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First published online 30 January 2008
doi: 10.1242/dev.010660
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Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, IL 60637, USA.
* Author for correspondence (e-mail: mkinkel{at}uchicago.edu)
Accepted 15 December 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cdx4, Cdx1a, Retinoic acid, Pancreas, β-cell, AP patterning
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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 β-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 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 loss-of-function embryos, we find that pancreatic β-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 β-cells and an increase in β-cell number during early pancreatogenesis. Knockdown of a second Cdx gene, cdx1a, in cdx4-deficient 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) 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/µl and cdx4 mRNA was
injected at 84 ng/µl. 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 µm 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 µm 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 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.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
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 β-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 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).
|
).
Together, these data suggest that Cdx4 function is required to correctly
localize the foregut.
Cdx4 is required for proper β-cell localization
Next, we examined the β-cell location defect in more detail. We used
insulin as a marker of differentiated β-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 β-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 β-cells
in live insulin:GFP transgenic embryos
(Huang et al., 2001) using
time-lapse imaging (data not shown). This analysis confirmed that the
β-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 β-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 β-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 β-cells and
functions in a dosage-dependent manner.
Cdx4 modulates β-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 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 β-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 β-cells than their siblings at these
timepoints (Fig. 4B). Thus, the
differences in β-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 β-cells.
Additionally, we observed that mutant embryos showed delayed convergence of
β-cells (Fig. 4C). For
wild-type and heterozygous embryos, the majority showed a single midline
cluster of β-cells at 19hpf, indicating that the initially bilaterally
located clusters of β-cells normally converge to the midline within a few
hours following the onset of insulin expression. By contrast, in
kgg mutants, only 33% of embryos showed a single cluster of
β-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
β-cell convergence.
|
;
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 β-cell development, using insulin expression to determine AP location of β-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 β-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 Cdx1a-deficient 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 β-cell number. We saw no significant increase in β-cell number at 19hpf, for either kgg siblings or mutants with cdx1a knockdown, relative to β-cell number in embryos deficient in Cdx4 only (Fig. 4, compare B with E). However, by 24hpf, there was a dramatic increase in β-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 β-cell number than embryos lacking Cdx4 alone. Additionally, the data show the importance of Cdx dosage from 24hpf, with β-cell number increasing as more Cdx expression is lost or knocked down. Finally, cdx1a knockdown resulted in a further delay in convergence of β-cells to form the islet (Fig. 4F). In summary, Cdx1a and Cdx4 function in concert to control the position and size of the pancreas.
|
). To
begin to distinguish between these possibilities, we labeled proliferative
endodermal cells with BrdU, and labeled β-cells with an anti-insulin
antibody. Our double-labeling strategy revealed that mature β-cells,
detected by the insulin antibody at 24hpf, were not co-labeled by BrdU
(n=13; data not shown), suggesting that proliferation of β-cells
at this developmental stage is absent or rare. The insulin antibody did not
label β-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 β-cell
number.
|
). 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 wild-type 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 β-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 Cdx4-deficient endoderm. Chimeric embryos were raised to 24hpf and probed for insulin expression. We found that when only the endoderm is Cdx4-deficient, β-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 `wild-type' location, as wild-type β-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.
|
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 Cdx4-deficent 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, β-cells are correctly localized (n=4/4; e.g. Fig. 6D).
|
).
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 β-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 β-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-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.
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 MO-injected 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 β-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 |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; van den Akker et al.,
2002). Similarly, Cdx1 and Cdx2 overexpression 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.
|
; 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 2-somite 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.
cdx4 is required to limit β-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 β-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 β-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 β-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 β-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 expression closely resembles the wild-type location of newly
differentiated β-cells at 16hpf. This suggests that in the cdx4
overexpression chimeras, β-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
β-cells fail to move posteriorly because they interpret their position as
already being in the posterior of the foregut.
|
).
Whereas Cdx1a-deficient kgg mutants have a pdx1-positive
domain that is expanded posteriorly compared with kgg mutants, the
β-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 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 cell-non-autonomously within adjacent mesoderm.
Interestingly, Cdx1a-deficient kgg mutants show an anterior expansion
of β-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 β-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 β-cells, versus anteriorly specified β-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 β-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-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 β-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.
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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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Argenton, F., Zecchin, E. and Bortolussi, M. (1999). Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo. Mech. Dev. 87,217 -221.[CrossRef][Medline]
Beck, F., Chawengsaksophak, K., Waring, P., Playford, R. J. and
Furness, J. B. (1999). Reprogramming of intestinal
differentiation and intercalary regeneration in Cdx2 mutant mice.
Proc. Natl. Acad. Sci. USA
96,7318
-7323.
Begemann, G., Schilling, T. F., Rauch, G. J., Geisler, R. and Ingham, P. W. (2001). The zebrafish neckless mutation reveals a requirement for raldh2 in mesodermal signals that pattern the hindbrain. Development 128,3081 -3094.[Medline]
Biemar, F., Argenton, F., Schmidtke, R., Epperlein, S., Peers, B. and Driever, W. (2001). Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet. Dev. Biol. 230,189 -203.[CrossRef][Medline]
Brennand, K., Huangfu, D. and Melton, D. (2007). All beta cells contribute equally to islet growth and maintenance. PLoS. Biol. 5, e163.[CrossRef][Medline]
Cano, D. A., Hebrok, M. and Zenker, M. (2007). Pancreatic development and disease. Gastroenterology 132,745 -762.[CrossRef][Medline]
Charite, J., de Graaff, W., Consten, D., Reijnen, M. J., Korving, J. and Deschamps, J. (1998). Transducing positional information to the Hox genes: critical interaction of cdx gene products with position-sensitive regulatory elements. Development 125,4349 -4358.[Abstract]
Chawengsaksophak, K., James, R., Hammond, V. E., Kontgen, F. and Beck, F. (1997). Homeosis and intestinal tumours in Cdx2 mutant mice. Nature 386,84 -87.[CrossRef][Medline]
Chen, Y., Pan, F. C., Brandes, N., Afelik, S., Solter, M. and Pieler, T. (2004). Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus. Dev. Biol. 271,144 -160.[CrossRef][Medline]
Clevers, H. (2006). Wnt/beta-catenin signaling in development and disease. Cell 127,469 -480.[CrossRef][Medline]
Davidson, A. J. and Zon, L. I. (2006). The caudal-related homeobox genes cdx1a and cdx4 act redundantly to regulate hox gene expression and the formation of putative hematopoietic stem cells during zebrafish embryogenesis. Dev. Biol. 292,506 -518.[CrossRef][Medline]
Davidson, A. J., Ernst, P., Wang, Y., Dekens, M. P., Kingsley, P. D., Palis, J., Korsmeyer, S. J., Daley, G. Q. and Zon, L. I. (2003). cdx4 mutants fail to specify blood progenitors and can be rescued by multiple hox genes. Nature 425,300 -306.[CrossRef][Medline]
Dessimoz, J., Opoka, R., Kordich, J. J., Grapin-Botton, A. and Wells, J. M. (2006). FGF signaling is necessary for establishing gut tube domains along the anterior-posterior axis in vivo. Mech. Dev. 123,42 -55.[CrossRef][Medline]
Devos, N., Deflorian, G., Biemar, F., Bortolussi, M., Martial, J. A., Peers, B. and Argenton, F. (2002). Differential expression of two somatostatin genes during zebrafish embryonic development. Mech. Dev. 115,133 -137.[CrossRef][Medline]
Gamer, L. W. and Wright, C. V. (1993). Murine Cdx-4 bears striking similarities to the Drosophila caudal gene in its homeodomain sequence and early expression pattern. Mech. Dev. 43,71 -81.[CrossRef][Medline]
Grandel, H., Lun, K., Rauch, G. J., Rhinn, M., Piotrowski, T., Houart, C., Sordino, P., Kuchler, A. M., Schulte-Merker, S., Geisler, R. et al. (2002). Retinoic acid signalling in the zebrafish embryo is necessary during pre-segmentation stages to pattern the anterior-posterior axis of the CNS and to induce a pectoral fin bud. Development 129,2851 -2865.[Medline]
Guo, R. J., Suh, E. R. and Lynch, J. P. (2004). The role of Cdx proteins in intestinal development and cancer. Cancer Biol. Ther. 3,593 -601.[Medline]
Hebrok, M. (2003). Hedgehog signaling in pancreas development. Mech. Dev. 120, 45-57.[CrossRef][Medline]
Ho, R. K. and Kane, D. A. (1990). Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors. Nature 348,728 -730.[CrossRef][Medline]
Huang, H., Vogel, S. S., Liu, N., Melton, D. A. and Lin, S. (2001). Analysis of pancreatic development in living transgenic zebrafish embryos. Mol. Cell. Endocrinol. 177,117 -124.[CrossRef][Medline]
Inoue, A., Takahashi, M., Hatta, K., Hotta, Y. and Okamoto, H. (1994). Developmental regulation of islet-1 mRNA expression during neuronal differentiation in embryonic zebrafish. Dev. Dyn. 199,1 -11.[Medline]
Jackman, W. R., Draper, B. W. and Stock, D. W. (2004). Fgf signaling is required for zebrafish tooth development. Dev. Biol. 274,139 -157.[CrossRef][Medline]
Joly, J. S., Maury, M., Joly, C., Duprey, P., Boulekbache, H. and Condamine, H. (1992). Expression of a zebrafish caudal homeobox gene correlates with the establishment of posterior cell lineages at gastrulation. Differentiation 50, 75-87.[CrossRef][Medline]
Kikuchi, Y., Agathon, A., Alexander, J., Thisse, C., Waldron,
S., Yelon, D., Thisse, B. and Stainier, D. Y. (2001).
casanova encodes a novel Sox-related protein necessary and sufficient for
early endoderm formation in zebrafish. Genes Dev.
15,1493
-1505.
Kim, H. J., Schleiffarth, J. R., Jessurun, J., Sumanas, S., Petryk, A., Lin, S. and Ekker, S. C. (2005). Wnt5 signaling in vertebrate pancreas development. BMC Biol. 3, 23.[CrossRef][Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Lin, J. W., Biankin, A. V., Horb, M. E., Ghosh, B., Prasad, N. B., Yee, N. S., Pack, M. A. and Leach, S. D. (2004). Differential requirement for ptf1a in endocrine and exocrine lineages of developing zebrafish pancreas. Dev. Biol. 270,474 -486.[CrossRef][Medline]
Lohnes, D. (2003). The Cdx1 homeodomain protein: an integrator of posterior signaling in the mouse. BioEssays 25,971 -980.[CrossRef][Medline]
Lyons, S. E., Shue, B. C., Lei, L., Oates, A. C., Zon, L. I. and Liu, P. P. (2001). Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes. Gene 281,43 -51.[CrossRef][Medline]
Martin, M., Gallego-Llamas, J., Ribes, V., Kedinger, M., Niederreither, K., Chambon, P., Dolle, P. and Gradwohl, G. (2005). Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice. Dev. Biol. 284,399 -411.[Medline]
Matsui, T., Raya, A., Kawakami, Y., Callol-Massot, C.,
Capdevila, J., Rodriguez-Esteban, C. and Izpisua Belmonte, J. C.
(2005). Noncanonical Wnt signaling regulates midline convergence
of organ primordia during zebrafish development. Genes
Dev. 19,164
-175.
Milewski, W. M., Duguay, S. J., Chan, S. J. and Steiner, D.
F. (1998). Conservation of PDX-1 structure, function, and
expression in zebrafish. Endocrinology
139,1440
-1449.
Molotkov, A., Molotkova, N. and Duester, G. (2005). Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232,950 -957.[CrossRef][Medline]
Mulley, J. F., Chiu, C. H. and Holland, P. W.
(2006). Breakup of a homeobox cluster after genome duplication in
teleosts. Proc. Natl. Acad. Sci. USA
103,10369
-10372.
Muncan, V., Faro, A., Haramis, A. P., Hurlstone, A. F., Wienholds, E., van Es, J., Korving, J., Begthel, H., Zivkovic, D. and Clevers, H. (2007). T-cell factor 4 (Tcf7l2) maintains proliferative compartments in zebrafish intestine. EMBO Rep. 8, 966-973.[CrossRef][Medline]
Mutoh, H., Sakurai, S., Satoh, K., Osawa, H., Hakamata, Y.,
Takeuchi, T. and Sugano, K. (2004). Cdx1 induced intestinal
metaplasia in the transgenic mouse stomach: comparative study with Cdx2
transgenic mice. Gut 53,1416
-1423.
Mutoh, H., Satoh, K., Kita, H., Sakamoto, H., Hayakawa, H., Yamamoto, H., Isoda, N., Tamada, K., Ido, K. and Sugano, K. (2005). Cdx2 specifies the differentiation of morphological as well as functional absorptive enterocytes of the small intestine. Int. J. Dev. Biol. 49,867 -871.[CrossRef][Medline]
Ober, E. A., Field, H. A. and Stainier, D. Y. (2003). From endoderm formation to liver and pancreas development in zebrafish. Mech. Dev. 120, 5-18.[CrossRef][Medline]
Oxtoby, E. and Jowett, T. (1993). Cloning of
the zebrafish krox-20 gene (krx-20) and its expression during hindbrain
development. Nucleic Acids Res.
21,1087
-1095.
Pilon, N., Oh, K., Sylvestre, J. R., Bouchard, N., Savory, J. and Lohnes, D. (2006). Cdx4 is a direct target of the canonical Wnt pathway. Dev. Biol. 289, 55-63.[CrossRef][Medline]
Prince, V. E., Moens, C. B., Kimmel, C. B. and Ho, R. K. (1998). Zebrafish hox genes: expression in the hindbrain region of wild-type and mutants of the segmentation gene, valentino. Development 125,393 -406.[Abstract]
Sakaguchi, T., Kuroiwa, A. and Takeda, H. (2001). A novel sox gene, 226D7, acts downstream of Nodal signaling to specify endoderm precursors in zebrafish. Mech. Dev. 107,25 -38.[CrossRef][Medline]
Shimizu, T., Bae, Y. K., Muraoka, O. and Hibi, M. (2005). Interaction of Wnt and caudal-related genes in zebrafish posterior body formation. Dev. Biol. 279,125 -141.[CrossRef][Medline]
Shimizu, T., Bae, Y. K. and Hibi, M. (2006).
Cdx-Hox code controls competence for responding to Fgfs and retinoic acid in
zebrafish neural tissue. Development.
133,4709
-4719.
Silberg, D. G., Sullivan, J., Kang, E., Swain, G. P., Moffett, J., Sund, N. J., Sackett, S. D. and Kaestner, K. H. (2002). Cdx2 ectopic expression induces gastric intestinal metaplasia in transgenic mice. Gastroenterology 122,689 -696.[CrossRef][Medline]
Skromne, I., Thorsen, D., Hale, M., Prince, V. E. and Ho, R.
K. (2007). Repression of the hindbrain developmental program
by Cdx factors is required for the specification of the vertebrate spinal
cord. Development. 134,2147
-2158.
Stafford, D. and Prince, V. E. (2002). Retinoic acid signaling is required for a critical early step in zebrafish pancreatic development. Curr. Biol. 12,1215 -1220.[CrossRef][Medline]
Stafford, D., Hornbruch, A., Mueller, P. R. and Prince, V. E. (2004). A conserved role for retinoid signaling in vertebrate pancreas development. Dev. Genes Evol. 214,432 -441.[Medline]
Stafford, D., White, R. J., Kinkel, M. D., Linville, A.,
Schilling, T. F. and Prince, V. E. (2006). Retinoids signal
directly to zebrafish endoderm to specify insulin-expressing β-cells.
Development 133,949
-956.
Subramanian, V., Meyer, B. I. and Gruss, P. (1995). Disruption of the murine homeobox gene Cdx1 affects axial skeletal identities by altering the mesodermal expression domains of Hox genes. Cell 83,641 -653.[CrossRef][Medline]
Sun, Z., Amsterdam, A., Pazour, G. J., Cole, D. G., Miller, M.
S. and Hopkins, N. (2004). A genetic screen in zebrafish
identifies cilia genes as a principal cause of cystic kidney.
Development 131,4085
-4093.
Tiso, N., Filippi, A., Pauls, S., Bortolussi, M. and Argenton, F. (2002). BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mech. Dev. 118, 29-37.[CrossRef][Medline]
Torban, E., Kor, C. and Gros, P. (2004). Van Gogh-like2 (Strabismus) and its role in planar cell polarity and convergent extension in vertebrates. Trends Genet. 20,570 -577.[CrossRef][Medline]
van den Akker, E., Forlani, S., Chawengsaksophak, K., de Graaff,
W., Beck, F., Meyer, B. I. and Deschamps, J. (2002). Cdx1 and
Cdx2 have overlapping functions in anteroposterior patterning and posterior
axis elongation. Development
129,2181
-2193.
Waxman, J. S. and Yelon, D. (2007). Comparison of the expression patterns of newly identified zebrafish retinoic acid and retinoid X receptors. Dev. Dyn. 236,587 -595.[CrossRef][Medline]
Westerfield, M. (1995). The Zebrafish Book: A Guide for the Laboratory use of Zebrafish (Danio rerio). Eugene: University of Oregon Press.
Wingert, R. A., Selleck, R., Yu, J., Song, H.-D., Chen, Z., Song, A., Zhou, Y., Thisse, B., Thisse, C., McMahon, A. P. et al. (2007). The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PloS Genet. 3,e189 .[CrossRef]
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