Transcriptional networks of pancreas endocrine

胰腺内分泌转录网络

• 批准号: 7056496
• 负责人: Christopher V Wright
• 金额: $ 36.1万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7056496
• 关键词: artificial chromosomes; cell differentiation; cell population study; cooperative study; developmental genetics; genetic transcription; histogenesis; homeobox genes; laboratory mouse; pancreatic islets; stem cells

The directed differentiation of stem, progenitor, or surrogate cells towards the pancreatic beta cell fate will require a detailed knowledge of how the program is normally triggered in vivo. Such knowledge could assess the fidelity of induced programs, and the quality/robustness of the final cell fate-both critical issues for cellular therapies for diabetes whether transplantation or regeneration based. The firmest information in this respect comes from genetic interference in model organisms, but very few studies have mapped the precise effect on cell lineage pathways. Our work is focused on obtaining a high-resolution characterization of the orchestrated interplay between transcription factors and intercellular signals during pancreas organogenesis. A stable foundation for long-term gains will be made by placing newly emergent transcription factors and signaling molecules into a functional framework in which well-understood genes act as "fixed" reference points. Our previous studies rationalize the homeobox pdx1 and bHLH ptf1a genes as compelling choices in this context, based upon their dramatic null phenotypes and role in both progenitor and differentiated cell lineages. They thus both afford opportunities for making many tools to dissect the function of other genes in mice. Despite much work on pdx1, and less on ptf1a, neither has been placed precisely in the transcriptional regulator network controlling endocrine specification and differentiation. To fill these gaps, and to pioneer analyses for known and emergent regulatory genes, we will: (1) Characterize endocrine differentiation when pdx1 is reduced or inactivated in specific progenitor classes or differentiated beta cells, analyzing cell lineage pathways, proliferation and maintenance of mature beta cells. (2) Create loxed cassette acceptor alleles of pdx1 via bacterial artificial chromosome engineering to allow the flexible, rapid insertion of markers and minigenes (e.g., recombinases, transcription factors, signaling molecules). We will test the effects of deleting pdx1 cis-regulatory motifs for transcription factors thought to be essential upstream regulators (with a later similar focus on ptf1a). (3) Create inducible ptf1a-CreERTm and floxed ptf1a alleles to connect the gene functionally to progenitor and committed cell behavior. Our function-based gene discovery program in zebrafish (separately funded) will define new loci that respond to or regulate pdx1 and ptf1a, and these will be imported into our analysis. Accurate mapping of cell lineages after gene disruption will hone our ideas on which transcription factors and signaling molecules to express, at what level (titer), and in what cell types, to trigger full beta cell differentiation.

干细胞、祖细胞或替代细胞定向分化为胰岛β细胞的命运将需要详细了解该程序是如何在体内正常触发的。这些知识可以评估诱导程序的保真度，以及最终细胞命运的质量/稳健性-这两个问题都是糖尿病细胞治疗的关键问题，无论是基于移植还是基于再生。在这方面最确凿的信息来自模式生物的遗传干扰，但很少有研究绘制出对细胞谱系路径的确切影响。我们的工作集中在获得胰腺器官发生过程中转录因子和细胞间信号之间协调相互作用的高分辨率表征。通过将新出现的转录因子和信号分子放入一个功能框架中，在这个框架中，众所周知的基因充当“固定的”参考点，将为长期收益奠定稳定的基础。我们之前的研究将同源框pdx1和bhlh ptf1a基因合理化为这一背景下的引人注目的选择，基于它们的 在祖细胞和分化细胞谱系中戏剧性的零表型和作用。因此，它们都为制造许多工具来剖析老鼠体内其他基因的功能提供了机会。尽管对PDX1的研究很多，而对ptf1a的研究较少，但两者都没有被准确地置于控制内分泌规范和分化的转录调控网络中。为了填补这些空白，并对已知的和新出现的调控基因进行先导分析，我们将：(1)表征当PDX1在特定情况下减少或失活时的内分泌分化 祖细胞分类或分化的贝塔细胞，分析成熟贝塔细胞的细胞谱系、增殖和维持。(2)通过细菌人工染色体工程创造PDX1的盒式受体等位基因，使标记和微基因(如重组酶、转录因子、信号分子)能够灵活、快速地插入。我们将测试删除PDX1顺式调控基序对被认为是 必要的上游监管机构(后来对ptf1a也有类似的关注)。(3)创建可诱导的ptf1a-CreERTm和开花的ptf1a等位基因，使基因在功能上与祖细胞连接，并承诺细胞行为。我们的斑马鱼基于功能的基因发现计划(单独资助)将定义对PDX1和ptf1a做出反应或调节的新基因座，这些基因将被引入我们的分析。基因破坏后细胞谱系的准确图谱将磨练我们的 关于表达哪些转录因子和信号分子、在什么水平(滴度)以及在什么细胞类型中触发完全的β细胞分化的想法。