Function of Nkx homeobox genes in enteroendocrine cell differentiation

描述（由申请方提供）：肠上皮的弥漫性内分泌细胞是体内最大的内分泌器官，具有重要的生理功能，如胃排空和运动、胰腺和胆汁分泌以及肠上皮的生长和修复。胃泌素，一种由胃窦G细胞分泌的肽激素，诱导胃酸分泌，是胃pH值的关键决定因素。关于肠道内分泌分化，特别是在胃中，知之甚少，尽管转录和信号传导途径开始成为焦点。同源结构域蛋白Nkx6.3控制G细胞的终末分化以及胃窦中产生生长抑素的D细胞;在Nkx6.3缺失小鼠中胃泌素和生长抑素水平分别升高和降低。尽管Nkx6.2与Nkx6.3在表达上有重叠，但在胃中尚未确定Nkx6.2的功能。本提案的目的是表征Nkx6.2和Nkx6.3在胃内分泌细胞分化中的个体和联合功能，并定义Nkx6.3指定G细胞的谱系途径。一个中心假设是，G和D细胞从一个共同的前体细胞分化，并且该内分泌谱系中的替代细胞命运由Nkx6.3的量决定，高水平促进G细胞分化。我还将研究Nkx6.2是否具有类似的内分泌细胞特异性，并在必要时可能补偿Nkx6.3。这些目标的实现将需要广泛的技术，包括RNA原位杂交，免疫组织化学，定量RT-PCR，并创建和分析的目标小鼠线表达他莫昔芬诱导Cre重组酶从内源性Nkx6.3基因座。因此，拟议的研究不仅将阐明胃内分泌细胞特化的分子机制，而且还将使我接触到一系列新的实验方法和实验室方法。总之，这些研究将有助于确定胃上皮内分泌细胞的分化途径，并延长我在学术儿科胃肠病学研究生涯的培训。公共卫生相关性：细胞谱系特化和分化通过复杂的转录因子级联发生，并且一些肠道转录因子与癌症和其他疾病相关;同样，肠道内分泌细胞的失调可能导致酸分泌、运动或消化障碍。Nkx6.3似乎在G细胞的最后指定步骤中起作用，G细胞是胃中酸产生的信号。这项工作将追踪Nkx6.3指定G细胞的途径，并确定同胞因子Nkx6.2是否具有相似的功能和/或补偿Nkx6.3的缺失。

DESCRIPTION (provided by applicant): The diffuse endocrine cells of the gut epithelium comprise the largest endocrine organ in the body and serve vital physiologic functions such as gastric emptying and motility, pancreatic and biliary secretion, and growth and repair of gut epithelium. Gastrin, a peptide hormone secreted by G cells of the antral stomach, induces acid secretion and is a key determinant of stomach pH. Little is known about gut endocrine differentiation, particularly in the stomach, although transcriptional and signaling pathways are beginning to come in focus. The homeodomain protein Nkx6.3 controls terminal differentiation of G cells as well as somatostatin-producing D cells in the stomach antrum; gastrin and somatostatin levels are respectively elevated and reduced in Nkx6.3-null mice. Although the close homolog Nkx6.2 overlaps in expression with Nkx6.3, a function for Nkx6.2 has not been established in the stomach. The aims of this proposal are to characterize the individual and joint functions of Nxk6.2 and Nkx6.3 in gastric endocrine cell differentiation and to define the lineage pathway through which Nkx6.3 specifies G cells. A central hypothesis is that G and D cells differentiate from a common precursor and that alternative cell fates in this endocrine lineage are determined by the amount of Nkx6.3, with high levels promoting G-cell differentiation. I will also examine if Nkx6.2 has similar endocrine cell-specifying properties and might compensate for Nkx6.3 when necessary. Accomplishment of these aims will require a breadth of techniques, including RNA in situ hybridization, immunohistochemistry, quantitative RT-PCR, and creation and analysis of a targeted mouse line expressing tamoxifen-inducible Cre recombinase from the endogenous Nkx6.3 locus. Thus, the proposed studies will not only shed light on molecular mechanisms of gastric endocrine cell specification but also expose me to a range of new experimental approaches and laboratory methods. Taken together, these studies will help define the differentiation pathways for gastric epithelial endocrine cells and extend my training for a research career in academic pediatric gastroenterology. PUBLIC HEALTH RELEVANCE: Cell lineage specification and differentiation occur through complex transcription factor cascades, and some gut transcription factors are linked to cancer and other disease; likewise, dysregulation of gut endocrine cells may lead to disorders of acid secretion, motility or digestion. Nkx6.3 seems to act in the final specification steps for G cells, which signal acid production in the stomach. This work will trace the pathway through which Nkx6.3 specifies G cells and also determine if the sibling factor Nkx6.2 has similar functions and/or compensate for absence of Nkx6.3.