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

描述(申请人提供)：内分泌胰腺由朗格汉斯的胰岛组成，负责维持血糖的动态平衡。胰岛由几种细胞类型组成，其中包括产生胰高血糖素的细胞。细胞、胰岛素分泌细胞、生长抑素分泌d细胞、胰多肽分泌PP细胞和胃促生长素分泌e细胞。糖尿病与胰岛素分泌细胞的丧失有关。目前的医学治疗集中于通过胰岛素替代和口服药物来控制血糖水平，以促进胰岛素的释放，抑制糖异生和抑制糖原分解。然而，在2000年，人们确定使用身体胰岛可以成功地将胰岛移植到糖尿病患者体内。尽管细胞替代疗法取得了成功，但供体胰岛的匮乏限制了其应用，并导致需要在实验室使用干细胞技术创造胰岛。目前的干细胞分化方案已经根据正常小鼠胰腺发育的研究进行了调整和改进，但尚未实现完全的细胞分化。因此，继续研究胰腺的正常发育对于创造更好的体外细胞分化方案，以产生可移植的细胞将是至关重要的。Nkx2.2是一种同源结构域转录因子，对几种类型的胰岛细胞的产生是必不可少的。Nkx2.2基因缺失小鼠不形成细胞，α细胞和PP细胞数量减少。相反，Nkx2.2缺失的胰岛充满了Ghrelin细胞，Ghrelin细胞是在ES细胞分化方案中形成的主要单激素胰岛细胞群。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起激活和抑制作用。目前，Nkx2.2蛋白的功能结构还不是很清楚，尽管已经描述了三个结构域，包括DNA结合同源结构域、Groucho共抑制物相互作用结构域(TN)和NK2特异性结构域(NK2-SD)，NK2-SD在家庭成员和跨物种中高度保守，但其功能尚不清楚。尽管NK2-SD结构域具有很高的同源性，但其功能尚不清楚。确定NK2-SD结构域在胰腺发育过程中的功能将有助于开发优化的细胞分化方案。具体目的：1.为了确定高度保守的Nkx2.2 NK2-SD结构域是否在体内介导Nkx2.2的功能，我将研究Nkx2.2-SD突变小鼠在胚胎发育过程中和出生后动物中的表型变化。2.为了了解NK2-SD结构域影响Nkx2.2介导的转录调控的机制，我将研究NK2-SD结构域与候选Nkx2.2靶启动子的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.