral
plate mesoderm at tail bud stages
(Fig. 4E,F).
Although Gata6 mRNA has been
detected in the laevis gastrula
endoderm by reverse transcriptasepolymerase chain reaction (Jiang
and Evans, 1996), the details of its
early expression pattern are undoc-
To isolate tropicalis orthologues
of the endoderm-specific, HMGbox transcription factor Sox17, we
screened our arrayed cDNA libraries
at high stringency with laevis Sox17␣
and Sox17␤ probes. We isolated
three tSox17␣ full-length cDNAs and
three full-length tSox17␤ cDNAs
(Table 1). Sequence comparison
showed that tSox17␣ was 93% and
89% identical at the amino acid
level to laevis Sox17␣1 and Sox17␣2,
respectively, whereas tSox17␤ was
80% identical to laevis Sox17␤.
In situ hybridization showed that
tSox17␣ and tSox17␤ are identically
expressed throughout all the deep
and superficial endoderm during
gastrulation (Fig. 5A–C,F–H), which is
consistent with their laevis counter
parts (Hudson et al., 1997). During
neurula and early tail bud stages,
tSox17␣ expression declines significantly in the anterior endoderm, except in the endoderm behind the
cement gland. Strong tSox17␣ expression persists in the endoderm
posterior to the liver diverticulum until late tail bud stages (Fig. 5D). By
late tail bud, tSox17␣ transcripts
are undetectable in most of the
endoderm but expression is maintained in the presumptive gall bladder region and the extreme posterior region, as has been observed in
laevis (Zorn and Mason, 2001). In addition, we observe tSox17␣ transcripts in what appear to be endothelial cells in the head and along
124 D'SOUZA ET AL.
visualize tFoxA1 mRNA in the deep
and superficial endoderm of the
midgastrula (Fig. 6A,B). At later
stages, tFoxA1 is expressed in a pattern identical to that described for
laevis (Fig. 6C–F).
Hex and Cerberus
Fig. 6. Xenopus tropicalis FoxA1. Whole-mount in situ hybridization analysis of tFoxA1
mRNA expression in tropicalis embryos. A: A vegetal view of a midgastrula embryo shows
tFoxA1 transcripts in the endoderm. B: A midsagittal section of the same embryo (dorsal to
the left) shows strong tFoxA1 expression in the chordomesoderm and weaker expression
throughout the endoderm. The dark staining in the animal cap is pigment. C: A dorsal view
(anterior up) of a stage 18 embryo shows tFoxA1 expression in the notochord and also in
ciliated cells of the epithilium. A lateral view of a tail bud embryo (D) and a cross-section
of the same embryo (E) shows tFoxA1 transcripts expressed throughout the endoderm, as
well as in the notochord and floor plate. F: Analysis of stage 40 embryos shows that tFoxA1
transcripts mark the endoderm late in development. st., embryonic stage.
the flank of the embryo (Fig. 5E). In
contrast to tSox17␣, tSox17␤ expression declines rapidly after gastrulation. However, like tSox17␣, a small
patch of endoderm cells behind the
cement gland maintains expression
even into tail bud stages.
The
dynamic
expression
of
tSox17␣/␤ that we observed between neurula and tail bud stages is
undocumented in laevis. We were
able to observe these expression domains in tropicalis because of the
superior penetration of probes with
the in situ procedure. When we
checked laevis embryos more carefully, we observed an identical expression pattern (data not shown).
The role of Sox17 in these tissues later
in development will be interesting to
examine.
FoxA1
Members of the FoxA forkhead
family of hepatic nuclear factors
(also known as HNF3 ␣/␤/␥) have
long been studied as key regulatory molecules and valuable markers of endoderm tissue. To identify
tropicalis FoxA family members, we
screened our arrayed cDNA library
filters with a laevis FoxA2 (HNF3␤)
probe at moderate stringency. Although we did not isolate tropicalis
FoxA2, we isolated several tropicalis
clones of the related FoxA1 (HNF3␣/
FKH2). One of the tFoxA1 clones was
full-length (Table 1) and 92% identical at the amino acid level to laevis
FoxA1 (Bolce et al., 1993). In tail bud
and larval stage laevis embryos,
FoxA1 is expressed throughout the
endoderm, in the notochord, floor
plate, and in ciliated cells of the epidermis. In addition, FoxA1 mRNA
has been detected in gastrula
endoderm by Northern blot but was
undetected by whole-mount in situ
hybridization (Bolce et al., 1993; R.
Harland, personal communication).
The superior penetration of probes
into tropicalis embryos allowed us to
The initial stages of endodermal patterning in laevis occur simultaneously with endoderm specification as observed by the asymmetric
expression of the genes Hex and
Cerberus in the anterior endoderm
(Zorn et al., 1999). To isolate tropicalis
orthologues of Hex, we screened our
arrayed cDNA library filters with a
laevis Hex probe and isolated two
putative full-length tHex clones
(Table 1). However, one clone
(tGas023e18) appeared to be an
unspliced transcript or had a cloning
artifact in the open reading frame.
Sequence analysis of the remaining
tHex cDNA indicated that it was 96%
identical to laevis Hex and the
amino acid level. We identified six
full-length tropicalis Cerberus sequences by database searching
(Table 1). One of these was completely sequenced and was 84%
identical to laevis Cerberus and the
amino acid level. In situ hybridization
analysis with tHex and tCerberus
probes demonstrated that the tropicalis genes were expressed in a pattern identical to their laevis counterparts (Bouwmeester et al., 1996;
Newman et al., 1997; Zorn et al.,
1999). tHex is expressed in the most
dorsoanterior endomesoderm of the
blastula and gastrula embryo (Fig.
7A–C; data not shown) and later is
restricted to the forming liver diverticulum (Fig. 7D). tCerberus is also expressed in the anterior endomesoderm of the early gastrula, but its
expression is also expanded laterally
around the margin at the endoderm/mesoderm boundary. By late
neurula, tCerberus mRNA is undetectable.
Concluding Note
Recently, a conserved molecular
pathway that regulates endoderm
specification has been deduced
(Stainier, 2002). Here, we have isolated most of those genes from the
ENDODERM GENES IN Xenopus tropicalis 125
calis. The tropicalis endoderm genes
that we isolated are expressed in a
manner almost identical to their laevis counterparts, suggesting that
they will have very similar functions.
Results obtained in the tropicalis system, therefore, are likely to be relevant to the laevis system and vice
versa. Finally, the high sequence
conservation between laevis and
tropicalis, coupled with the in-depth
EST sequencing, allowed us to find
most of the genes that we looked for
in the databases. As a result, we
were able to easily and quickly assemble an entire molecular pathway in tropicalis and this is likely to
hold true for any genes one wishes
to study.
EXPERIMENTAL PROCEDURES
Embryos
X. tropicalis embryos were obtained
by either natural mating or in vitro
fertilizations, according to the protocol from the University of Virginia
tropicalis Web site (http://faculty.
virginia.edu/xtropicalis/). X. tropicalis
husbandry details can also be found
in Khokha et al. (2002). Embryos
were staged according to the normal table of development for Xenopus laevis (Nieuwkoop and Faber,
1994).
cDNA Library Screening
Fig. 7. Xenopus tropicalis Hex and Cerberus. In situ hybridization analysis of tropicalis
embryos with tHex (A–D) and tCerberus (E–G) antisense probes. The top row shows vegetal
views (dorsal up), and the second row shows lateral views (dorsal left) of tHex (A,B) and
tCerberus (E,F) expression in the anterior endoderm of the early gastrula. Note tCerberus is
more broadly expressed than tHex. C,G: Midsagittal sections (dorsal left) of gastrula
embryos, hybridized in whole-mount with tHex and tCerberus probes. D: A cleared tail bud
embryo (anterior right) shows tHex transcripts localized to the presumptive liver region. st.,
embryonic stage.
diploid Xenopus tropicalis, because
we believe that “loss-of-function”
analysis by antisense oligos and possibly forward genetics will work better in this species than in the
pseudotetraploid Xenopus laevis. In
addition, tropicalis retains all of the
classic embryologic advantages enjoyed by Xenopus laevis (Amaya et
al., 1998; Khokha et al., 2003). We
show that the key components of
the endoderm development path-
way are conserved in tropicalis, but
as expected, there are not multiple
redundant copies of each gene as
found in laevis. For example, there
are only three Mix-like genes in tropicalis, whereas there are seven in laevis. This reduced complexity should
make functional analyses significantly easier in tropicalis. We show
that the standard laevis in situ hybridization protocol gives superior results for endodermal genes in tropi-
The details of the tropicalis cDNA library construction, arraying, and EST
project will be described in detail
elsewhere and are available upon
request. Briefly, polyA⫹ mRNA was
isolated from gastrula and neurula
tropicalis embryos. Oligo-dT–primed
cDNA was directionally cloned into
pCS107 and electroporated into
Escherichia coli,resulting in the gastrula (tGas) and neurula (tNeu)
cDNA libraries. Clones (55,000) from
each of these libraries were robotically arrayed by the RZPD, and highdensity filters were produced. These
same libraries were given to the
Sanger Centre, UK, for the tropicalis
EST project. X. tropicalis clones were
either isolated by screening the filters
or by searching the publicly available EST database at NCBI and the
Sanger Centre (http://www.sanger.
ac.uk/Projects/X_tropicalis/). For fil-
126 D'SOUZA ET AL.
ter screening, the full-length coding
regions of X. laevis Sox17␣ (pSKSox17␣, KpnI/SacI), Sox17␤ (pSKSox17␤, EcoRI/NotI; Zorn et al., 1999),
Hex (pSK-Hex, EcoRI/XhoI; Newman
et al., 1997), Cerberus (pBS-Cerberus, EcoRI/XhoI; Bouwmeester et al.,
1996), and Hnf3␤/FoxA2 (pCS2-XFD3,
EcoRI/NotI, a gift from Dr. Knoechel),
were gel-isolated, radiolabeled by
random priming, and hybridized at
high stringency in Church’s buffer.
We only pursued cDNAs and ESTs
that appeared to encode full-length
clones: that is, they had the putative
start of translation based on the laevis sequence. A summary of the accession numbers and clone identification numbers of all the ESTs and
full-length cDNAs identified in this
study are presented in Table 1. The
clones described in this study, as well
as any clones from the Sanger Centre tropicalis EST project, will be available from the HGMP through the
MCR Geneservice, UK.
In Situ Hybridization
Table 1 lists the restriction enzymes
used to linearize each clone to produce antisense digoxygenin-11-UTP
RNA probes using T7 RNA polymerase (Ambion T7 MEGA script kits).
Whole-mount in situ hybridizations
were performed in home-made baskets by using the standard laevis protocol (Sive et al., 2000) with minor
modifications to improve penetration of endodermal probes. Embryos
were incubated with anti-digoxigenin antibody (1/2,000) overnight at
4°C (overnight with the antibody is
essential to get efficient penetration
into the deep endoderm). The following day, embryos were washed
12 ⫻ 30 min in Maleic acid buffer
with a final wash at 4°C overnight.
The following day, embryos were
equilibrated in alkaline phosphatase
buffer and the chromogenic reaction, with either nitro blue tetrazolium/5-bromo-4-chloro-3-indoxyl
phosphate (NBT/BCIP) or BM purple
allowed to proceed for several days
at 15°C. After photography of the
whole-mounts, embryos were embedded in 7% low-gelling temperature agarose and 30-micron sections
were cut on a Vibratome and
TABLE 2. Conservation between Xenopus tropicalis and X. laevis Genesa
tropicalis
Gene
laevis
Gene
Nucleotide Identity in the
Open Reading Frame (%)
Amino Acid
Identity (%)
tVegT
tMixer
tMix
VegT
Mixer/Mix3
Mix.1
Mix.2
Bix1/Mix4
Bix2/Milk
Bix3
Bix4
Gata6a
Gata6b
Sox17␣1
Sox17␣2
Sox17␤
FoxA1
Hex
Cerberus
93
87
85
81
88
85
83
84
91b
93b
93
91
85
89
92
88
93
78
74
75
78
74
75
73
94b
94b
93
89
80
92
96
84
tBix
tGata6
tSox17␣
tSox17␤
tFoxA1
tHex
tCerberus
a
The values shown only reflect the regions common between tropicalis and
laevis.
b
tropicalis gata6 appears to use an alternative upstream ATG like mouse and
human, the reported laevis cDNAs do not contain these sequences; therefore, it
is assumed that the downstream ATG is used (Brewer et al., 1999).
mounted in 90% glycerol/Tris buffered saline.
ACKNOWLEDGMENTS
We thank Jen Ashurst, Amanda McMurray, Liz Huckle, Ruth Taylor, Jane
Rogers, and Richard Durbin at the
Wellcome Trust Sanger Centre, UK,
for enthusiastically undertaking the
tropicalis EST project. This work was
supported by grants from the Wellcome Trust UK (J.S., E.A., N.P., A.Z.)
and the NIH (A.Z.).
REFERENCES
Alexander J, Stainier DYR. 1999. A molecular pathway leading to endoderm formation in zebrafish. Curr Biol 9:1147–
1157.
Amaya E, Offield MF, Grainger RM. 1998.
Frog genetics: Xenopus tropicalis jumps
into the future. Trends Genet 14:253–
255.
Bolce ME, Hemmati-Brivanlou A, Harland
RM. 1993. XFKH2, a Xenopus HNF-3␣ homologue, exhibits both activin-inducible and autonomous phases of expression in early embryos. Dev Biol 160:
413– 423.
Bouwmeester T, Kim S, Sasai Y, Lu B, De
Robertis EM. 1996. Cerberus is a headinducing secreted factor expressed in
the anterior endoderm of Spemann’s
organizer. Nature 382:595– 601.
Brewer A, Gove C, Davies A, McNulty C,
Barrow D, Koutsourakis M, Farzaneh F,
Pizzey J, Bomford A, Patient R. 1999. The
human and mouse GATA-6 genes utilize two promoters and two initiation
codons. J Biol Chem 274:38004 –38016.
Butler K, Zorn AM, Gurdon JB. 2001. Nonradioactive in situ hybridization to Xenopus tissue sections. Methods 23:303–
312.
Casey ES, Tada M, Fairclough L, Wylie
CC, Heasman J, Smith JC. 1999. Bix4 is
activated directly by VegT and mediates endoderm formation in Xenopus
development. Development 126:4193–
4200.
Clements D, Friday RV, Woodland HR.
1999. Mode of action of VegT in mesoderm and endoderm formation. Development 126:4903– 4911.
Ecochard V, Cayrol C, Rey S, Foulquier F,
Caillol D, Lemaire P, Duprat AM. 1998.
A novel Xenopus mix-like gene milk involved in the control of the endomesodermal fates. Development 125:2577–
2585.
Gove C, Walmsley M, Nijjar S, Bertwistle D,
Guille M, Partington G, Bomford A, Patient R. 1997. Over-expression of Gata-6
in Xenopus embryos blocks differentiation of heat precursors. EMBO J 16:355–
368.
Hasegawa M, Hiraoka Y, Hagiuda J,
Ogawa M, Aiso S. 2002. Expression and
characterization of Xenopus laevis SRYrelated cDNAs, xSox17alpha1, xSox17alph2, xSox17alpha and xSox17beta.
Gene 15:163–172.
Heasman J. 2002. Morpholino oligos:
making sense of antisense? Dev Biol
243:209 –214.
Heasman J, Kofron M, Wylie C. 2000.
Beta-catenin signaling activity dis-
ENDODERM GENES IN Xenopus tropicalis 127
sected in the early Xenopus embryo: a
novel antisense approach. Dev Biol
222:124 –134.
Henry GL, Melton DA. 1998. Mixer, a homeobox gene required for endoderm
development. Science 281:91–96.
Henry GL, Brivanlou IH, Kessler DS, Hemmati-Brivanlou A, Melton DA. 1996.
TGF-␤ signals and a prepattern in Xenopus laevis endodermal development. Development 122:1007–1015.
Horb ME, Thomsen GH. 1997. A vegetally
localized T-box transcription factor in
Xenopus eggs specifies mesoderm and
endoderm and is essential for embryonic mesoderm formation. Development 124:1689 –1698.
Hudson C, Clements D, Friday RV, Stott D,
Woodland HR. 1997. Xsox17alpha and
-beta mediate endoderm formation in
Xenopus. Cell 91:397– 405.
Jiang Y, Evans T. 1996. The Xenopus
GATA-4/5/6 genes are associated with
cardiac specification and can regulate cardiac-specific transcription during embryogenesis. Dev Biol 174:258 –
270.
Jones CM, Kuehn MR, Hogan BL, Smith
JC, Wright CV. 1995. Nodal-related signals induce axial mesoderm and dorsalize mesoderm during gastrulation.
Development 121:3651–3662.
Joseph EM, Melton DA. 1997. Xnr4: a Xenopus nodal-related gene expressed
in the Spemann organizer. Dev Biol 184:
367–372.
Khokha MK, Chung C, Bustamante EL,
Gaw LWK, Trott KA, Yeh J, Lim N, Lin J,
Taverner N, Amaya E, Papalopulu N,
Smith JC, Zorn AM, Harland RM, Grammer TC. 2002. Techniques and probes
for the study of Xenopus tropicalis development. Dev Dyn 225:499 –510.
Kofron M, Demel T, Xanthos J, Lohr J, Sun
B, Sive H, Osada S, Wright C, Wylie C,
Heasman J. 1999. Mesoderm induction
in Xenopus is a zygotic event regulated
by maternal VegT via TGF␤ growth factors. Development 126:5759 –5770.
Lustig KD, Kroll KL, Sun EE, Kirschner MW.
1996. Expression cloning of a Xenopus
T-related gene (Xombi) involved in mesodermal patterning and blastopore
lip formation. Development 122:4001–
4012.
Newman CS, Chia F, Krieg PA. 1997. The
XHex homeobox gene is expressed
during development of the vascular
endothelium: overexpression leads to
an increase in vascular endothelial cell
number. Mech Dev 66:83–93.
Nieuwkoop PD, Faber J. 1994. Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of
the development from the fertilized
egg till the end of metamorphosis. New
York: Garland Publishing, Inc.
Nutt SL, Bronchain OJ, Hartley KO,
Amaya E. 2001. Comparison of morpholino based translational inhibition
during the development of Xenopus
laevis and Xenopus tropicalis. Genesis
30:110 –113.
Rosa FM. 1989. Mix.1, a homeobox mRNA
inducible by mesoderm inducers, is expressed mostly in the presumptive
endodermal cells of Xenopus embryos.
Cell 57:965–974.
Sive HL, Grainger RM, Harland RM. 2000.
Early development of Xenopus laevis:
a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press.
Stainier DYR. 2002. A glimpse into the molecular entrails of endoderm formation.
Genes Dev 16:893–907.
Stennard F, Carnac G, Gurdon JB. 1996.
The Xenopus T-box gene, Antipodean,
encodes a vegetally localised maternal mRNA and can trigger mesoderm
formation. Development 122:4179 –
4188.
Sun BI, Bush SM, Collins-Racie LA, LaVallie
ER, DiBlasio-Smith EA, Wolfman NM,
McCoy JM, Sive HL. 1999. derriere: a
TGF-beta family member required for
posterior development in Xenopus. Development 126:1467–1482.
Tada M, Casey ES, Fairclough L, Smith JC.
1998. Bix1, a direct target of Xenopus
T-box genes, causes formation of ven-
tral mesoderm and endoderm. Development 125:3997– 4006.
Takahashi S, Yokota C, Takano K, Tanegashima K, Onuma Y, Goto J,
Asashima M. 2000. Two novel nodal related genes initiate early inductive
events in Xenopus Nieuwkoop initiative. Development 127:5319 –5329.
Vize PD. 1996. DNA sequences mediating
the transcriptional response of the
Mix.2 homeobox gene to mesoderm
induction. Dev Biol 177:226 –231.
Weber H, Symes CE, Walmsley MEs, Rodaway AR, Patient RK. 2000. A role for
GATA5 in Xenopus endoderm specification. Development 127:4345– 4360.
Wells JM, Melton DA. 1999. Vertebrate
endoderm development. Annu Rev
Cell Dev Biol 15:393– 410.
Wylie CC, Snape A, Heasman J, Smith JC.
1987. Vegetal pole cells and commitment to form endoderm in Xenopus
laevis. Dev Biol 119:496 –502.
Xanthos JB, Kofron M, Wylie C, Heasman
J. 2001. Maternal VegT is the initiator
of a molecular network specifying
endoderm in Xenopus laevis. Development 128:167–180.
Yasuo H, Lemaire P. 1999. A two-step
model for the fate determination of
presumptive endodermal blastomeres
in Xenopus embryos. Curr Biol 9:869 –
879.
Zhang J, King ML. 1996. Xenopus VegT
RNA is localized to the vegetal cortex
during oogenesis and encodes a novel
T-box transcription factor involved in
mesodermal patterning. Development
122:4119 – 4129.
Zhang J, Houston DW, King ML, Payne C,
Wylie C, Heasman J. 1998. The role of
maternal VegT in establishing the primary germ layers in Xenopus embryos.
Cell 94:515–524.
Zorn AM, Mason J. 2001. Gene expression
in the embryonic Xenopus liver. Mech
Dev 103:153–157.
Zorn AM, Butler K, Gurdon JB. 1999. Anterior endomesoderm specification in Xenopus by Wnt/␤-catenin and TGF-␤ signalling pathways. Dev Biol 209:282–297.