Abstract
Introduction: Functional pancreatic α and β cells are necessary for glucose homeostasis. The molecular mechanisms of the generation and maturation of these cells are still unclear.
Our study is focused on the role of transcription factor Isl1 during pancreas development. Material and Methods: We generated a transgenic mouse model of conditional knockout of the Isl1 gene using the Cre-loxP system (Neurod1-Cre, Isl1f/f) with fluorescent reporter tdTomato.
RNA-seq, Cut&Tag-seq, and RT-qPCR were performed on FACS-sorted cells. We used immunohistochemistry and confocal microscopy to evaluate the cellular pancreatic endocrine phenotype. for a better insight into the anatomical 3D microenvironment, we performed light sheet fluorescent microscopy.
Bioinformatic tools were used to identify ISL1 binding sites (HOMER) and to deconvolve cell subtypes based on single-cell transcriptomic dataset (CibersortX). Results: Isl1CKO mice show diabetic phenotype with significant neonatal hyperglycemia that increased with age.
Isl1CKO showed complete loss of α cell lineage and disrupted architecture of the islets of Langerhans. β cell mass was significantly reduced compared to controls. RNA-seq data show dysregulated transcriptome in Isl1CKO- downregulated genes important α and β cell markers and regulators, upregulation of endocrine progenitor specific genes.
These data were confirmed by RT-qPCR. The Cut&Tag method that uncovers the epigenetic landscape revealed altered silencing by H3K27me3 histone modification in the promoter regions in genes essential for endocrine cell differentiation.
HOMER analysis indicates that downregulated genes with H3K27me3 enrichment, such as Mafa and Eya1 (mature β cell markers), and upregulated genes without H3K27me3 modification, like Vim, Fev and Pbxip1 (endocrine progenitor cell marker), contain ISL1 binding motif. Conclusions: Isl1 is a critical transcription and epigenetic modulator for the generation and maturation of fully functional α and β cells.
More studies, especially single-cell transcriptomics, are needed to understand molecular pathways governed by ISL1. Supported by GACR (GA19-07378S) and institutional support of CAS (RVO:86652036).