RESEARCH ARTICLE
919
Development 135, 919-929 (2008) doi:10.1242/dev.010660
Cdx4 is required in the endoderm to localize the pancreas
and limit ␤-cell number
Mary D. Kinkel*, Stefani C. Eames, Martha R. Alonzo and Victoria E. Prince
Cdx transcription factors have crucial roles in anteroposterior patterning of the nervous system and mesoderm. Here we focus on
the role of cdx4 in patterning the endoderm in zebrafish. We show that cdx4 has roles in determining pancreatic ␤-cell number,
directing midline convergence of ␤-cells during early pancreatic islet formation, and specifying the anteroposterior location of
foregut organs. Embryos deficient in cdx4 have a posteriorly shifted pancreas, liver and small intestine. The phenotype is more
severe with knockdown of an additional Cdx factor, cdx1a. We show that cdx4 functions within the endoderm to localize the
pancreas. Morpholino knockdown of cdx4 specifically in the endoderm recapitulates the posteriorly shifted pancreas observed in
cdx4 mutants. Conversely, overexpression of cdx4 specifically in the endoderm is sufficient to shift the pancreas anteriorly. Together,
these results suggest a model in which cdx4 confers posterior identity to the endoderm. Cdx4 might function to block pancreatic
identity by preventing retinoic acid (RA) signal transduction in posterior endoderm. In support of this, we demonstrate that in cdx4deficient embryos treated with RA, ectopic ␤-cells are located well posterior to the normal pancreatic domain.
INTRODUCTION
In recent years, many molecular signals have been identified that are
crucial for the early steps of pancreas development. We currently
understand pancreatogenesis as a process in which mesodermal
signals elicit a program of differentiation in the adjacent endoderm.
Known signals include FGFs, BMPs, retinoic acid (RA), activins
and Sonic hedgehog (Shh) (Cano et al., 2007). A variety of studies
has shown that the timing and location of these signals must be
tightly controlled for correct pancreas development along the
anteroposterior (AP) axis. For example, in zebrafish, treatment with
exogenous RA produces anterior ectopic ␤-cell differentiation
(Stafford and Prince, 2002), and disinhibition of BMP signaling
enlarges the pancreatic domain, whereas Bmp2b deficiency reduces
the pancreatic domain (Tiso et al., 2002). In mice, pancreas
development requires inhibition of Shh signals from the region of
the notochord that overlies the endodermal domain of pancreatic
precursors (Hebrok, 2003). Studies such as these have yielded
important insights into how the pancreatic endoderm is induced.
However, our current understanding of endodermal patterning is
limited, relative to our more detailed knowledge of patterning in the
other germ layers.
There is intense interest in Cdx transcription factors because of
their function as modulators of AP patterning in all three germ
layers. In a variety of contexts, Cdx factors have been shown to
function downstream of RA, FGFs and Wnts, and, in turn, to convey
these signals by directly regulating Hox gene expression (Lohnes,
2003). All vertebrates have three Cdx genes: Cdx1, Cdx2 and Cdx4
in tetrapods, and cdx1a, cdx1b and cdx4 in zebrafish (Mulley et al.,
2006). In the endoderm, Cdx factors are crucial for patterning the
intestine along the AP axis as well as along the crypt-villus axis.
Loss of mouse Cdx2 leads to development of gastric epithelium in
more-posterior intestinal domains and, conversely, overexpression
Department of Organismal Biology and Anatomy, The University of Chicago,
1027 East 57th Street, Chicago, IL 60637, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 15 December 2007
of Cdx2 or Cdx1 transforms gastric epithelium to an intestinal fate
(Beck et al., 1999; Mutoh et al., 2004; Mutoh et al., 2005; Silberg et
al., 2002). In light of their ability to transform epithelia, it is not
surprising that both Cdx1 and Cdx2 have been studied intensively
for their roles in gastric and intestinal cancers (Guo et al., 2004). By
contrast, little is known about the role of a third Cdx family member,
Cdx4, in patterning the endoderm. However, recent studies in
zebrafish have begun to reveal crucial roles for Cdx4 in patterning
the ectoderm and mesoderm (Davidson et al., 2003; Shimizu et al.,
2005; Shimizu et al., 2006; Skromne et al., 2007).
In the zebrafish ectoderm, cdx4 is required to establish the
boundary between hindbrain and spinal cord territories (Shimizu et
al., 2006; Skromne et al., 2007). A second Cdx factor, Cdx1a,
exhibits partial functional redundancy with Cdx4 such that embryos
deficient in both factors have enhanced phenotypes (Shimizu et al.,
2006; Skromne et al., 2007). Loss of Cdx4 results in posterior
expansion of segmented hindbrain at the expense of spinal cord.
Conversely, overexpressing cdx4 has a posteriorizing effect
(Skromne et al., 2007). Loss of zebrafish Cdx4 also disrupts
mesoderm patterning; for example, the anterior limits of expression
of kidney markers are shifted posteriorly, and hematopoiesis is
disrupted (Davidson et al., 2003; Wingert et al., 2007). Again, this
phenotype is exacerbated with additional removal of Cdx1a function
(Davidson and Zon, 2006). Thus, cdx4 and cdx1a are crucial for
patterning both the ectoderm and mesoderm.
Here, we study zebrafish cdx4 function in the endoderm and show
that cdx4 has multiple roles in patterning the foregut. In Cdx4 lossof-function embryos, we find that pancreatic ␤-cells are mislocated
toward the posterior, and this is indicative of a more general AP
patterning defect in which the entire foregut is shifted posteriorly.
Using targeted cell transplantations, we show that cdx4 functions
directly within the endoderm to localize the pancreas. Morpholino
knockdown of cdx4 specifically in the endoderm is sufficient to
shift the pancreas posteriorly. Conversely, endoderm-specific
overexpression of cdx4 shifts the pancreas anteriorly. In addition to
these general AP regionalization defects, we also find that kugelig
(kgg; cdx4) mutants exhibit delayed midline convergence of ␤-cells
and an increase in ␤-cell number during early pancreatogenesis.
Knockdown of a second Cdx gene, cdx1a, in cdx4-deficient
DEVELOPMENT
KEY WORDS: Cdx4, Cdx1a, Retinoic acid, Pancreas, ␤-cell, AP patterning
RESEARCH ARTICLE
embryos results in a more severe phenotype. Thus cdx4, together
with cdx1a, is important for localizing the pancreas and modulating
the size of the islet.
MATERIALS AND METHODS
Embryo collection and genotyping
Zebrafish were maintained following standard procedures (Westerfield,
1995) and staged as described (Kimmel et al., 1995). Embryos were
collected from the wild-type *AB line and from a locally obtained pet-store
line, kgghi2188A (Sun et al., 2004), Tg(insulin:GFP) (Huang et al., 2001) and
Tg(gut:GFP) (Ober et al., 2003). Genotyping of kgghi2188A siblings was
performed using primers: mutant forward, 5⬘-TGATCTCGAGTTCCTTGGGAGGGTCTCCTC; wild-type forward, 5⬘-CGTAATTCCTATCAGTGGATGAGC; and shared reverse, 5⬘-CCAGTCACTGACTTCAACACCTTC.
Microinjections
Capped mRNAs encoding zebrafish Cdx4 and Sox32 were synthesized
using mMachine mMessage (Ambion), following the manufacturer’s
protocol. sox32 mRNA was injected at 40 ng/␮l and cdx4 mRNA was
injected at 84 ng/␮l. Antisense morpholinos (Gene Tools) for Cdx4
(Davidson et al., 2003), Cdx1a (Shimizu et al., 2005; Skromne et al., 2007)
and Sox32 (Stafford et al., 2006) were used as previously described.
Cell transplantation
Transplantation was performed as previously described (Ho and Kane, 1990;
Stafford et al., 2006). For transplants in which Sox32 was used to target
reagents to the endoderm, donor embryos were co-injected at the one-cell stage
with sox32 mRNA, cdx4 mRNA or Cdx4 MO, and 40 kDa lysinated
fluorescein dextran (Molecular Probes). Hosts were injected at the one-cell
stage with Sox32-MO. At 4hpf, ~25-40 cells from donor embryos were
transplanted along the blastoderm margin of stage-matched host embryos. The
resulting chimeras were raised to 24hpf and fixed in 4% paraformaldehyde.
In situ hybridization, immunohistochemistry and imaging
In situ probes were used as previously described (Prince et al., 1998), using
probes for cdx4 (Joly et al., 1992), cebpa (Lyons et al., 2001), glucagon and
somatostatin 2 (Argenton et al., 1999), somatostatin 1 (Devos et al., 2002),
insulin (Milewski et al., 1998), islet1 (Inoue et al., 1994), trypsin (Biemar et
al., 2001), pdx1 (Lin et al., 2004) and krox20 (also known as egr2 – ZFIN)
(Oxtoby and Jowett, 1993). In situs for pax9 were performed with
modifications as previously described (Jackman et al., 2004). For sections,
embryos were embedded in Durcupan (Fluka) and cut at 5.5 ␮m using a
Sorvall MT-2 ultramicrotome. Immunohistochemistry was performed
following standard protocols. Somites were labeled using mouse
monoclonal anti-myosin antibody (1:100) (Developmental Studies
Hybridoma Bank), followed by an AlexaFluor 488-conjugated secondary
antibody (1:2000) (Molecular Probes), or followed by the Vectastain
Universal ABC Kit (Vector Labs) using the secondary antibody at 1:500.
ABC labeling was followed by either tyramide labeling (Perkin Elmer) using
the manufacturer’s protocol, or by labeling with an avidin-conjugated
AlexaFluor 546 at 1:1000 (Molecular Probes). To analyze the AP location
of endodermal expression domains, embryos were deyolked, flat-mounted
and photographed under brightfield and fluorescence using a Zeiss
Axioskop. To analyze the location of endodermal cdx4, paraxial and lateral
plate mesoderm was trimmed off, and embryos were mounted laterally and
photographed under brightfield and fluorescence, as above. Images were
merged and analyzed using Adobe Photoshop.
Histology
Larvae were fixed in 10% neutral buffered formalin, embedded in 1% low
melting temperature agarose, and processed for paraffin embedding.
Sections were cut at 4 ␮m and stained with Hematoxylin and Eosin
following standard protocols.
BrdU treatment
Mutant and sibling embryos at 19hpf and 24hpf were manually
dechorionated, then incubated with 10 mM BrdU (5-Bromo-2⬘Deoxyuridine, Sigma) in embryo medium and 15% DMSO for 1 hour at
Development 135 (5)
28.5°C. Following treatment, embryos were washed three times with
embryo medium and fixed in 4% paraformaldehyde. Antibody labeling was
performed using a standard protocol with the addition of a 1-hour incubation
in 2N HCl following enzymatic digestion. The BrdU antibody was used at
1:100 (G3G4, Developmental Studies Hybridoma Bank), AlexaFluor 546
secondary at 1:2000 (Molecular Probes), insulin antibody at 1:1000 (Dako)
and AlexaFluor 488 at 1:2000 (Molecular Probes). For BrdU treatment and
antibody labeling, mutants and siblings were processed in the same tubes.
Retinoic acid treatment
Embryos were incubated at 28.5°C in the dark in embryo medium containing
10–6 M RA (Sigma) for 1 hour. The RA was removed by repeated washing
with embryo medium. Embryos were grown to 24hpf and fixed in 4%
paraformaldehyde.
RESULTS
cdx4 and pdx1 are expressed in overlapping
gradients
Vertebrates express Cdx4 in all three germ layers during
development (Davidson et al., 2003; Gamer and Wright, 1993; Joly
et al., 1992). In zebrafish, the presumptive endoderm begins to
express cdx4 during late epiboly (Joly et al., 1992), but details of its
subsequent endodermal expression pattern are lacking. We therefore
investigated whether zebrafish cdx4 is expressed in the endoderm
during early pancreas development. In situ hybridizations on
embryos from tailbud stage [10 hours post-fertilization (hpf)] to the
20-somite stage (19hpf) revealed cdx4 expression in all three germ
layers (Fig. 1 and data not shown). Sectional analysis revealed
robust expression of cdx4 within the endoderm (Fig. 1A⬘,B⬘). We
found that endodermal expression is in a gradient from high
posteriorly to low anteriorly, and extends further anteriorly with
time, approaching the foregut (Fig. 1G-J). We compared the location
of cdx4 to that of pdx1, a foregut marker of pancreatic and anterior
intestinal precursors (Fig. 1C-J). pdx1 expression was localized
exclusively to the endoderm, in a gradient that is high anteriorly and
low posteriorly. These reciprocal gradients of pdx1 and cdx4
expression were non-overlapping until ~16hpf (Fig. 1C-J and data
not shown). At this stage, a small number of scattered cdx4-positive
cells were found within the domain of pdx1-positive cells and this
number increased at 18hpf.
Cdx4 functions in endodermal AP patterning
The cdx4 gene plays an important role in regionalization of neural
ectoderm and mesoderm. We hypothesized that this gene might
similarly be important for endoderm regionalization. As cdx4
expression is localized to the posterior part of the embryo (Joly et
al., 1992), we postulated that it might play a role in setting the
boundary between the posteriorly located intestine and the foregut.
To explore this possibility, we compared various foregut markers in
wild-type embryos and in kgg (cdx4) mutants (Figs 2, 3). In situ
hybridization analysis of markers for pancreatic ␤-cells (insulin), ␣cells (glucagon) and ␦-cells (somatostatin 1), as well as a general
marker of endocrine pancreas cells (islet1), demonstrated that the
entire endocrine pancreas is shifted posteriorly in kgg mutants (Fig.
2A-D, Fig. 3 and data not shown). Interestingly, mutants did not
express the ␦-cell marker somatostatin 2 (sst2) at 48hpf (data not
shown), although by 72hpf we detected two embryos (of 17) with
five and eight sst2-positive cells, respectively, versus ~20 cells in
wild types (data not shown). Markers for the exocrine pancreas
(trypsin) and the liver (cebpa) were also shifted posteriorly in kgg
mutants, as was expression of pdx1 (Fig. 2E-L). In addition to
showing a posterior shift in expression, the mutant pdx1 domain was
expanded anteroposteriorly, typically lying adjacent to somites 1-7
DEVELOPMENT
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Cdx4 and pancreas development
RESEARCH ARTICLE
921
Fig. 1. cdx4 is expressed in the endoderm during
zebrafish pancreas development. (A-B⬘) Dashed
lines in A,B indicate approximate plane of section in
A⬘,B⬘. Arrow indicates endoderm. (C-J) Locations of
expression domains determined by photographing
each embryo under brightfield for either pdx1 or
cdx4 expression and under fluorescence to reveal
myosin expression in the somites. Images were
merged (not shown) to determine the AP location of
pdx1 or cdx4 expression. (C-F) Dorsal views of pdx1
expression. Brackets indicate somite level.
(G-J) Lateral views of cdx4 expression. Bracket
indicates somite adjacent to the anterior-most region
of the cdx4 domain. To image endodermal cdx4
expression, the paraxial and lateral plate mesoderm
were removed. Magnification: 200⫻ in C-J.
Cdx4 is required for proper ␤-cell localization
Next, we examined the ␤-cell location defect in more detail. We
used insulin as a marker of differentiated ␤-cells, and began our
analysis at 16hpf, shortly after the onset of expression (Biemar et al.,
2001). In wild-type embryos, we found that insulin was initially
expressed in bilateral domains in the anterior trunk, as previously
reported (Biemar et al., 2001; Kim et al., 2005). Using anti-myosin
antibody to visualize somites, we found that ␤-cells are typically
located adjacent to somites 0-2 at 16hpf (Fig. 3A,A⬘). At subsequent
stages, the bilateral insulin domains resolved into a single, midline
domain that shifts posteriorly with time (Fig. 3B-D), such that by
48hpf the wild-type insulin domain was located adjacent to somites
4-5. We visualized the posterior movement and convergence of
individual ␤-cells in live insulin:GFP transgenic embryos (Huang
et al., 2001) using time-lapse imaging (data not shown). This
analysis confirmed that the ␤-cells undergo movement [as
previously described (Kim et al., 2005)].
In kgg mutants, the insulin expression domain was located more
posteriorly than in wild types at each stage (Fig. 3I-L), such that by
48hpf the mutant expression domain was adjacent to somites 6-7.
This domain is shifted relative to the wild-type insulin domain by
two somite widths. As with wild types, time-lapse imaging of kgg
mutants transgenic for insulin:GFP confirmed that the posterior
shift of insulin expression is caused by ␤-cell movement (data not
shown). We further found that most siblings of kgg homozygous
mutant embryos express insulin in an intermediate location relative
to wild types and homozygous mutants from 19hpf onwards. PCR
genotyping of these siblings confirmed that kgg heterozygous
embryos exhibit a gene dosage effect (Fig. 3F-H and data not
shown). However, at 16hpf, the gene dosage effect is not yet
apparent; at this stage, we could not detect any consistent difference
in AP location of insulin expression between wild type and siblings
(P=0.3075, ␹2 test for trend). By contrast, the posterior shift in the
homozygous mutant insulin domain was detectable as early as
16hpf. Statistical analysis revealed a significant difference in the AP
location of the insulin domain, with kgg mutant ␤-cells showing a
trend for being clustered more posteriorly compared with both wild
types (P=0.0039) and siblings (P=0.0006, ␹2 test for trend). In
summary, we conclude that cdx4 is required to correctly localize ␤cells and functions in a dosage-dependent manner.
Cdx4 modulates ␤-cell number and midline
convergence
In addition to determining the location of the pancreas along the AP
axis, we also quantitated the size of the insulin expression domain.
At 16hpf, the size and location of the insulin expression domain are
more variable than at later timepoints. We could not detect a
statistically significant difference in domain size between kgg
mutants and wild types and could not designate a modal domain
DEVELOPMENT
at 19hpf. By contrast, the wild-type pdx1 domain at 19hpf typically
extended from just anterior to the first somite, to somite 4 (Fig. 2,
compare G-J). In 50% (17/34) of mutants, the pdx1 domain was
bifurcated posteriorly (Fig. 2H).
In contrast to the posterior shift of foregut gene markers, the
expression domain of pharyngeal endoderm marker pax9 was
unaltered in kgg mutants, indicating that this more-anterior region
of the endoderm is unaffected (see Fig. S1 in the supplementary
material). We hypothesized that the expanded mutant endoderm
between the pax9-positive pharynx and the intestine would be
esophageal; histology of the kgg larval digestive tract supports that
endoderm anterior to the intestine is esophageal in nature, whereas
the epithelium immediately posterior to the fifth gill arch has
ambiguous identity (see Fig. S2 in the supplementary material).
Additionally, at 7 days post-fertilization (dpf), kgg larvae have a
posteriorly shifted esophageal-intestinal junction that lies adjacent
to somites 5-6. By contrast, in 7dpf wild types this junction is
adjacent to somites 2-3, as recently reported for 4dpf larvae
(Muncan et al., 2007). Together, these data suggest that Cdx4
function is required to correctly localize the foregut.
922
RESEARCH ARTICLE
Development 135 (5)
mutants, only 33% of embryos showed a single cluster of ␤-cells by
19hpf (Fig. 3J, Fig. 4C), and bilateral domains of insulin were still
evident at 24hpf (29%). We conclude that cdx4 is also required for
the normal temporal and spatial pattern of ␤-cell convergence.
size. Fig. 3A,E,I show representative insulin expression patterns at
this stage, but do not necessarily show the most common expression
pattern. For example, the kgg mutant insulin domain had an equal
likelihood of being one, two or three somite widths in size.
Comparisons of the domain sizes of all three genotypes at 16hpf
revealed no statistically significant difference in AP length
(P=0.411, ␹2 test) or in ␤-cell number. By contrast, at 19 and 24hpf,
we observed that the kgg mutant insulin domain was larger in the AP
axis than that in heterozygotes or wild types (summarized in Fig.
4A). Cell counts, performed on clutchmates that had been fixed
simultaneously to ensure stage-matched embryos, confirmed that the
kgg mutants had more ␤-cells than their siblings at these timepoints
(Fig. 4B). Thus, the differences in ␤-cell number and domain
location observed in mutants are not apparent until several hours
after the first appearance of insulin expression. These results suggest
that cdx4 plays a role in the proliferation or specification (or both)
of ␤-cells. Additionally, we observed that mutant embryos showed
delayed convergence of ␤-cells (Fig. 4C). For wild-type and
heterozygous embryos, the majority showed a single midline cluster
of ␤-cells at 19hpf, indicating that the initially bilaterally located
clusters of ␤-cells normally converge to the midline within a few
hours following the onset of insulin expression. By contrast, in kgg
Altered cell proliferation is not the primary cause
of the kgg pancreatic phenotype
Next we attempted to address the mechanism by which Cdx4
modulates ␤-cell number. Experiments shown in Fig. 2G-J
demonstrated that the pdx1-positive field is expanded in kgg mutants
during early pancreas development. As pdx1-positive cells include
the pancreas progenitors, this suggests that ␤-cells might be more
numerous in mutants either due to increased specification or to
increased proliferation of the precursors. Additionally, ␤-cells might
also be expanded owing to increased proliferation of the ␤-cells
themselves (Brennand et al., 2007). To begin to distinguish between
DEVELOPMENT
Fig. 2. Foregut gene expression is shifted posteriorly in cdx4–/–
zebrafish embryos. (A,B) glucagon (gcga) expressed by ␣-cells in
endocrine pancreas. (C,D) somatostatin 1 (sst1) expressed by ␦-cells in
endocrine pancreas. (E,F) trypsin (tryp) expressed by acinar cells in
exocrine pancreas. (G-J) pdx1 expressed by pancreatic precursors and
small-intestine precursors. (K,L) cebpa expressed in liver. Numbers
indicate somites. Dorsal views. Magnification: 100⫻, except G-J at
200⫻.
Cdx1a functions redundantly with Cdx4 to
establish the pancreatic domain
Studies on cdx1a function in the ectoderm and mesoderm have
established that deficiency in both Cdx1a and Cdx4 results in a more
severe phenotype than loss of Cdx4 alone (Davidson and Zon, 2006;
Shimizu et al., 2005; Shimizu et al., 2006). We therefore tested
whether cdx4 cooperates with cdx1a during pancreas development.
Morpholino (MO) knockdown of cdx1a had no overt effect on
endoderm patterning (data not shown), similar to findings in other
germ layers. We next asked whether Cdx1a deficiency in a kgg
background results in a more severe pancreas phenotype than loss
of Cdx4 alone.
We used Cdx1a-MO to knockdown cdx1a expression in kgg
mutants and siblings. We found that in double-deficient embryos at
19hpf and 24hpf, the pdx1 domain is further expanded, and is more
posteriorly located, than in embryos deficient in Cdx4 only
(summarized in Fig. 4D and compare Fig. 5B,D with Fig. 2H,J).
cdx1a knockdown had a similar, though less dramatic, effect on kgg
siblings (compare Fig. 5A,C with Fig. 2G,I). Next, we examined the
effect of Cdx double-deficiency on ␤-cell development, using insulin
expression to determine AP location of ␤-cells. Interestingly, cdx1a
knockdown in kgg mutants caused the insulin domain to expand
anteriorly, but not posteriorly, compared with kgg mutants at 19 and
24hpf (Fig. 5F,H and compare Fig. 4A,D). The anterior expansion of
insulin expression in Cdx double-deficient embryos suggests that ␤cells differentiate adjacent to anterior somites as in wild-type embryos,
but are delayed in moving posteriorly. As with pdx1 expression, there
was a similar, but less dramatic, effect on insulin expression in Cdx1adeficient kgg siblings (Fig. 5E,G and Fig. 4A,D).
Because the pdx1 domain was further expanded in the AP axis in
response to Cdx double-deficiency, we counted insulin-positive cells
to determine whether there was a corresponding increase in ␤-cell
number. We saw no significant increase in ␤-cell number at 19hpf,
for either kgg siblings or mutants with cdx1a knockdown, relative to
␤-cell number in embryos deficient in Cdx4 only (Fig. 4, compare
B with E). However, by 24hpf, there was a dramatic increase in ␤cell number for Cdx1a-deficient kgg mutants (Fig. 4E). Cell counts
and statistical analysis (Table 1) revealed that embryos deficient in
both Cdx4 and Cdx1a have a greater increase in ␤-cell number than
embryos lacking Cdx4 alone. Additionally, the data show the
importance of Cdx dosage from 24hpf, with ␤-cell number
increasing as more Cdx expression is lost or knocked down. Finally,
cdx1a knockdown resulted in a further delay in convergence of ␤cells to form the islet (Fig. 4F). In summary, Cdx1a and Cdx4
function in concert to control the position and size of the pancreas.
Cdx4 and pancreas development
RESEARCH ARTICLE
923
these possibilities, we labeled proliferative endodermal cells with
BrdU, and labeled ␤-cells with an anti-insulin antibody. Our doublelabeling strategy revealed that mature ␤-cells, detected by the insulin
antibody at 24hpf, were not co-labeled by BrdU (n=13; data not
shown), suggesting that proliferation of ␤-cells at this
developmental stage is absent or rare. The insulin antibody did not
label ␤-cells at 19hpf, indicating that the translated product is below
detection level. In general, our BrdU labeling revealed extremely
limited proliferation of cells within the pre-pancreatic field, with no
obvious differences between wild-type and mutant embryos (data
not shown). We conclude that although proliferation may be subject
to Cdx4 regulation, this cannot be the primary mode through which
the cdx4 gene limits ␤-cell number.
Cdx4 functions within the endoderm to localize
the pancreas
As we found that cdx4 is expressed in the endoderm during
timepoints critical for pancreas specification and development, we
asked whether Cdx4 functions in the endoderm to localize the
pancreas. These experiments relied on a cell transplantation
approach to target (or exclude) reagents specifically to (or from) the
endoderm (Stafford et al., 2006). This approach utilizes Sox32,
which is necessary and sufficient to specify endoderm (Kikuchi et
al., 2001; Sakaguchi et al., 2001). We previously demonstrated that
cell transplantations from Sox32-expressing donors can restore the
endoderm, including insulin expression, of hosts injected with
Sox32 morpholino (Sox32-MO) (Stafford et al., 2006). Here we
performed additional control experiments to test whether
endodermal cell transplantation restores insulin expression in the
correct AP location. We blocked endoderm development in wildtype hosts using Sox32-MO, transplanted endodermal precursors
from sox32 mRNA-expressing donors, and allowed the chimeras to
develop to 24hpf. Then we probed for insulin expression and
determined the AP location of the domain. Similar to unmanipulated
embryos, the insulin domain was located either adjacent to somite
2-4 (n=3) or somite 3-4 (n=2), indicating a normal ␤-cell location
(data not shown). However, development might be slightly delayed,
as suggested by the embryos that expressed insulin adjacent to
somite 2-4, which is consistent with a more immature islet.
To test whether Cdx4 functions in the endoderm, host embryos
deficient in Sox32 received endoderm from donors injected with a
fluorescein-dextran lineage tracer, Cdx4 MO and sox32 mRNA
(schematized in Fig. 6A). These chimeric embryos develop with
normal gross morphology, and combine host-derived wild-type
mesoderm and ectoderm with donor-derived FITC-labeled Cdx4deficient endoderm. Chimeric embryos were raised to 24hpf and
DEVELOPMENT
Fig. 3. Cdx4 localizes the pancreas in a dosage-dependent fashion. (A-L) In situ hybridization for insulin expression (purple), photographed in
brightfield. A,E,I additionally show krox20 expression in rhombomere 5 and neural crest cells (arrows). (A⬘-C⬘,E⬘-G⬘,I⬘-K⬘) Myosin antibody staining
(green fluorescence) to reveal somites. Brightfield and fluorescence images were merged for analysis and are shown separately for clarity. Bracket
indicates the somite adjacent to the posterior limit of insulin expression. Arrowheads in I indicate posterior ectopic krox20-positive neural crest cells
(out of focus) in 16hpf mutants. The asterisk in K indicates the boundary between somite 6 and 7. Dorsal views, anterior to the left. Magnification:
100⫻ for 16hpf panels; all others 200⫻.
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RESEARCH ARTICLE
Development 135 (5)
probed for insulin expression. We found that when only the
endoderm is Cdx4-deficient, ␤-cells are localized in unusually
posterior positions (n=6/9; e.g. Fig. 6B). Our criteria were that the
insulin domain spanned at least three somite widths and extended
posteriorly to somite 5 or further, consistent with the insulin pattern
observed in kgg mutant embryos lacking Cdx4 in all three germ
layers. Three chimeric embryos had insulin expression adjacent to
somites 4-5, and we considered these pancreases to be in a ‘wildtype’ location, as wild-type ␤-cells can occasionally be found
adjacent to somites 4-5 at 24hpf (data not shown). However, it
should be noted that cdx4 heterozygotes typically express insulin
adjacent to somites 4-5 at 24hpf.
We also employed an alternative technique to target Cdx4 MO
to the endoderm. We injected one cell of a wild-type embryo at
the 32-cell stage with FITC, Cdx4 MO and sox32 mRNA, and
grew the embryos to 24hpf. In those specimens in which the
FITC label was confined to the endoderm, we found that most
embryos had insulin expression that resembled the mutant
pattern (n=17/23), consistent with our cell transplantation data.
Embryos typically expressed insulin at somites 3-6 or 4-6,
although some expressed insulin in much larger domains, for
example from somites 1-6. Four embryos had ␤-cells adjacent to
somites 4-5, and we conservatively scored these as ‘wild-type’,
as above.
DEVELOPMENT
Fig. 4. Cdx4 has roles in determining ␤-cell localization, cell number and in regulating ␤-cell convergence to the midline during early
pancreatogenesis. (A) Summary of Fig. 3 data showing average (modal) insulin domain locations for post-16hpf timepoints relative to somite
number (shown as numbered boxes). mut, kgg mutants (cdx4–/–); sib, siblings of kgg mutants, including both heterozygotic and wild-type (wt)
zebrafish clutchmates. (B) The number of ␤-cells increases more rapidly in cdx4–/– embryos than in siblings (mean±s.d.). (C) Midline convergence of
␤-cells is delayed in cdx4–/– embryos. y-axis indicates percentage of embryos in which ␤-cells have converged to the midline. (D) Summary of Fig. 5
data showing modal insulin and pdx1 domain locations for Cdx1a-MO-injected kgg mutants and siblings. (E) ␤-cell number increases further in
Cdx1a-MO-injected kgg siblings and mutants (mean±s.d.). (F) Midline convergence is further delayed in Cdx1a-MO-injected kgg siblings and
mutants. y-axis indicates percentage of embryos in which ␤-cells have converged to the midline. Note than in E and F, mutant and sibling data from
B and C are included for ease of comparison. Sample sizes for A-C were as follows. Wild type (wt): 16hpf, n=38; 19hpf, n=36; 24hpf, n=60; 48hpf,
n=17. Siblings (sib): 16hpf, n=38; 19hpf, n=82 for A and C, n=45 for B; 24hpf, n=80 for A and C, n=40 for B; 48hpf, n=33. Mutant (mut): 16hpf,
n=23; 19hpf, n=30; 24hpf, n=24; 48hpf, n=23. Sample sizes for D-F were as follows. insulin data, siblings+Cdx1a-MO (sib+MO): 19hpf, n=24;
24hpf, n=16. Mutant+Cdx1a-MO (mut+MO): 19hpf, n=22; 24hpf, n=10. Sample sizes for D were as follows. pdx1 data, wild type: 19hpf, n=22;
24hpf, n=9. Siblings+Cdx1a-MO (sib+MO): 19hpf, n=15; 24hpf, n=17. Mutant+Cdx1a-MO (mut+MO): 19hpf, n=16; 24hpf, n=20.
Cdx4 and pancreas development
RESEARCH ARTICLE
925
Table 1. ␤-cell number comparisons between Cdx phenotypes
Comparisons
mut vs sib
sib vs sib+MO
sib vs mut+MO
mut vs sib+MO
mut vs mut+MO
mut+MO vs sib+MO
19 hpf
n
24 hpf
n
P<0.001
NS
P<0.001
P<0.05
NS
P<0.001
30, 45
45, 24
45, 22
30, 24
30, 22
22, 24
P<0.001
P<0.05
P<0.001
NS
P<0.001
P<0.001
24, 40
40, 16
40, 10
24, 16
24, 10
10, 16
Two-way ANOVA and Bonferroni post-tests.
MO, Cdx1a morpholino.
NS, not significant.
Reciprocal experiments were performed in which host embryos
deficient in both Cdx4 and Sox32 received Cdx4-positive donor
endoderm (schematized in Fig. 6C). These embryos show typical
kgg mutant gross morphology, and combine host-derived Cdx4deficent mesoderm and ectoderm, with donor-derived FITC-labeled
Cdx4-positive endoderm. We found that in these specimens, in
which only the endoderm cells are Cdx4-positive, ␤-cells are
correctly localized (n=4/4; e.g. Fig. 6D).
To further test whether cdx4 functions only in the endoderm to
localize the pancreas, we performed transplantation experiments to
manipulate Cdx4 expression in paraxial mesoderm. In these
experiments, cells were transplanted to the paraxial mesoderm by
placing donor-derived cells close to the host blastoderm margin,
again as previously described (Stafford et al., 2006). Transplanted
cells contributed to at least five somites in the anterior trunk on one
side of the chimeric embryos. In chimeras combining wild-type host
cells with donor-derived Cdx4-deficient paraxial mesoderm, the ␤cells showed a wild-type location at 24hpf (n=16/16, data not
shown). Similarly, in chimeric embryos combining Cdx4-deficient
host cells with donor-derived wild-type paraxial mesoderm, the ␤cells showed a kgg mutant location at 24hpf (n=4/4, data not shown).
Taken together, these transplantation experiments suggest that Cdx4
functions directly within the endoderm to localize the pancreas.
Finally, to test whether Cdx4 has the capacity to confer posterior
fate directly to the endoderm, we generated embryos overexpressing
cdx4 mRNA throughout this germ layer. Our expectation was that
overexpression of Cdx4 would posteriorize more-anterior endoderm
structures. We transplanted FITC-labeled endoderm cells from
embryos previously injected with cdx4 mRNA to endoderm-
Cdx4 maintains posterior endodermal identity
Previous work has demonstrated that RA is necessary and sufficient
to specify the pancreas in vertebrates (Chen et al., 2004; Martin et
al., 2005; Molotkov et al., 2005; Stafford et al., 2004; Stafford and
Prince, 2002). When wild-type zebrafish embryos are treated with
RA, endodermal cells express insulin throughout the anterior
endoderm, ectopic to the normal expression domain (Stafford and
Prince, 2002). Additionally, expressing a dominant active RA
receptor throughout the endoderm also produces anterior ectopic
insulin expression (Stafford et al., 2006). Interestingly, neither of
these manipulations is sufficient to induce posterior ectopic insulin.
We therefore hypothesized that posterior cdx4-positive endoderm is
not competent to respond to RA signaling. To test this, we combined
cdx4 knockdown with RA treatments. Uninjected and Cdx4 MOinjected specimens were treated with RA for 1-hour intervals during
gastrulation stages, and assayed for insulin expression at 24hpf.
Uninjected RA-treated embryos expressed ectopic insulin only in
anterior domains, as previously reported (Fig. 7A) (Stafford and
Prince, 2002). By contrast, in Cdx4-deficient RA-treated embryos
we observed a small number of ectopic insulin-expressing cells well
posterior to the expected location of the pancreas, in addition to
ectopic anterior insulin. (Fig. 7B,C). We observed more ectopic ␤cells, located further towards the posterior, in those embryos treated
at earlier stages (Fig. 7D). We conclude that the normal posterior
expression of Cdx4 functions to prevent posterior endodermal
precursors from responding to RA signaling.
DISCUSSION
cdx4 is required to correctly localize foregut
organs
Cdx genes play a role in AP patterning of all three germ layers. Lossand gain-of-function studies have demonstrated that disruption of
Cdx1 and/or Cdx2 expression in mice results in homeotic
transformations along the axial skeleton (Subramanian et al., 1995;
van den Akker et al., 2002). Similarly, Cdx1 and Cdx2 overexpression
DEVELOPMENT
Fig. 5. Cdx4 and Cdx1a function redundantly. (A-D) The pdx1
domain is further expanded in zebrafish kgg mutants and siblings
following morpholino (MO) knockdown of cdx1a. (E-H) The insulin
domain is further expanded, and ␤-cells show a delay in converging to
form the islet, following cdx1a knockdown in kgg mutants and siblings.
Numbers indicate somites. Dorsal views. Magnification: 200⫻.
deficient hosts (schematized in Fig. 6E). In the resultant chimeras,
Cdx4 is expressed in anterior endoderm, where cdx4 transcripts are
not normally detected. As predicted, we found that in these embryos
insulin expression was shifted anteriorly by 2 to 3 somites at 24hpf
(n=6/9; Fig. 6F). Of the three chimeras that expressed insulin in the
wild-type location, adjacent to somites 3-4, two expressed insulin in
anterior trunk locations as well, indicating a partial shift. In a single
chimeric specimen raised to 48hpf, imaging of the gut:GFP
transgene revealed that the entire pancreas (endocrine and exocrine),
as well as the intestinal bulb and liver bud, was shifted anteriorly
(data not shown). We conclude that endodermal cdx4 functions
similarly to Cdx genes in other germ layers: namely, that
overexpression in anterior domains causes posteriorization of fates.
926
RESEARCH ARTICLE
Development 135 (5)
Fig. 6. Cdx4 functions in the endoderm to localize the
pancreas. (A,C,E) Cell transplantation strategy. Donor and
host zebrafish embryos were injected at the one-cell stage
with the indicated reagents. At the sphere stage, donor cells
were transplanted to host blastoderm margin. (B) cdx4
knockdown in endoderm is sufficient to shift the pancreas
posteriorly. (D) Wild-type endoderm rescues the pancreas
location in a cdx4-knockdown host. (F) Cdx4 overexpression
in endoderm results in insulin expression anterior to the
normal pancreatic domain. Embryos at 24hpf, dorsal view.
Magnification: 100⫻.
cdx4 is required to limit ␤-cell number and has a
role in midline convergence
Examination of insulin expression in cdx4 mutant embryos revealed
that at 19hpf, shortly after the onset of expression, the insulin
domain is abnormally expanded along the AP axis. This expansion
is characterized by an increase in the number of insulin-positive ␤cells in cdx4 mutant embryos compared with wild type. As our cell
proliferation analysis did not reveal any obvious differences in
proliferation rates between wild-type and kgg mutant embryos, we
suggest that the primary role for cdx4 is in limiting the specification
of ␤-cells. Interestingly, at 19hpf, the size of the pdx1 expression
domain, which labels pancreatic and intestinal precursors, is also
significantly expanded in mutants, suggesting an expanded
progenitor pool. The early excess of ␤-cells might be at the expense
of ␦-cells, as we could not detect somatostatin 2 expression in
mutants until 72hpf, at which time a small number of positive cells
were detected in a few embryos. In the pancreas, subsets of ␦-cells
have been reported, with some cells expressing both somatostatins
and others expressing only one (Devos et al., 2002). However,
because the number of somatostatin 1-positive ␦-cells is increased,
as are numbers of other differentiated endocrine pancreas
derivatives, it is more likely that the field of endocrine precursors is
generally expanded in the absence of Cdx4 function.
The excess ␤-cell number in kgg mutants at 19 and 24hpf is
associated with a delay in midline convergence of these cells. This
is consistent with a similar report of a midline convergence delay for
angioblasts in kgg mutants (Davidson et al., 2003). Both cell number
and midline convergence may be modulated by Cdx4, which is a
direct Wnt target (Pilon et al., 2006). Wnts are involved in both cell
proliferation and convergence movements (Clevers, 2006)
(reviewed by Torban et al., 2004), and Wnt signaling is implicated
in midline convergence of foregut precursors (Kim et al., 2005;
Matsui et al., 2005).
cdx4 functions within the endoderm during
pancreas development
We have used cell transplantation experiments to demonstrate that
cdx4 functions within the endoderm to localize the pancreas.
Additionally, we showed that overexpression of cdx4 throughout the
endoderm shifts the pancreas anteriorly. This is consistent with Cdx4
function in other vertebrates. Previous studies in chick showed that
FGF treatment resulted in an anterior shift in Cdx-B (chick Cdx4),
which in turn resulted in anterior shifts in downstream endoderm
gene expression (Dessimoz et al., 2006). Similarly, overexpressing
Cdx4 in mice resulted in anteriorly shifted Hoxb8 expression in the
neural tube and somites (Charite et al., 1998). We found that
overexpression of cdx4 in endoderm shifted the pancreas anteriorly,
such that insulin was expressed anterior to the somites or adjacent
to the first somite at 24hpf. This anteriorly localized insulin
DEVELOPMENT
produces intestinal homeosis in mice (Beck et al., 1999;
Chawengsaksophak et al., 1997; Mutoh et al., 2004; Silberg et al.,
2002). Recently, studies in zebrafish have shown that deficiency of
cdx4 results in a posteriorly shifted hindbrain/spinal cord boundary
(Shimizu et al., 2006; Skromne et al., 2007), as well as a posterior shift
in the boundary between anterior angioblasts and posterior
hematopoietic progenitors (Davidson et al., 2003). Here, we have
shown that cdx4 has a role in establishing the AP location of the
foregut. In loss-of-function studies, we show that cdx4-deficient
embryos have posteriorly shifted foregut organs, including the
endocrine and exocrine pancreatic buds, the liver and the intestine.
Examination of ␤-cell location during the first 48 hours of pancreas
development revealed that there is a gene dosage effect on islet
location by 19hpf. Specifically, at 48hpf, the wild-type islet is located
adjacent to somites 4-5; with loss of one cdx4 copy the islet shifts
posteriorly by one somite, and with loss of two copies the islet shifts
posteriorly by two somites so that it lies adjacent to somites 6-7 at
48hpf. The intermediate phenotype in heterozygotes is consistent with
previous studies of mouse Cdx genes that showed dosage effects for
axial skeletal patterning and for intestinal patterning (Beck et al., 1999;
Chawengsaksophak et al., 1997; Mutoh et al., 2004; Subramanian et
al., 1995; van den Akker et al., 2002). A gene dosage effect for cdx4
has not been previously reported in zebrafish.
We observed a posterior shift for all foregut organ markers tested,
including somatostatin 1, glucagon, islet1, trypsin, cebpa and pdx1,
indicating that cdx4 has a role in setting the posterior boundary of the
foregut. Interestingly, a similar 2-somite posterior shift of ectoderm
marker gene expression, including neuronal markers and Hox genes,
was recently reported in cdx4-deficient embryos (Skromne et al.,
2007). Disruptions in Cdx1 and Cdx2 in mice are known to cause
shifts in Hox domains in the mesoderm, with concomitant homeotic
transformations of the vertebrae (Subramanian et al., 1995; van den
Akker et al., 2002). In zebrafish, loss of cdx4 again produces a 2somite posterior shift in Hox expression domains in the mesoderm (I.
Skromne, personal communication). It is likely that endodermal Hox
domains are also shifted posteriorly when cdx4 is lost. Future studies
will determine the timing and location of specific Hox expression in
zebrafish endoderm.
Fig. 7. Retinoic acid elicits posterior insulin expression in cdx4
morphants. (A-C) Zebrafish embryos treated at shield stage and fixed
at 24hpf. Brackets indicate the normal position of the pancreas in
untreated wild-type and cdx4 morphant embryos. (D) Percentage of
embryos expressing posterior ectopic insulin in response to RA
treatment plus cdx4 knockdown. Average cell numbers (±s.d.) by
treatment timepoint were as follows: 4.3hpf, 3.4±1.9; 5.3hpf, 2.6±1.7;
6hpf, 2.3±1.8; 7hpf, 1.7±0.6; 8hpf, 1.4±0.5. Sample sizes for RAtreated embryos, without Cdx4 MO injection, were a minimum of 25
embryos per timepoint. Bars represent the combined results of two
independent experiments. Dorsal views, anterior to left. Magnification:
100⫻.
expression closely resembles the wild-type location of newly
differentiated ␤-cells at 16hpf. This suggests that in the cdx4
overexpression chimeras, ␤-cells are specified normally but fail to
move posteriorly owing to their location in an environment of
already high Cdx4 expression. Our expression analysis showed that
cdx4 is expressed in posterior endoderm and excluded from anterior
foregut during the earliest stages of pancreas development, prior to
the onset of insulin expression. Subsequently, cdx4 is expressed at
low levels more anteriorly, in a salt-and-pepper pattern, throughout
much of the pdx1-positive (and insulin-positive) domain. We
suggest that in cdx4 overexpression chimeras, the newly
differentiated ␤-cells fail to move posteriorly because they interpret
their position as already being in the posterior of the foregut.
Cdx4 and Cdx1a function redundantly during
pancreas development
Our studies on Cdx1a/Cdx4-deficient embryos revealed that Cdx
genes function redundantly to localize the pancreas and modulate ␤cell number. At 24hpf, a Cdx dosage effect on ␤-cell number was
observed, such that cell number increased as Cdx dosage decreased,
suggesting that these two genes exhibit partial functional
redundancy. A similar Cdx dosage effect was reported for
mesoderm-derived blood cell precursors in the intermediate cell
mass (Davidson and Zon, 2006). Whereas Cdx1a-deficient kgg
mutants have a pdx1-positive domain that is expanded posteriorly
compared with kgg mutants, the ␤-cell location expands anteriorly
rather than posteriorly. As reducing Cdx1a activity did not allow the
pancreatic islet to shift further posterior than in the cdx4/kgg mutant,
we suggest that additional mechanisms operate in the posterior of
the embryo to control the location of the pancreas. Although a third
Cdx gene, cdx1b (Mulley et al., 2006), has been described for
zebrafish, our preliminary data show that this gene is not expressed
during the first 48 hours of development (M.D.K., M.R.A. and
V.E.P., unpublished), making it an unlikely candidate to modulate
RESEARCH ARTICLE
927
pancreas position. We therefore suggest that Cdx-independent
mechanisms also play a role in establishing the posterior limit of the
pancreas.
During gastrulation, cdx1a is expressed in marginal cells and
becomes restricted to the posterior tailbud during somitogenesis, but
expression in the endoderm earlier than 48hpf has not been reported
(Davidson and Zon, 2006). We were unable to detect endodermal
transcripts between the 5-somite stage and 24hpf (our unpublished
results), stages when critical steps in AP patterning of the endoderm
are taking place. Thus, whereas the endoderm expresses cdx4, and
our cell transplantations demonstrate that cdx4 functions within the
endoderm to localize the pancreas, cdx1a is likely to function cellnon-autonomously within adjacent mesoderm. Interestingly, Cdx1adeficient kgg mutants show an anterior expansion of ␤-cells
compared with kgg mutants. This suggests that in addition to the
endodermal role of Cdx4, Cdx1a and Cdx4 might function together
within the mesoderm to further refine ␤-cell number and location.
The mesoderm is a source of various signals that pattern the
endoderm; the expanded domain of pdx1-positive precursors and
insulin-positive cells in cdx1a/cdx4-deficient embryos is consistent
with a model in which Cdx deficiency alters expression of key
mesodermal signals.
cdx4 prevents insulin expression in posterior
endoderm
We have established that RA signaling from anterior paraxial
mesoderm is required for pancreas specification, which requires
precise control of the RA signals generated in the mesoderm and
received by the endoderm (Stafford et al., 2006). However, the RA
synthesis enzyme Raldh2 (also known as Aldh1a2 – ZFIN) is
expressed along the trunk mesoderm in domains that extend
posterior to the pancreatic domain (Begemann et al., 2001; Grandel
et al., 2002), and RA receptors are expressed throughout the
posterior endoderm (Waxman and Yelon, 2007). It is thus likely that
the RA-degrading Cyp26 enzymes are also important for
modulating RA signals during pancreas development. Both Raldh2
and Cyp26a1 are regulated by Cdx factors, and their expression
domains are shifted posteriorly in response to Cdx1a/Cdx4
deficiency during early somitogenesis (Shimizu et al., 2006). Such
shifts might underlie the subsequent shift in foregut expression
markers that we have observed.
Additionally, we have shown that Cdx4 has a role in preventing
insulin expression in posterior endoderm. cdx4 morphant embryos
treated with RA responded by expressing insulin throughout the AP
extent of the endoderm, including regions posterior to the trunk and
notochord. These results are consistent with a model in which high
levels of cdx4 expression in the posterior renders endodermal cells
unable to respond to RA signals, thus maintaining a posterior
identity. Interestingly, early-stage RA treatments proved most
effective at producing posterior insulin-expressing cells, perhaps
suggesting that at later stages additional mechanisms block RA
signaling in the posterior. Our RA-treatment experiments do not
distinguish between models in which RA acts directly on posterior
endoderm to specify ␤-cells, versus anteriorly specified ␤-cells
moving too far posteriorly into the cdx4-deficient environment.
However, as insulin-positive cells are located many somite widths
posterior to the normal location, we favor the first interpretation.
Although cdx4 prevents insulin expression in posterior endoderm,
our cdx4-mRNA overexpression experiments demonstrated that
anteriorly expressed cdx4 is nevertheless compatible with ␤-cell
differentiation in the anterior-most region of the trunk. This can be
attributed to the fact that cdx4 is normally expressed in a posterior-
DEVELOPMENT
Cdx4 and pancreas development
RESEARCH ARTICLE
to-anterior gradient, with a low expression level in the
foregut/pancreatic domain of wild-type embryos. In the
overexpression assay, the level of ectopic cdx4 expression in the
anterior-most trunk is likely to be consistent with the low expression
level observed in the wild-type foregut, and thus is compatible with
␤-cell differentiation in this context.
We have shown that Cdx4 is a crucial factor in localizing the
pancreas and in limiting its size. Although the mesoderm expresses
Cdx4, it is endodermal Cdx4 that is required for localizing the
pancreas. By contrast, Cdx1a functions in mesoderm to influence
adjacent endoderm. Our work reveals that Cdx genes are important
regulators of AP patterning in all three germ layers.
We thank Can Gong and Christy Schmehl for their expert histological work.
Louis Choi, Matthew Rowe and Elizabeth Sefton provided excellent fish care
and technical support. Ru Yi Teow lent his confocal microscopy expertise. Isaac
Skromne provided invaluable insights and suggestions throughout the course
of this work and provided useful comments on the manuscript. The BrdU and
myosin monoclonal antibodies developed by Stephen J. Kaufman and Helen
M. Blau, respectively, were obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the NICHD and maintained
by The University of Iowa, Department of Biological Sciences, Iowa City,
IA 52242. This work was supported by NIH NICHD fellowship
#1F32HD050031 to M.D.K., JDRF grant #1-2003-257 to V.E.P. and NIH NIDDK
grant #DK-064973 to V.E.P.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/919/DC1
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Cdx4 and pancreas development