Fig. 2.

Physiological changes and molecular abnormalities in islets of Langerhans associated with diabetic phenotype of Isl1CKO. a Blood glucose levels of Isl1CKO (n = 59 pups), heterozygous (HET, NeuroD1^Cre/Isl1^f/+; n = 77 pups), and controls (n = 138 pups) fed ad libitum after birth at P0-P3. Violin plots indicate median (middle line), 25th, and 75th percentile (dotted lines). Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (****P < 0.0001). b Total pancreatic insulin content per pancreatic tissue (ng/mg) at P0 (n = 6 pancreases per genotype), P15 (n = 13 pancreases per control, 14 pancreases per Isl1CKO), and in the adult mice (n = 19 pancreases per control, n = 16 pancreases per Isl1CKO) fed ad libitum. Data are presented as mean ± SEM, Student’s t test (*P = 0.017, ****P < 0.0001). c-f Representative sections from the control and Isl1CKO pancreas immunostained for glucagon (GCG), insulin (INS), PDX1 (marker of β cells), or alpha amylase (marker for exocrine cells) at P0 demonstrate reduced endocrine tissue and abnormalities in the formation of pancreatic islets in Isl1CKO with lower production of INS, missing GCG⁺ cells, and reduced expression of PDX1. g, h Immunolabeling for proliferating cell nuclear antigen Ki67 (red) shows proliferating GCG⁺ and INS⁺ cells in the islets of the Isl1CKO and control pancreas at P0. i Total number of INS⁺ and GCG⁺ cells in 80-μm sections of the pancreas (n = 5 pancreases per genotype), and the percentage of INS⁺ cells expressing Ki67 (n = 5 mice per genotype) and phosphorylated histone H3 (pHH3; n = 4 mice per genotype). See also Additional file 1: Fig. S3. Data are presented as mean ± SD, unpaired t test (****P < 0.0001, ***P < 0.001, ns = not significant). j-o Immunostaining of α- and β-cell markers (PDX1, INS, GCG, PAX6, and NKX6.1) in the pancreatic sections at P9 shows abnormalities in β cells, including reduced production of INS, variable expression levels of PDX1 with some cells expressing INS but not PDX1 (arrowheads in k indicate INS⁺ cells without PDX1 expression). GCG producing α cells are lost in the Isl1CKO islets of Langerhans. Arrows in m indicate the unusual GCG⁺ cells with a missing expression of PAX6, a marker of both β and α cells. p, q At P35, compare to the control pancreatic islets, the expression of INS and PDX1 is reduced, and GCG⁺ producing cells are missing in the Isl1CKO islets. r, s Representative confocal microscopy images of double staining with anti-PDX1 and anti-ISL1 antibodies show that the expression of ISL1 is not detected in nuclei of endocrine cells of pancreatic islets of Isl1CKO in contrast to the control. Arrowheads indicate nuclei with ISL1 and PDX1 co-expression in the control pancreatic islet. Note reduced PDX1 expression in Isl1CKO. Scale bars: 50 μm. HS, Hoechst nuclear staining