Nkx2.2 in Pancreatic Development: The Role of the NK2-SD Domain

描述（由申请人提供）：由朗格汉斯胰岛组成的内分泌胰腺负责维持血糖稳态。胰岛由几种细胞组成，其中包括产生胰高血糖素的细胞。细胞、产生胰岛素的细胞、产生生长抑素的d细胞、产生胰腺多肽的PP细胞和产生生长素的e细胞。糖尿病与胰岛素生成细胞的减少有关。目前的医学治疗主要是通过胰岛素替代以及促进胰岛素释放、抑制糖异生和抑制糖原分解的口服药物来控制血糖水平。然而，在2000年，确定胰岛可以成功地移植到糖尿病患者使用尸体胰岛。尽管细胞替代疗法已经取得了成功，但供体胰岛的缺乏限制了其应用，并导致需要在实验室中使用干细胞技术制造胰岛。目前的干细胞分化方案已经适应和改进，以响应正常小鼠胰腺发育的研究，但完全的细胞分化尚未实现。因此，对正常胰腺发育的持续研究对于创建更好的体外细胞分化方案以产生可移植细胞至关重要。Nkx2.2是一种同源结构域转录因子，对几种胰岛细胞类型的产生至关重要。Nkx2.2缺失小鼠不形成¿细胞，a和PP细胞数量减少。相反，Nkx2.2无效的胰岛充满了生长素细胞，这是胚胎干细胞分化方案中形成的主要的单激素胰岛细胞群。因此，了解Nkx2.2的功能和调控对于制定合适的胰岛细胞分化方案非常重要。Sussel实验室的研究表明，Nkx2.2在不同的发育时间点和不同的细胞环境中作为激活因子和抑制因子发挥作用。目前，Nkx2.2蛋白的功能结构尚未得到很好的表征，尽管已经描述了三个结构域，包括DNA结合同源结构域、groucho共抑制因子相互作用结构域（TN）和nk2特异性结构域（NK2-SD），这些结构域在家族成员和物种之间高度保守，但其功能尚不清楚。尽管具有高度同源性，但NK2-SD结构域的功能尚不清楚。表征胰腺发育过程中NK2- SD结构域的功能将有助于优化细胞分化方案的发展。具体目标：1；为了确定高度保守的Nkx2.2NK2-SD结构域是否在体内介导Nkx2.2功能，我将研究Nkx2.2NK2-SD突变小鼠在胚胎发育期间和出生后动物的表型变化。2. 为了了解NK2-SD结构域影响Nkx2.2介导的转录调控的机制，我将描述NK2-SD结构域在候选Nkx2.2靶启动子上的DNA结合和转录活性。

DESCRIPTION (provided by applicant): The endocrine pancreas, composed of the islets of Langerhans, is responsible for the maintenance of blood glucose homeostasis. Several cell types comprise the islets and include glucagon-producing . cells, insulin- producing ¿ cells, somatostatin-producing d cells, pancreatic polypeptide-producing PP cells, and ghrelin- producing e cells. Diabetes mellitus is associated with a loss of insulin-producing ¿ cells. Current medical treatment is focused on management of blood glucose levels through insulin replacement as well as oral medications that facilitate insulin release, inhibit gluconeogenesis, and inhibit glycogenolysis. However, in 2000, it was determined that islets can be successfully transplanted into diabetic patients using cadaveric islets. Although cell replacement therapies have been successful, the paucity of donor islets has limited its application and has led to the need to create islets in the laboratory using stem cell technologies. Current stem cell differentiation protocols have been adapted and improved in response to studies of normal mouse pancreas development, however complete ¿ cell differentiation has yet to be achieved. Thus, continued investigation into normal pancreas development will be of utmost importance for creating better in vitro ¿ cell differentiation protocols for the generation of transplantable cells. Nkx2.2 is a homeodomain transcription factor that is essential for the production of several islet cell types. Nkx2.2 null mice do not form ¿ cells and have reduced numbers of a and PP cells. Instead, the Nkx2.2 null islets are filled with ghrelin cells, the predominant mono-hormonal islet cell population that forms in the ES cell differentiation protocols. Therefore, knowledge of Nkx2.2 function and regulation will be important for the development of proper islet cell differentiation protocols. Studies from the Sussel lab have indicated that Nkx2.2 functions as an activator and a repressor at different developmental time points and in different cellular contexts. At this time, the functional structure of the Nkx2.2 protein is not well characterized, although three domains have been described, including the DNA binding homeodomain, the groucho co-repressor interaction domain (TN) and the NK2-specific domain (NK2-SD), which is highly conserved amongst family members and across species, but its function is unknown. Despite its high homology, the function of the NK2-SD domain is unknown. Characterization of the function of the NK2- SD domain during pancreas development will facilitate the development of optimized ¿ cell differentiation protocols. Specific aims: 1. To determine whether the highly conserved Nkx2.2 NK2-SD domain mediates Nkx2.2 function in vivo, I will investigate the phenotypic changes associated with Nkx2.2NK2-SD mutant mice during embryonic development and in postnatal animals. 2. To understand the mechanism by which the NK2-SD domain influences Nkx2.2 mediated transcriptional regulation, I will characterize the DNA binding and transcriptional activities of the NK2-SD domain on candidate Nkx2.2 target promoters.