Understanding liver bud emergence, formation and potential

描述（由申请人提供）：肝脏是一种重要的再生器官，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是如何形成的，对于开发各种肝脏疾病和损伤的临床疗法非常重要。目前，在了解肝脏发育的初始阶段方面存在重大差距。这个项目的目标是获得一个更深入的了解小鼠肝芽，形态上不同的结构，窝藏肝祖细胞。肝芽从内胚层出现的过程是所有内胚层衍生器官的典型过程，包括肺、胰腺和甲状腺，使用来自邻近组织的分泌信号。目的1是检测候选信号通路在肝出芽中的作用。为了下调肝前体群体中的目标途径，使用了两种利用全胚胎培养的方法：设计用于下调局部内胚层结构域中的候选途径的构建体的电穿孔和直接向培养基中添加抑制剂。然后将操作过的胚胎培养到肝芽发育阶段。如果一个候选者在出芽中起作用，那么这个过程将被破坏，并且从所得表型推断其作用。将产生一种新的表达Cre的小鼠系，以测试经验证的候选物在体内的作用。目的2是检验两个肝前体细胞群是不同前体细胞的假设。命运映射实验已经证明，肝脏来源于两个离散的细胞群体，它们对肝芽的贡献不同。初步数据表明，这两个群体的分子和形态彼此不同，并从其余的内胚层。为了评估两种肝脏前体细胞群和第三种非肝脏前体细胞群之间的分子差异，将产生并比较来自单独确认的细胞库的转录谱。目的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，肝母细胞是多能的，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展示肝脏如何生长，以及两种肝细胞类型是否来自共同的肝芽前体。结合这三个目标将提供关于正常肝脏发育的新信息，这将极大地有助于理解肝脏疾病，并有助于体外诱导肝细胞和肝脏器官形成的研究。 公共卫生相关性：拟议工作的目标是了解在发育过程中引起和支持肝脏特化的分子机制，并探索早期肝芽细胞如何对成年器官做出贡献。实现这些目标将导致对肝脏个体发育和肝脏再生的新见解，这将加深我们对肝脏疾病中出现问题的理解，为治疗设计提供新的方向，包括从胚胎干细胞生产肝细胞。

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.