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Developmental Biology 285 (2005) 211 – 223 www.elsevier.com/locate/ydbio

sox4b is a key player of pancreatic a cell differentiation in zebrafish

Anastasia Mavropoulosa, Nathalie Devosa, Fre´de´ric Biemara,1, Elisabetta Zecchinb, Francesco Argentonb, Helena Edlundc, Patrick Motted, Joseph A. Martiala, Bernard Peersa,*

aLaboratoire de Biologie Mole´culaire et de Ge´nie Ge´ne´tique, Center of Biomedical Integrative Genoproteomics (CBIG), Universite´ de Lie`ge, Institut de Chimie, Baˆtiment B6, 4000 Lie`ge (Sart-Tilman), Belgium bDipartimento di Biologia, Universita’ di Padova, Via U. Bassi 58b, 35131 Padova, Italy cUmea Center for Molecular Medicine, Umea University, SE-901 87 Umea, Sweden dLaboratoire de Biologie Cellulaire Ve´ge´tale, Cellule d’Appui Technologique en Microscopie (CATl), Universite´ de Lie`ge, Institut de Botanique, Baˆtiment B22, 4000 Lie`ge (Sart-Tilman), Belgium

Received for publication 15 February 2005, revised 2 June 2005, accepted 13 June 2005

Abstract

Pancreas development relies on a network of transcription factors belonging mainly to the Homeodomain and basic Helix–Loop–Helix families. We show in this study that, in zebrafish, , a member of the SRY-like HMG-box (SOX) family, is required for proper endocrine cell differentiation. We found that two orthologous to mammalian Sox4 are present in zebrafish and that only one of them, sox4b,is strongly expressed in the pancreatic anlage. Transcripts of sox4b were detected in mid-trunk endoderm from the 5-somite stage, well before the onset of expression of the early pancreatic pdx-1. Furthermore, by fluorescent double in situ hybridization, we found that expression of sox4b is mostly restricted to precursors of the endocrine compartment. This expression is not maintained in differentiated cells although transient expression can be detected in a cells and some h cells. That sox4b-expressing cells belong to the endocrine lineage is further illustrated by their absence from the pancreata of slow-muscle-omitted mutant embryos, which specifically lack all early endocrine markers while retaining expression of exocrine markers. The involvement of sox4b in cell differentiation is suggested firstly by its up-regulation in mind bomb mutant embryos displaying accelerated pancreatic cell differentiation. In addition, sox4b knock-down leads to a drastic reduction in glucagon expression, while other pancreatic markers including insulin, somatostatin, and trypsin are not significantly affected. This disruption of a cell differentiation is due to down-regulation of the arx gene specifically in the pancreas. Taken together, these data demonstrate that, in zebrafish, sox4b is expressed transiently during endocrine cell differentiation and plays a crucial role in the generation of a endocrine cells. D 2005 Elsevier Inc. All rights reserved.

Keywords: SOX; ; Pancreas; Zebrafish; Development; Glucagon

Introduction synthesize and release more than 20 digestive enzymes into the duodenum through the pancreatic ductal system. The The pancreas is a compound exocrine and endocrine endocrine pancreas, consisting of individual islets of gland playing crucial roles in the regulation of metabolism. Langerhans scattered within the exocrine tissue, releases The exocrine acini forming the major part of the pancreas into the bloodstream the hormones involved notably in glucose homeostasis (Edlund, 2002; Slack, 1995). These islets are composed of four distinct cell types, a, h, y, and PP, respectively producing glucagon, insulin, somatostatin, * Corresponding author. Fax: +32 43662968. and the pancreatic polypeptide. E-mail address: [email protected] (B. Peers). 1 Present address: Department of Molecular and Cell Biology, Division of Pancreas organogenesis has been studied notably in Genetics and Development, 16 Barker Hall, University of California, mouse. During embryogenesis, the pancreas develops as Berkeley, CA 94720, USA. two protrusions, a dorsal one and a ventral one, emerging

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2005.06.024 212 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 from the foregut epithelium. Signals from adjacent tissues, Interestingly, studies in mouse, zebrafish, and frog point to including the notochord, the vascular tissue, and the lateral a role for Sox genes such as casanova (cas/sox32) and plate mesoderm, induce a complex network of transcrip- sox17 in endoderm induction and specification (Alexander tional regulatory factors leading cells within these primor- et al., 1999; Henry and Melton, 1998; Kanai-Azuma et al., dia to differentiate and give rise to the exocrine and 2002; Kikuchi et al., 2001). Some studies report the endocrine tissues and ductal system of the mature organ expression of SOX genes in murine pancreatic cell lines (Hebrok, 2003; Kumar et al., 2003; Lammert et al., 2003). (Katoh, 2002; Saitoh and Katoh, 2002) and in the human Gene targeting experiments have emphasized the critical and Xenopus, pancreata (Lee and Saint-Jeannet, 2003; role of several transcription factors of the Homeodomain Piper et al., 2002). More recently, Lioubinski et al. (2003) and basic Helix–Loop–Helix families, such as Pdx-1 and showed that several SOX genes are expressed during Ngn3, in pancreas development (Jensen, 2004; Wilson pancreas development in mouse. Despite these expression et al., 2003). data, the function of SOX genes in this organ has not been More recently, investigators have begun to focus on investigated so far. pancreas development in teleost models such as zebrafish In the present study, we have investigated the expression (Argenton et al., 1999; Biemar et al., 2001) and medaka and role of sox4 in pancreas development in zebrafish. We (Assouline et al., 2002). In zebrafish as in mouse, pancreas show that one of the two SOX4 orthologous genes identified development involves the emergence of two primordia. We in zebrafish, sox4b, is expressed in the developing pancreas. have previously shown that the pancreatic anlage arises We additionally provide evidence that sox4b is a very early through the fusion of two rows of endodermal cells marker of the pancreas anlage, as its expression is detected marked by pdx-1 expression at the 10-somite stage before the one of the pancreatic homeobox gene pdx-1. (Biemar et al., 2001). This domain first generates a dorsal Finally, we show that sox4b is primarily required for bud detectable 24 h post-fertilization (hpf). A second differentiation of glucagon cells. pancreatic primordium subsequently emerges about 34 hpf, ventrally and anteriorly with respect to the first one. The dorsal bud generates the bulk of endocrine cells whereas Materials and methods the ventral bud gives rise to the pancreatic duct, the exocrine tissue, and some endocrine cells. The two buds Zebrafish maintenance, mutant lines and transgenic lines, merge about 44 hpf to form the final pancreas (Field et al., and retinoic acid treatments 2003). The transcriptional regulatory mechanisms controlling Zebrafish (Danio rerio) were raised and cared for pancreas development seem to be conserved among according to standard protocols (Westerfield, 1995). Wild- vertebrates. Homologs of genes encoding several tran- type embryos from the AB strain were used and staged scription factors required in the mouse pancreas, includ- according to Kimmel (Kimmel et al., 1995). Homozygous ing pdx-1, , nkx2.2, and .2, are also expressed mutants were obtained by mating heterozygous fish for the during pancreas development in zebrafish (Biemar et al., slow-muscle-omitted, smub641(Barresi et al., 2000); mind 2001). Moreover, recent reports based on morpholino- bomb, mibta52b (Haddon et al., 1998); and neckless, nlsi26 mediated gene knock-down in zebrafish point to a (Begemann et al., 2001) alleles, respectively. conserved role for pdx-1, ptf1a, and hb9 in the pancreas For exogenous retinoic acid (RA) treatments, 95% development in vertebrates (Huang et al., 2001; Lin et epiboly embryos were treated as described by Stafford and al., 2004; Wendik et al., 2004; Yee et al., 2001; Zecchin Prince (2002). et al., 2004). The SRY-like HMG-box (SOX) family comprises tran- Cloning and sequencing of the zebrafish sox4 genes scription factors characterized by a highly conserved DNA-binding domain, the HMG box. Sox are Using the SSAHA server, we found two assemblies, found in all metazoans, with 20 members identified in z06s067965 and z06s06440A, respectively containing the humans. These proteins have been assigned to 8 groups, A genomic sequences of sox4a and sox4b (www.sanger.ac. to H, based on sequence similarity inside and outside the uk). The fb82f08.y1 Expressed Sequenced Tag (EST) was HMG box (Bowles et al., 2000; Schepers et al., 2002). Sox obtained from the RZPD (www.rzpd.de, Accession genes show diverse and dynamic patterns of expression n-AI584726, AI584225). sox4b cDNA was cloned by RT- throughout embryogenesis and in a variety of adult tissue PCR with a pair of primers encompassing the predicted types (Pevny and Lovell-Badge, 1997; Prior and Walter, complete coding sequence (upstream primer: 5V-GCGTG- 1996; Wegner, 1999). Mutations in several of these genes CTGTCAACGGGGAAGGATA; downstream primer: 5V- revealed critical roles for members of this family in the GTCTCGTGTCGAGCACCGGGAAA) on mRNA extrac- development of various tissues such as in sex determi- ted from adult zebrafish, according to the SuperScripti nation, heart, bones, and lymphocyte development (Foster System instructions. Betaine at a final concentration of 2 M et al., 1994; Schilham et al., 1996; Sudbeck et al., 1996). was added to the samples. They were subjected to 30 cycles A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 213 of 30 s at 94-C, 45 s at 55-C, and 90 s at 68-C, followed by FISH imaging a final 10-min extension at 68-C. The 1447 bp RT-PCR product was cloned into the pCRIIRTOPOR vector Confocal imaging was performed by using a Leica (Invitrogen), and sequenced. TCS SP2 inverted confocal laser microscope (Leica The amino acid phylogenetic tree was generated by using Microsystems, Germany). Digitized images were acquired ClustalX (Thompson et al., 1997). using a 10Â (NA 0.4) or 63Â (NA 1.5) Plan-Apo water- immersion objective at 1024 Â 1024 pixel resolution. Radiation hybrid mapping For multicolor imaging, FITC was visualized by using an excitation wavelength of 488 nm and the emission To map sox4b on the LN54 hybrid panel, we used a pair light was dispersed and recorded at 500 to 535 nm. Cy3 of primers amplifying a fragment of the 3V region of sox4b: was detected by using an excitation wavelength of 543 upstream primer: 5V-CAGAGCCCCGAGAAGAAGAAGC; and the fluorescence emission was dispersed and downstream primer: 5V-CTCACCGGGCGGCGTATTAT. recorded at 560 to 650 nm. The acquisition was set up PCR results on the 93 radiation hybrid DNA samples were to avoid any cross-talk of the two fluorescence emis- submitted to http://dir.nichd.nih.gov/lmg/lmgdevb.htm for sions. Series of optical sections were carried out to analysis and placement on the zebrafish map. analyze the spatial distribution of fluorescence, and for each embryo, they were recorded with a Z-step ranging Single and fluorescent double whole-mount in situ between 0.5 and 1.0 Am. Image processing, including hybridization on zebrafish embryos background subtraction and projection of Z-stacks, was performed with Leica software (version 2.5). Captured Single hybridizations and detections were carried out as images were exported as TIFF format files and further previously described (Hauptmann and Gerster, 1994)on processed using Adobe Photoshop 7.0 for figure mount- wild-type embryos. Anti-sense RNA probes were prepared ing and labeling purpose. by transcribing linearized cDNA clones with SP6, T7, or T3 polymerase using digoxigenin or fluorescein labeling mix Morpholino and mRNA injections in zebrafish (Roche). Fluorescent double hybridizations were performed by The morpholino oligonucleotides (Gene Tools) used adapting various fluorescent in situ hybridization protocols were: mo1soxb, GACTCAGTCTGATTGCACACAGTCC, to the zebrafish (Denkers et al., 2004; Zhou and Vize, 2004). that targets the 5VUTR, its 5-mismatch version mo1mis, Before the hybridization, zebrafish embryos were incubated GACTGAGTGTGATTCCAGACACTCC, mo2sox4b, in 2% H2O2 during 40 min for endogenous peroxydase TGCTGCTGCTGGATCTCTGGAGCAT, that targets the inactivation, just prior to proteinaseK treatment. For the first 25 bases of the coding sequence, and the morpholino hybridization, anti-sense probes were prepared using digox- standard (mosd) provided by Gene Tools. Approximately 2 igenin labeling mix (Roche) or DNP-11-UTP ribonucleo- ng morpholino diluted in Danieau solution was injected into tides (TSAi Plus system, Perkin Elmer). The embryos the yolk of wild-type embryos. Rhodamin dextran was were blocked in 100 mM Tris–HCl pH 7.5, 150 mM NaCl added at 0.5% to the samples to check injection efficiency. (TNT buffer) with 0.5% Blocking Reagent (Perkin Elmer). The sox4bDC plasmid was constructed from the For the detection, we used pre-absorbed HRP-coupled anti- pCRIIRTOPOR-sox4b plasmid. In a first step, we sub- digoxigenin (Roche) or HRP-coupled anti-DNP antibodies cloned the sox4b fragment released by EcoRI digestion into (Perkin Elmer). The embryos were then extensively washed the pCS2+ vector. The C-terminal region of sox4b was in TNT buffer. The revelation was performed by incubating deleted by BsrGI and XhoI double digestion, followed by a embryos during 40 min in tyramide-FITC and tyramide-Cy3 Klenow treatment prior to ligation. This construction prepared according to Peter Vize’s protocol (Zhou and Vize, generates a containing only the first 187 residues 2004) at a final dilution of 1/50 in 1Â Amplification of Sox4b. Reagent (Perkin Elmer). Embryos were then conserved in Capped mRNAs were transcribed from the NotI-line- TNT buffer. arized expression plasmids (pCS2+-sox4bDC and pCS2+- The sox4a probe was generated from KpnI-linearized GFP) and the HindIII-linearized pT7TS-shh (diIorio et al., fb82f08, using SP6 RNA polymerase. The sox4b probe was 2002) with the mMessage mMACHINE transcription kit synthesized by transcription from pCRIIRTOPOR-sox4b (Ambion) according to the manufacturer’s instructions. linearized with HindIII and using T7 RNA polymerase. For microinjection, mRNAs were diluted in Danieau The other probes used were: pdx-1 and preproinsulin solution to a final concentration of 500 ng/Al. GFP at a (Milewski et al., 1998), glucagon (Argenton et al., 1999), concentration of 50 ng/Al was added to the injected samples, somatostatin2 (Devos et al., 2002), isl1 (Korzh et al., 1993), to check injection efficiency. Approximately 0.9 ng of neuroD (Korzh et al., 1998), nkx2.2 (Barth and Wilson, mRNA was microinjected into the yolk of zebrafish 1995), pax6.2 (Krauss et al., 1991), arx (Miura et al., 1997), embryos at the one-cell stage using published procedures and trypsin (Biemar et al., 2001). (Westerfield, 1995). 214 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223

Results genome for a SOX4 ortholog. Two sequences highly similar to mammalian SOX4 were identified. One of them, The zebrafish sox4a and sox4b genes are orthologs of corresponding to the Expressed Sequence Tag (EST) human SOX4 fb82f08, was named sox4a. We amplified the second cDNA by RT-PCR on adult zebrafish mRNA and named it sox4b. In a RT-PCR screen using degenerate primers, we The zebrafish sox4a and sox4b cDNAs each contain a single identified Sox4 as one member of the SOX family expressed open reading frame. The encoded putative proteins comprise in murine pancreatic cell lines. Subsequent expression respectively 363 and 342 amino acids. Sequence alignment pattern analysis showed that Sox4 is expressed in the showed that the two zebrafish Sox4 proteins share 70% developing mouse pancreas, as reported by Lioubinsky and identity over their entire sequence. The HMG domain co-workers (A.M. and H.E., unpublished results and displays more than 95% identity with all vertebrate Sox4 Lioubinski et al., 2003). To investigate its expression and sequences analyzed (Fig. 1A). The C-terminal region, pancreatic function in zebrafish, we searched the zebrafish previously shown to contain a transactivation domain in

Fig. 1. (A) Alignment of vertebrate Sox4 peptidic sequences. The 79 amino acids HMG box is indicated by a line. The residues that are identical in all species are shaded in yellow, and those conserved in just some of them are shaded in blue. Dr: Danio rerio, Hs: Homo sapiens, Mm: Mus musculus, Xl: Xenopus laevis. The HMG box and the C-terminal region present a high level of similarity among all the vertebrates. (B, C) Phylogenetic and mapping analysis. (B) Phylogenetic tree of group C Sox proteins. Numbers on branches are the number of times, in one thousand runs, that the two clades branched as sisters. The results show with very high bootstrap support, that zebrafish Sox4 proteins belong to the Sox4 clade. The accession numbers for the sequences are as follows: XlSox4, Xenopus laevis Sox4 (AAG17026); OmSox24, Oncorhynchus mykiss Sox24 (BAA24575); MmSox4, Mus musculus Sox4 (XP_109322); HsSOX4, Homo sapiens SOX4 (I38240); DrSox4a, Danio rerio Sox4a (AY369081); DrSox4b, Danio rerio Sox4b (AY369082); GgSox11, Gallus gallus Sox11 (BAA25297); HsSOX11, Homo sapiens SOX11 (AAB08518); MmSox11, Mus musculus Sox11 (AAB82425); RnSox11, Rattus norvegicus Sox11 (NP_445801); DrSox11a, Danio rerio Sox11a (NP_571411); DrSox11b, Danio rerio Sox11b (NP_571412); HsSOX12, Homo sapiens SOX12 (NP_008874). (C) Conserved syntenies in Sox4 genes chromosomal regions. The comparison of zebrafish 19 (Dr 19), chromosome 16 (Dr 16), and human (Hs 6) shows that several human gene homologs are either present on Dr 19 (; ck2b; neu1; abcf1; ppp1r10; gtf2h4), or on Dr 16 (mgc44669; vmp; hist1h3b; bat5; b 5-tubulin), demonstrating that the zebrafish sox4 genes are both the orthologs of human SOX4. A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 215 mouse (van de Wetering et al., 1993), is also well conserved among the different species. To establish whether the two newly identified genes are true orthologs of human SOX4, we performed phylogenetic and mapping analyses. A phylogenetic tree constructed with group-C SOX proteins of different vertebrates shows that both zebrafish Sox4 proteins fit well into the Sox4 clade with a very high bootstrap value (Fig. 1B). This supports their assignment as zebrafish sox4 genes. To determine whether they arose from tandem or genome duplication, we investigated their chromosomal location. With the help of the LN54 hybrid panel, the sox4a gene has been mapped previously to chromosome 19 (Dr 19) (Hukriede et al., 1999). Using the same hybrid panel, we mapped sox4b to chromosome 16 (Dr 16), very close to the marker z9520 (positioned at 0cR from z9520 by the RH Instant Mapper Software, Hukriede et al., 1999). In humans, SOX4 is located on chromosome 6 (Hs 6). A comparison of loci close to the sox4 genes in humans and zebrafish revealed syntenies between human chromosome 6 and zebrafish 16 and 19 (Fig. 1C): in addition to SOX4,Hs 6 bears 11 markers located on either Dr 19 (e2f3; ck2b; neu1; abcf1; ppp1r10; gtf2h4)orDr16(mgc44669; vmp; hist1h3b; bat5; b 5-tubulin). This demonstrates that the zebrafish sox4 genes are orthologs of human SOX4 and that they likely arose through the partial genome duplication that occurred in the ray-finned lineage before teleost radiation (Postlethwait et al., 2000).

The sox4b gene is expressed early in the pancreatic Fig. 2. Expression of sox4a and sox4b genes during zebrafish development. Whole-mount in situ hybridization with sox4a- (left panels) and sox4b- primordium, in cells committed to an endocrine fate (right panels) digoxigenin-labeled anti-sense probes at various stages of zebrafish development (from the 5-somite stage to 24 hpf). (A–D) Dorsal The expression patterns of sox4a and sox4b, determined views, anterior to the top. (E, F) Dorsal views, anterior to the left. (G–I) by whole-mount in situ hybridization at various stages of dorsal views (G, H) and lateral view (I) with anterior to the left of yolk-free A A development, were found to be highly dynamic and distinct. embryos. A scale bar = 200 m, G scale bar = 200 m in G, H, and I. Arrowhead in panel B: mid-trunk endoderm. Arrow in panel E: dorsal A thorough description will be presented elsewhere. Fig. 2 neural tube; arrow in panel I: floor plate. The notochord (N) is indicated in presents some sites of expression of both genes. For panels G and I. As soon as the 5-somite stage (B), sox4b starts to be instance, we found that sox4a is expressed in several tissues expressed in some scattered cells of the mid-trunk endoderm (arrowhead). such as the lateral plate mesoderm and hindbrain (Figs. 2A, As somitogenesis goes on, the expression persists in this region (D, F C, E, and unpublished results). However, even after long arrowhead) until 24 hpf (H, I). By contrast, sox4a is expressed in various tissues such as the lateral plate mesoderm (A, arrowhead), the hindbrain (C, staining reaction, the level of expression of sox4a in the arrowhead), the neural crest (E, arrow) but expression in the pancreatic pancreatic region is hardly above the limit of detection of region is hardly above the detection limit (G, star). our assays (Fig. 2G). sox4b, in contrast, was found to be strongly expressed in the pancreatic anlage, in addition to other tissues, such as the nervous system, where sox4b hpf (see also Figs. 3P, Q, and data not shown). This timing transcripts were found from the 12-somite stage on of expression suggests that sox4b is expressed in endocrine (unpublished results). cells, as they are generated well before the exocrine tissue. Transcripts of sox4b were detected as early as the 5- However, we observed that sox4b-expressing cells did not somite stage (5s) in a few mid-trunk endodermal cells in the cluster in the first pancreatic bud, but remained scattered prospective pancreatic region (Fig. 2B). At the 12-somite within the pancreatic anlage. stage, the sox4b-expressing cells are arranged in a one-cell- In order to identify in which pancreatic cell type sox4b is thick V-shaped domain (Fig. 2D). This disposition mimics expressed, we set up a protocol of fluorescent in situ that of the early pancreatic genes pdx-1 (Biemar et al., hybridization (FISH, see Materials and methods). Fig. 3 2001). sox4b expression persists in this region until 24 hpf presents optical sections of fluorescent double in situ (Figs. 2F, H, and I). From then on, sox4b expression hybridization performed with sox4b and various pancreatic decreases in this region and was no longer detectable at 40 markers. At 20 hpf, we found that sox4b is expressed in a 216 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223

Fig. 3. Expression of sox4b in pancreas observed by FISH. (A–L) Expression of either pdx-1, neuroD, nkx2.2, isl1 (left column), or sox4b (central column) in 20 hpf embryos. The right column presents the overlay of the respective left and middle panels. (M, N) Overlay of sox4b- and either insulin- (M) or somatostatin2 (N) expression in 20 hpf embryos. (O, P) Expression of glucagon (O) and sox4b (P) in 36 hpf embryos. (Q) Overlay of panels O and P. (Q) Overlay of sox4b- and insulin expression in 26 hpf embryos. All the panels are optical sections of ventrally viewed yolk-free embryos (A–Q) and laterally viewed yolk-free embryo (R), with anterior to the left. sox4b was detected with a digoxigenin-labeled anti-sense probe and a tyramide-Cy3 substrate and the other markers with DNP-labeled anti-sense probes and tyramide-FITC substrate (see Materials and methods). A–N scale bars = 50 Am; O–Q scale bars = 25 Am. The arrowheads point to cells where sox4b is co-expressed with another marker, arrows in panels P and Q point to cells expressing only sox4b. sub-set of the pdx-1 domain (Figs. 3A–C, supplementary We then investigated the expression of sox4b and the data S1). This is particularly visible in some cells strongly pancreatic hormones. At 20 hpf, most sox4b and insulin expressing sox4b (arrowheads in Figs. 3A–C). transcripts are distinct, although a few cells co-expressing To determine whether sox4b-expressing cells might be both markers can be found (Fig. 3M, supplementary endocrine precursors, we performed double staining with dataS5). At the same stage, sox4b cells do not co-express endocrine markers such as nkx2.2, neuroD, and isl1, three somatostatin (Fig. 3N, supplementary dataS6). We per- genes known to be involved in the differentiation of formed a FISH at 36 hpf, when the glucagon cells are endocrine cells in mouse (Ahlgren et al., 1997; Sander et numerous (Fig. 3O). At this stage, sox4b expression is al., 2000; Sussel et al., 1998). Figs. 3D–F show a very good decreasing (Fig. 3P). We observed that most glucagon cells, overlap between neuroD and sox4b, notably in the anterior express generally low levels of sox4b (arrowheads in Figs. part of the pancreatic anlage where both genes are strongly 3O–Q, supplementary data S7 and arrows in Figs. 3P and expressed (arrowheads in Figs. 3D–F, supplementary data Q). This suggests that sox4b expression is decreasing in S2). We also observed a partial overlap between sox4b with these cells, as they are completing differentiation. either nkx2.2 (Figs. 3G–I, supplementary data S3) or isl1 We finally investigated the position of the sox4b (Figs. 3J–L, supplementary data S4). expression domain relative to the anterior bud. For that A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 217 purpose, we performed a FISH at 26 hpf, when the dorsal pancreatic bud emerges and begins to form an islet, and sox4b is still strongly expressed. Fig. 3R (and supplemen- tary data S8) shows that the sox4b cells are located ventrally to the insulin cells present in the developing islet. These data suggest that sox4b is expressed in the pancreatic gut epithelium and is switched off when the differentiated endocrine cells are located in the pancreatic islet. Altogether, these data show that sox4b is expressed early in a subset of cells of the pancreatic anlage, some of which having already acquired an endocrine fate.

Expression of sox4b responds to signaling pathways controlling pancreas development

In zebrafish, three signaling pathways have been shown to control pancreas development: the retinoic acid, Hedgehog, and Delta-Notch pathways. We therefore analyzed relationships between these signaling pathways and sox4b expression. Retinoic acid (RA) signaling was recently shown to be involved in pancreas specification (Chen et al., 2004; Stafford et al., 2004; Stafford and Prince, 2002). RA supplied to wild- type embryos during gastrulation results in dramatic expan- sion of pancreatic marker expression towards the anterior regions. In addition, the neckless (nls) mutant in which RA synthesis is affected fails to express pancreatic and liver Fig. 4. Expression of sox4b in response to affected retinoic acid, Hedgehog markers. To test whether sox4b expression might be similarly and Delta/Notch signaling. Dorsal views of whole-mount in situ hybrid- controlled by RA signaling, we applied exogenous RA to ization for sox4b expression. Anterior to the left; B and F scale bars = 100 late-gastrulation-stage wild-type embryos. In the treated Am in A–D panels and in panels E–G, respectively. Arrowheads and embryos, sox4b expression dramatically spread from the brackets indicate sox4b expression in pancreas and in neural crest, mid-trunk endoderm to more anterior regions (Figs. 4A and respectively. Panels A and B present yolk-free embryos. e: eye. Exogenous RA leads to an anterior shift of sox4b expression in pancreas, as described B). In contrast, nls mutant embryos showed no sox4b for pancreatic markers (A: control embryo, B: RA-treated embryos). In the expression in the pancreas (Figs. 4C and D), although nls mutant, defective in RA synthesis, sox4b expression is absent from the expression of this gene was unaffected in non-endodermal pancreatic region (C: wild-type sibling embryo, D: nls mutant embryo). Up- tissues (i.e. cranial neural crest). Thus, RA signaling delimits regulation of sox4b is observed in the mib mutant embryos (E: wild-type the region of sox4b expression along the antero-posterior sibling, F: mib mutant embryo), defective in the Delta-Notch signaling pathway. Alterations of the Hedgehog signaling affect also sox4b axis, as showed for other pancreatic markers. expression in pancreas (G–J). In the smu mutant embryos, in which the Studies in mouse and zebrafish have highlighted that endocrine part of pancreas fails to develop, sox4b is absent (G: wild-type Notch signaling is required to delay pancreatic cell differ- sibling, H: smu mutant embryo). In addition, injection of shh mRNA entiation from a pool of precursor cells (Apelqvist et al., induces the over-expression of sox4b (I: GFP mRNA-injected embryo, J: 1999; Hald et al., 2003; Jensen et al., 2000; Miyamoto et al., shh mRNA-injected embryo), as previously shown for endocrine pancreatic markers. 2003; Murtaugh et al., 2003). Inactivation of Notch pathway components in mouse leads to an acceleration of endocrine cell differentiation. More recently, it has been shown that Recent reports have demonstrated that Hedgehog signal- Notch signaling is also involved in exocrine cell differ- ing is required in zebrafish for endocrine cell specification entiation in mouse as well in zebrafish (Esni et al., 2004). (diIorio et al., 2002; Roy et al., 2001). The slow-muscle- Esni et al. (2004) showed that, in the zebrafish mind bomb omitted (smu) mutant, in which the Hedgehog co- (mib) mutant, impaired in Notch signal transduction, the Smoothened is inactivated, lacks expression of dorsal bud exocrine cell differentiation is accelerated. Indeed, the bulk markers, while the level of expression of exocrine markers of exocrine precursor cells appears to start differentiating such as trypsin is unaffected (Zecchin et al., 2004). In together, rather than progressively. Interestingly, we ob- addition, over-expression of sonic hedgehog (shh) in wild- served in mib mutant embryos a dramatic increase of the type embryos leads to an increase in endocrine cells (diIorio number of cells expressing sox4b at the 15-somite stage, as et al., 2002). Using a similar approach, we investigated how compared to wild-type embryos (Figs. 4E and F). These data sox4b expression is affected when Hedgehog signaling is indicate that sox4b expression is downstream of the Notch altered. In smu mutant embryos, no sox4b expression was signaling. detected in the pancreatic primordium (Figs. 4G and H), 218 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 although the gene continued to be expressed in the anterior sense morpholino oligonucleotides, mo1sox4b and neural tube. Injection of shh mRNA into wild-type embryos mo2sox4b, which respectively target the 5VUTR and ATG resulted in over-expression of sox4b in the pancreatic region of sox4b mRNA. We confirmed by in vitro assays that these (Figs. 4I and J), as previously described for endocrine two morpholinos blocked efficiently and specifically the genes. Together, these data suggest that sox4b is expressed translation of sox4b mRNA (data not shown). As negative in cells of the endocrine lineage. controls, we used the 5-mismatch morpholino of mo1sox4b These results show that sox4b lies downstream from the (mo1mis) and the unrelated-sequence morpholino mosd retinoic acid, Hedgehog, and Notch pathways in the (Gene Tools). Neither of these affected in vitro translation of pancreas regulatory gene network. sox4b mRNA (data not shown). Embryos were injected with 2 ng morpholino and raised until 30 hpf or 3 dpf. The The sox4b gene is crucial to glucagon cell differentiation expression of different pancreatic markers was then ana- lyzed qualitatively and/or quantitatively (summarized in To gain insights into the function of sox4b, we examined Table 1). the effects of its inactivation in wild-type embryos. First, we Knock-down of sox4b led to a drastic decrease in injected into 1- to 2-cell-stage embryos two different anti- glucagon-expressing cells, as compared to control embryos

Table 1 Quantitative and qualitative analyses of injection experiments Injection Probe Cell number compared to average wild-type expression (%) Embryos number 100–90% 90–50% 50–10% <10% – glucagon 30 (66%) 16 (34%) – – 46 Mosd glucagon 6 (22%) 19 (70%) 1 (4%) 1 (4%) 27 Mo1mis glucagon 23 (50%) 13 (28%) 8 (18%) 2 (4%) 46 Mo1sox4b glucagon 2 (3%) 5 (7%) 10 (15%) 48 (75%) 64 Mo2sox4b glucagon 4 (19%) 3 (14%) 7 (33.5%) 7 (33.5%) 21 Gfp glucagon 5 (25%) 10 (50%) 5 (25%) – 20 Sox4bDC glucagon – 4 (8%) 22 (41%) 27 (51%) 53 – insulin 19 (73%) 7 (27%) – – 26 Mosd insulin 6 (46%) 6 (46%) 1 (8%) – 13 Mo1mis insulin 8 (32%) 13 (52%) 4 (16%) – 25 Mo1sox4b insulin 12 (30%) 18 (45%) 8 (20%) 2 (5%) 40 – sst2 8 (30%) 18 (70%) – – 26 Mosd sst2 3 (18%) 14 (82%) – – 17 Mo1mis sst2 6 (30%) 13 (65%) 1 (5%) – 20 Mo1sox4b sst2 8 (19.5%) 25 (61%) 7 (17%) 1 (2.5%) 41 – arx 6 (40%) 7 (47%) 2 (13%) – 15 Mosd arx 14 (43%) 11 (33%) 8 (24%) – 33 Mo1mis arx 2 (17%) 7 (58%) – 3 (25%) 12 Mo1sox4b arx 7 (13%) 8 (14%) 9 (16%) 41 (57%) 55

Injection Probe Expression level compared to average wild-type expression (%) Embryos 100–90% 90–50% 50–10% <10% number – pax6.2 9 (70%) 3 (23%) 1 (7%) – 13 Mosd pax6.2 ––––– Mo1mis pax6.2 21 (52%) 17 (41%) 3 (7%) – 41 Mo1sox4b pax6.2 31 (70%) 12 (27%) 1 (3%) – 44 – neuroD 16 (89%) 2 (11%) – – 18 Mosd neuroD 15 (49%) 16 (51%) – – 31 Mo1mis neuroD 9 (35%) 11 (42%) 4 (15%) 2 (7%) 26 Mo1sox4b neuroD 25 (45%) 21 (37.5%) 8 (14%) 2 (3.5%) 56 – pdx-1 12 (63%) 7 (37%) – – 19 Mosd pdx-1 ––––– Mo1mis pdx-1 15 (32%) 31 (68%) – – 46 Mo1sox4b pdx-1 20 (36%) 35 (64%) – – 55 – trypsin 20 (95%) – 1 (5%) – 21 Mosd trypsin ––––– Mo1mis trypsin 16 (84%) 3 (16%) – – 19 Mo1sox4b trypsin 20 (87%) 2 (9%) 1 (4%) – 23 Each embryo was analyzed quantitatively at 30 hpf for pancreatic markers expression and classified according to phenotype severity: unchanged cell number compared to the average wild-type expression in non injected embryos (100–90%), mild or severe phenotype (90–50%; 50–10%), or disappearance of the cells (<10%). For pax6.2, neuroD, pdx-1, and trypsin (analyzed at 3 dpf), the number of cells was approximately estimated given their large number. A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 219

(Figs. 5A, B, and W). In 75% of the mo1sox4b morphants, expression were not significantly affected (Figs. 5C–F, W, no more than one glucagon cell, if any, was detected (Table and Table 1). Yet in about 50% of the analyzed embryos, the 1). Injection of mo2sox4b leads also to an absence of insulin and somatostatin cells remained scattered and failed glucagon expression in 33% of the injected embryos (Figs. to cluster in the endocrine islet (Figs. 5C and E). We also 5I and J). This lower of mo2sox4b effect could reflect analyzed the exocrine pancreatic tissue 3 days post- differential efficiency between the two morpholinos. In fertilization (dpf) by examining expression of the trypsin contrast to glucagon, levels of insulin and somatostatin marker. This marker showed no significant change in its

Fig. 5. sox4b is required for the differentiation of a cells. (A–V) Expression of various pancreatic markers in wild-type control embryos (first and third columns) or sox4b knock-down embryos (second and fourth columns), analyzed at 30 hpf (except trypsin that was analyzed at 3 dpf). Compared to the controls (A and I), glucagon expression dramatically decreases in embryos injected with either mo1sox4b or mo2sox4b (B and J). The injection of the mRNA encoding Sox4deltaC leads to the same phenotype (K and L) while the injection of gfp mRNA has not any effect. By contrast, the expression of insulin (C, D), somatostatin (E, F) is not much affected although the cells are scattered. In addition to glucagon decrease, sox4b knock-down causes a similar phenotype on arx expression specifically in the pancreas (panels M and N and arrowhead in O and P), without any effect on the floor plate nor the somites (thin and thick arrows in panels O and P, respectively). By contrast, the levels of expression of pax6.2 (Q, R), neuroD (S, T), pdx-1 (U, V), and trypsin (G, H) are not significantly affected. All the panels present yolk-free embryos. Except panels O and P that are lateral views, all the panels are dorsal views with the anterior to the left. B, N, and P scale bars = 50 Am; H scale bar = 100 Am. In panels O and P, arrowhead: dorsal bud, thin arrow: floor plate, thick arrow: somites. W: quantitative analysis of endocrine markers in mo1mis or mo1sox4b morphant embryos at 30 hpf. The knock-down of sox4b leads, on average, to the loss of 82% and 55% of glucagon and arx cells. The average number of cells of the other markers analyzed is poorly affected. Y bars correspond to the standard error. See Table 1 for embryo numbers. 220 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 expression level in sox4b morphant embryos (see brackets al., 2000; Hunt and Clarke, 1999; Wotton et al., 1995). The in Figs. 5G and H), indicating that the differentiation of the generation of knock-out mice established the involvement exocrine cells remains unaffected. of Sox4 in heart and lymphocytes development (Schilham et In parallel with the injection of morpholinos, we used al., 1996). In a recent report, Lioubinski et al. (2003) another strategy to block Sox4b activity. As previously described the expression Sox4 in the developing mouse described in the mouse, Sox4 consists of two separable pancreas. In the present study, we provide functional data on domains, the HMG box and a C-terminal transactivation the role of Sox4 in endocrine pancreatic cell differentiation, domain (van de Wetering et al., 1993), both of which are using zebrafish as model system. well conserved among vertebrates (see Fig. 2). We reasoned We identified the two zebrafish orthologs of mammalian that a mutant protein lacking the transactivation domain SOX4, which probably derive from the genome duplication would act as an antimorph by binding to its target sites and that occurred after divergence of the ray-finned fish and hence blocking – or reducing the activity of – endogenous tetrapod lineages (Amores et al., 1998). We found that both Sox4b. A similar approach has been used to block genes present quite distinct expression patterns: only sox4b efficiently the activity of the Sox18 factor in mouse and is expressed in the pancreas, while sox4a is expressed in of the Sox9 factor in chick (Moniot et al., 2004; Pennisi et various other tissues. This suggests that after the genome al., 2000a,b). We therefore generated a deletion mutant duplication, both gene copies have been conserved thanks to named sox4bDC encoding a Sox4b protein lacking trans- a partitioning of the ancestral domains (and activation properties. Injection of sox4bDC mRNA into functions) between them. This is an example of sub- wild-type embryos resulted also in a severe reduction in functionalization, also displayed by several fish genes glucagon-expressing cells (Figs. 5K and L and data not (Cresko et al., 2003). shown). These results confirm that sox4b expression is sox4b appears in the mid-trunk endoderm well before necessary for the development of glucagon cells. pdx-1, which is expressed in all pancreatic precursors. Knock-out experiments in mouse have shown a require- Several lines of evidence indicate that sox4b-expressing ment for transcription factors such as Arx and Pax6 during cells belong to the endocrine lineage only. Firstly, sox4b a cell differentiation. Arx, a homeobox-containing gene, is appears early in the region of the pancreatic dorsal bud, expressed in endocrine precursor cells. It is required for a which generates only endocrine cells (Field et al., 2003). cell fate acquisition while it represses h and y cell destiny Secondly, sox4b expression partially overlaps with that of (Collombat et al., 2003). We observed that zebrafish arx is several pancreatic endocrine markers, such as neuroD, also expressed in the pancreatic a cells (Figs 5M, O, and nkx2.2,andisl1. Thirdly, sox4b behaves like all endocrine unpublished results), in addition to its previously described markers of the dorsal bud in response to the Hedgehog expression in the floor plate of the neural tube and in the signaling. Altogether, these observations indicate that somites (Miura et al., 1997). As shown in Figs. 5M–P and sox4b-expressing cells belong to the endocrine lineage. W, knock-down of sox4b resulted in a loss of arx By contrast with pdx-1, nkx2.2, isl1, pax6.2, and hb9 expression, specifically in the region of the pancreas. More genes, whose expressions are maintained in pancreas than 50% of the morphant embryos were found to be totally throughout zebrafish embryogenesis (Biemar et al., 2001; devoid of arx-expressing cells in this region (Table 1), while Wendik et al., 2004), sox4b is transiently expressed in the expression in the floor plate and somites was unaffected pancreatic primordium. In mouse, one important gene (Figs. 5O and P). In mouse, the paired-homeodomain Pax6 transiently expressed in endocrine precursors is the bHLH gene is expressed in all endocrine cell types and it is crucial Ngn3 gene (Apelqvist et al., 1999; Gradwohl et al., 2000). to the differentiation of glucagon-producing cells (St-Onge Yet no zebrafish ngn3 gene expressed in the pancreatic et al., 1997). In zebrafish, the orthologous gene pax6.2 is anlage has been described (Wang et al., 2001). Thus, sox4b also expressed in pancreatic endocrine cells (Biemar et al., is so far the only gene transiently expressed in endocrine 2001). In contrast to arx expression, pax6.2 expression was cells of zebrafish pancreas. The zebrafish sox4b- and the not significantly affected in sox4b morphant embryos (Figs. murine Ngn3 genes display similar characteristics: both are 5Q and R) nor were levels of neuroD, nkx2.2,orpdx-1 transiently expressed in endocrine precursor cells, respond expression (Figs. 5S–V and Table 1). to Notch signaling, and are involved in endocrine cell Together, these results show that sox4b is involved in the differentiation. Thus, it is possible that both genes are differentiation of glucagon cells, acting upstream from arx involved in the same regulatory cascade. in the pancreatic regulatory cascade, and downstream, or in During the course of pancreas development in zebrafish, parallel to pax6.2. the dorsal pancreatic bud emerges from the pancreatic gut epithelium. A putative mechanism for the formation of the dorsal bud is the delamination of the endocrine precursor Discussion cells from the gut epithelium, followed by a migration and aggregation of these cells that will generate the pancreatic Sox4, a member of group C SOX, is expressed in islet. We observed that at 26 hpf the sox4b cells form a different tissues during embryonic and adult life (Cheung et domain located ventrally to the emerging islet. This suggests A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223 221 that sox4b is expressed in the endocrine precursor cells as control of cell death and proliferation in sox4b mor- they are still located in the pancreatic gut epithelium but is phant versus control embryos are required to verify this turned off when they are clustered into the islet of hypothesis. Langerhans. In summary, our results show that sox4b is an early and In mouse, disruption of Notch signaling leads to an transient marker of the pancreatic epithelium and is involved acceleration of endocrine and exocrine cell differentiation in the differentiation of a cells, the last differentiating (Apelqvist et al., 1999; Hald et al., 2003; Jensen et al., endocrine cell type. However, on the sole basis of the 2000; Miyamoto et al., 2003; Murtaugh et al., 2003). markers analyzed in this study, we cannot exclude the Recently, Esni et al. (2004) showed that, in zebrafish as possibility that sox4b could also be involved in other well as in mouse, Notch signaling is involved in exocrine mechanisms of pancreas development, as suggested by the cell differentiation. Similarly, accelerated differentiation of early onset of sox4b expression. We indeed noticed a failure endocrine cells is observed in mib mutant embryos of the endocrine cells to aggregate into a compact islet in (Wolfgang Driever and F.A, manuscript in preparation). many of the sox4b morphant embryos. Thus, sox4b could Interestingly, we found that sox4b is up-regulated at mid- play a role in the migration of endocrine cells and their somitogenesis, while it is expressed at comparable levels convergence into the mature islet. in mutant and wild-type embryos at later stages (this sox4b might also be involved in the differentiation of study and data not shown). All together, these data other pancreatic hormone cell types, role being comple- suggest that in zebrafish Notch signaling is involved in mented by other Sox genes in the sox4b knock-down. The endocrine cell differentiation, and that sox4b is expressed first candidates that come to mind are and , the in cells engaged in the differentiation process. The FISH other genes of group C SOX-family gene. In mouse, Sox4 experiments were also informative regarding the putative and Sox11 display overlapping expression patterns in the role of sox4b in pancreas cell differentiation. Indeed, embryonic pancreas (Lioubinski et al., 2003), but neither of although sox4b transcripts partially overlap with several the two zebrafish orthologs of mouse Sox11, sox11a, and endocrine markers, in particular neuroD, the situation is sox11b, is expressed in the developing pancreas (de Martino different in a, h, and y. Somatostatin cells do not express et al., 2000), neither the zebrafish sox12 ortholog gene sox4b, but a few insulin cells do, and glucagon cells (unpublished results). So far, ongoing expression analyses express low levels of sox4b. This suggests that sox4b is of several other zebrafish sox genes have failed to identify transiently expressed during endocrine cell differentiation. another SOX-family gene expressed in the zebrafish According to this hypothesis, the sox4b/glucagon- (or pancreas (unpublished results). An exhaustive analysis of insulin)-positive cells could represent some endocrine the SOX family in zebrafish is needed to determine whether cells in which the differentiation process is not yet other genes of this family are involved in pancreas completed. development in this organism. A final demonstration that sox4b is required for the differentiation of endocrine cells is provided by analysis of sox4b knock-down. Injection of anti-sense morpholino Acknowledgments or of mRNA encoding a truncated form of Sox4b leads to a dramatic decrease in the number of glucagon cells. We thank Pr. W. Driever for providing us the neckless This effect is accompanied by dramatic down-regulation mutants and for useful advices and discussions. We thank of arx expression in morphant embryos, specifically in Dr. A. Chitnis and Dr. Z. Varga for generously providing us the pancreatic anlage. We have observed that zebrafish the mind bomb and slow-muscle-omitted zebrafish strains. arx is expressed in a cells. Thus, the phenotype resulting We are grateful to Christelle Dekairelle for technical from sox4b knock-down indicates not only that sox4b assistance, to Christine De Mulder for precious help during acts upstream from arx in the process of a cell morphants analysis, and Drs. M. Voz, H. Pendeville- differentiation, but also that the zebrafish arx and mouse Samain, and Didier Stainier for helpful discussion during Arx genes probably have similar functions. Experiments are manuscript preparation. This work was supported by a grant currently performed in our laboratory to determine the from the EC, Fifth and Sixth Frameworks (QLRT-1999- actual function of arx in zebrafish. In contrast, the level of 00149 to B.P., H.E, F.A.; LSHB-CT-2005-521145 to B.P.), pax6.2 expression is not significantly affected, suggesting from the Belgian State Program on ‘‘Interuniversity Poles of that sox4b acts downstream or in parallel to it. Attraction’’ (SSTC, PAI p5/35), from Fund for Scientific In addition to pax6.2, the expression level of neuroD, Medical Research (Belgium), from ‘‘Actions Concerte´es’’ of nkx2.2, and pdx-1 appeared unaffected by sox4b knock- the University of Lie`ge, and by FRFC grant 2.4542.00 and down. Thus, the absence of glucagon would not be due the Special Fund for Research to P.M.. A.M. holds a to cell death or arrested cell divisions, but rather to a doctoral fellowship from the ‘‘Fonds pour la Formation a`la blocking of a cell differentiation. However, as the Recherche dans l’Industrie et l’Agriculture’’ (F.R.IA.) and number of a cells is quite small compared to the total B.P. is ‘‘Chercheur Qualifie´’’ from the ‘‘Fonds National pour number of endocrine cells, an accurate count and a la Recherche Scientifique’’ (F.N.R.S.). 222 A. Mavropoulos et al. / Developmental Biology 285 (2005) 211–223

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