Understanding liver bud emergence, formation and potential

了解肝芽的出现、形成和潜力

• 批准号: 7993310
• 负责人: KIMBERLY D TREMBLAY
• 金额: $ 35.12万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7993310
• 关键词: Adult; Architecture; Biliary; Biological; Bone Morphogenetic Proteins; Cells; Childhood; Clinical; Culture Media; Data; Development; Duct (organ) structure; Ductal Epithelial Cell; Electroporation; Embryo; Embryo Culture Techniques; Endoderm; Epithelial; Event; Gene Expression; Genetic; Genetic Recombination; Genetic Techniques; Germ Layers; Goals; Growth; Hepatic; Hepatocyte; Histological Techniques; In Vitro; Individual; Injury; Knowledge; Label; Lateral; Lead; Liver; Liver Regeneration; Liver diseases; Lung; Maps; Molecular; Morphogenesis; Mus; Natural regeneration; Nature; Organ; Organogenesis; Pancreas; Pathway interactions; Phase; Phenotype; Play; Population; Primitive foregut structure; Process; Production; Reporter; Research Design; Role; Signal Pathway; Signal Transduction; Staging; Stem cells; Structure; Surface; System; Testing; Therapeutic; Thyroid Gland; Time; Tissues; Transforming Growth Factors; Tube; Work; cell type; design; design and construction; embryo culture; embryonic stem cell; in vivo; inhibitor/antagonist; insight; interest; novel; precursor cell; progenitor; public health relevance; regenerative; research study

DESCRIPTION (provided by applicant): The liver is a vital regenerative organ that is composed of two cell types: hepatocytes and duct cells. Understanding how these cells are formed during regeneration and development are important in developing clinical therapies for the myriad of liver diseases and injuries. Currently, there is a significant gap in understanding the initial stages of liver development. The goal of this project is to gain a more thorough understanding of the murine liver bud, the morphologically distinct structure that harbors the liver progenitors. The liver bud emerges from the endoderm in a process that is typical of all endoderm-derived organs including the lung, pancreas and thyroid, using secreted signals from adjacent tissues. Aim 1 is to test the role of candidate signaling pathways in liver budding. To down-regulate the pathway of interest in the liver precursor population, two approaches utilizing whole embryo culture are used: electroporation of constructs designed to downregulate candidate pathways in localized endodermal domains and the addition of inhibitors directly to the culture media. The manipulated embryos are then cultured through the liver bud phase of development. If a candidate plays a role in budding then this process will be disrupted and its role inferred from the resultant phenotype. A novel Cre-expressing mouse line will be produced to test the role of validated candidates in vivo. Aim 2 is to test the hypothesis that the two liver precursor populations are distinct precursors. Fate mapping experiments have demonstrated that the liver is derived from two discreet populations of cells that contribute differently to the liver bud. Preliminary data demonstrate that the two populations are molecularly and morphologically distinct from one another and from the remainder of the endoderm. To assess the molecular differences between the two liver precursor populations and a third non-liver precursor, transcriptional profiles derived from pools of individually confirmed cells will be produced and compared. Aim 3 is designed to test the potential of individual liver bud cells, also termed hepatoblasts. Indirect evidence suggests that hepatoblasts are multipotent, giving rise to both hepatocytes and ductal cells. We propose a genetic marking strategy, utilizing an endoderm-specific CreER line and the R26R reporter, to produce single recombination events in the liver bud that turns on the reporter in that cell and all of its descendants. This retrospective lineage analysis will demonstrate how the liver grows and if the two liver cell-types are derived from a common liver bud precursor. Combined these three Aims will provide novel information on normal liver development that will greatly aid in understanding diseases of the liver and contribute to studies designed to induce hepatocytes and hepatic organogenesis in vitro. PUBLIC HEALTH RELEVANCE: The goal of the proposed work is to understand the molecular mechanisms that cause and support liver specification during development and to explore how the early liver bud cells contribute to the adult organ. Accomplishing these goals will lead to novel insights into liver ontogeny and liver regeneration that will deepen our understanding of what has gone wrong in liver disease, offering new directions for therapeutic design, including the production of hepatocytes from embryonic stem cells.

描述（由申请人提供）：肝脏是一个重要的再生器官，由两种细胞类型组成：肝细胞和导管细胞。了解这些细胞在再生和发育过程中如何形成对于开发针对多种肝脏疾病和损伤的临床疗法非常重要。目前，对肝脏发育初始阶段的了解还存在很大差距。该项目的目标是更全面地了解小鼠肝芽，即含有肝脏祖细胞的形态独特的结构。肝芽从内胚层中出现的过程是所有内胚层衍生器官（包括肺、胰腺和甲状腺）的典型过程，利用来自邻近组织的分泌信号。目标 1 是测试候选信号通路在肝出芽中的作用。为了下调肝脏前体群体中感兴趣的途径，使用了两种利用全胚胎培养的方法：电穿孔旨在下调局部内胚层区域中的候选途径的构建体以及直接向培养基中添加抑制剂。然后将经过处理的胚胎培养至肝芽发育阶段。如果候选者在出芽过程中发挥作用，那么该过程将被破坏，并且其作用可以从所得的表型中推断出来。将生产一种表达 Cre 的新型小鼠品系，以测试经过验证的候选物在体内的作用。目标 2 是检验两个肝脏前体群体是不同前体的假设。命运图谱实验表明，肝脏源自两个离散的细胞群，它们对肝芽的贡献不同。初步数据表明，这两个群体在分子和形态上彼此不同，并且与内胚层的其余部分不同。为了评估两个肝脏前体群体和第三个非肝脏前体群体之间的分子差异，将产生并比较来自单独确认的细胞池的转录谱。目标 3 旨在测试单个肝芽细胞（也称为成肝细胞）的潜力。间接证据表明成肝细胞是多能的，可产生肝细胞和导管细胞。我们提出了一种遗传标记策略，利用内胚层特异性 CreER 系和 R26R 报告基因，在肝芽中产生单个重组事件，从而打开该细胞及其所有后代中的报告基因。这种回顾性谱系分析将证明肝脏如何生长以及两种肝细胞类型是否源自共同的肝芽前体。这三个目标的结合将提供有关正常肝脏发育的新信息，这将极大地有助于了解肝脏疾病，并有助于体外诱导肝细胞和肝器官发生的研究。 公共健康相关性：拟议工作的目标是了解在发育过程中引起和支持肝脏规范的分子机制，并探索早期肝芽细胞如何对成体器官做出贡献。实现这些目标将为肝脏个体发育和肝脏再生带来新的见解，从而加深我们对肝脏疾病问题的理解，为治疗设计提供新的方向，包括从胚胎干细胞生产肝细胞。