© 2007 Nature Publishing Group http://www.nature.com/naturegenetics
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Fgf10 regulates hepatopancreatic ductal system patterning
and differentiation
P Duc Si Dong1, Chantilly A Munson1, William Norton2,5, Cecile Crosnier3, Xiufang Pan4, Zhiyuan Gong4,
Carl J Neumann2 & Didier Y R Stainier1
During organogenesis, the foregut endoderm gives rise to the
many different cell types that comprise the hepatopancreatic
system, including hepatic, pancreatic and gallbladder cells, as
well as the epithelial cells of the hepatopancreatic ductal
system that connects these organs together and with the
intestine. However, the mechanisms responsible for
demarcating ducts versus organs are poorly understood. Here,
we show that Fgf10 signaling from the adjacent mesenchyme is
responsible for refining the boundaries between the
hepatopancreatic duct and organs. In zebrafish fgf10 mutants,
the hepatopancreatic ductal epithelium is severely dysmorphic,
and cells of the hepatopancreatic ductal system and adjacent
intestine misdifferentiate toward hepatic and pancreatic fates.
Furthermore, Fgf10 also functions to prevent the differentiation
of the proximal pancreas and liver into hepatic and pancreatic
cells, respectively. These data shed light onto how the
multipotent cells of the foregut endoderm, and subsequently
those of the hepatopancreatic duct, are directed toward
different organ fates.
The hepatopancreatic ductal system, consisting of the extrahepatic
duct (EHD), cystic duct, common bile duct (CBD) and extrapancreatic duct (EPD), joins the liver, gallbladder and pancreas with the
intestine through the hepatopancreatic ampulla (HPA). We found that
the hepatopancreatic ductal system is organized in zebrafish as it is in
amniotes (Fig. 1a–c). The CBD and EPD converge into the HPA,
which connects to the intestine. Likewise, the cystic duct and EHD
converge into the CBD. As in mammalian embryos, the hepatopancreatic ductal system in zebrafish develops from cells that lie between
the emerging liver and ventral pancreas (data not shown). The
developmental and structural similarities we observed between the
hepatopancreatic ductal system of zebrafish and amniotes warrant the
use of this model system for studying hepatopancreatic organogenesis.
The hepatopancreatic ducts and organs are morphologically and
genetically distinct. Morphologically, the hepatopancreatic ducts are
epithelial structures that do not comprise pancreatic or hepatic cells
(Fig. 1a–c). Neogenesis of pancreatic endocrine and exocrine cells is
observed at the first branch of the intrapancreatic duct (Fig. 1b).
Similarly, hepatocytes are found in the proximal liver, distal to
the EHD. Genetically, we found that the transcription factor Prox1,
which is necessary for development of the mouse liver and pancreas1,2,
was expressed in the zebrafish pancreas, liver and distal EHD at 56
and 80 h post-fertilization (hpf) (Supplementary Fig. 1 online).
Conversely, Prox1 expression was low or not detected in the gallbladder, cystic duct, CBD, HPA, EPD and intestinal tube. Hepatocyte nuclear factor (Hnf) 4a, a transcription factor required for
mouse hepatocyte differentiation3, was expressed in the zebrafish
liver and intestine (Supplementary Fig. 1 online). Hnf4a expression
was absent from the pancreas, gallbladder, entire hepatopancreatic
ductal system and anterior intestine. Therefore, morphological borders between the hepatopancreatic ducts and organs demarcate
expression domains of Prox1, Hnf4a and other pancreas and liver
differentiation markers.
To identify genes regulating patterning of the hepatopancreatic
system, we examined the surrounding mesenchymal cells and
observed that they expressed the LIM homeodomain protein Islet1
(Isl1). Isl1-positive mesenchymal cells surrounded the hepatopancreatic ductal system and the region of the intestine between the
junction of the swimbladder, anteriorly, and the anterior intestinal
bulb, posteriorly (Fig. 1d–g). However, we did not find any Isl1positive cells around the pancreas or liver (Fig. 1d–g). This
localization suggested that Isl1-positive mesenchymal cells might
regulate patterning and differentiation of the hepatopancreatic ductal
system. As fibroblast growth factor 10 (Fgf10) has been shown to be
coexpressed with Isl1 and to signal from the mesenchyme to the
endoderm in mouse4, we used a mutation in fgf10 (daetbvbo)5
to examine its role in regulating hepatopancreatic ductal system
development in zebrafish.
At 56 hpf, fgf10 was expressed in the mesenchyme surrounding
the developing hepatopancreatic ductal system and the gut region
1Department
of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics and Human Genetics, and the Diabetes Center, University of California, San
Francisco, 1550 Fourth Street, San Francisco, California 94158, USA. 2Developmental Biology Program, European Molecular Biology Laboratory, Meyerhofstrasse 1,
D-69117 Heidelberg, Germany. 3Vertebrate Development Laboratory, Cancer Research UK, London WC2 A3PX, UK. 4Department of Biological Sciences, The National
University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. 5Present addresses: ZEN Neurogenetics Group, GSF Forschungszentrum, Neuherberg D-85758,
Germany (W.N.) and Department of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio 45229, USA (X.P.).
Correspondence should be addressed to D.Y.R.S. (didier_stainier@biochem.ucsf.edu).
Received 9 November 2006; accepted 15 December 2006; published online 28 January 2007; doi:10.1038/ng1961
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between the junction of the swimbladder, anteriorly, and the anterior
intestinal bulb, posteriorly (Fig. 1h,i). We observed this mesenchymal
expression of fgf10 from 54 to 80 hpf. Of note, the lack of fgf10 and
Isl1 expression around the liver and pancreas suggests that neither
gene is expressed at a detectable level in the mesenchyme surrounding
these outgrowths of the foregut endoderm. In mouse, Fgf10 and Isl1
appear to be coexpressed in the mesenchyme surrounding the foregut
endoderm and not around the ventral pancreatic bud4,6. In zebrafish,
Isl1 is clearly expressed in the mesenchyme adjacent to the hepatopancreatic ductal system and the intestinal tube in the region between
the swimbladder and intestinal bulb (Fig. 1d–g). Furthermore, the
gene encoding Fgfr2, which has been suggested to be the primary
receptor for Fgf10 (refs. 7,8), is expressed in the embryonic endoderm
in zebrafish9 (Supplementary Fig. 2 online), suggesting that endodermal cells can receive Fgf10 signals. Consistent with these patterns of
fgf10 and fgfr2 expression, we found that the endodermal fgf10 mutant
phenotypes were primarily localized to the hepatopancreatic ductal
system and the intestinal tube between the swimbladder and intestinal
bulb, as described below.
To analyze the morphology of the hepatopancreatic ductal system in
greater detail, we used monoclonal antibody 2F11 (ref. 10) because it
strongly marks the entire hepatopancreatic ductal system as well as the
gallbladder (Fig. 2a,b). 2F11 also moderately marks the intrahepatic
Figure 1 fgf10 expression in mesenchyme adjacent to the hepatopancreatic
ductal system. (a,b) Three-dimensional rendering of the hepatopancreatic
system showing 80-hpf Tg(elastase:GFP); Tg(lfabp:dsRed) larva labeled with
monoclonal antibody 2F11 (blue) to mark hepatopancreatic ductal system
(including the gall bladder, GB) and somatostatin (Sst) to mark the principal
islet of the pancreas (P). Liver (L) expresses the lfabp:dsRed transgene.
Exocrine pancreas expresses the elastase:GFP transgene. (b) Higher magnification of a, highlighting structure and organization of the hepatopancreatic
ductal system. Arrow indicates Sst-positive cell at the border between
proximal pancreas and distal extrapancreatic duct. (c) Illustration of
hepatopancreatic ductal system (extrapancreatic duct, EPD red;
hepatopancreatic ampulla, HPA, green; common bile duct, CBD, blue; cystic
duct, CD, orange; extrahepatic duct, EHD, yellow). (d,e) Three-dimensional
rendering of hepatopancreatic system of a 56-hpf Tg(gutGFP)s854 embryo
labeled for Isl1, which is expressed in mesenchymal cells and pancreatic
endocrine cells. (e) Higher magnification of d showing the Isl1-positive
mesenchymal cells surrounding the hepatopancreatic ductal system.
(f) Z-focal plane of embryo in e showing Isl1-positive mesenchymal cells
(arrows) surrounding developing hepatopancreatic duct but not pancreas
or liver. Arrowheads indicate neogenic endocrine cells coexpressing Isl1
and Tg(gutGFP)s854. (g) More dorsal Z-focal plane of the embryo in e with
Isl-positive mesenchymal cells surrounding developing intestinal tube (I).
Yellow asterisks indicate swim bladder duct; white asterisks indicate
intestinal bulb (d,e,h,i). (h,i) fgf10 in situ hybridization of a 56-hpf
Tg(gutGFP)s854 embryo immunofluorescently labeled for GFP showing fgf10
expression in mesenchymal cells around the hepatopancreatic ductal system
and intestinal tube but not the pancreas or liver (arrows), similar to Isl1
expression. (i) Higher magnification of h.
and intrapancreatic ducts. The elevated level of 2F11 staining in the
hepatopancreatic ductal system does not extend into the intestine.
However, 2F11 does mark scattered enteroendocrine cells and goblet
cells in the intestine10. Unlike wild-type larvae, in which all the
components of the hepatopancreatic ductal system are discernable,
the hepatopancreatic ductal system of fgf10 mutants is severely
dysmorphic, with no clear morphological distinction between the
CBD and other components of the hepatopancreatic ductal system.
The CBD is severely reduced in length (and sometimes appears
absent) and has a globular rather than tubular shape. This defect
causes the EPD, cystic duct and EHD to connect directly to the HPA
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Figure 2 fgf10 regulates the morphogenesis of the hepatopancreatic duct
and intestinal tube. (a–f) 80-hpf wild-type (a,c,e) and fgf10 mutant (b,d,f)
larvae. (a,b) Three-dimensional rendering of the hepatopancreatic ductal
system marked by 2F11. (b) fgf10 mutant hepatopancreatic ductal system
is severely reduced and dysmorphic. (c,d) Z-focal planes through the
intestinal tube stained with 2F11 and Hnf4a antibodies; arrowheads point
to the posterior junction area between the EPD/HPA and the intestinal tube.
Note that 2F11-positive cells extend into the intestine to be continuous with
the intestinal tube in fgf10 mutants (d), unlike in wild-type larvae (c). Also,
the anterior domain of Hnf4a expression is shifted posteriorly in fgf10
mutants (d) compared with the wild-type (c). (e,f) Z-focal planes through the
intestinal tube stained for pan-Cadherin (Cad) and Isl1 showing that fgf10
mutant intestinal tubes are thinner (f) than wild-type tubes (e). Arrows point
to the proximal swimbladder; yellow asterisks mark the area where the HPA
is connected to the intestine.
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Figure 3 fgf10 represses pancreatic and hepatic organ differentiation, as assessed by Prox1 and Hnf4a expression. (a,b) Three-dimensional rendering of
hepatopancreatic system of 60-hpf wild-type (a) and fgf10 mutant (b) embryos stained for Prox1. Arrows point to the presumptive hepatopancreatic ducts;
arrowheads point to the intestinal tube posterior to the hepatopancreatic system. Note the presence of ectopic Prox1-positive cells in the ductal system and
intestine of fgf10 mutants. (c–j) 80-hpf wild-type (c,e,g) and fgf10 mutant (d,f,h,i,j) larvae stained with antibodies to Prox1, 2F11 and Hnf4a shown at
various focal planes throughout the hepatopancreatic system. (d,f,h) Cells misexpress Prox1 (arrows) in the EPD (d), CBD (f) and adjacent intestinal tube (h)
of fgf10 mutant larvae but not wild-type (c,e,g) larvae. Cells misexpressing Hnf4a in the proximal pancreas of fgf10 mutants (i,j) are also positive for 2F11
and Prox1 (arrowhead; white). (k,l) Three-dimensional rendering of the hepatopancreatic system of 100-hpf Tg(elastase:GFP); Tg(lfabp:dsRed) wild-type (k)
and fgf10 mutant (l) larvae stained with 2F11. fgf10 mutant larvae (l), unlike their wild-type siblings (k), show ectopic hepatic tissue in their proximal
pancreas (arrow).
(Fig. 2). In most mutants, the EPD and EHD were reduced in size.
These structures appeared thinner and shorter, with no discernable
lumens (Fig. 2b). In rare cases, the EPD or EHD was completely
absent, leaving the pancreas or liver disconnected from the intestine
(Supplementary Fig. 3 online). Furthermore, elevated 2F11 staining
in the hepatopancreatic ductal system of fgf10 mutants extended
continuously into the intestine (Fig. 2d), suggesting that the HPA
was expanded beyond its normal boundary. Alternatively, the intestinal cells adjacent to the HPA might be abnormally differentiating into
hepatopancreatic ductal cells. When we examined the intestinal
expression of Hnf4a, we found that in wild-type larvae, the anterior
boundary of Hnf4a expression in the intestine extended between five
to ten cell lengths anterior of the junction of the HPA and the gut
(Fig. 2c). However, in fgf10 mutants, the anterior boundary of Hnf4a
expression was shifted to the posterior of the HPA (Fig. 2d). These
data suggest that the portion of the intestine adjacent and anterior to
the HPA requires Fgf10 signaling for expression of Hnf4a. Alternatively, the expanded hepatopancreatic ductal cells are displacing the
Hnf4a-positive cells of the intestine in fgf10 mutants. Furthermore,
the area of the intestinal tube between the swimbladder and intestinal
bulb, which is surrounded by Isl1-positive mesenchyme, was markedly
thinner and apparently not lumenized in fgf10 mutants at 80 hpf
(Fig. 2e,f). A thinner gut tube has also been previously reported in
mouse Fgf10 mutants11. Thus, Fgf10 signaling has an essential function in the morphogenesis of the hepatopancreatic ductal system and
the organ-forming area of the intestinal tube.
To examine the consequences of a lack of Fgf10 signaling on
differentiation of the hepatopancreatic system, we examined expression of genes that are differentially expressed between the hepatopancreatic ducts and organs. In 60-hpf wild-type embryos, Prox1 was
expressed in both the developing liver and pancreas but was undetectable in most of the presumptive hepatopancreatic ductal system
(Fig. 3a). At 80 hpf, when the branches of the hepatopancreatic ductal
system were clearly distinct, Prox1 was mostly absent from these
structures, although we did detect some expression in the distal
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EPD and detected low levels in the distal EHD in some cases. In fgf10
mutants, Prox1 was misexpressed in most cells of the hepatopancreatic
duct, including the EPD and CBD (Fig. 3a–f). We were surprised to
find Prox1 expression in cells of the intestine, posterior to where the
HPA joins the intestine (Fig. 3a,b,g,h). This finding correlates with
our observation of ectopic 2F11-positive cells in the intestinal tube of
fgf10 mutants (Fig. 2d). Together, these data indicate that this part of
the intestinal tube in fgf10 mutants is more similar to hepatopancreatic system cells. The misexpression of Prox1 in the hepatopancreatic
ductal system and intestine suggests that the boundaries among the
hepatopancreatic organs, ducts and intestine are obscured in fgf10
mutants. We also found ectopic Hnf4a expression in fgf10 mutants: in
approximately one-third of more than 25 larvae examined, Hnf4a was
misexpressed in one to three cells in the proximal pancreas and
hepatopancreatic ductal system (Fig. 3i,j and data not shown). All
Hnf4a-misexpressing cells in the proximal pancreas and hepatopancreatic ductal system also coexpressed Prox1 (Fig. 3i,j). Furthermore, a
subset of the cells in the intestine that misexpressed Prox1 also
coexpressed Hnf4a (Fig. 3h,i). This coexpression of Prox1 and
Hnf4a, which, in wild-type endoderm, we observed only in hepatocytes (Fig. 3c,e,g), suggests that cells in the proximal pancreas,
hepatopancreatic ductal system and adjacent intestinal regions of
fgf10 mutants are differentiating toward liver fates. Indeed, when we
assessed at a later stage the expression of a dsRed transgene driven by a
liver fatty acid binding protein (lfabp) promoter12, which is normally
restricted to cells of the liver (n 4 60 larvae examined; Fig. 3k), we
observed that one-third of the fgf10 mutants (n 4 30) misexpressed
Tg(lfabp:dsRed) in a patch of cells in the proximal pancreas (Fig. 3l).
These data indicate that hepatocytes differentiate in the proximal
pancreas of fgf10 mutants.
Most pancreatic endocrine cells, as marked by Tg(nkx2.2a:
mEGFP)13,14 and Isl1 (ref. 6) expression, that are localized outside
of the principal islet cluster were found in the proximal pancreas,
suggesting that islet cell neogenesis occurs immediately distal to the
EPD (Fig. 4a). The EPD, along with the rest of the hepatopancreatic
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Figure 4 fgf10 represses endocrine cell differentiation throughout the hepatopancreatic system.
(a–f) Three-dimensional rendering of the hepatopancreatic system of 80-hpf Tg(nkx2.2a:mGFP)
wild-type (a) and fgf10 mutant (b–f) larvae stained for Isl1 and Cad. (b) fgf10 mutant with large
clusters of Tg(nkx2.2a:mGFP)-positive and Isl1-positive endocrine cells associated with the
hepatopancreatic ductal system (arrow) and adjacent intestinal tube (arrowhead). (c,d) Higher
magnification of b (shown in d without the green channel to emphasize Isl-positive cells that are
delaminated and clustered in the intestine (arrowheads)). (e,f) Focal plane through an fgf10
mutant EHD (asterisk marks the distal EHD) and proximal liver (L); arrowheads point to
nkx2.2:GFP-positive and Isl1-positive endocrine cells in the liver. Only green and red channels of
e are shown in f. (g,h) Three-dimensional rendering of the hepatopancreatic system of 80-hpf
wild-type (g) and fgf10 mutant (h) larvae stained for Isl1, Ins and Cad. Ins-positive cells are
found in the EPD (arrow) in fgf10 mutants. (i,j) Three-dimensional rendering of the
hepatopancreatic system of 96-hpf Tg(nkx2.2a:mGFP) wild-type (i) and fgf10 mutant (j) larvae
stained with antibodies to somatostatin (Sst) and 2F11. (j) Sst-positive cells are seen in the
cystic duct/EHD (arrowhead) and EPD (arrow) in fgf10 mutants. White lines indicate the border
between the proximal pancreas and distal EPD.
ductal system, was adjacent to the fgf10-expressing mesenchyme
(Fig. 1h–i). These observations prompted us to test whether fgf10
signaling might be inhibiting endocrine differentiation in the EPD. In
wild-type larvae, we generally did not find endocrine cells in the
hepatopancreatic ductal system, although in approximately one-quarter of larvae (n 4 60 larvae examined), we did find one or two
endocrine cells in the distal EPD (Fig. 4a). In fgf10 mutants, we found
a marked increase in the number of Tg(nkx2.2a:mEGFP)-positive and
Isl1-positive cells in the EPD, often observing more than ten endocrine
cells distributed all along the EPD. When we examined earlier-stage
embryos (56 hpf), we found that the ectopic endocrine cells appeared
to originate from the hepatopancreatic duct (data not shown),
suggesting that the observed 80-hpf phenotype is not caused by a
migration defect. Furthermore, the principal islet cluster often
appeared larger in fgf10 mutants (Fig. 4a,b). We found similar results
when we examined expression of neurod, which encodes a transcription factor required for pancreatic endocrine cell differentiation and
survival in mouse15 (data not shown). We were surprised to observe
Tg(nkx2.2a:mEGFP)-positive and Isl1-positive cells in the CBD, HPA
and even in the adjacent intestine and proximal liver of fgf10 mutant
larvae (Fig. 4b–f), suggesting that these tissues are competent to
generate pancreatic cells. In wild-type larvae, as neogenic pancreatic
endocrine cells differentiated, they delaminated and appeared to
migrate toward the principal islet and sometimes toward each other
to form small clusters of endocrine cells (data not shown). We did not
observe such behavior with intestinal enteroendocrine cells. In fgf10
mutants, many of the ectopic endocrine cells appeared to have
delaminated, often in clusters, from the hepatopancreatic ducts and
intestine, suggesting that these cells behave more like pancreatic
endocrine cells than intestinal enteroendocrine cells. To determine
whether the ectopic endocrine cells observed in fgf10 mutants could
further differentiate, we examined the expression of insulin and
somatostatin, hormones produced by pancreatic endocrine cells. In
fgf10 mutants, unlike their wild-type siblings (Fig. 4g), a subset of the
Isl1-positive cells in the hepatopancreatic ductal system coexpressed
insulin (Fig. 4h). Similarly, some cells in the hepatopancreatic ductal
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system of fgf10 mutants also expressed somatostatin, unlike their wildtype siblings (Fig. 4i,j). Therefore, fgf10 signaling represses endocrine
cell differentiation primarily in the EPD but also in the rest of the
hepatopancreatic ductal system, the adjacent intestine and even the
proximal liver.
We observed excessive differentiation in the hepatopancreatic
system of fgf10 mutants, indicating that Fgf10 functions to repress
differentiation in this subset of endodermal organs. In contrast to
these results, it was originally reported that there was no excessive
differentiation of pancreatic cells in Fgf10 mutant mice11. But consistent with our data, misexpression of Fgf10 in mouse did appear to
block pancreas differentiation16,17. These conflicting data in mouse,
together with our findings in zebrafish, warrant re-examination of the
Fgf10 mouse mutant, particularly for hepatopancreatic system differentiation phenotypes.
In addition, we observed ectopic differentiation of endocrine cells in
the EPD and hepatic cells in the proximal pancreas of fgf10 mutants.
Data from mice suggested that maintenance of Pdx1 expression by
Fgf10 is required for maintenance of pancreatic progenitor cells,
potentially by inhibiting differentiation and promoting proliferation16,17. Consistent with this hypothesis, and providing a possible
explanation for the presence of ectopic endocrine cells in the EPD and
hepatic cells in the proximal pancreas of fgf10 mutants, we found that
Pdx1 expression in the EPD and proximal pancreas was lost by 80 hpf
(Supplementary Fig. 4 online). However, Pdx1 expression in the
endocrine pancreas and intestine did not seem to be affected.
Furthermore, our observation of ectopic expression of Tg(lfabp:dsRed)
in zebrafish fgf10 mutants and the presence of putative Pdx1 binding
sites in the lfabp regulatory element18 supports the possibility that
Fgf10 represses liver differentiation in the proximal pancreas by
maintaining pdx1 expression.
We are just beginning to understand how the developing foregut
endodermal cells are allocated to become distinct tissues of the
hepatopancreatic system. Others19 have shown that the transcription
factor Ptf1a functions to prevent presumptive pancreatic cells
from being incorporated into the intestine and differentiating into
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intestinal cells. The results shown here indicate that the domains
of organ differentiation are still plastic in the region of the hepatopancreatic ductal system and proximal liver and pancreas even after
organ specification and initial outgrowth. We propose a model
whereby different bilateral factors promoting liver and pancreas
development are repressed at the midline, where the hepatopancreatic ductal system, proximal liver and proximal pancreas
differentiate. This model is consistent with our observations of
misdifferentiation of cells of the intestine, hepatopancreatic ductal
system, proximal liver and proximal pancreas in fgf10 mutants.
However, in wild-type embryos and larvae, where Fgf10 signaling is
present at the midline, these organ-promoting factors are unable to
direct liver or pancreas differentiation in the hepatopancreatic
system, thereby making the organ-forming and duct-forming
domains distinct. Although Ptf1a has been proposed to be positively
regulated by Fgf10 in mouse4, we did not find any reduction
in ptf1a expression in zebrafish fgf10 mutants (data not shown),
suggesting that the misdifferentiation observed is not due to
ptf1a downregulation.
Although pancreas and liver development has received much
attention, the work presented here emphasizes the importance of
studying these organs in the context of the surrounding tissues.
By examining cell differentiation in detail in the pancreas, liver,
hepatopancreatic ductal system and intestine, we have uncovered
a previously unknown role for Fgf10 in patterning the organforming region of the endoderm. Furthermore, by using highresolution, three-dimensional fluorescence imaging, we show that
the zebrafish model is very effective for whole-mount analyses of
the entire hepatopancreatic system. The extensive morphological
and genetic similarities of the hepatopancreatic system between
zebrafish and amniotes validate the use of this model to study
human diseases affecting the liver, pancreas and hepatopancreatic
ductal system.
The widespread misdifferentiation present in fgf10 mutants demonstrates the marked plasticity of cells in the proximal liver and pancreas,
hepatopancreatic ductal system and adjacent intestine and raises the
question of whether there are common progenitors for these organs.
Indeed, there may be an overlooked progenitor cell population in the
hepatopancreatic ductal system that can differentiate into both pancreas and liver cells. Future studies focusing on the hepatopancreatic
ductal system and proximal pancreas and liver will probably lead to
further insights into how organ progenitor cells are specified and
regulated. A more profound understanding of how endodermal cells
are allocated to become distinct cell types should facilitate their
manipulation for use in transplant therapy for diseases such as
diabetes and liver failure.
METHODS
Zebrafish strains. Embryos and adult fish were raised and maintained
under standard laboratory conditions. We used the following mutant and
transgenic lines: daedalus (daetbvbo)5, Tg(gutGFP)s854 (ref. 20), Tg(elastase:GFP),
Tg(lfabp:dsRed)12 and Tg(nkx2.2a:mEGFP)13.
In situ hybridization and immunohistochemistry. Whole-mount in situ
hybridizations were performed as previously described21 using the fgf10
(ref. 5) and fgfr2 (ref. 9) probes, followed by fluorescent immunohistochemistry and imaging using a Zeiss Axioplan microscope. We used the following
antibodies: rabbit polyclonal antibody against GFP (rabbit, 1:200; Torrey Pines
Biolabs), rabbit polyclonal antibody against Prox1 (rabbit, 1:1,000; Chemicon),
mouse monoclonal antibody against Islet1/2 (1:10; Developmental Studies
Hybridoma Bank, clone 39.4D5), rabbit polyclonal antibody against
pan-Cadherin (1:5,000; Sigma), mouse monoclonal 2F11 (ref. 10) (1:1,000;
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gift from J. Lewis, Cancer Research UK), goat polyclonal antibody against
Hnf4a (1:100, Santa Cruz Biotechnology), guinea pig polyclonal antibody
against insulin (1:500; Biomeda), rabbit polyclonal antibody against somatostatin (1:1,000; ICN Biomedicals), guinea pig polyclonal antibody against
zebrafish Pdx1 (1:200; gift from C. Wright, Vanderbilt University) and
fluorescently conjugated Alexa antibodies from Molecular Probes. fgf10 mutant
embryos were selected based on their underdeveloped pectoral fin phenotype.
Mutant and wild-type siblings were stained in the same tube. The embryos were
fixed with 2% formaldehyde in 0.1 M PIPES, 1.0 mM MgSO4 and 2 mM EGTA
overnight at 4 1C. The yolk was manually removed and the embryos were
blocked for 1 h in PBS with 4% BSA and 0.3% Triton X-100. Both primary and
secondary antibodies were incubated overnight. Washes were done with 0.3%
Triton X-100 in PBS for 3 h to remove excess antibodies. Samples were
mounted in Vectashield (Vector Labs) and imaged using a Zeiss LSM5 Pascal
confocal microscope.
Note: Supplementary information is available on the Nature Genetics website.
ACKNOWLEDGMENTS
We thank A. Ayala, S. Waldron and N. Zvenigorodsky for help with the fish;
C. Wright, M. Hebrok, M. German, S. Curado, E. Ober, T. Sakaguchi, M. Bagnat
and A. Schlegel for discussions and/or critical reading of the manuscript;
C. Wright and J. Lewis for antibodies and B. Appel (Vanderbilt University) for
the Tg(nkx2.2a:mEGFP) line. P.D.S.D. was supported by the Juvenile Diabetes
Research Foundation (32002643) and the Larry L. Hillblom Foundation (2005
1G). C.A.M. was supported by a US National Science Foundation predoctoral
fellowship. This work was supported in part by grants from the US National
Institutes of Health (National Institute of Diabetes and Digestive and Kidney
Diseases), the Juvenile Diabetes Research Foundation and the Packard Foundation
(D.Y.R.S.). Requests for materials should be addressed to D.Y.R.S.
(didier_stainier@biochem.ucsf.edu).
AUTHOR CONTRIBUTIONS
P.D.S.D. made the original observation, generated hypotheses, designed
experiments, analyzed fgf10 mutants and made the figures; C.A.M. analyzed
fgf10 and neuroD expression, performed phenotype assessment and drew the
illustration; W.N. and C.J.M. provided fgf10 mutants prior to publication;
C.C. provided 2F11 antibodies prior to publication; X.P. and Z.G. provided
Tg(lfabp:dsRed) zebrafish prior to publication; D.Y.R.S. oversaw the studies
and P.D.S.D., C.A.M. and D.Y.R.S. wrote the manuscript with feedback from
the other authors.
COMPETING INTERESTS STATEMENT
The authors declare that they have no competing financial interests.
Published online at http://www.nature.com/naturegenetics
Reprints and permissions information is available online at http://npg.nature.com/
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