Fig. 3.

Hnf1b is required for pancreas development in vitro and in vivo. (A) MO-mediated knockdown of Hnf1b in pancreatic explants. To demonstrate the specificity of the MO effect, RNA encoding a hormone-inducible version of Hnf1β (Hnf1b-GR) was co-injected and explants were treated with the GR inducer dexamethasone (DEX) together with RA at the equivalent of the gastrula stage. At the equivalent of stages 31 and 39, total RNA was isolated from ∼30 explants per condition and subjected to RT-PCR. Detection was of endogenous (endo) and injected Hnf1b (inj.), as well as for the marker genes indicated. The Hnf1b loss-of-function phenotype and its rescue was observed for four independent biological replicates. (B) Four-cell stage embryos were injected with RNA encoding β-galactosidase (glb1) and either Hnf1b-MO or a control-MO. At stage 32, embryos from two independent biological replicates were used for WMISH against Pdx1 and Ptf1a and a real-time PCR analysis for Pdx1, Ptf1a and Insulin. The graph indicates the fold change in tested markers in relation to Odc. ctr, uninjected embryos. (C) Four-cell stage embryos were injected with RNA encoding β-galactosidase alone or in combination with Hnf1b-GR RNA. At the gastrula stage, embryos were treated with dexamethasone (DEX) to induce Hnf1β function. WMISH against Pdx1 and Ptf1a at stage 32 is shown. Boxplots display the range of the percentage area of Pdx1 and endodermal Ptf1a domains in the endoderm observed in embryos from two independent biological replicates (see Fig. S7). By the use of ImageJ (https://imagej.net), Pdx1 and Ptf1a-positive areas were measured (orange dotted line) as a ratio of the whole endoderm (green dotted line). Values above the upper whisker, which is set at 1.5× interquartile range above the third quartile, are indicated as maximum outliers (°). (P-values in an unpaired Student's t-test **<0.01, ***<0.001).