Transcriptional networks of pancreas endocrine cell differentiation

胰腺内分泌细胞分化的转录网络

• 批准号: 7924803
• 负责人: Christopher V Wright
• 金额: $ 38.82万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7924803
• 关键词: Acinar Cell; Address; Adopted; Affect; Alleles; Animal Model; Antibiotic Resistance; Antibodies; Area; Bacterial Artificial Chromosomes; Behavior; Beta Cell; Breeding; Cell Differentiation process; Cell Lineage; Cells; Commit; Complement; Data; Developmental Biology; Diagnostic; Dissection; Dose; ES Cell Line; Elements; Embryo; Endocrine; Engineering; Foundations; Funding; Gene Silencing; Genes; Genetic; Homeobox; Hybrids; In Vitro; Integrase; Islets of Langerhans; Knock-in Mouse; Knowledge; Lead; Life; Maintenance; Mammals; Maps; Metaplastic; Molecular Profiling; Mus; Natural regeneration; Organ; Organogenesis; Pancreas; Pathway interactions; Pattern; Phenotype; Proteins; Regulation; Regulator Genes; Resolution; Role; Signal Transduction; Signaling Molecule; Sorting - Cell Movement; Staging; Structure of beta Cell of islet; System; Testing; Time; Tissues; Transcription factor genes; Transcriptional Regulation; Transplantation; Work; Zebrafish; adult stem cell; base; cell behavior; cell type; cofactor; design; diabetes mellitus therapy; flexibility; gain of function; gene discovery; gene function; in vivo; insight; overexpression; pancreas development; progenitor; programs; recombinase; regenerative; research study; spatiotemporal; stem; tool; transcription factor; transdifferentiation

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、祖细胞或替代细胞向胰腺β细胞命运的定向分化将需要详细了解该程序在体内通常如何触发。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——无论是基于移植还是基于再生，这都是糖尿病细胞疗法的关键问题。这方面最可靠的信息来自模型生物体的遗传干扰，但很少有研究描绘出对细胞谱系途径的精确影响。我们的工作重点是获得胰腺器官​​发生过程中转录因子和细胞间信号之间精心策划的相互作用的高分辨率特征。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，在该框架中，充分理解的基因充当“固定”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盒 pdx1 和 bHLH ptf1a 基因合理化为在此背景下的令人信服的选择，基于它们的 显着的无效表型以及在祖细胞和分化细胞谱系中的作用。因此，它们都提供了制造许多工具来剖析小鼠其他基因功能的机会。尽管对 pdx1 的研究较多，而对 ptf1a 的研究较少，但两者都没有被精确地置于控制内分泌规范和分化的转录调节网络中。为了填补这些空白，并开拓对已知和新出现的调控基因的分析，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。 祖细胞类别或分化的 β 细胞，分析细胞谱系途径、成熟 β 细胞的增殖和维持。 (2) 通过细菌人工染色体工程创建 pdx1 的锁定盒受体等位基因，以允许灵活、快速地插入标记和小基因（例如重组酶、转录因子、信号分子）。我们将测试删除 pdx1 顺式调控基序对被认为是转录因子的影响 重要的上游监管机构（后来类似地关注 ptf1a）。 (3) 创建诱导型 ptf1a-CreERTm 和 floxed ptf1a 等位基因，以将基因功能与祖细胞和定型细胞行为联系起来。我们基于功能的斑马鱼基因发现计划（单独资助）将定义响应或调节 pdx1 和 ptf1a 的新基因座，并将这些基因座导入我们的分析中。基因破坏后细胞谱系的准确绘制将磨练我们的能力 关于哪些转录因子和信号分子要表达、以什么水平（滴度）以及在什么细胞类型中表达，以触发完全 β 细胞分化的想法。