Additional file 1 of ISL1 controls pancreatic alpha cell fate and beta cell maturation
Creators: name: Bohuslavova, Romana
name: Fabriciova, Valeria
name: Lebrón-Mora, Laura
name: Malfatti, Jessica
name: Smolik, Ondrej
name: Valihrach, Lukas
name: Benesova, Sarka
name: Zucha, Daniel
name: Berkova, Zuzana
name: Saudek, Frantisek
name: Evans, Sylvia M
name: Pavlinkova, Gabriela
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig. S1. Efficient deletion of ISL1 in the Isl1CKO developing pancreas. Representative whole-mount immunolabeling of the pancreas of tdTomato reporter control-Ai14 and Isl1CKO-Ai14 embryos during the primary transitions (E10.5 and E11.5) and the secondary transition (E13.5) shows ISL1 expression in the Neurod1Cre positive domain visualized by tdTomato expression. The pancreatic epithelium is delineated by the expression of PDX1. Scale bars: 50 μm. Fig. S2. Diabetic phenotype of Isl1CKO. a The average blood glucose levels over time (from 1 week to 5 weeks of age) in females and males mice fed ad libitum. The 5 weeks of age female mice had blood glucose unmeasurable (above 35 mmol/l), the 5 week of age males show a high variability with 7 mice with blood glucose unmeasurable, and 3 with 0.8, 7.9 and 31.9 mmol/l. Data are presented as mean ± SD, analyzed by VA (****P &lt; 0.0001). b The average body weight of adult female and male mice. Data are presented as mean ± SEM, Student’s t test. c Glucose tolerance test plotted using glucose vs time in heterozygous (Neurod1Cre/Isl1flox+/−), and control mice. Data are presented as mean ± SD, analyzed by twoway Anova with Bonferroni post-hoc analysis for glucose vs time. d Blood glucose concentration in adults fed ad libitum, 6-8 weeks of age. Only measurable levels of glucose (&lt; 35 mmol/l) are shown for Isl1CKO. Data are presented as mean ± SEM, Student’s t test (****P &lt; 0.0001). e The weight of pancreas of adult mice. Total pancreas weights of 244 ± 78 mg (n = 22) in adult Isl1CKO mutants compared to those of controls (211 ± 57 mg, n = 21). Data are presented as mean ± SEM, Student’s t test. Fig. S3. Immunolabeling for the marker of cellular mitosis, phosphorylated histone H3 (pHH3). Representative sections from the control and Isl1CKO pancreas immunostained for insulin (INS) and PDX1 (marker of β cells) at P0 and E17.5. Scale bars: 50 μm. Fig. S4. H3K4me3 and H3K27me3 CUT&amp;Tag-seq analyses of E14.5 pancreatic endocrine cells. a The functional enrichment analysis of the differentially expressed genes in Isl1CKO was performed using g: Profiler (Gene Ontology: MF, molecular function; BP, biological processes). b A UMAP overview of nine cell clusters of different pancreatic cell types used as a reference for the cell type deconvolution analysis (van Gurp et al., 2019). c The deconvolved cell cluster proportions in E14.5 endocrine population from our bulk RNA-seq data of Isl1CKO and control. d The UCSC browser view of whole genome showing H3K4me3 and H3K27me3 peaks in control and Isl1CKO endocrine cells at E14.5 based on CUT&amp;Tag-seq analyses. The mapped read counts distributed across all chromosomes are in comparable read depth for control and Isl1CKO samples. e Bar plot showing percentage of H3K4me3 and H3K27me3 peaks at promoter regions (± 3 kb from TSS), gene body regions, and intergenic regions. f Pie chart illustrating the proportion of differentially expressed genes that differentially exhibited one or both H3K4me3 and H3K27me3 marks at their promoter regions from pairwise comparison of Isl1CKO and control pancreatic endocrine cells at E14.5. Fig. S5. H3K4me3 and H3K27me3 CUT&amp;Tag-seq analyses of P9 pancreatic endocrine cells. a The UCSC browser view of whole genome showing H3K4me3 and H3K27me3 peaks in control and Isl1CKO endocrine cells at P9 based on CUT&amp;Tag-seq analyses. The mapped read counts distributed across all chromosomes are in comparable read depth for control and Isl1CKO samples. b Bar plot showing percentage of H3K4me3 and H3K27me3 peaks at promoter regions (± 3 kb from TSS), gene body regions, and intergenic regions of endocrine cells of the P9 pancreas. Fig. S6. ISL1 binding CUT&amp;Tag-seq analyses of E14.5 pancreatic endocrine cells. a Pie chart showing genomic distribution of ISL1 loci in E14.5 pancreatic endocrine cells. b The most enriched Gene Ontology (GO) biological processes for genes bound by ISL1 and with Isl1CKO-specific H3K27me3 depositions. Fig. S7. Gating strategy used to isolate tdTomato+ cells. Representative example to show gating to purify live and individual tdTomato+ cells for RNA-seq and CUT&amp;Tag-seq. Table S1. Primer sequences for genotyping. Table S2. Primer sequences for RT-qPCR. Table S3. List of antibodies.