Quantification and statistical analysis

Data are presented as mean (SD). E12.5 lumen discontinuity quantifications were performed on whole-mount-immunostained tissues, where pancreata were analyzed for isolated foci of Muc1 covering ≈80% of the tissue (n = 2 per genotype). Statistical analysis for lumen discontinuity assessment was performed using one-way ANOVA in GraphPad Prism software. E14.5 lumen discontinuity quantifications were performed on 30-µm cryosections, where at least 25 tips per pancreas were analyzed for isolated foci of Muc1 (n = 3 per genotype). The E18.5 core area was measured on head regions of pancreata whole-mount-stained for DBA. The core region was identified by using the Filament Tracer function in Imaris, and the area of this region was calculated using ImageJ measurements (n = 4 per genotype). The core area on E12.5 explants cultured for 6 d was identified by using Filament Tracer. The Surface function was used to create a separate channel for the core region with different color assignment, and this channel was overlaid with the nonmasked surface to visualize the core and the periphery in different colors. The E18.5 Sox9⁺ core volume was calculated on head regions of pancreata whole-mount-stained for DBA and Sox9. The core region was identified by using the Filament Tracer function in Imaris, and the Surface function was used to create a separate channel for the core region within the Sox9 channel. The core Sox9⁺ volume was calculated using the Surface function within this channel created for the core (n = 2 wild-type; n = 3 DKO). Quantification of endocrine volume at P0 was performed on whole-mount insulin/glucagon-immunostained tissues. Head regions of pancreata were analyzed. To account for the size difference between wild-type and DKO pancreata, z-stack images of whole head regions were taken regardless of the area that they occupied (two z-stacks at 10× magnification and 0.5× digital zoom for wild type and one z-stack at 10× magnification and 0.5× digital zoom for DKO). The Surface function of Imaris was used to calculate the overall volume of insulin/glucagon-positive cells. The summed volume of two z-stacks for wild type was compared with one z-stack for DKO. The endocrine area in explants was analyzed in whole-mount-immunostained tissue using the Surface function in Imaris (n = 11 wild type; n = 3 DKO). E17.5 endocrine proliferation was assessed on pH3/endocrine marker-costained sections, where at least five sections per pancreas were analyzed (n = 3 per genotype). The E18.5 endocrine yield in the core versus the periphery was analyzed on pancreata whole-mount-immunostained for Neurog3 and Sox9, where at least three different areas for each region (the core or periphery) were analyzed per tissue (n = 2 per genotype). Sox9⁺ cells were counted using the Spots function in Imaris. Neurog3⁺ cells were counted manually. The endocrine yield was calculated as Neurog3⁺ cell number divided by Sox9⁺ cell number multiplied by 100 to represent percentage. The overall endocrine yield at E15.5 or E17.5 was assessed on sections where Neurog3⁺ and Sox9⁺ were counted manually. Neurog3⁺ cell numbers were analyzed by counting Neurog3⁺ cells on sections and normalizing these numbers to the DAPI⁺ pancreatic surface area (the obtained numbers were multiplied by 10⁶ for convenience). The endocrine volume at E15.5 was assessed using the same method as at E18.5 except the entire pancreas was analyzed. Unless multiple comparisons were made, all statistical analyses were performed using two-tailed unpaired Student's t-test in GraphPad Prism software. Multiple comparison analyses are described in the relevant Materials and Methods sections. P < 0.05 was considered statistically significant.