Abstract：
OBJECTIVE: Strategies to augment functional beta cell mass include directed differentiation of stem cells towards a beta cell fate, which requires extensive knowledge of transcriptional programs governing endocrine progenitor cell differentiation in vivo. We aimed to study the contributions of the Brahma-related gene-1 (BRG1) and Brahma (BRM) ATPase subunits of the SWI/SNF chromatin remodelling complex to endocrine cell development.
METHODS: We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1Δendo;Brm+/-) or homozygous (double knockout: DKOΔendo) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos.
RESULTS: Brg1Δendo;Brm+/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKOΔendo mice were not recovered at weaning; however, postnatal day 6 DKOΔendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKOΔendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3)+ cells was reduced in Brg1Δendo;Brm+/- and DKOΔendo mutants.
CONCLUSIONS: Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion.
METHODS: Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369.
METHODS: We generated mice with endocrine progenitor-specific Neurog3-Cre BRG1 removal in the presence of heterozygous (Brg1Δendo;Brm+/-) or homozygous (double knockout: DKOΔendo) BRM deficiency. Whole-body metabolic phenotyping, islet function characterisation, islet quantitative PCR and histological characterisation were performed on animals and tissues postnatally. To test the mechanistic actions of SWI/SNF in controlling gene expression during endocrine cell development, single-cell RNA-seq was performed on flow-sorted endocrine-committed cells from embryonic day 15.5 control and mutant embryos.
RESULTS: Brg1Δendo;Brm+/- mice exhibit severe glucose intolerance, hyperglycaemia and hypoinsulinaemia, resulting, in part, from reduced islet number; diminished alpha, beta and delta cell mass; compromised islet insulin secretion; and altered islet gene expression programs, including reductions in MAFA and urocortin 3 (UCN3). DKOΔendo mice were not recovered at weaning; however, postnatal day 6 DKOΔendo mice were severely hyperglycaemic with reduced serum insulin levels and beta cell area. Single-cell RNA-seq of embryonic day 15.5 lineage-labelled cells revealed endocrine progenitor, alpha and beta cell populations from SWI/SNF mutants have reduced expression of Mafa, Gcg, Ins1 and Ins2, suggesting limited differentiation capacity. Reduced Neurog3 transcripts were discovered in DKOΔendo endocrine progenitor clusters, and the proliferative capacity of neurogenin 3 (NEUROG3)+ cells was reduced in Brg1Δendo;Brm+/- and DKOΔendo mutants.
CONCLUSIONS: Loss of BRG1 from developing endocrine progenitor cells has a severe postnatal impact on glucose homeostasis, and loss of both subunits impedes animal survival, with both groups exhibiting alterations in hormone transcripts embryonically. Taken together, these data highlight the critical role SWI/SNF plays in governing gene expression programs essential for endocrine cell development and expansion.
METHODS: Raw and processed data for scRNA-seq have been deposited into the NCBI Gene Expression Omnibus (GEO) database under the accession number GSE248369.
摘要：
目的：增加功能β细胞量的策略包括干细胞向β细胞命运的定向分化，这需要在体内控制内分泌祖细胞分化的转录程序的广泛知识。我们旨在研究SWI/SNF染色质重塑复合物的Brahma相关基因1（BRG1）和Brahma（BRM）ATPase亚基对内分泌细胞发育的贡献。
方法：我们在存在杂合（Brg1Δendo;Brm+/-）或纯合（双敲除：DKOΔendo）BRM缺陷的情况下，产生了内分泌祖细胞特异性Neurog3-CreBRG1去除的小鼠。全身代谢表型，胰岛功能表征，在出生后对动物和组织进行胰岛定量PCR和组织学表征。为了测试SWI/SNF在内分泌细胞发育过程中控制基因表达的机制作用，对来自胚胎第15.5天对照和突变胚胎的流式分选内分泌定向细胞进行单细胞RNA-seq.
结果：Brg1Δendo;Brm/-小鼠表现出严重的葡萄糖不耐受，高血糖和低胰岛素血症，产生的，在某种程度上,从胰岛数量减少；阿尔法减少，β和δ细胞团；胰岛胰岛素分泌受损；胰岛基因表达程序改变，包括MAFA和尿皮质素3（UCN3）的减少。DKOΔendo小鼠在断奶时没有恢复；然而，出生后第6天DKOΔendo小鼠出现严重高血糖,血清胰岛素水平和β细胞面积降低.胚胎第15.5天谱系标记细胞的单细胞RNA-seq显示内分泌祖细胞，SWI/SNF突变体的α和β细胞群的Mafa表达减少，Gcg,Ins1和Ins2，表明分化能力有限。在DKOΔendo内分泌祖细胞簇中发现了减少的Neurog3转录本，Brg1Δendo;Brm/-和DKOΔendo突变体中神经原蛋白3（NEUROG3）细胞的增殖能力降低。
结论：来自发育中的内分泌祖细胞的BRG1缺失对出生后的葡萄糖稳态有严重影响，两个亚基的丧失阻碍了动物的生存，两组都表现出胚胎激素转录本的改变。一起来看,这些数据突出了SWI/SNF在控制内分泌细胞发育和扩增所必需的基因表达程序中的关键作用.
方法：scRNA-seq的原始数据和处理数据已保存到NCBI基因表达综合（GEO）数据库中，登录号为GSE248369。
方法：我们在存在杂合（Brg1Δendo;Brm+/-）或纯合（双敲除：DKOΔendo）BRM缺陷的情况下，产生了内分泌祖细胞特异性Neurog3-CreBRG1去除的小鼠。全身代谢表型，胰岛功能表征，在出生后对动物和组织进行胰岛定量PCR和组织学表征。为了测试SWI/SNF在内分泌细胞发育过程中控制基因表达的机制作用，对来自胚胎第15.5天对照和突变胚胎的流式分选内分泌定向细胞进行单细胞RNA-seq.
结果：Brg1Δendo;Brm/-小鼠表现出严重的葡萄糖不耐受，高血糖和低胰岛素血症，产生的，在某种程度上,从胰岛数量减少；阿尔法减少，β和δ细胞团；胰岛胰岛素分泌受损；胰岛基因表达程序改变，包括MAFA和尿皮质素3（UCN3）的减少。DKOΔendo小鼠在断奶时没有恢复；然而，出生后第6天DKOΔendo小鼠出现严重高血糖,血清胰岛素水平和β细胞面积降低.胚胎第15.5天谱系标记细胞的单细胞RNA-seq显示内分泌祖细胞，SWI/SNF突变体的α和β细胞群的Mafa表达减少，Gcg,Ins1和Ins2，表明分化能力有限。在DKOΔendo内分泌祖细胞簇中发现了减少的Neurog3转录本，Brg1Δendo;Brm/-和DKOΔendo突变体中神经原蛋白3（NEUROG3）细胞的增殖能力降低。
结论：来自发育中的内分泌祖细胞的BRG1缺失对出生后的葡萄糖稳态有严重影响，两个亚基的丧失阻碍了动物的生存，两组都表现出胚胎激素转录本的改变。一起来看,这些数据突出了SWI/SNF在控制内分泌细胞发育和扩增所必需的基因表达程序中的关键作用.
方法：scRNA-seq的原始数据和处理数据已保存到NCBI基因表达综合（GEO）数据库中，登录号为GSE248369。
