Zecchin et al., 2007 - Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish. Developmental Biology   301(1):192-204 Full text @ Dev. Biol.

Fig. 1  Inhibition of Notch signalling increases differentiation of beta-cells. In situ hybridizations showing insulin (A–D) somatostatin (PPSS2) (E–H) and glucagon (I–L) expression in mib mutants and DAPT-treated embryos. The pancreatic area is depicted. Insulin and somatostatin expression, in blue, is increased in mib mutants and DAPT-treated embryos (C, D, G, H) compared with controls (A, B, E, F). Conversely, glucagon expression is reduced or absent (compare K, L with I, J). Embryos in panels A, E, I and B, F, J should be compared with embryos in panels C, G, K and D, H, L, respectively. Embryos have been hybridized at 72 hpf and are presented in a ventral view with anterior to the left. Scale bar in panel A is 50 μm.

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Stage: Protruding-mouth

Fig. 2  Block of Notch signalling decreases the number of exocrine cells in the pancreas. In situ hybridizations showing reduction of mnr2a (A–D), ptf1a (E–H) and trypsin (I–L) expression in mib mutants (C, G, K) and DAPT-treated embryos (D, H, L). The pancreatic area is depicted. Embryos in panels A, E, I and B, F, J should be compared with embryos in panels C, G, K and D, H, L, respectively. Embryos have been hybridized at 72 hpf and are presented in ventral view with anterior to the left. Scale bar in panel A is 50 μm.

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Stage: Protruding-mouth

Fig. 3  Endoderm development in mib mutants. In situ hybridizations showing expression of pdx1, pax6.2, neuroD and ngn3 in the endoderm of mib mutant embryos and controls. Panels A–D: pdx1 is expressed at normal levels in the gut (arrowheads) of mib embryos and their wild type siblings, whereas its expression is stronger in the endocrine pancreas (p) of mib mutants (B, D) when compared with controls (A, C). Panels E–H: the expression of neuroD and pax6.2, both active in endocrine precursors, is stronger in the endocrine pancreas (p) of mib mutants (F, H) when compared with controls (E, G). The gut (g) is outlined. Panels I–J: ngn3 is expressed ectopically in the gut (g, also outlined) of mib mutants. Panels K–N: neuroD and pax6.2 are expressed ectopically in the gut (g, indicated by arrows and outlined) of mib mutants (L, N). The endocrine pancreas (arrowhead) is indicated. Embryos are presented in ventral (A–J) or lateral (K–N) views with anterior to the left (A–J) or to the right (K–N). Scale bar is 50 μm.

Fig. 4  Increase of enteroendocrine cell differentiation in mib mutants and DAPT-treated embryos. Panels A, B: in situ hybridization with a GFP antisense probe showing increased activity of the glucagon promoter in the gut (black arrows) of a glucagon:GFP (glu:GFP) transgenic zebrafish line treated with DAPT at 30 hpf (B). Panels C, D: nk2.2a:GFP transgenic embryo showing an increase of enteroendocrine cells (white arrows) when treated with DAPT at 30 hpf (D). The pancreatic duct (d) and the endocrine islet (arrowhead) are indicated. Panels E–H: glucagon expression in the gut (g) and pancreas (black arrowhead) of wt (E, G) and mib mutants (F, H) analyzed at 96 hpf. In panels G–H the gut is outlined. Panels I, J: expression of the peptide transporter pept1 in wt (I) and mib mutant embryos (J). Embryos are in lateral (C–F), ventral (A, B) or dorsal (G–J) views with anterior to the right. Scale bar is 50 μm.

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Stage: Day 4

Fig. 5  Expression of Notch ligands in the pancreatic primordium. Panel A: deltaA expression in the endoderm of an embryo at the 7-somite stage. Panel B: expression of neuroD in the endoderm of an embryo at the 7-somite stage. Panels C, D, F: expression of insulin (red) and deltaA (blue) in the endoderm of zebrafish embryos. Panel E: expression of pdx1 (red) and deltaA (blue) in the endoderm of zebrafish embryos at 24 hpf. Panels G, H: expression at 28 hpf of jagged1b (blue) in cells located around the insulin (red) producing cells. Panels I, J: expression of insulin (red) and jagged2 (blue) in the endoderm of zebrafish embryos at 36 hpf. The pancreatic primordium is indicated with a black arrowhead. “n” indicates the notochord; “2s” indicates the position of the second somite. Embryos are in ventral (A, B, G, I), lateral (C–F) views with anterior to the left or transversal vibratome sections (H, J) Scale bar in panel A is 50 μm.

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Stage Range: 5-9 somites to Prim-25

Fig. 6  Effects of morpholino-mediated knock down of Notch ligands on the differentiation of pancreatic cell populations. Panels A–D: insulin and glucagon expression at 24 hpf in control and deltaA-morphant embryos. glucagon-expressing cells are decreased in number in deltaA morphants (D). Panels E–H: glucagon expression in the pancreas (E, F) and gut (G, H) is increased in jagged1b-morphant embryos (F, H) compared with controls (E, G). Panels I–J: trypsin expression is decreased in jagged1b morphant embryos. Panels K–N: expression of endocrine and exocrine markers in jagged2-morphant embryos. insulin (red) expression is increased and trypsin (blue) expression decreased in jagged2-morphants (panels L and L′ blow up) compared with controls (panels K and K′ blow up). glucagon expression profile in jagged2-morphants is enlarged (N) reflecting the increased size of the beta-cell cluster. Embryos are in ventral view with anterior to the left. Stages are indicated at the bottom right of each frame. The scale bar is 50 μm.

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Stage Range: Prim-5 to Day 4

Fig. 7  Expression of pdx1, insulin, somatostatin (PPSS2), glucagon, ptf1a and trypsin in control (hsp70:Gal4 neg; UAS:NICDpos or hsp70:Gal4pos; UAS:NICDneg) and NICD-expressing (hsp70:Gal4pos; UAS:NICDpos) embryos heat shocked at different time points. Markers analyzed are indicated at the top of each subset of panels, while the time point of the first heat shock (HS) is indicated on the left. For each gene expression test, mono-genic control embryos (hsp70:Gal4 neg or UAS:NICDneg) are on the left and bi-genic embryos (hsp70:Gal4pos and UAS:NICDpos) are on the right. pdx1 (A–C); precursors, expressing pdx1, are increased in number when bi-genic embryos are heat shocked at 20 hpf or before (A′, B′, C′) indicating that Notch activation leads to proliferation of precursors. ins and sst (D–I); differentiation of insulin- and somatostatin-producing cells is completely blocked only when bi-genic embryos are heat shocked at 12 hpf (D′ and G′). glu (J–L); differentiation of glucagon-producing cells is completely blocked when bi-genic embryos are heat shocked at 20 hpf or before (J′ and K′) and severely impaired in embryos heat shocked at 30 hpf (L′). ptf1a and try (M–P); bigenic embryos heat shocked at 30 hpf fail to differentiate the exocrine pancreas (M′, N′, O′, P′). The stage at which the embryos were analyzed is indicated within each frame. Individual embryos were genotyped by PCR for presence or absence of each transgene. Embryos are in ventral views with anterior to the left.

Fig. 8  Expression of insulin, somatostatin (PPSS2), glucagon, pax6.2 and pdx1 in ins:Gal4;UAS:NICD bi-genic embryos. Mono-genic control embryos (ins:Gal4 neg and UAS:NICD pos) are on the left and bi-genic embryos (ins:Gal4 pos and UAS:NICD pos) are on the right. Expression of NICD under the control of the insulin promoter leads to the lack of insulin-producing cells but somatostatin and glucagon producing cells are present similarly to control. Genes whose expression is analyzed are indicated on the left while the genotypes are indicated on the top. Embryos are in ventral views with anterior to the left. Individual embryos were genotyped by PCR for presence or absence of each transgene. Scale bar is 50 μm.

Fig. 9  Model describing the role of Notch-ligands in pancreatic cell differentiation. The timeline is on the left and relevant stages of pancreatic differentiation are indicated: beta-cells differentiating at 12-somite and alpha-cell differentiating at 20-somite stage will contribute to the first bud while the second bud, differentiating at 33 hpf, is supposed to form the exocrine tissue and some endocrine cells. The role of each Notch ligand in limiting the number of differentiating precursors is indicated.

Fig. S1 jagged2 knockdown increases the number of insulin expressing cells. Expression of insulin in control (jag2mismMO) and jagged2-morphants analyzed at 48 hpf.

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Stage: Long-pec

Fig. S2 Effects of knock down mediated by morpholinos directed against splicing of Notch ligands on the differentiation of pancreatic cell populations. Morpholinos are indicated on top of each group and probes on the left. glucagon expression in the pancreas is increased in jag1bspliMO-morphant embryos compared with mismatched controls (see also RT-PCR on Fig. S3). Moreover, expression of insulin in is increased while trypsin expression is decreased in jag2spliMO-morphants compared with mismatched controls. Embryos are in ventral view with anterior to the left. Stages are indicated at the bottom right of each frame.

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Stage Range: Long-pec to Day 4

Fig. S3 RT-PCR on Jagged mRNAs. A; RT-PCR on embryos injected with jag1spliMO and jag2spliMO and controls. M = molecular weight marker, 1 = embryos injected with splicing-morpholino, 2 = embryos injected with mismatched morpholino, 3 = non-injected controls. B; RT-PCR of jagged genes on embryos at different stages of development. M = molecular weight marker, 1 = 1–2 cell stage, 2 = high stage, 3 = 50% epiboly stage, 4 = 80% epiboly stage, 5 = 5 somite stage, 6 = 16–18 hpf, 7 = 24 hpf, 8 = adult, - = negative control.

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Stage Range: 1-cell to Adult

Fig. S4 Forced Notch signaling blocks exocrine differentiation. A–B: expression of ptf1a in control (hsp70:Gal4 neg;UAS:NICD pos or hsp70:Gal4 pos;UAS:NICD neg) and NICD-expressing (hsp70:Gal4 pos;UAS:NICD pos) embryos heat shocked at 24 hpf and analyzed at 32 and 44 hpf. Embryos are in ventral views with anterior to the left. Individual embryos were genotyped by PCR for presence or absence of each transgene.

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Stage Range: Prim-15 to High-pec

Fig. S5 Forced Notch signaling blocks endocrine differentiation. Expression of pax6.2 (A–B2) and neuroD (C–D2) in control (hsp70:Gal4 neg; UAS:NICD pos or hsp70:Gal4 pos; UAS:NICD neg) and NICD-expressing (hsp70:Gal pos; UAS:NICD pos) embryos heat shocked at 12 or 20 hpf and analyzed respectively at 24 and 30 hpf. The analyzed gene is indicated at the top of each subset of panels. Embryos are in ventral views with anterior to the left. Individual embryos were genotyped by PCR for presence or absence of each transgene.

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Stage Range: Prim-5 to Prim-15
Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 301(1), Zecchin, E., Filippi, A., Biemar, F., Tiso, N., Pauls, S., Ellertsdottir, E., Gnugge, L., Bortolussi, M., Driever, W., and Argenton, F., Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish, 192-204, Copyright (2007) with permission from Elsevier. Full text @ Dev. Biol.