Fig. 6

Robo signalling activity is restricted to pancreatic progenitors. a Pancreas gross morphology at E18.5. Robo^PaΔ (Pdx1-Cre;Robo1^-/-;Robo2^fl/fl) shows pancreatic hypoplasia. Asterisk (*) indicates the head of the pancreas, which is mostly affected. Blue dotted line delineates the whole pancreas; duo duodenum, sp spleen, st stomach. Scale bars, 1 mm. b Quantification of pancreas weight in control (CTRL; n = 6), Robo^PaΔ (n = 4) and single Robo1 KO (n = 5) E18.5 mouse embryos. Error bars represent ± s.e.m. *Two-tailed Student’s t-test P < 0.05. c Conditional inactivation of Robo genes in pancreatic progenitors recapitulates the pancreatic size defect of Robo1/2 KO pancreas at E9.5. Measurement of Pdx1⁺ ventral pancreatic bud volume (μm³) in E9.5 CTRL, Robo1/2 KO and Robo^PaΔ embryos. Pdx1⁺ vp volume measurement was performed using the surface detection tool in Imaris. Error bars represent ± s.e.m. *Two-tailed Student’s t-test P < 0.05. d Representative IF images of E9.5 CTRL and Robo^PaΔ cryosections stained for Albumin (Alb), Pdx1 and Prox1. Hoechst (Hoe) was used as nuclear counterstain. Arrowheads indicate Prox1⁺/Alb⁺ liver progenitors in hepatic cords. In dashed box, Pdx1^-, Prox1⁺ and Alb⁺ cells within the ventral pancreatic epithelium (vp) of KO embryos. Insets show split channels of boxed area at higher magnification. Scale bars, 50 µm. e RT-qPCR analysis evaluating pancreatic gene expression in mESC differentiation into pancreatic endoderm (PE) in the absence or presence of Robo2-Fc. Data are represented as relative fold change. Values shown are mean ± s.e.m. (n = 3). *P < 0.05, two-tailed unpaired t-test for differentiated PE versus undifferentiated ESC; ^#P < 0.05 for differentiated PE versus PE+Robo2-Fc. ND not detected. f Representative IF images of PE and PE+Robo2-Fc stained with Pdx1 and Hoechst (Hoe) as nuclear counterstain. Scale bars, 20 µm. g Percentage of Pdx1⁺ cells in PE and PE+Robo2-Fc cultures was calculated by counting Pdx1⁺ cells relative to total number of Hoechst⁺ nuclei per well. Error bars represent ±s.e.m. *Two-tailed Student’s t-test P < 0.05