Regulation of Exocrine Pancreatic Progenitors by Ptf1a-p48

描述(申请人提供)：胰腺前体细胞是糖尿病细胞替代治疗的重要来源，也可能是胰腺癌的起源细胞。在调节胰腺发育和分化的众多转录因子中，碱性螺旋-环-螺旋转录因子ptf1a-p48的表达代表了胰腺前体细胞的特征。几个实验室，包括我们自己的，已经确定了ptf1a-p48在胰腺发育过程中的不同早期和晚期影响。在发育早期，ptf1a-p48在新生的胰腺上皮细胞中广泛表达，在那里它是确定胰腺特性以及早期生长和形态发生所必需的。在发育后期，ptf1a-P48被限制在外分泌室，在那里它诱导细胞周期退出和腺泡细胞分化。然而，ptf1a-P48发挥不同发育阶段特异性效应的机制尚不清楚。我们现在建议利用我们研究小鼠和斑马鱼胰腺发育的独特能力，全面描述早期和晚期ptf1a-p48的功能。这项工作将基于以下中心假设：第一，ptf1a-P48对胰腺发育的早期和晚期影响是通过完全不同的靶基因介导的：第二，ptf1a-P48分子的不同结构域可能是诱导这些早期和晚期效应所必需的；第三，ptf1a-P48靶基因可能包括编码和非编码元件，包括功能上重要的microRNA。为了验证这些假说，将追求以下特定目标：1)利用新开发的体内分析方法，确定ptf1a-P48分子的离散结构域，负责介导其对胰腺发育的发育阶段特异性影响；2)通过全基因组芯片分析鉴定新的ptf1a-P48靶基因，并确定与这些基因表达变化相关的功能获得和功能丧失表型；3)在发育中的小鼠和斑马鱼胰腺中鉴定和鉴定新的ptf1a-P48调控的microRNA。总之，这些研究将确定ptf1a-p48在小鼠和斑马鱼胰腺发育过程中发挥多重影响的机制。在这样做的过程中，我们很可能还将发现重要的新的胰腺规范、形态发生和分化的调节因子。通过阐明这种重要的转录因子在调节胰腺祖细胞池中的作用，这些研究将有助于最终在胰腺癌和糖尿病背景下对这些细胞进行治疗操作。 公共卫生相关性：胰腺前体细胞是糖尿病细胞替代治疗的重要来源，也可能是胰腺癌的起源细胞。该方案中的研究重点是胰腺转录因子Ptf1a-P48如何调节胰腺前体细胞的初始规格、生长和分化。我们最近完成了两个全基因组范围的基因筛选，寻找可能作用于Ptf1a-P48下游的基因。在此过程中，我们已经确定了一些额外的编码和非编码基因，这些基因可能在调节胰腺祖细胞池中发挥重要作用。使用小鼠和斑马鱼模型系统，我们现在计划从功能上表征这些新的Ptf1a-P48靶基因。通过更好地阐明Ptf1a-P48如何调控前体细胞的生长和分化，这些研究将有助于最终在胰腺癌和糖尿病的背景下对胰腺前体细胞进行治疗操作。

DESCRIPTION (provided by applicant): Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Among the many transcription factors regulating pancreatic development and differentiation, expression of the basic helix-loop-helix transcription factor, ptf1a-p48, represents the defining feature of pancreatic progenitor cells. Several laboratories, including our own, have identified distinct early and late effects of ptf1a-p48 during pancreatic development. Early in development, ptf1a-p48 is widely expressed in the emerging pancreatic epithelium, where it is required for specification of pancreatic identity, as well as for early growth and morphogenesis. Later in development, ptf1a-p48 becomes restricted to the exocrine compartment, where it induces cell cycle exit and acinar cell differentiation. However, the mechanisms by which ptf1a-p48 exerts its different developmental stage-specific effects remain unknown. We now propose to comprehensively characterize both early and late ptf1a-p48 functions, taking advantage of our unique ability to study pancreas development in both mouse and zebrafish. This work will be based on the following central hypotheses: First, that the early and late effects of ptf1a-p48 on pancreatic development are mediated through entirely different sets of ptf1a-p48 target genes; second, that different structural domains of the ptf1a-p48 molecule may be required for induction of these early and late effects; and third, that ptf1a-p48 target genes may include both coding and non-coding elements, including functionally important microRNA's. To test these hypotheses, the following Specific Aims will be pursued: 1) To identify discrete domains of the ptf1a-p48 molecule responsible for mediating its developmental stage-specific effects on pancreatic development, using newly developed in vivo assays; 2) To identify novel ptf1a-p48 target genes through genome-wide ChIP-on-chip analysis, and determine gain-of-function and loss-of-function phenotypes associated with altered expression of these genes; and 3) To identify and functionally characterize novel ptf1a-p48-regulated microRNA's in developing mouse and zebrafish pancreas. Together, these studies will determine the mechanisms by which ptf1a-p48 exerts its multiple influences in developing mouse and zebrafish pancreas. In so doing, it is likely that we will also identify important new regulators of pancreatic specification, morphogenesis and differentiation. By clarifying the role of this important transcription factor in regulating the pancreatic progenitor pool, these studies will contribute to the eventual therapeutic manipulation of these cells in the context of pancreatic cancer and diabetes. Public Health Relevance: Pancreatic progenitor cells represent an important resource for cell replacement therapy in diabetes, and may also represent the cell-of-origin for pancreatic cancer. Studies in this proposal focus on how Ptf1a-p48, a pancreatic transcription factor, regulates the initial specification, growth and differentiation of pancreatic progenitor cells. We have recently completed two genome-wide screens for genes that may act downstream of Ptf1a-p48. In so doing, we have identified a number of additional coding and non-coding genes that may play important roles in regulating the pancreatic progenitor pool. Using both mouse and zebrafish model systems, we now plan to functionally characterize these novel Ptf1a-p48 target genes. By better clarifying how Ptf1a-p48 regulates progenitor cell growth and differentiation, these studies will contribute to the eventual therapeutic manipulation of pancreatic progenitors in the context of pancreatic cancer and diabetes.