Inhibition of DachB expression by morpholino antisense oligonucleotides. Expression of insulin (A–C), somatostatin (D–F) and glucagon (G–I) was analyzed by whole-mount in situ hybridization at 30 hpf embryos injected with the morpholino oligonucleotides shown: a standard control (A, D, G), a mismatch control with 4 bases altered from the complimentary dachb sequence (B, E, H), and an oligonucleotide complimentary to a dachb splice junction (C, F, I). All panels present ventral views of yolk-free embryos with anterior to the left. In panel J, the numbers of cells expressing the hormones shown were assessed in morpholino-injected embryos at 30 hpf. Each data point represents the mean ± standard error of at least 40 embryos for each condition. ***p<0.001 compared with embryos injected with morpholino 4-mismatch control by Student’s t test. Scale bar, 50µm.

Expression of Dach1 in the mouse pancreas. Immunofluorescence staining for Dach1 is shown in red at E12.5 (A–D), E15.5 (E, F), and adult (G – J). Double immunofluorescence staining for transcription factor Pdx1 or islet hormones (green) was performed for Pdx1 (B) glucagon (D, F and H), insulin (E and G), somatostatin (I), and pancreatic polypeptide (PP, in J). Scale bar, 100µm.

Pancreas development in the absence of Dach1 at E18.5. Hematoxylin-eosin staining demonstrates the morphology of the pancreas in Dach1^+/+ (A) and Dach1^−/− (B) mouse embryos. Scale bar, 500 µm. Immunofluorescent co-staining for insulin (C, D; red), glucagon (C, D; green), somatostatin (E, F; green) and pancreatic polypeptide (PP; E, F; red) was performed on the pancreas in Dach1^+/+ (C, E) and Dach1^−/− (D, F) mouse embryos at E18.5. Scale bar, 100 µm. Immunofluorescent co-staining for insulin (green) and Ki67 (red) was performed on the pancreas in Dach1^+/+ (G) and Dach1^−/− (H) mouse embryos at E18.5. Scale bar: 100 µm. I. Insulin- and glucagon-positive cells from Dach1^+/+ (black bars) and Dach1^−/− (white bars) embryos were counted and expressed as the total number of cells per total pancreatic area. J. Percentage of β-cells replicating in Dach1^+/+ (black bars) and Dach1^−/− (white bars) embryos at E18.5 was assessed by counting the number of cells co-staining for insulin and Ki67 and dividing by the total number of cells staining for insulin; and the percent of pancreatic progenitor cells replicating in Dach1^+/+ (black bars) and Dach1^−/− (white bars) embryos at E12.5 was assessed by counting the number of cells co-staining for Pdx1 and Ki67 and dividing by the total number of cells staining for Pdx1. Each data point represents the mean of 4 embryos ± standard error of the mean.*p < 0.02, **p < 0.01, and ***p < 0.001 compared with Dach1^+/+ embryos by Student’s t test.

Expression of dachb in the zebrafish pancreas. Whole-mount in situ hybridization was performed for dachb (A–K, blue) and insulin (I, red), somatostatin (J, red), and glucagon (K, red) at the stages of zebrafish development shown. Ventral views (A–D;I–K), and lateral views (E–H) are shown with anterior to the left. On all the panels the yolk was manually removed from the embryos. The notochord (n) and the lateral line (ll) have been indicated in panels A, E and C. Black arrowheads: dachb-expressing cell in the pancreatic region. Scale bar, 50µm.

Expression of Dach mRNA in the mouse. RT-PCR was performed with gene specific primers with RNA purified from isolated mouse tissues at the indicated ages in A, and from the mouse pancreatic ductal cell line mPAC L20 infected with adenovirus shown in B. Products were not amplified in the absence of RT (data not shown).

Expression of Dach1with islet transcriptional regulators in the mouse pancreas at E15.5. Immunofluorescence staining for Dach1 is shown in red (A, C, D, F, G, I). Double immunofluorescence staining for islet transcription factors (green) was performed for Pdx1 (B, C), Nkx6.1 (E, F) and Neurogenin3 (H, I). Nuclei expressing both proteins appear yellow (C, F, I). Examples of co-staining nuclei in panels G–I are indicated with white arrows. Scale bar, 50µm.

Cell cycle regulation by Dach1. A. Immunofluorescent co-staining was performed for cyclin-dependent kinase inhibitor p27Kip1 (red) and insulin (green) on sectioned pancreas from Dach1^+/+ and Dach1^−/− mouse embryos at E18.5. Scale bar, 25µm. B. The intensity of p27Kip1 protein expression in insulin-positive cells in E18.5 pancreases from Dach1^+/+ (black bar) and Dach1^−/− (white bar) embryos was quantified and expressed as the intensity normalized to cell area.*p<0.0001. C. Chromatin IP studies were performed by immunoprecipitating cross-linked chromatin with antiserum against Dach1, with control IgG or without antiserum. Fragments of the mouse genes shown were amplified by PCR from the precipitates or the input DNA. Each data point represents the mean of quantification in 4 embryos ± standard error of the mean.