Genetic and physiologic regulation of pig islet development and function

摘要 对啮齿动物的研究从根本上提高了我们对胰岛细胞的理解 了解人类胰腺的啮齿动物模型的发展和功能及其局限性 形成和疾病加剧了人们对实验系统的兴趣，这些实验系统更密切地反映了 人胰腺发育与胰岛功能。我们的研究揭示了 研究猪胰岛的发育，包括多种保守特征的证据 猪和人的胰岛发育，在老鼠身上没有观察到。这包括表达 控制人类胰岛β细胞命运特征的转录因子，如six2和six3 和功能。我们已经建立了一个可靠的框架来获取猪的胰腺和胰岛 并描述了分子、细胞、信号和遗传特征 发育中的猪α，β和δ细胞从胎儿到新生儿阶段，包括第一个详细的 这些细胞的发育转录组。此外，我们的团队还生成了一些 首次使用CRISPR/Cas9靶向进行基因编辑。这些进步激励了以下几个方面 目标： 目的1.阐明猪胰岛细胞发育和功能成熟的遗传结构 目的2.鉴定猪胰岛SIX2和SIX3表达的天然调控因子 目的3.调查猪HNF1A杂合性突变的表型 为了实现我们的目标，我们组建了一支出色的合作调查团队， 在发育生物学、猪遗传学、胰岛生物学、基因组学和 糖尿病研究。这项工作应该允许以前无法实现的基因和 猪胰岛细胞的生理调控机制。这包括确定监管机构 连接染色质动力学和基因转录以控制胰岛α，β和δ细胞的特征 命运和功能，以及创造更好地概括人类疾病的MODY新模型 遗传学、发病机制和治疗学。我们的工作应该创造新的灵活的实验 从生理学角度研究胰岛细胞发育和功能成熟的研究范式 和病理阶段，这是一个显著的优势，扩大了我们的提案对 人类健康。

Abstract Studies in rodents have fundamentally advanced our understanding of pancreatic islet cell development and function, but limitations of rodent models for understanding human pancreas formation and disease have intensified interest in experimental systems that more closely reflect human pancreas development and islet function. Our studies have revealed advantages for investigating islet development in pigs, including evidence for multiple conserved features of islet development in pigs and humans, not observed in mice. This includes expression of transcription factors like SIX2 and SIX3 which govern hallmark features of human islet β cell fate and function. We have developed a reliable framework for procuring pig pancreata and islets from any developmental stage, and described molecular, cellular, signaling and genetic features of developing pig α, β, and δ cells from fetal to neo-natal stages, including the first detailed developmental transcriptome of these cells. In addition, our team has generated some of the first gene-edited pigs using CRISPR/Cas9 targeting. These advances motivate the following Aims: Aim 1. Elucidate the genetic architecture of pig islet cell development and functional maturation Aim 2. Identify native regulators of pig islet SIX2 and SIX3 expression Aim 3. Investigate phenotypes of HNF1A heterozygous mutation in pigs To achieve our aims, we assembled a superb team of collaborating investigators with complementary expertise in developmental biology, pig genetics, islet biology, genomics and diabetes research. This work should allow previously unattainable investigation of genetic and physiological mechanisms regulating pig islet cells. This includes identification of regulatory features that connect chromatin dynamics and gene transcription to control islet α, β and δ cell fate and function, and creation of new models of MODY that better recapitulate human disease genetics, pathogenesis and therapeutics. Our work should create new flexible experimental paradigms to investigate development and functional maturation of islet cells from physiological and pathological stages, a striking advantage that broadens the impact of our proposal on human health.