Understanding liver bud emergence, formation and potential

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治疗方法非常重要。目前，在了解肝脏发育的初始阶段有很大的差距。这个项目的目标是获得一个更彻底的了解小鼠肝芽，形态上独特的结构，窝藏肝祖细胞。肝芽利用邻近组织分泌的信号从内胚层中出现，这一过程是所有内胚层来源器官（包括肺、胰腺和甲状腺）的典型过程。目的1是测试候选信号通路在肝脏出芽中的作用。为了下调肝前体群体中感兴趣的通路，采用了两种利用全胚胎培养的方法：电穿孔设计用于下调局部内胚层区域的候选通路，以及直接在培养基中添加抑制剂。然后，经过处理的胚胎在肝芽发育阶段进行培养。如果一个候选者在萌芽中起作用，那么这个过程将被破坏，其作用将从结果表型推断出来。将产生一种新的表达cre的小鼠系，以测试验证候选物在体内的作用。目的2是检验两个肝前体群体是不同的前体的假设。命运图谱实验已经证明，肝脏来源于对肝芽贡献不同的两个离散的细胞群。初步数据表明，这两个种群在分子和形态上彼此不同，也与内胚层的其余部分不同。为了评估两种肝前体群体和第三种非肝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。目的3是测试个体肝芽细胞（也称为肝母细胞）的潜能。间接证据表明，肝母细胞具有多能性，可同时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，从而开启该细胞及其所有后代的报告基因。这一回顾性谱系分析将证明肝脏是如何生长的，以及这两种肝细胞类型是否来源于一个共同的肝芽前体。将这三个目标结合起来，将提供关于正常肝脏发育的新信息，这将极大地帮助理解肝脏疾病，并有助于体外诱导肝细胞和肝器官发生的研究。

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.