Expression of calsenilin in the zebrafish pancreas. A: Search for new genes expressed in the pancreas: zebrafish at 24 hours postfertilization (hpf) were cohybridized with digoxigenin-labeled antisense probes (in blue) corresponding to the gene to be tested and fluorescein-labeled antisense insulin probe (in red). Calsenilin that was coexpressed with insulin was kept for further analysis. B: Expression of calsenilin during zebrafish development. Whole-mount in situ hybridization was performed with digoxigenin-labeled antisense calsenilin and insulin probes at different stages of development (21-somites, 24 hpf, 29 hpf, 32 hpf, 39 hpf). Arrows point to insulin-positive regions.

Coexpression of calsenilin and pancreatic hormones. Double fluorescent in situ hybridization in zebrafish embryos. Zebrafish at 24 hours postfertilization (hpf) and 30 hpf were cohybridized with digoxigenin-labeled antisense calsenilin probe in combination with either DNP-labeled antisense insulin, glucagon, or somatostatin2 probes. Calsenilin was revealed with tyramide-Cy3 substrate (in red) while insulin, glucagon, and somatostatin2 were revealed with tyramide-fluorescein isothiocyanate (in green). Images on the right column represent superposition of the respective left and middle confocal panels.

Expression of calsenilin in mutants with affected retinoic acid synthesizing aldehyde dehydrogenase (RALDH; nls) and Notch (mib) signaling. Whole-mount in situ hybridization with digoxigenin-labeled antisense insulin and calsenilin probes on wild-type (WT), nls mutant defective in retinoic acid (RA) synthesis and mib mutants defective in the Delta-Notch pathway. Black and white arrows indicate insulin and calsenilin expression in the pancreas, respectively.

Pancreatic endocrine development is affected in calsenilin knockdown embryos. A: In vitro efficacy of morpholino antisense oligonucleotides directed against calsenilin. B: Whole-mount in situ hybridization on embryos injected with MO-calsenilin, MO3-calsenilin or with 5-misMO-calsenilin control antisense oligonucleotides. Zebrafish embryos at 24 hours postfertilization (hpf) or 40 hpf were hybridized with a digoxigenin-labeled antisense insulin probe. Quantification of insulin-positive cells either in 5-misMO-calsenilin, MO-calsenilin, or MO3-calsenilin morphants at stage 24 hpf. In knockdown embryos, the number of insulin-expressing cells was strongly decreased. ***P < 0.0001 C: Whole-mount in situ hybridization with digoxigenin-labeled antisense sst2, pdx1, isl1, and pax6.2 in MO-calsenilin and control 5-misMO-calsenilin embryos at 24 hpf. Note that in MO-calsenilin morphants, pancreatic endocrine cells were dispersed and did not associate to form an islet. Quantification of sst2-, isl1-, and Pax6.2-positive cells in wild-type or MO-calsenilin morphants at stage 24 hpf. *P < 0.01; ***P < 0.0001