Zebrafish rfx6 is expressed in pancreatic endocrine progenitors as well as in mature endocrine cells. (A) Lateral view of fluorescent wholemount in situ hybridization (WISH) performed on stage 19S embryos with an rfx6 antisense probe (arrowhead). (B) Ventral view of WISH with an rfx6 probe on 24 hpf embryos. (C-N) Lateral views of the pancreas area from embryos analyzed by double-fluorescent WISH, anterior to the left, dorsal to the top. (C) The expression domains of isl1 and sox4b are distinct. (D-F) Relative location of the hormone-expressing cells in relation to the neurod-, isl1- and sox4b-expressing cells. Hormone expression was detected by WISH using a cocktail of insulin, glucagon, somatostatin and ghrelin probes. The diagram shows the respective locations of sox4b-, isl1-, neurod-, and hormone-positive cells within the pancreatic area in a lateral view at 24 hpf. (G-I) At 18S, rfx6 and sox4b expression domains completely overlap, whereas at 24 hpf and 30 hpf sox4b is maintained only in the ventral part of rfx6 domain. (J-L) The expression pattern of rfx6 completely overlaps the pancreatic neurod expression pattern. (M,N) The isl1-expressing cells are located in the dorsal part of the rfx6 expression domain at 24 hpf and 30 hpf.

Impaired endocrine cell differentiation, accumulation of islet progenitor cells and failure of insulin-expressing cells clustering in rfx6 morphants in the zebrafish. (A-X) Ventral views of the pancreas area from embryos analysed by WISH, anterior to the left. (A-L) Hormone expression in the control and rfx6 morphants at 30 hpf. (M-X) Pancreatic expression of isl1, sox4b and neuroD at 24 hpf and arx at 30 hpf in the control and rfx6 morphants. (Y-ZII) Lateral views of the pancreas area from 30 hpf embryos analysed by double-fluorescent WISH for sox4b and isl1, anterior to the left, dorsal to the top. Quantifications (right side of figure) represent the number of positive cells per embryo for controls and morphants. Asterisks (*) indicate that the difference between cell number in control and morphants is statistically significant by Student′s t-test (P<0.001). Scale bar: 14 μm in A-L; 18 μm in M-X.

rfx6 is exclusively expressed in the pancreas area during early zebrafish development. (A) General view of embryo stained by fluorescent wholemount in situ hybridization (WISH) for the expression of rfx6. (B-H) Pancreatic expression starts around 17 hpf, peaks around 24 hpf and persists at least until 72 hpf. Ventral views, anterior to the left. Scale bars:10μm. Panels A and D are the same as panels A and B in Fig. 6.

rfx6 is expressed in all of the hormone-expressing cells in zebrafish. Ventral views of the pancreas area from 30 hpf embryos analysed by double-fluorescent wholemount in situ hybridization (WISH), anterior to the left. The right column presents the overlay of the left and middle panels.

Acetylated-tubulin labelling reveals ciliated cells within the developing pancreas of wild-type and morphant zebrafish. Experiments to visualize primary cilia were performed on transgenic zebrafish embryos tg(pax6:GFP) where pancreatic endocrine cells can be detected by GFP expression (Delporte et al., 2008). (A,B) Pancreatic cell labelling using the tg(pax6:GFP) transgenic line underlines the developing pancreas in control (A) and rfx6 Mo2 morphants (B). (C,D) Magnifications of the area in white boxes in A and B with the acetylated-tubulin-labelled cilia in red and pax6:GFP in green. Arrows in C and D point to cilia. The same cilia are marked with a grey dot visible in Movies 1 and 2. Scale bars: 10 mm in A,B; 2 mm in C,D.