Zebrafish sox9b is crucial for hepatopancreatic duct development and pancreatic endocrine cell regeneration.
ABSTRACT Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells are derived from pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis, labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentiation in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine cells deriving from pancreatic ducts in zebrafish.
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ABSTRACT: Despite the importance of tendons and ligaments for transmitting movement and providing stability to the musculoskeletal system, their development is considerably less well understood than that of the tissues they serve to connect. Zebrafish have been widely used to address questions in muscle and skeletal development, yet few studies describe their tendon and ligament tissues. We have analyzed in zebrafish the expression of several genes known to be enriched in mammalian tendons and ligaments, including scleraxis (scx), collagen 1a2 (col1a2) and tenomodulin (tnmd), or in the tendon-like myosepta of the zebrafish (xirp2a). Co-expression studies with muscle and cartilage markers demonstrate the presence of scxa, col1a2 and tnmd at sites between the developing muscle and cartilage, and xirp2a at the myotendinous junctions. We determined that the zebrafish craniofacial tendon and ligament progenitors are neural crest derived, as in mammals. Cranial and fin tendon progenitors can be induced in the absence of differentiated muscle or cartilage, although neighboring muscle and cartilage are required for tendon cell maintenance and organization, respectively. By contrast, myoseptal scxa expression requires muscle for its initiation. Together, these data suggest a conserved role for muscle in tendon development. Based on the similarities in gene expression, morphology, collagen ultrastructural arrangement and developmental regulation with that of mammalian tendons, we conclude that the zebrafish tendon populations are homologous to their force-transmitting counterparts in higher vertebrates. Within this context, the zebrafish model can be used to provide new avenues for studying tendon biology in a vertebrate genetic system.Development 05/2014; 141(10):2035-45. · 6.27 Impact Factor
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ABSTRACT: Pancreatic adenocarcinoma, one of the worst malignancies of exocrine pancreas, is a solid tumor with increasing incidence and mortality in industrialized countries. It is usually driven by oncogenic Kras point mutations and evolves into a highly aggressive metastatic carcinoma due to secondary gene mutations and specific signaling pathways unbalance. To examine in vivo the effects of Kras(G12D) during pancreatic cancer progression and time correlation with cancer signaling pathways activities, we have generated a zebrafish model of Pancreatic adenocarcinoma in which eGFP-Kras(G12D) expression was specifically driven to the pancreatic tissue by using the GAL4/UAS conditional expression system. Outcrossing the inducible oncogenic Kras(G12D) line with transgenic zebrafish reporters harboring specific signaling responsive elements of transcriptional effectors, we were able to follow TGFβ, Notch, Bmp and Shh activities during tumor development. Zebrafish transgenic lines expressing eGFP-Kras(G12D) showed normal exocrine pancreas development till 3 weeks post fertilization (wpf). From 4 to 24 wpf we observed several degree of acinar lesions, characterized by an increase of mesenchymal cells and mixed acinar/ductal features followed by progressive bowel and liver infiltrations finally bringing to highly aggressive carcinoma. Moreover, live imaging analysis of the exocrine pancreatic tissue revealed an increasing number of Kras positive cells and progressive activation of TGFβ; and Notch pathways. Increase of TGFβ, following Kras(G12D) activation, was confirmed in a concomitant model of medulloblastoma (MDB). Notch and Shh signaling activities during tumor onset were different between MDB and pancreatic adenocarcinoma indicating a tissue specific regulation of cell signaling pathways. Moreover, our results shows that a living model of pancreatic adenocarcinoma joined with cell signaling reporters is a suitable tool to describe in vivo the signaling cascades and molecular mechanisms involved in tumor development and a potential platform to screen for novel oncostatic drugs.Disease Models and Mechanisms 05/2014; · 5.54 Impact Factor
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ABSTRACT: To gain insight into liver and pancreas development, we investigated the target of 2F11, a monoclonal antibody of unknown antigen, widely used in zebrafish studies for labeling hepatopancreatic ducts. Utilizing mass spectrometry and in vivo assays, we determined the molecular target of 2F11 to be Annexin A4 (Anxa4), a calcium binding protein. We further found that in both zebrafish and mouse endoderm, Anxa4 is broadly expressed in the developing liver and pancreas, and later becomes more restricted to the hepatopancreatic ducts and pancreatic islets, including the insulin producing ß-cells. Although Anxa4 is a known target of several monogenic diabetes genes and its elevated expression is associated with chemoresistance in malignancy, its in vivo role is largely unexplored. Knockdown of Anxa4 in zebrafish leads to elevated expression of caspase 8 and Δ113p53, and liver bud specific activation of Caspase 3 and apoptosis. Mosaic knockdown reveal that Anxa4 is required cell-autonomously in the liver bud for cell survival. This finding is further corroborated with mosaic anxa4 knockout studies using the CRISPR/Cas9 system. Collectively, we identify Anxa4 as a new, evolutionarily conserved hepatopancreatic factor that is required in zebrafish for liver progenitor viability, through inhibition of the extrinsic apoptotic pathway. A role for Anxa4 in cell survival may have implications for the mechanism of diabetic ß-cell apoptosis and cancer cell chemoresistance.Developmental Biology 08/2014; · 3.64 Impact Factor
Zebrafish sox9b is crucial for hepatopancreatic duct development and
pancreatic endocrine cell regeneration
Isabelle Manfroida,n, Aure ´lie Ghayea, Franc -ois Nayea, Nathalie Detrya, Sarah Palma,
Luyuan Panb, Taylur P. Mab, Wei Huangc, Meritxell Rovirac, Joseph A. Martiala,
Michael J. Parsonsc, Cecilia B. Moensb, Marianne L. Voza, Bernard Peersa
aUnit of Molecular Biology and Genetic Engineering, Giga-Research, University of Li? ege, 1 avenue de l’Hˆ opital B34, B-4000 Sart-Tilman, Belgium
bHoward Hughes Medical Institute, Division of Basic Science, Fred Hutchinson Cancer Research Center, B2-152, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA
cDepartment of Surgery, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
a r t i c l e i n f o
Received 1 November 2011
Received in revised form
5 April 2012
Accepted 6 April 2012
Available online 17 April 2012
Beta cell regeneration
a b s t r a c t
Recent zebrafish studies have shown that the late appearing pancreatic endocrine cells are derived from
pancreatic ducts but the regulatory factors involved are still largely unknown. Here, we show that the
zebrafish sox9b gene is expressed in pancreatic ducts where it labels the pancreatic Notch-responsive
cells previously shown to be progenitors. Inactivation of sox9b disturbs duct formation and impairs
regeneration of beta cells from these ducts in larvae. sox9b expression in the midtrunk endoderm
appears at the junction of the hepatic and ventral pancreatic buds and, by the end of embryogenesis,
labels the hepatopancreatic ductal system as well as the intrapancreatic and intrahepatic ducts. Ductal
morphogenesis and differentiation are specifically disrupted in sox9b mutants, with the dysmorphic
hepatopancreatic ducts containing misdifferentiated hepatocyte-like and pancreatic-like cells. We also
show that maintenance of sox9b expression in the extrapancreatic and intrapancreatic ducts requires
FGF and Notch activity, respectively, both pathways known to prevent excessive endocrine differentia-
tion in these ducts. Furthermore, beta cell recovery after specific ablation is severely compromised in
sox9b mutant larvae. Our data position sox9b as a key player in the generation of secondary endocrine
cells deriving from pancreatic ducts in zebrafish.
& 2012 Elsevier Inc. All rights reserved.
Stimulating in vivo regeneration of insulin-producing cells
(beta cells) from endogenous precursors offers great interest in
the treatment of diabetes mellitus. It has long been thought that
endogenous beta cell precursors reside in adult pancreatic ducts
(Bonner-Weir et al., 2004). However, this hypothesis has been
recently challenged by lineage tracing studies in mouse using two
ductal markers, Hnf1b (Solar et al., 2009) and Sox9 (Kopp et al.,
2011; Solar et al., 2009), which have failed to highlight endocrine
differentiation from pancreatic ducts after birth. In contrast, in
zebrafish, there is accumulating evidence of ductal origin of
endocrine cells after embryogenesis (Dong et al., 2007, 2008;
Field et al., 2003; Parsons et al., 2009; Wang et al., 2011). Thus, it
is crucial to understand how beta cells are generated from ducts
in zebrafish if we hope to stimulate this pathway in mammals to
be able to provide diabetes patients with endogenous sources of
Zebrafish and mammalian pancreas consist of endocrine islets
embedded in a large exocrine tissue. Their development share
many key steps and players (Kinkel and Prince, 2009 for review).
For example, they form as two embryonic buds from the pdx1-
expressing domain of the endoderm adjacent to the hepatic
primordium. During zebrafish development, the pdx1 domain
gives rise to the dorsal pancreatic bud which generates the first
endocrine cells from 15 hpf, and to the ventral pancreatic anlage
from 32 hpf (Biemar et al., 2001; Field et al., 2003; Roy et al.,
2001). At the end of embryogenesis (3 days post fertilisation;
3 dpf), the zebrafish pancreas consists of a principal endocrine
islet mostly derived from the dorsal bud surrounded by the
exocrine tissue derived from the ventral pancreas and composed
of acini and ducts. Along the extrapancreatic duct (EPD), between
2 and 5 dpf, additional endocrine cells appear that will join
the principal islet (Dong et al., 2007, 2008; Field et al., 2003;
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/developmentalbiology
0012-1606/$-see front matter & 2012 Elsevier Inc. All rights reserved.
Abbreviations: BMP, bone morphogenic protein; EHD, extrahepatic duct;
EPD, extrapancreatic duct; FGF, fibroblasts growth factor; GB, gall bladder;
HNF, hepatocyte nuclear factor; HPD, hepatopancreatic ductal system; IHD,
intrahepatic ducts; IPD, intrapancreatic ducts; SOX9, SRY-related HMG box
transcription factor 9
nCorresponding author. Fax: þ32 4 366 41 98.
E-mail address: Isabelle.firstname.lastname@example.org (I. Manfroid).
Developmental Biology 366 (2012) 268–278
Pisharath et al., 2007) and, along the intrapancreatic ducts (IPD),
secondary endocrine islets will differentiate after 5 dpf (Parsons et al.,
2009; Wang et al., 2011) from a population of pancreatic progenitors,
the Notch-responsive cells (PNCs), aligned along the IPD. Collectively,
these late endocrine cells, derived from the ventral pancreatic bud,
constitute the second wave of endocrine cell differentiation. This will
ultimately contribute to the majority of the endocrine mass as the
dorsal bud-derived islet cells are quiescent (Hesselson et al., 2009).
Zebrafish also display a remarkable capacity to regenerate beta cells
after targeted cell ablation in young larvae and adults (Curado et al.,
2007; Moss et al., 2009; Pisharath et al., 2007). Whether this
regeneration occurs from pancreatic ducts remains unknown.
Similar to the organisation in amniotes, the pancreatic ducts in
zebrafish form a complex branched network which is anatomically
connected to the intestine and the hepatic ducts through the
hepatopancreatic ductal system (HPD) (Dong et al., 2007). In
mammals, SOX9 is expressed in all these ductal structures from
embryogenesis to adulthood (Antoniou et al., 2009; Carpentier et al.,
2011; Furuyama et al., 2011; Kopp et al., 2011; Seymour et al., 2007)
but its function in their ontogenesis is still poorly explored. In the
mouse liver, SOX9 is the earliest marker of intrahepatic ductal cells
(also called cholangiocytes) and is involved in the maturation of
these ducts (Antoniou et al., 2009). In the mouse pancreas, SOX9 is
detected in the progenitor cells of the pancreatic anlagen (Lynn
et al., 2007; Seymour et al., 2007) and its inactivation causes
pancreas hypoplasia with severe reduction of all pancreatic cell
types, endocrine, acinar and ductal (Seymour et al., 2007). However,
its function specifically in pancreatic ductal development remains
unknown, as well as its role in the formation of the HPD. In the
present study, we show that zebrafish sox9b gene expression defines
a novel population of progenitors partially overlapping the adjacent
hepatic and pancreatic anlagen and is later expressed in the
intrahepatic, intrapancreatic ducts and in the HPD system. Using a
new sox9b mutant, the sox9bfh313, we demonstrate that sox9b plays
an essential role not only in intrahepatic duct morphogenesis but
also in morphogenesis and differentiation of the intrapancreatic
ducts and of the HPD system. Finally, we show that sox9b is essential
for endocrine cell formation from the pancreatic ducts and for beta
Material and methods
Embryos and adult fish were raised and maintained under
standard conditions. We used the following transgenic and mutant
lines: sox9bfh313identified by TILLING (Draper et al., 2004) (http://
www.zfishtilling.org/zfish/index.php), sox9bb971(Yan et al., 2005),
Tg(ptf1a:egfp)jhl(Godinho et al., 2005), daedalus (daetbvbomutant
allele of fgf10) (Norton et al., 2005), Tg(Tp1:hmgb1-mCherry) (Parsons
et al., 2009) and Tg(ins:nfsB-mCherry) (Pisharath et al., 2007).
Adult sox9bfh313heterozygous and mutant embryos in their
progeny were identified by genotyping.
Sorting of GFPþcells by FACS
Fluorescent cells were isolated from pancreas dissected from
adult Tg(Tp1:hmgb1-eGFP) and Tg(ptf1a:eGFP) zebrafish. Flow
cytometry was performed using a FACSAria (Becton Dickinson)
EdU injection and labelling
The Click-iT EdU Alexa Fluor555 Imaging Kit (Invitrogen) was
performed following the manufacturer’s protocol. Briefly, 5 nl of EdU
solution (1mM EdU, 2% DMSO, 0.1% phenol red) were injected into
the yolk of 3 or 4 dpf larvae which were further incubated 60 min and
fixed in 2% PFA. Larvae were then processed for 2F11-duct immuno-
detection and nuclei staining with DRAQ7 (Biostatus Limited).
Whole mount in situ hybridisation and immunohistochemistry
Fluorescent and colorimetric whole mount in situ hybridisation
was performed as previously described (Hauptmann and Gerster,
1994; Manfroid et al., 2007) with the following probes: sox9b (Yan
et al., 2005), neurod (Korzh et al., 1998), pdx1 (Milewski et al., 1998),
cp (Korzh et al., 2001), try (Biemar et al., 2001), ptf1a (Zecchin et al.,
2004), tfa (Mudumana et al., 2004) and prox1a (Glasgow and
Whole mount immunohistochemistry was described in Dong
et al. (2007). We used the following antibodies: polyclonal rabbit
anti-Prox1 (1:1000, Chemicon), polyclonal guinea pig against
zebrafish Pdx1 (1:200, gift from C. Wright), polyclonal goat anti-
HNF4a (1:100, Santa Cruz Biotechnology), monoclonal mouse
2F11 (1:1000, Abcam ab71286) (Crosnier et al., 2005), rabbit
polyclonal anti-Pax6a/b (1:500, kind gift of F. Biemar and
D. Georlette), mouse monoclonal anti-Nkx6.1 (1:15, developed
by O.D. Madsen and obtained from the Developmental Studies
Hybridoma Bank, F55A1), chicken anti-GFP (1:1000, Aves Labs),
rabbit polyclonal anti-SOX9 (1:500, gift from Silvana Guioli)
(Morais da Silva et al., 1996), mouse monoclonal anti-mCherry
(1:400, anti-DsRed from Clontech) and fluorescently conjugated
AlexaFluor antibodies (Invitrogen).
Fluorescent images were acquired with a Leica SP2 or Olympus
FluoView FV1000 confocal microscopes.
Genotyping of sox9b, fgf10 and fgf24 mutants
Genotyping was performed on genomic DNA extracted from
adult tails or tails obtained from embryos processed through
in situ hybridisation or immunohistochemistry. The ikarus muta-
tion in the fgf24 locus generates a restriction site for the AccI
50-CTGTCAGTCCCACAGCAGTGGACCA-30and reverse 50-CCATGTA-
GATTTTATTACATGTAGGT-30primers (615 pb) digested by AccI
produces two fragments (185 and 430 pb) in the mutants. The
daedalus mutation in fgf10 generates a SNP that was identified by
the TaqMan SNP Genotyping Assays (Applied Biosystem). The
region encompassing the mutation was amplified with the for-
ward primer dae-SNp1F 50-CCGAGCTCCAGGACAATGTG-30and
reverse primer dae-SNp1R 50-GCAGGACAGACGGAACCA-30. Allelic
discrimination wasperformed by
50-CCCTTAGTCACTTTCCATTT-30(wild type allele) and dae-SNp1M2
FAM primer 50-CCTTAGTCACTTaCCATTT-30(mutant allele) according
to the manufacturer.
The sox9bfh313mutation creates a SfcI restriction site. A 440 pb
PCR fragment containing the mutated site was amplified with
forward 50-TGTCCGGAGCTCCGAGCCCGAG-30and reverse 50-ACT-
CATCAGTGCCCTTACTMAGTGTG-30primers. Upon digestion, the
presence of the mutation generated two fragments (166 and
Treatments with pharmacological inhibitors and beta cell
ablation and recovery
Wild type embryos were treated with 5 mM SU5402 (Calbio-
chem) from 54 to 72 hpf or with 100 mM g-secretase inhibitor
DAPT from 3 to 4 or 6 dpf (Calbiochem). DMSO was used in
For specific beta cell ablation, Tg(ins:nfsB-mCherry); sox9b com-
pound larvae were treated at 56 hpf with 7.5 mM Metronidazole
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
(Sigma) with 0.1% DMSO for 24 h as previously described (Pisharath
et al., 2007). This concentration does not cause any adverse effect in
the zebrafish larvae. After several washes with fish water, some
larvae were fixed in 1% PFA to check mCherry fluorescence and
ablation efficiency. The other larvae were grown up to 6.5 dpf, fixed
in 2% PFA and processed for 2F11 immunohsitochemistry (revealed
with anti-mouse AlexaFluor488, Invitrogen). As we could not easily
detect mCherry fluorescence after IHC process, insulin immunode-
tection was performed along with 2F11, and was revealed by anti-
guinea pig AlexaFluor555 (Invitrogen). Larvae were then genotyped
for identification of sox9b mutants and examined with confocal
Zebrafish sox9b is first expressed in the hepatopancreatic endoderm
and then in the entire hepatopancreatic ductal system
While expression of the zebrafish sox9a and sox9b genes has
been reported at the level of the neural crest, pharyngeal arches,
otic vesicles, somites, central nervous system and gonads
(Akiyama et al., 2005; Chiang et al., 2001; Cresko et al., 2003;
Piper et al., 2002; Yan et al., 2005), no data was available so far on
their expression in the liver and in pancreas. Hence, we analysed
in detail the endodermal expression of the two zebrafish Sox9
paralog genes by in situ hybridisation. sox9b transcripts were
detected in the hepatopancreatic region in contrast to sox9a
which was never detected in the midtrunk endoderm at all
examined stages. From 24 hpf, sox9b is expressed at the base of
the dorsal pancreas and, just anteriorly, in the region of the
hepatic primordium and of the prospective ventral pancreas
(abbreviated HVP for Hepatic-Ventral Pancreatic primordia)
(Fig. 1A, D, E). While expression at the level of the dorsal
pancreatic bud is transient, over the next day of development,
sox9bþ cells outline a duct-like structure linking the liver and
pancreas from 2 dpf (Fig. 1B, C). To define more precisely the
structures expressing sox9b, double fluorescent labelling was
performed between 28 and 80 hpf with hepatic and pancreatic
markers. At 28 hpf, sox9b expression in the HVP region overlaps
the caudal part of the prox1aþ hepatic domain (Fig. 1D) and the
anterior part of the pdx1þ pancreatic domain (Fig. 1E), indicating
that sox9b is expressed at the boundary of the two domains. At
that stage, some prox1aþ/pdx1þ cells were detected at this
Fig. 1. sox9b expression in the HPD system, IPD and IHD throughout zebrafish embryonic development. (A) sox9b transcript is detected in the region of the hepatic and
ventral pancreatic primordia (HVP). The yellow arrowhead points to the dorsal pancreas (DP). (B,C) During embryogenesis, sox9b expression delineates a network
connecting the liver and pancreas. Yellow dotted lines delimit the pancreas and the liver primordia. (D) Confocal section through sox9b and prox1a expression in the HVP
domain at 28 hpf. The brackets highlight the overlap between both expression domains. (E) Confocal section through sox9b and pdx1 adjacent and partially common
expression domains. (F) Immunodetection of Prox1 and Pdx1 revealing a partial overlap (bracket). (G) sox9b expression in the ventral pancreas of Tg(ptf1a:eGFP) embryos.
(H) Confocal projection of sox9b and the hepatocyte marker cp expression in the hepatic bud at 52 hpf. (I, J) Comparison at 3 dpf of sox9b expression in the liver labelled by
cp (I) with the pattern of the IHD labelled with the ductal marker 2F11 and Prox1 (the IHDs appear yellow) (J). sox9b expression is restricted to cp-cells (I, arrows) and
displays a highly similar pattern to 2F11þ cholangiocytes (J). (K) At this stage, in the pancreatic acinar tissue labelled with ptf1a, sox9b is restricted to IPD (inset) and EPD
cells. (L) Detection of SOX9 protein in the Notch-responsive cells (PNCs) along the IPD of Tg(Tp1:hmgb1-mCherry) transgenic larvae at 5 dpf. (M) sox9b mRNA detection
followed by immunolabelling with the ductal maker 2F11 demonstrating sox9b expression in the IHD and in the HPD system, notably in the gall bladder (GB, arrowhead).
The dotted lines delineate the border of the liver and pancreas with the HPD. Note that the IPD could not be detected by 2F11 immunolabelling following in situ
hybridisation. EHD, extrahepatic duct; EI, endocrine islet; EPD, extrapancreatic duct; GB, gall bladder; HB, hepatic bud; HVP, hepatic and ventral pancreatic domain; IHD,
intrahepatic ducts; IPD, intrapancreatic ducts; OV, otic vesicle; VP: ventral pancreas. Scale bar¼40 mm except H, I and J¼20 mm.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
boundary (Fig. 1F). Double staining at 2 dpf with the ventral
pancreatic marker ptf1a (Fig. 1G) and the hepatic marker cerulo-
plasmin (cp) (Fig. 1H) revealed that the sox9bþ cells form the
prospective HPD system located in the proximal part of the
ventral pancreatic and hepatic buds and making a junction
between these two buds (see also Fig. 1B). Within the pancreatic
and hepatic buds, sox9b is largely excluded from ptf1aþ cells
(prospective acinar cells) and cpþ hepatocytes, and is expressed
in the prospective ductal cells (Fig. 1G, H). At 3 dpf, the sox9b cells
within the liver are distinct from the cp labelled hepatocytes
(Fig. 1I) and create a pattern similar to the IHD revealed with the
ductal 2F11 antibody (Fig. 1J) (Crosnier et al., 2005; Lorent et al.,
2010). Furthermore, sox9b and 2F11 domains perfectly overlap in
the HPD system connecting the liver and pancreas, showing that
sox9b is expressed in the EHD, EPD and the gall bladder (GB)
(Fig 1M). Within the pancreas (Fig. 1K, L), sox9b was also detected
in the IPD that surrounds the principal islet (labelled with neurod;
Fig.1L) and extends posteriorly in the pancreatic tail.
In conclusion, these data indicate that sox9b is first expressed
in a subset of progenitors encompassing the border between the
liver progenitor domain and the prospective ventral pancreas,
and, at later stages of development, its expression delineates the
biliary and pancreatic ductal network and the HPD system that
connects them to the intestine.
sox9b promotes morphogenesis and differentiation of the hepatic,
pancreatic and hepatopancreatic ductal system
Since the previously reported sox9bb971mutant allele (Yan
et al., 2005) contains a large deletion comprising genes adjacent
to sox9b which could interfere with the analysis of sox9b function,
we investigated the role of sox9b in liver and pancreas develop-
ment by analysing the phenotype of a new sox9b mutant allele.
This mutant, sox9bfh313, harbours a nonsense mutation (K68n) in
the first exon generating a short truncated protein lacking all the
conserved functional domains and therefore should not be func-
tional. Furthermore, SOX9b protein could not be detected by
immunostaining (data not shown) indicating that no alternative
start codon is used downstream the mutation. In contrast to the
sox9bb971deletion mutants (Yan et al., 2005), the general mor-
phology of the new sox9bfh313mutant embryos is identical to wild
type. At 3 dpf, the global morphology and the size of the liver and
the pancreas in the sox9bfh313mutants are indistinguishable from
the wild type organs, as revealed by the transferrin a (tfa) and
trypsin (try) markers (Fig. 2A, B). In addition, the endocrine islet
derived from the dorsal pancreatic bud also appeared unaffected
(data not shown). Analysis of prox1a, pdx1 and ptf1a expression at
24 and 34 hpf also indicated normal specification of the hepatic
and ventral pancreatic domains (data not shown). In contrast, as
depicted in Fig. 2C and D, while Prox1 immunolabelling at 3 dpf
confirmed that the overall morphology of the liver and pancreas is
normal in sox9b mutants as well as the number of hepatocytes
and pancreatic acinar cells, the 2F11 staining revealed dys-
morphic ducts within the liver, pancreas and in the HPD system
connecting the two organs to the intestine in all larvae examined.
These defects were already observed at 2.5 dpf (data not shown)
and they were more obvious at 7 dpf when hepatic and pancreatic
ductal systems are completed and functional (compare the liver
in Fig. 2 G, G0and H, H0, and the pancreas in Fig. 2G00and H00). In
the liver of wild type embryos and larvae, while IHD cells
(2F11þ) are contacting one another to create a well defined
and thin IHD network (Fig. 2E0at 3 dpf and Fig. 2G, G0at 7 dpf),
they remain clustered together in the mutants and form thicker
and fewer cellular interconnections (Fig. 2F0at 3 dpf and Fig. 2H,
H0at 7 dpf, see asterisks).
In the pancreas, the antero-posterior alignment of IPD cells
and the extent of their migration driving primary pancreatic duct
formation within the tail of the pancreas appeared normal
(compare Fig. 2C and D). In contrast, these cells display much
weaker staining of the ductal marker 2F11 at 3 dpf compared to
Fig. 2. Affected morphogenesis of the IPD, IHD and HPD system in sox9b mutants. (A, B) Acinar (try) and hepatocyte (tfa) differentation as well as the global morphology of
the larvae are similar in wild type larvae (A) and in sox9b mutants (B) at 84 hpf. (C, D) Three dimensional rendering of the liver and pancreas at 84 hpf, labelled by Prox1,
and of the entire ductal network highlighted by 2F11. The insets represent the 2F11 staining in the liver. White arrows in D and inset point at the disrupted connections
between cholangiocytes in sox9b mutants. (E, F) Close-up of the HPD system connecting the pancreas and the liver to the intestine. Ectopic Prox1þ cells are detected
throughout the HPD system (red arrow). (E0, F0) Less interconnecting ducts are detected in the IHD sox9b mutants. (E00, F00) 3-D rendering of the pancreas showing weaker
2F11 labelling in the IPD of sox9b mutant (F00) compared to wild type (E00). (G, H). 3-D rendering at 7 dpf of ductal 2F11 labelling in the liver (IHD) in wild type (G) and sox9b
mutants (H). (G0, H0) Higher magnification showing thicker connections between IHD cells (asterisk). (G00, H00) 3-D rendering at 7 dpf of ductal 2F11 labelling in the pancreas
(IPD) in wild type (G0) and sox9b mutants (H0). GB, gall bladder; HPD, hepatopancreatic ductal system; IHD, intrahepatic ducts; IPD, intrapancreatic ducts. Scale
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
wild type ducts (Fig. 2E00, F00). At 7 dpf, the primary pancreatic
ductal network is discontinuous presenting masses of aggregated
cells forming cyst-like structures; the ductal branching leading to
the formation of first and second order pancreatic ducts are also
clearly affected (Fig. 2G00, H00). In contrast, the acinar tissue is
normal in the mutants (data not shown).
In addition to anomalies of the IHD and IPD, the HPD system
joining the pancreas and liver to the intestine is also dysmorphic
in sox9b mutants as it is dilated and harbours a smaller GB
compared to wild type larvae (Fig. 2E, F). The HPD system also
shows weaker 2F11 staining than in wild-type embryos and
displays ectopic Prox1þ cells (red arrow in Fig. 2F0) at 3 dpf
(see below). All these observations reveal that sox9b is required
not only for the morphogenesis of the IHD but also for morpho-
genesis and differentiation of the IPD and HPD system.
To examine more closely the differentiation defect of the HPD
in sox9b mutants, we analysed several markers at 3 dpf. As
depicted in Fig. 3, while these ducts in wild type larvae are
labelled by 2F11 only and not by Prox1 and HNF4a, the
dysmorphic sox9b?/? HPD contains misdifferentiated cells at
3 dpf (see Fig. 3A, B for 3D projections of the HPD system and
Fig. 3A0and B0–B000for z-planes through the same region). Indeed,
hepatic-like cells (coexpressing Prox1 and HNF4a) were found in
most mutant embryos within these structures (71% of embryos,
n¼14), notably proximal to the pancreas (green arrowheads in
Fig. 3B and B00). Similarly, ectopic Pax6þ pancreatic endocrine
cells could be detected distal from the pancreas (Fig. 3C, D, white
arrowheads), notably in the EHD and GB and even within the liver
(66% of mutant embryos, n¼12). Moreover, the pancreatic ductal
marker Nkx6.1 is ectopically expressed throughout the HPD
system in all mutants examined (compare Fig. 3A, A0and
Fig. 3B–B000). Indeed, whereas in wild type larvae high level of
Nkx6.1 expression was normally detected in the IPD only (see
yellow cells within the pancreas in Fig. 3A, A0as IPD cells also
express Prox1 which is revealed in green), Nkx6.1þ cells were
also found in all parts of the HPD in sox9b mutants, notably within
the junction with the intestine as well as near and within the
liver, i.e. locations far from the pancreas (yellow arrowheads in
Fig. 3B, B0–B000). Ectopic pancreatic acinar cells were never
detected in the HPD system of the mutants (data not shown).
Moreover, a significant number of ectopic Nkx6.1þ cells in the
HPD also exhibit different levels of HNF4a, demonstrating com-
pletely aberrant differentiation of HPD duct cells resulting in
chimeric identities (pink arrowheads pointing at triple Nkx6.1/
Prox1/HNF4a positive cells in Fig. 3B and B000).
As the anomalies of hepatic and pancreatic ducts could
partially be due to a reduced number of duct cells, we counted
cells in both organs in sox9b mutants at 3 and 4 dpf. The number
of IHD cells and hepatocytes appeared globally unaffected in
mutant livers compared to wild type. In contrast, there is a
decreased number of IPD cells (94715 duct cells in n¼11 wild
type pancreas versus 58710 cells in n¼10 mutants at 4 dpf)
while the amount of acinar cells is unaffected, as was already
suggested by normal tryspin expression at 3 dpf. No significant
difference in proliferation (EdU and PH3 labelling, Supplemental
Table 1) and apoptosis (Tunel labelling, data not shown) of duct
cells could be detected in the liver and pancreas between wild
type and sox9b mutants. Similarly, hepatocyte and acinar cell
Fig. 3. Misdifferentiation of the HPD system in sox9b mutants. (A, B) 3D-rendering (stack) at 80 hpf of the liver, pancreas and HPD system immunolabelled with Prox1,
HNF4a and the pancreatic ductal marker Nkx6.1. (A0, B0–B000) Z-planes through the same larvae as in A and B. Prox1 with HNF4a label hepatocytes while Prox1 together
with Nkx6.1 strongly labels the IPD. In sox9b mutants (B and B0–B000), the HPD system becomes labelled with the three markers and display different ectopic cell types (see
the colour code at the bottom). Dotted lines delimitate organs and ducts boundaries. (C, D) 3-D rendering of the HPD system at 84 hpf labelled with the ductal marker 2F11
and the endocrine marker Pax6. Ectopic endocrine cells along the HPD and in the liver are indicated by white arrowheads. EHD, extrahepatic duct; EPD, extrapancreatic
duct; HPD, hepatopancreatic ductal system; IPD, intrapancreatic ducts. Scale bar¼20 mm.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
proliferation and apoptosis are not affected in the mutant. These
data indicate that sox9b is not critical for duct cell proliferation
and survival. Overall, our results suggest that it is required
for establishment of proper connections between duct cells in
the liver and pancreas as well as for duct cell differentiation in the
pancreas and HPD system.
The sox9bfh313mutant allele is not embryonic lethal but the
sox9b mutants hardly reach adulthood. Indeed, the analysis of the
survival rate in the progeny of sox9b heterozygote matings
revealed only one surviving homozygous mutant out of 81 fish
after 6 months, and 3 homozygotes out of 31 fish after 2.5 months.
Furthermore, the overall size of the fish is dramatically reduced in
the mutants (average sox9b mutant length 1.770.36 cm com-
pared with 2.4270.48 cm for wild type fish at 2.5 months).
sox9b early expression is activated by the FGF and BMP signalling
In order to identify extrinsic factors required for the activation
of sox9b expression in the hepatopancreatic domain at 24 hpf, we
examined signalling pathways known to be involved in the
specification of the ventral pancreas and the liver in zebrafish.
Specification of the liver bud has been previously shown to
depend on the action of WNT2bb (Ober et al., 2006). Also, we
previously reported that the FGF signalling, namely FGF10 and
FGF24, is essential for ventral pancreas specification (Manfroid
et al., 2007) while BMP2a is important for both liver and ventral
pancreas specification (Naye et al., 2012). All these extrinsic
factors are secreted from the lateral plate mesoderm adjacent to
the prospective endodermal hepatic and ventral pancreatic
domain (here referred to as HVP). In the double fgf10; fgf24
mutants, sox9b expression is indeed lost in the HVP at 30 hpf
(Fig. 4A, B). Similarly, sox9b expression was undetectable in the
HVP of bmp2a morphants (Fig. 4C, D). In contrast, sox9b expres-
sion is not affected in the wnt2bb mutants (data not shown).
These data demonstrate that the induction of sox9b in the
hepatopancreatic domain at 24 hpf is controlled by FGF and
BMP signals released by the lateral plate mesoderm.
Maintenance of ductal sox9b expression requires FGF and
A previous study has reported that fgf10 controls HPD differ-
entiation and maintains borders between the HPD system, the
pancreas and the liver, and that the HPD system in fgf10 mutants
displays misdifferentiated cells (Dong et al., 2007). Given the similar
defects in sox9b mutants and in the fgf10 mutants at 3 dpf, we asked
whether fgf10 controls sox9b expression in the HPD system. sox9b
early expression in the HVP is correctly induced in fgf10 mutant at
30 hpf (data not shown). At 3 dpf, however, sox9b expression was
barely detected in the HPD system in fgf10 mutants (Supplementary
Fig. 1A, B) though its expression is normal in the hepatic and
pancreatic ductal network. To more precisely assess the role of FGF
signalling on sox9b expression after these ductal structures are
specified, wild type embryos were treated with the FGF inhibitor
SU5402 from 54 to 72 hpf (Fig. 5A, B and A0, B0). In treated embryos,
sox9b expression is strongly diminished in the EPD (Fig. 5A0, B0). As
this treatment has been shown to induce endocrine differentiation
from the EPD (Chung et al., 2010), the endocrine marker neurod was
also analysed with sox9b expression (Fig. 5A, B). Interestingly, the
ductal sites where sox9b expression is lost display massive endo-
crine differentiation. As sox9b mutants also harbour ectopic endo-
crine cells, these data strongly suggest that sox9b labels in the HPD
system progenitors that are able to undergo endocrine differentia-
tion. To analyse further similitude between sox9b and fgf10 mutants,
Nkx6.1 expression was examined in the fgf10 mutant and compared
with Pax6, as ectopic endocrine differentiation was previously
reported in the HPD system of this mutant (Dong et al., 2007). Like
in sox9b mutants, Nkx6.1 is ectopically expressed throughout the
HPD system, notably within the junction with the intestine, and
within the liver of fgf10 mutants (Fig. 5C, D). Some ectopic Pax6þ
endocrine cells were also detected in this region. These observations
showed that (i) the HPD system of the fgf10 and sox9b mutants
displays a similar phenotype and (ii) sox9b expression is severely
compromised in this structure in fgf10 mutants, suggesting that
Sox9b could mediate, at least partially, the action of FGF10 in
patterning and differentiating the HPD system.
Recent studies have established that the IPD contain pancrea-
tic Notch-responsive cells (PNCs) that correspond to pancreatic
progenitors giving rise to late endocrine cells. PNCs are easily
detected by expression of mCherry in the transgenic line
Tg(Tp1:hmgb1-mCherry) (Parsons et al., 2009). Sox9 immunode-
tection was performed on Tg(Tp1:hmgb1-mCherry) larvae at 5 dpf
and revealed that pancreatic ductal Sox9b is localised in the PNCs
(Fig. 5G–G00). As PNCs disappear upon Notch signalling inhibi-
tion concomitant with an increase in endocrine differentiation
(Parsons et al., 2009), we examined whether sox9b may be
dependent on Notch signalling in the IPD by incubating wild type
larvae with the g-secretase inhibitor DAPT from 72 to 96 hpf. As
depicted in Fig. 5E–F0, loss of sox9b expression in parts of the IPD
of treated larvae was detected with concomitant robust increase
in neurodþ endocrine cells at ductal sites where sox9b expression
disappears. Collectively, our results indicate that sox9b expression
marks the PNCs which, upon Notch inhibition, lose their sox9b
expression and undergo endocrine differentiation. In contrast, the
same DAPT treatment did not impair sox9b expression in the IHD
(data not shown) showing that Notch is not required to maintain
sox9b expression in the IHD.
To determine whether sox9b expression still labels PNCs in the
adult, its expression was analysed by Q-PCR in pancreatic Notch-
responsive cells isolated by FACS from Tg(Tp1:hmgb1-eGFP) adult
fish. In the adult zebrafish pancreas, Notch activity persists in
centroacinar cells (Parsons et al., 2009). In mouse, these cells
display progenitor function and express Sox9 (Rovira et al., 2010).
sox9b expression was specifically found in Notch-responsive
GFPþ cells while it was not detected at all in ptf1aþ acinar cells
isolated from Tg(ptf1a:eGFP) adult fish (Fig. 5H). Conversely, the
acinar marker amylase was not detected in GFPþ cells. This
suggests that sox9b expression, like its murine orthologue, is
localised in putative pancreatic progenitors in the adult.
Fig. 4. sox9b early hepatopancreatic expression is activated by FGF and BMP and is
maintained in the ducts by FGF and Notch. (A, B) sox9b early expression (30 hpf) in
the hepatic and ventral pancreatic primordia (HVP) is not activated in compound
fgf10; fgf24 mutants (C, D) Similarly, sox9b is not induced in bmp2a morphants.
Note that sox9b in the dorsal pancreatic bud (DP) is not affected.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
sox9b is required for late endocrine cells formation and for
beta cell regeneration in larvae
In order to investigate the role of sox9b in differentiation of
late endocrine cells, we used two experimental strategies, one
stimulating precocious endocrine differentiation in the IPD by
Notch inhibition as described above (Parsons et al., 2009) and the
other triggering beta cell recovery upon their ablation (Pisharath
et al., 2007). In the first approach, wild type and sox9b mutants
were exposed to DAPT or DMSO from 3 to 6 dpf and endocrine
differentiation in secondary islets along the IPD was examined
using Pax6 and 2F11 markers at 6 dpf (Fig. 6A–D). In wild type
larvae and sox9b mutants treated with DMSO, a few secondary
endocrine cells were observed (Fig. 6A, B). In contrast, blocking
Notch signalling with DAPT in wild type larvae caused signifi-
cantly increased formation of secondary islets while only a few
endocrine cells were detected in sox9b mutants (Fig. 6C, D).
In the second approach, we used the Tg(ins:nfsB-mCherry) line
encoding in beta cells a Nitroreductase enzyme which converts
Metronidazole (Met) in cytotoxins (Pisharath et al., 2007). This
allows the specific and conditional beta cell ablation following
incubation of larvae with Met. To compare the recovery capacity
of beta cells in wild type and sox9b mutants, Tg(ins:nfsB-mCherry)
larvae and sox9bfh313mutants harbouring the (ins:nfsB-mCherry)
transgene were treated with Met from 56 to 80 hpf to eliminate
the dorsal bud-derived as well as the first ‘‘late’’ beta cells.
Ablation was verified just after the treatment (Supplementary
Fig. 2). While untreated wild type transgenic larvae display at
Fig. 5. Concomitant maintenance of ductal sox9b expression with repression of endocrine differentiation by FGF and Notch signalling. (A, B) Expression at 3 dpf of sox9b
with the endocrine marker neurod in wild type embryos treated from 54 to 72 hpf with DMSO (A) or with the FGF inhibitor SU5402 (B). Confocal planes containing the EPD
are shown. (A0, B0) sox9b expression only is shown. (C, D) Ectopic Nkx6.1þ and endocrine Pax6 cells are found in the HPD of fgf10 mutants, as in sox9b mutants. Yellow
dotted lines demarcate organ boundaries. (E–F0) 3-D rendering of the pancreas showing sox9b and neurod expression at 4 dpf at the level of the IPD in wild type embryos
treated from 72 to 96 hpf with DMSO (E, E0) or with the Notch inhibitor DAPT (F, F0). (E0, F0) sox9b expression alone is shown. (G–G00) Detection of SOX9 protein in the Notch-
responsive cells (PNCs) revealed with anti-mCherry in Tg(Tp1:hmgb1-mCherry) transgenic larvae at 5 dpf. (H) Quantitative RT-PCR analyses of sox9b and amylase expression
in centroacinar and acinar cells isolated from the pancreas of adult Tg(Tp1:hmgb1-eGFP) (Tp1þ) and Tg(ptf1a:eGFP) (ptf1aþ) fish, respectively. EPD, extrapancreatic duct;
HPD, hepatopancreatic ductal system; IPD, intrapancreatic duct. Scale bar¼20 mm.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
least 20 beta cells clustered in the principal islet with intense
mCherry fluorescence, treated larvae, as previously reported
(Pisharath et al., 2007), have only very few beta cells which are
also more spherical and less fluorescent, and small cell debris are
often found outside the islet thereby supporting efficient ablation.
The larvae were allowed to recover for 3 days after ablation in
order to assess beta cell recovery (Fig. 6E–H). While the principal
islet appeared completely regenerated in 32.5% of wild type
larvae (n¼40), sox9b mutants (n¼18) never show normal number
of beta cells. Although there is a variation of beta cell regeneration
among wild type and sox9b?/? larvae, the number of larvae
displaying absence of regeneration was dramatically increased in
sox9b mutants (72.2%) compared to wild type (20%). Partial beta
cell recovery was observed in 47.5% (5–10 beta cells/islet) wild
type and in 27.8% sox9b mutants (1–5 beta cells/islet). This
demonstrates a highly compromised recovery in sox9b mutants.
Taken together, these two approaches demonstrate that sox9b is
required for the formation of secondary islets along the IPD and
for beta cells regeneration.
The present work unveils an important function of zebrafish
sox9b in hepatopancreatic duct development, in endocrine cell
formation and in beta cell regeneration from pancreatic ducts. We
show that sox9b expression appears in cells covering the interface
between the hepatic and ventral pancreatic primordia and later
delineates the hepatopancreatic ductal system as well as the
intra-pancreatic and hepatic ducts. sox9b is essential for morpho-
genesis of the whole hepatopancreatic ducts and prevents pan-
creatic, hepatic and intestinal misdifferentiation in the HPD
system. We also show that sox9b expression is induced at
24 hpf by the combined action of fgf10 and fgf24 and by bmp2a,
two signalling pathways important for ventral pancreas and liver
specification. In larvae, its ductal expression is maintained by FGF
signalling in the HPD system and by Notch in the IPD. Finally, we
show that, in the IPD, sox9b expression marks the PNCs in the
zebrafish larva and adult, and that it is essential for the formation
of secondary islets along the IPD and for beta cell regeneration in
the principal islet following specific cell ablation in larvae.
sox9b marks the HPD, IPD and IHD throughout development
In wild type embryos, sox9b expression at 28 hpf draws an
interesting pattern in the hepatopancreatic domain. Indeed, sox9b
expression overlaps the hepatic and pancreatic primordia thereby
labelling a subset of cells across each side of these two progenitor
domains. As sox9b is detected in all hepatopancreatic ducts at
later stages, this pattern suggests that the sox9bþ cells at 28 hpf
are HPD progenitors. Recent data in mouse showed that the
extrahepatobiliary system (here referred to as HPD) shares a
common origin with the ventral pancreas (Spence et al., 2009).
These progenitors express Sox17 which later becomes restricted
to the gall bladder. In zebrafish, sox17 also marks the gall bladder
at late developmental stages (from ?40 hpf) but its expression is
not detected at 28 hpf between the hepatic and pancreatic
primordia like sox9b. Based on our data, we propose that this
sox9b early expression may define a progenitor cell population
which gives rise to the HPD in addition to the IHD and IPD. Cell
fate experiments will be required to test this hypothesis.
sox9b governs morphogenesis and differentiation of the IPD, IHD
and of the HPD system
According to the restricted expression of sox9b in all hepato-
sox9bfh313zebrafish larvae revealed defects specifically in these
ducts and not in the pancreatic acinar tissue and the first
endocrine cells nor in hepatocytes. Indeed, the pancreas of
sox9bfh313larvae display fewer, dysmorphic ducts with lower
2F11 staining. This phenotype is much less severe than the one
reported for the Sox9 pancreatic knockout mice that have a drastic
pancreatic hypoplasia with reduced number of acinar, ductal and
endocrine cells. In mouse, it is likely that the essential function of
Sox9 in the maintenance of the pool of pancreatic progenitors
precludes the analysis of a specific requirement in duct formation
at later stages. Inversely, the defects in the liver are more drastic
in zebrafish sox9bfh313mutant larvae compared to Sox9 hepatic
knockout mice. Indeed connections between duct biliary cells
Fig. 6. sox9b is required for late endocrine cell formation in the IPD and for islet
beta cell recovery upon ablation in larvae. (A–D) Immunodetection at 6 dpf of
endocrine cell differentiation (Pax6) along the IPD labelled with 2F11 in wild type
(A) and sox9b mutant larvae (B). Larvae were treated with DMSO (A, B) or DAPT
(C, D) from 3 to 6 dpf. Secondary isolated endocrine cells are detected (small
arrowheads) in wild type and sox9b mutant control larvae. The dotted line
demarcates the intestine from the pancreatic region. (C, D) The massive increase
in secondary islets resulting from DAPT treatment of wild type larvae (large
arrowheads in C) is not observed in sox9b mutants which still display isolated
endocrine cells along the IPD (small arrowheads in D). (E–H) Beta cell recovery in
Tg(ins:nfsB-mCherry); sox9b larvae upon Metronidazole (Met) mediated ablation.
Pancreatic ducts were labelled with 2F11 and beta cells were revealed by Insulin
(Ins) immunodetection as mCherry fluorescence was undetectable after the
process of immunofluorescence. (E, F) 6.5 dpf Tg(ins:nfsB-mCherry) wild type
(E) and sox9b mutant larvae (F) 3 days (80 h) after DMSO treatment from 56 to
80 hpf. (G, H). Tg(ins:nfsB-mCherry) wild type (G) and sox9b mutant (H) larvae 3
days after Met exposure. Scale bar¼20 mm.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
leading the formation of the IHD network are severely disrupted
at 7 dpf, when the biliary system is completed and functional in
wild type larvae, while hepatic duct maturation is only delayed in
the mouse Sox9 knockout and bile ducts are normal 5 weeks after
birth (Antoniou et al., 2009). This emphasises the benefit to
examine two animal models to gain complete understanding of
the function of a regulatory gene. The phenotypic differences
between the Sox9 loss-of-function in mouse and zebrafish are
probably due to the involvement of other factors able to com-
pensate one another in the pancreas or liver.
In the zebrafish liver, IHD development does not proceed via a
ductal intermediate as described in mouse (Lorent et al., 2004).
Crucial steps of zebrafish IHD morphogenesis, occur between
2.5 and 4 dpf, and involve formation of a network by the first
contiguous IHD cells which then undergoes extensive morphoge-
netic remodelling: IHD cells adopt a stellate morphology, migrate
and separate from one another starting to arrange in a network,
the connections between them narrow, they proliferate exten-
sively and lumen forms. Our data show that sox9b regulates
formation of intercellular connections but not proliferation. It
would be interesting to explore whether duct cell migration and
lumen formation are also affected in the mutants.
Despite differences in the IHD morphogenesis and anatomy
between species, there is strong conservation of the molecular
processes leading IHD development, including the Notch path-
way. Particularly, it has been shown in zebrafish that Notch
signalling is required for IHD remodelling (Lorent et al., 2010).
In mouse, a link between Sox9 and Notch in biliary development
has been suggested based on the observation of the expression
pattern of Hes1, a mediator of Notch signalling, is affected in Sox9
hepatic knockout (Antoniou et al., 2009). In zebrafish, it is not
clear whether sox9b and Notch signalling cooperate in IHD
morphogenesis as (i) Notch appears to regulate more aspects of
duct remodelling than sox9b and (ii) sox9b is still well expressed
in the IHD following DAPT treatment (data not shown). Overall,
our data suggest a role of Sox9 in IHD maturation being conserved
through the vertebrates and the relation between Sox9 and
Notch signalling in zebrafish as well as in mouse remains to be
In the zebrafish pancreas, duct morphogenesis is not well
understood though it has been proposed that pancreatic ducts
arise from ductal progenitor cells in situ rather than arising from
reiterative branching of the pancreatic epithelium, like in mam-
malian pancreas (Yee et al., 2005). As our results reveal that sox9b
is involved in pancreatic duct cell differentiation and morphogen-
esis, it would be interesting to determine whether Sox9 function
in pancreatic duct development is also conserved in mammals.
Taken together, our results uncovering the importance of sox9b in
both pancreatic and hepatic duct morphogenesis suggest com-
mon mechanisms involved in duct development in both organs.
We also demonstrate that sox9b is essential for the proper
morphogenesis and differentiation of the HPD system connecting
the liver and pancreatic ducts to the intestine. Indeed, in sox9b
mutants, the HPD structures are dysmorphic and misdifferen-
tiated as highlighted by (i) enlarged and less well morphologically
defined HPD structures, (ii) smaller gall bladder, and (iii) presence
of ectopic hepatocyte and pancreatic markers and lower ductal
marker 2F11 staining. Ectopic pancreatic acinar differentiation
was never detected in the HPD of sox9b mutants suggesting that
these ducts do not have the competence to differentiate into all
cell types or that signalling pathways from adjacent tissues have a
repressive role on acinar differentiation from these progenitors.
Alternatively, one could argue that ectopic endocrine cells and
hepatocytes along the HPD have migrated from the pancreas and
the liver. However, the presence of chimeric differentiated cells
(expressing endocrine pancreatic and hepatic markers together in
the same cells) within the HPD system in sox9b mutants argues
for genuine misdifferentiation of HPD cells. Misdifferentiation in
the HPD system at 3 dpf in the absence of sox9b indicates that
sox9b is required to maintain ductal identity and the boundaries
between organs in the HPD. In mouse, ectopic pancreas in the
HPD system as well as GB agenesis have been described in Hes1
knockout embryos (Sumazaki et al., 2004). However, while only
ectopic endocrine cells were detected in the HPD of sox9b
mutants, both ectopic endocrine and exocrine tissues were found
in the HPD of Hes1 mice. In contrast, the defects of the HPD
system presented by sox9b mutants are mostly similar to the
zebrafish fgf10 mutant, strongly suggesting that sox9b and fgf10
are acting in the same regulatory pathway. This is further
supported by the loss of sox9b expression in the HPD system of
The ductal defects described here for the sox9bfh313nonsense
mutation were also observed in the previously described
sox9bb971mutant (data not shown) (Yan et al., 2005). However,
this sox9bb971mutant is not the adequate tool to address the
specific function of sox9b as (i) this allele contains a large deletion
removing sox9b but also sox8 and pvalb1, 4, 8 (Yokoi et al., 2009)
and (ii) the global morphology of sox9bb971mutants was altered
and many organs were hypoplasic such as the pancreas and liver,
but also the intestine and swim bladder which both do not
express sox9b. Moreover, we can assume that sox9bfh313is a null
mutant as the compound sox9bfh313; sox9bb971mutants (obtained
by crossing sox9bfh313and sox9bb971heterozygous) present the
same defects as sox9bfh313homozygous (data not shown), and
SOX9 protein could not be detected in the sox9bfh313homozygous.
sox9b is required for ‘‘late’’ endocrine cells formation and
regeneration in the larva
In zebrafish larvae, the first ‘‘late’’ endocrine cells derive from
the EPD at around 3 dpf (Field et al., 2003). The failure of sox9b
mutants to restore the beta cell mass in the principal islet after
ablation indicates that sox9b is required for the capacity of ductal
cells to generate the secondary transition of endocrine cells. It is
plausible that sox9b is crucial to preserve the progenitor pluripo-
tency of ductal cells. Previous studies have reported Sox9 expres-
sion in adult human and mouse HPD system (Furuyama et al.,
2011) and demonstrated that the HPD cells of adult human
donors express SOX9 and can generate hepatic and pancreatic
cell types in vitro (Cardinale et al., 2011). Hence we can speculate
that sox9b inactivation leads to a premature cell differentiation
causing a depletion of progenitor cells in the EPD. This hypothesis
is supported by the presence of misdifferentiated cells in the HPD
of sox9b mutants.
A role of sox9b in maintenance of the progenitor state is
further strengthened by our observation that FGF and Notch
signalling, both required to maintain progenitor identity (Dong
et al., 2007; Wang et al., 2011), also maintain sox9b expression in
the EPD and IPD, respectively. Furthermore, we show that sox9b
marks the PNCs in the larva and that it mediates Notch repressive
activity on endocrine differentiation in the IPD. These PNCs
constitute progenitors that have recently been identified along
the IPD and have been shown to be able to contribute to adult
endocrine cells in zebrafish (Parsons et al., 2009; Wang et al.,
2011). Our results showing that sox9b is required for IPD devel-
opment suggest that sox9b is important to establish pancreatic
progenitors in the IPD. In the adult, PNCs correspond to centroa-
cinar cells, a cell type closely associated to terminal ducts that has
been shown in mouse to be capable of endocrine differentiation
in vitro (Rovira et al., 2010). Thus, our observation that sox9b
expression localises in PNCs in adult zebrafish suggests that sox9b
could play progenitor function in beta cell genesis in adult
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
pancreas. Future experiments will be needed to explore this
Acknowledgements and Funding
We thank C. Wright, J. Lewis, F. Biemar and D. Georlette for
kindly providing antibodies, S. Leach for Tg(ptf1a:eGFP) transgenic
line, J.H. Postlethwait and C. Neumann for mutants strains. The
authors thank Dr. S. Ormenese and the ‘‘GIGA-Cell Imaging’’
facility, and Dr. M. Winandy and the ‘‘GIGA-Zebrafish Facility’’.
I.M. was supported by the FNRS-FRS and by the Action de
Recherches Concerte ´es (University of Li? ege). B.P. and M.L.V. are
Chercheurs qualifie ´s FNRS. N.D. and V.V.B. are funded by the
WALEO (Re ´gion Wallonne). F.N. has a postdoctoral fellowship
from the University of Li? ege. This work was funded by the Belgian
State’s ‘‘Interuniversity Attraction Poles’’ Program (SSTC, PAI) and
by the 6th European Union Framework Program (BetaCellTherapy
Integrated Project). TILLING of sox9b was supported by NIH
HG002995 to CBM. CBM is an Investigator of the Howard Hughes
Medical Institute. MJP and WH are funded by NIH grants
DK080730 and DK090816.
Appendix A. Supporting information
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.ydbio.2012.04.002.
Akiyama, H., Kim, J.E., Nakashima, K., Balmes, G., Iwai, N., Deng, J.M., Zhang, Z.,
Martin, J.F., Behringer, R.R., Nakamura, T., de Crombrugghe, B., 2005. Osteo-
chondroprogenitor cells are derived from Sox9 expressing precursors. Proc.
Natl. Acad. Sci. USA 102, 14665–14670.
Antoniou, A., Raynaud, P., Cordi, S., Zong, Y., Tronche, F., Stanger, B.Z., Jacquemin,
P., Pierreux, C.E., Clotman, F., Lemaigre, F.P., 2009. Intrahepatic bile ducts
develop according to a new mode of tubulogenesis regulated by the transcrip-
tion factor SOX9. Gastroenterology 136, 2325–2333.
Biemar, F., Argenton, F., Schmidtke, R., Epperlein, S., Peers, B., 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.
Bonner-Weir, S., Toschi, E., Inada, A., Reitz, P., Fonseca, S.Y., Aye, T., Sharma, A.,
2004. The pancreatic ductal epithelium serves as a potential pool of progenitor
cells. Pediatr. Diabetes 5 (Suppl. 2), 16–22.
Cardinale, V., Wang, Y., Carpino, G., Cui, C.B., Gatto, M., Rossi, M., Berloco, P.B.,
Cantafora, A., Wauthier, E., Furth, M.E., Inverardi, L., Dominguez-Bendala, J.,
Ricordi, C., Gerber, D., Gaudio, E., Alvaro, D., Reid, L., 2011. Multipotent stem/
progenitor cells in human biliary tree give rise to hepatocytes, cholangiocytes
and pancreatic islets. Hepatology.
Carpentier, R., Suner, R.E., van Hul, N., Kopp, J.L., Beaudry, J.B., Cordi, S., Antoniou,
A., Raynaud, P., Lepreux, S., Jacquemin, P., Leclercq, I.A., Sander, M., Lemaigre,
F.P., 2011. Embryonic ductal plate cells give
periportal hepatocytes, and adult liver progenitor cells. Gastroenterology
141, 1432–1438. (e4).
Chiang, E.F., Pai, C.I., Wyatt, M., Yan, Y.L., Postlethwait, J., Chung, B., 2001. Two sox9
genes on duplicated zebrafish chromosomes: expression of similar transcrip-
tion activators in distinct sites. Dev. Biol. 231, 149–163.
Chung, W.S., Andersson, O., Row, R., Kimelman, D., Stainier, D.Y., 2010. Suppression
of Alk8-mediated Bmp signaling cell-autonomously induces pancreatic beta-
cells in zebrafish. Proc. Natl. Acad. Sci. USA 107, 1142–1147.
Cresko, W.A., Yan, Y.L., Baltrus, D.A., Amores, A., Singer, A., Rodriguez-Mari, A.,
Postlethwait, J.H., 2003. Genome duplication, subfunction partitioning, and
lineage divergence: Sox9 in stickleback and zebrafish. Dev. Dyn. 228, 480–489.
Crosnier, C., Vargesson, N., Gschmeissner, S., Ariza-McNaughton, L., Morrison, A.,
Lewis, J., 2005. Delta-Notch signalling controls commitment to a secretory fate
in the zebrafish intestine. Development 132, 1093–1104.
Curado, S., Anderson, R.M., Jungblut, B., Mumm, J., Schroeter, E., Stainier, D.Y.,
2007. Conditional targeted cell ablation in zebrafish: a new tool for regenera-
tion studies. Dev. Dyn. 236, 1025–1035.
Dong, P.D., Munson, C.A., Norton, W., Crosnier, C., Pan, X., Gong, Z., Neumann, C.J.,
Stainier, D.Y., 2007. Fgf10 regulates hepatopancreatic ductal system patterning
and differentiation. Nat. Genet. 39, 397–402.
rise to cholangiocytes,
Dong, P.D., Provost, E., Leach, S.D., Stainier, D.Y., 2008. Graded levels of Ptf1a
differentially regulate endocrine and exocrine fates in the developing pan-
creas. Genes Dev. 22, 1445–1450.
Draper, B.W., McCallum, C.M., Stout, J.L., Slade, A.J., Moens, C.B., 2004. A high-
throughput method for identifying N-ethyl-N-nitrosourea (ENU)-induced
point mutations in zebrafish. Methods Cell Biol. 77, 91–112.
digestive system in zebrafish. II. Pancreas morphogenesis. Dev. Biol. 261,
Furuyama, K., Kawaguchi, Y., Akiyama, H., Horiguchi, M., Kodama, S., Kuhara, T.,
Hosokawa, S., Elbahrawy, A., Soeda, T., Koizumi, M., Masui, T., Kawaguchi, M.,
Takaori, K., Doi, R., Nishi, E., Kakinoki, R., Deng, J.M., Behringer, R.R., Nakamura,
T., Uemoto, S., 2011. Continuous cell supply from a Sox9-expressing progenitor
zone in adult liver, exocrine pancreas and intestine. Nat. Genet. 43, 34–41.
Glasgow, E., Tomarev, S.I., 1998. Restricted expression of the homeobox gene prox
1 in developing zebrafish. Mech. Dev. 76, 175–178.
Godinho, L., Mumm, J.S., Williams, P.R., Schroeter, E.H., Koerber, A., Park, S.W.,
Leach, S.D., Wong, R.O., 2005. Targeting of amacrine cell neurites to appro-
priate synaptic laminae in the developing zebrafish retina. Development 132,
Hauptmann, G., Gerster, T., 1994. Two-color whole-mount in situ hybridization to
vertebrate and Drosophila embryos. Trends Genet. 10, 266.
Hesselson, D., Anderson, R.M., Beinat, M., Stainier, D.Y., 2009. Distinct populations
of quiescent and proliferative pancreatic beta-cells identified by HOTcre
mediated labeling. Proc. Natl. Acad. Sci. USA 106, 14896–14901.
Kinkel, M.D., Prince, V.E., 2009. On the diabetic menu: zebrafish as a model for
development and function. Bioassays 31, 139–152.
Kopp, J.L., Dubois, C.L., Schaffer, A.E., Hao, E., Shih, H.P., Seymour, P.A., Ma, J.,
Sander, M., 2011. Sox9þ ductal cells are multipotent progenitors throughout
development but do not produce new endocrine cells in the normal or injured
adult pancreas. Development 138, 653–665.
Korzh, S., Emelyanov, A., Korzh, V., 2001. Developmental analysis of ceruloplasmin
gene and liver formation in zebrafish. Mech. Dev. 103, 137–139.
Korzh, V., Sleptsova, I., Liao, J., He, J., Gong, Z., 1998. Expression of zebrafish bHLH
genes ngn1 and nrd defines distinct stages of neural differentiation. Dev. Dyn.
Lorent, K., Moore, J.C., Siekmann, A.F., Lawson, N., Pack, M., 2010. Reiterative use of
the notch signal during zebrafish intrahepatic biliary development. Dev. Dyn.
Lorent, K., Yeo, S.Y., Oda, T., Chandrasekharappa, S., Chitnis, A., Matthews, R.P.,
Pack, M., 2004. Inhibition of Jagged-mediated Notch signaling disrupts zebra-
fish biliary development and generates multi-organ defects compatible with
an Alagille syndrome phenocopy. Development 131, 5753–5766.
Lynn, F.C., Smith, S.B., Wilson, M.E., Yang, K.Y., Nekrep, N., German, M.S., 2007.
Sox9 coordinates a transcriptional network in pancreatic progenitor cells.
Proc. Natl. Acad. Sci. USA 104, 10500–10505.
Manfroid, I., Delporte, F., Baudhuin, A., Motte, P., Neumann, C.J., Voz, M.L., Martial,
J.A., Peers, B., 2007. Reciprocal endoderm-mesoderm interactions mediated by
fgf24 and fgf10 govern pancreas development. Development 134, 4011–4021.
Milewski, W.M., Duguay, S.J., Chan, S.J., Steiner, D.F., 1998. Conservation of PDX-1
structure, function,and expression
Morais da Silva, S., Hacker, A., Harley, V., Goodfellow, P., Swain, A., Lovell-Badge, R.,
1996. Sox9 expression during gonadal development implies a conserved role
for the gene in testis differentiation in mammals and birds. Nat. Genet. 14,
Moss, J.B., Koustubhan, P., Greenman, M., Parsons, M.J., Walter, I., Moss, L.G., 2009.
Regeneration of the pancreas in adult zebrafish. Diabetes 58, 1844–1851.
Mudumana, S.P., Wan, H., Singh, M., Korzh, V., Gong, Z., 2004. Expression analyses
of zebrafish transferrin, ifabp, and elastaseB mRNAs as differentiation markers
for the three major endodermal organs: liver, intestine, and exocrine pancreas.
Dev. Dyn. 230, 165–173.
Naye, F., Voz, M.L., Detry, N., Hammerschmidt, M., Peers, B., Manfroid, I., 2012.
Essential roles of zebrafish bmp2a, fgf10, and fgf24 in the specification of the
ventral pancreas. Mol. Biol. Cell 23, 945–954.
Norton, W.H., Ledin, J., Grandel, H., Neumann, C.J., 2005. HSPG synthesis by
zebrafish Ext2 and Extl3 is required for Fgf10 signalling during limb develop-
ment. Development 132, 4963–4973.
Ober, E.A., Verkade, H., Field, H.A., Stainier, D.Y., 2006. Mesodermal Wnt2b
signalling positively regulates liver specification. Nature 442, 688–691.
Parsons, M.J., Pisharath, H., Yusuff, S., Moore, J.C., Siekmann, A.F., Lawson, N., Leach,
S.D., 2009. Notch-responsive cells initiate the secondary transition in larval
zebrafish pancreas. Mech. Dev. 126, 898–912.
Piper, K., Ball, S.G., Keeling, J.W., Mansoor, S., Wilson, D.I., Hanley, N.A., 2002. Novel
SOX9 expression during human pancreas development correlates to abnorm-
alities in Campomelic dysplasia. Mech. Dev. 116, 223–226.
Pisharath, H., Rhee, J.M., Swanson, M.A., Leach, S.D., Parsons, M.J., 2007. Targeted
ablation of beta cells in the embryonic zebrafish pancreas using E. coli
nitroreductase. Mech. Dev. 124, 218–229.
Rovira, M., Scott, S.G., Liss, A.S., Jensen, J., Thayer, S.P., Leach, S.D., 2010. Isolation
and characterization of centroacinar/terminal ductal progenitor cells in adult
mouse pancreas. Proc. Natl. Acad. Sci. USA 107, 75–80.
Roy, S., Qiao, T., Wolff, C., Ingham, P.W., 2001. Hedgehog signaling pathway is
essential for pancreas specification in the zebrafish embryo. Curr. Biol. 11,
D.Y.,2003.Formation of the
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278
Seymour, P.A., Freude, K.K., Tran, M.N., Mayes, E.E., Jensen, J., Kist, R., Scherer, G.,
Sander, M., 2007. SOX9 is required for maintenance of the pancreatic
progenitor cell pool. Proc. Natl. Acad. Sci. USA 104, 1865–1870.
Solar, M., Cardalda, C., Houbracken, I., Martin, M., Maestro, M.A., De Medts, N., Xu,
X., Grau, V., Heimberg, H., Bouwens, L., Ferrer, J., 2009. Pancreatic exocrine
duct cells give rise to insulin-producing beta cells during embryogenesis but
not after birth. Dev. Cell 17, 849–860.
Spence, J.R., Lange, A.W., Lin, S.C., Kaestner, K.H., Lowy, A.M., Kim, I., Whitsett, J.A.,
Wells, J.M., 2009. Sox17 regulates organ lineage segregation of ventral foregut
progenitor cells. Dev. Cell 17, 62–74.
Sumazaki, R., Shiojiri, N., Isoyama, S., Masu, M., Keino-Masu, K., Osawa, M.,
Nakauchi, H., Kageyama, R., Matsui, A., 2004. Conversion of biliary system to
pancreatic tissue in Hes1-deficient mice. Nat. Genet. 36, 83–87.
Wang, Y., Rovira, M., Yusuff, S., Parsons, M.J., 2011. Genetic inducible fate mapping
in larval zebrafish reveals origins of adult insulin-producing beta-cells.
Development 138, 609–617.
Yan, Y.L., Willoughby, J., Liu, D., Crump, J.G., Wilson, C., Miller, C.T., Singer, A.,
Kimmel, C., Westerfield, M., Postlethwait, J.H., 2005. A pair of Sox: distinct and
overlapping functions of zebrafish sox9 co-orthologs in craniofacial and
pectoral fin development. Development 132, 1069–1083.
Yee, N.S., Lorent, K., Pack, M., 2005. Exocrine pancreas development in zebrafish.
Dev. Biol. 284, 84–101.
Yokoi, H., Yan, Y.L., Miller, M.R., BreMiller, R.A., Catchen, J.M., Johnson, E.A.,
Postlethwait, J.H., 2009. Expression profiling of zebrafish sox9 mutants reveals
that Sox9 is required for retinal differentiation. Dev. Biol. 329, 1–15.
Zecchin, E., Mavropoulos, A., Devos, N., Filippi, A., Tiso, N., Meyer, D., Peers, B.,
Bortolussi, M., Argenton, F., 2004. Evolutionary conserved role of ptf1a in the
specification of exocrine pancreatic fates. Dev. Biol. 268, 174–184.
I. Manfroid et al. / Developmental Biology 366 (2012) 268–278