NEUROD1 reinforces endocrine cell fate acquisition in pancreatic development

NEUROD1 is a transcription factor that helps maintain a mature phenotype of pancreatic beta cells. Disruption of Neurod1 during pancreatic development causes severe neonatal diabetes; however, the exact role of NEUROD1 in the differentiation programs of endocrine cells is unknown. Here, we report a crucial role of the NEUROD1 regulatory network in endocrine lineage commitment and differentiation. Mechanistically, transcriptome and chromatin landscape analyses demonstrate that Neurod1 inactivation triggers a downregulation of endocrine differentiation transcription factors and upregulation of non-endocrine genes within the Neurod1-deficient endocrine cell population, disturbing endocrine identity acquisition. Neurod1 deficiency altered the H3K27me3 histone modification pattern in promoter regions of differentially expressed genes, which resulted in gene regulatory network changes in the differentiation pathway of endocrine cells, compromising endocrine cell potential, differentiation, and functional properties. Mouse pancreatic islets were isolated (and pooled) from four mice per each genotype (control and Neurod1CKO) by perfusion of the common bile duct with Collagenase. Digestion of islets to single cell was performed with trypsin supplemented with Actinomycin D. Single-cell suspension was used for library preparation:743 Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10 x Genomics, Pleasanton, CA) was sed to prepare the sequencing libraries, according to the manufacturer's protocols.NEUROD1是一种可维持胰腺β细胞成熟表型的转录因子（transcription factor）。胰腺发育过程中Neurod1的功能缺失会引发严重的新生儿糖尿病，但NEUROD1在内分泌细胞分化程序中的确切作用仍未明确。本研究揭示了NEUROD1调控网络在内分泌谱系定型与分化进程中的关键作用。机制层面，转录组与染色质景观分析结果显示，在Neurod1缺陷的内分泌细胞群中，Neurod1失活会导致内分泌分化相关转录因子表达下调，而非内分泌基因表达上调，进而干扰内分泌细胞身份的建立。Neurod1缺失会改变差异表达基因启动子区域的H3K27me3组蛋白修饰模式，致使内分泌细胞分化通路中的基因调控网络发生改变，损害内分泌细胞的发育潜能、分化能力与功能特性。本研究通过向胆总管灌注胶原酶（Collagenase），从每种基因型（对照组与Neurod1CKO）的4只小鼠体内分离并混合胰腺胰岛。随后使用添加了放线菌素D（Actinomycin D）的胰蛋白酶将胰岛消化为单细胞悬液。单细胞悬液用于文库制备：依照制造商操作规程，采用743 Chromium Next GEM Single Cell 3' Reagent Kits v3.1（10 x Genomics，美国加利福尼亚州普莱森顿）制备测序文库。