Myt1 Function for Endocrine Islet Development & Fuction

描述（由申请人提供）：脊椎动物胰腺有两个腺体：外分泌腺泡和胰管，由外分泌胰腺组成，分泌消化酶并将其运送到十二指肠；四种内分泌细胞（13、5、PP细胞）组成朗格汉斯内分泌胰岛，分泌内分泌激素，调节多种生理过程。与胰腺有关的疾病包括胰腺癌和糖尿病，前者是癌症死亡的主要原因之一，后者在美国折磨着超过1600万人。因此，胰腺相关研究不仅揭示了指导器官发生的基本机制，而且对人类健康具有重要意义。尽管在阐明胰腺发育的分子和细胞机制方面做出了越来越多的努力并取得了快速进展，但对导致胰腺细胞分化的调节相互作用知之甚少。例如，虽然已知Notch信号通过控制基本环-螺旋-环蛋白NGN3的表达参与内分泌-外分泌细胞命运的决定，但尚不清楚还有哪些其他因素与NGN3协同诱导成熟内分泌细胞分化，也不知道需要哪些因素来维持适当的胰岛功能。为了解决这些问题，这位首席研究员成功地利用了一种暂时控制的细胞标记技术来鉴定成熟内分泌胰岛的祖细胞。我们发现在胰腺芽中瞬时表达Ngn3标志内分泌祖细胞。通过GFP标记分离表达ngn3的细胞，并使用基于微阵列的分析检查其基因表达谱，发现了几个可能在内分泌发育中起作用的候选基因。我们的初步数据表明，这些候选基因之一的产物髓磷脂转录因子I （Mytl，产生两个MYT1亚型）可能与NGN3相互作用，并参与内分泌分化。在本课题中，我们将首先开发myt1肽抗体，广泛研究Mytl在胰腺不同阶段的表达。然后，我们将利用一种暂时控制的功能获得方法来研究MYT1在NGN3的作用下为胰腺细胞分化为成熟胰岛细胞提供能力的假设。具体来说，我们将在不同阶段的胰腺细胞中共同表达Mytl和Ngn3，以确定它们是否协同诱导胰岛细胞成熟。此外，功能丧失方法，包括siRNA或基因敲除，将被用于在胚胎或成年阶段选择性地灭活Mytl

DESCRIPTION (provided by applicant): The vertebrate pancreas has two glands: exocrine acini and pancreatic ducts comprise the exocrine pancreas, which secretes and transports digestive enzymes to the duodenum; four types of endocrine cells ( 13,5, and PP cells) form endocrine islets of Langerhans, which secrete endocrine hormones to regulate a variety of physiological processes. Disease related to the pancreas includes pancreatic cancer, one of the leading causes of cancer death, and diabetes mellitus, which afflicts over 16 million individuals in the United States. As a result, pancreas-related studies not only shed light on the basic mechanisms that direct organogenesis, but also bear strong implications for human health. Despite the increased efforts and rapid progress in elucidating the molecular and cellular mechanisms underlying pancreatic development, little is known about the regulatory interactions that lead to pancreatic cell differentiation. For example, although it is known that Notch signaling participates in endocrine-exocrine cell fate determination by controlling the expression of a basic loop-helix-loop protein, NGN3, it is not known what other factors cooperate with NGN3 to induce mature endocrine cell differentiation nor what factors are needed to maintain proper islet function. In order to address these questions, this principal investigator has successfully utilized a temporally controlled cell marking technique to identify progenitor cells for mature endocrine islets. We found that transient Ngn3 expression in the pancreatic buds marks endocrine progenitor cells. Isolation of the Ngn3-expressing cells by GFP tagging and examination of their gene expression profiles using microarray-based analysis has revealed several candidate genes that may play roles in endocrine development. Our preliminary data indicate that the products of one of these candidate genes, myelin transcription factor I (Mytl, which produces two MYT1 isoforms), potentially interact with NGN3 and are involved in endocrine differentiation. In this proposal, we will first develop MYT 1 peptide antibodies to extensively study Mytl expression in the pancreas at different stages. Then we will utilize a temporally controlled gain-of-function approach to investigate the hypothesis that MYT1 provides competence to pancreatic cells to differentiate to mature islet cells in response to NGN3. Specifically, we will co-express Mytl and Ngn3 in pancreatic cells at different stages to determine whether they cooperate to induce islet cell maturation. In addition, loss of function approaches, including siRNA or knockout, will be utilized to selectively inactivate Mytl at embryonic or adult stages to examine its function for endocrine development and function. These fundamental studies not only identify molecular cascades for organogenesis, but also reveal crucial factors that can be used to induce endocrine differentiation or regeneration, so that functional islet ceils can be induced for relief of diabetic symptoms.