NEUROD1 is critical for the initial cell identity determination and differentiation of the endocrine progenitors in the pancreas [RNA-seq]

While diabetes incidence is gradually rising worldwide, novel therapeutical approaches are required in the search for a replacement of dysfunctional endocrine tissue, especially beta-cells. A promising potential lies in direct cellular reprogramming of similar cell types sharing common multipotent progenitors. With the knowledge of molecular mechanisms determining the endocrine cell fate commitment and endocrine lineage differentiation, other endocrine cells contained within the islets of Langerhans or closely related cells could be trans- or dedifferentiated either in situ or in vitro with their subsequent transplantation into the patient. NEUROD1 is a transcription factor situated on the base of the gene regulatory network of the developing endocrine precursors, directly downstream of endocrine lineage master regulator NEUROG3. While NEUROG3 initiates a rapid cascade of chromatin reorganization in numerous bivalent promoters resulting in spatiotemporal gene expression leading to differentiation of all endocrine cell subtypes from pancreatic multipotent progenitors, the role of NEUROD1 has yet to be clarified. Notably, NEUROD1 can induce neuronal program through pioneering and chromatin remodelling in other cell types. In this study, we performed advanced molecular and phenotypic analyses in early endocrine-specific Neurod1-deficient mouse model, including RNA and CUT&Tag sequencing and lightsheet microscopy, to gain insight into the NEUROD1-regulated transcription network driving endocrine lineage cell fate commitment. Besides a postnatal diabetic phenotype, disrupted endocrine differentiation and associated altered islet architecture, we observed an apparent elevation in the non-endocrine gene expression pattern and uncovered alterations in the epigenetic landscape of characteristic genes, specifically in the H3K4me3 and H3K27me3 distribution. Therefore, we provide evidence that NEUROD1 is critical player responsible for the establishment of the endocrine cell identity, which further affects endocrine development and may serve as a potent proendocrine reprogramming driver. 8 samples of 100 FACS-sorted tdTomato+ endocrine cells were analyzed (4 control samples and 4 mutant samples).全球糖尿病发病率正逐步攀升，当前亟需开发新型治疗手段，以替代功能异常的内分泌组织——尤其是β细胞（beta-cells）。其中一项颇具应用前景的策略，是对共享共同多能祖细胞（multipotent progenitors）的同类细胞开展直接细胞重编程（cellular reprogramming）。若能明确调控内分泌细胞命运决定（endocrine cell fate commitment）与内分泌谱系分化（endocrine lineage differentiation）的分子机制，便可在原位（in situ）或体外（in vitro）将朗格汉斯岛（islets of Langerhans）内的其他内分泌细胞或密切相关细胞进行转分化或去分化，随后将其移植回患者体内。 NEUROD1是位于发育中内分泌前体细胞基因调控网络（gene regulatory network）核心层级的转录因子（transcription factor），其直接处于内分泌谱系主调控因子NEUROG3的下游。NEUROG3可在众多二价启动子（bivalent promoters）中触发快速的染色质重塑（chromatin reorganization）级联反应，进而引发时空特异性基因表达（spatiotemporal gene expression），最终使胰腺多能祖细胞（pancreatic multipotent progenitors）分化为所有内分泌细胞亚型；而NEUROD1的具体作用仍有待阐明。值得注意的是，NEUROD1可通过在其他细胞类型中发挥先驱因子功能与染色质重塑，诱导神经元程序的激活。 本研究针对早期内分泌特异性Neurod1敲除小鼠模型开展了先进的分子与表型分析，包括RNA测序（RNA sequencing）、CUT&Tag测序以及光片显微镜（lightsheet microscopy）成像，以期深入解析调控内分泌谱系细胞命运决定的NEUROD1依赖型转录调控网络。除观察到出生后糖尿病表型、内分泌分化异常以及伴随的胰岛结构改变外，我们还发现非内分泌基因表达谱出现显著上调，并揭示了特征性基因的表观遗传景观（epigenetic landscape）发生改变，尤其是组蛋白H3赖氨酸4三甲基化（H3K4me3）与组蛋白H3赖氨酸27三甲基化（H3K27me3）的分布异常。据此，我们证实NEUROD1是建立内分泌细胞身份的关键调控因子，其进一步影响内分泌发育过程，有望成为强效的促内分泌重编程驱动因子。 本研究共分析了8份经荧光激活细胞分选（FACS, Fluorescence-Activated Cell Sorting）得到的tdTomato阳性内分泌细胞样本，其中对照组与突变组各4份。