Fig. 7

E-box binding proteins but not Ngn3 and NeuroD are required for beta-cell specific hb9 expression. (A–F) Similar expression of hb9 mRNA at 24 hpf in motoneurons (MN in A–C) and in the pancreatic islet (pancreas in D–F) of uninjected wild type (wt) embryos (A, D) and embryos injected with ngn3 (B, E) or a combination of ngn3 and neuroD morpholinos (C, F). Different focus planes of the same embryos are shown in dorsal view with anterior to the left. (G) Countings of hb9 positive cells in the pancreas of the wild type (wt, n = 21), ngn3 (MO^ngn3, n = 32) and ngn3/neuroD (MO^ngn3/neuroD, n = 22) morphants at 24 hpf. (H) Schematic drawing of the hb^Em:GFP construct in which both E-boxes (red line) were mutated by site-directed mutagenesis (origin and mutated sequences are shown with consensus sequences being underlined). Numbers on the right summarize the results of transient reporter assays in which a single embryo out of 120 GFP positive embryos (n) showed reporter expression within the pancreas (P). (I) GFP expression in Tg[hb9^Em:GFP] embryos at 28 hpf in whole embryos and in embryos stained with antibodies for GFP (green, expression corresponds to motor axons), insulin (blue) and islet (red). The right images shown are confocal image projections with an overlay at the right. Scale bar corresponds to 100 μm (live embryo in I) and 20 μm (A–F, confocal images in I).