Understanding liver bud emergence, formation and potential

描述(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，对肝脏发育的初始阶段的理解存在着很大的差距。这个项目的目标是更彻底地了解小鼠的肝芽，这是一种在形态上截然不同的结构，其中含有肝脏前体。利用邻近组织的分泌信号，肝芽从内胚层出来，这是包括肺、胰腺和甲状腺在内的所有内胚层器官的典型过程。目的1是测试候选信号通路在肝脏萌发中的作用。为了下调肝脏前体群体中的感兴趣途径，利用全胚胎培养的两种方法：电穿孔构建的结构，旨在下调局部内胚层区域的候选途径，以及直接向培养液中添加抑制剂。然后，经过处理的胚胎在发育的肝芽阶段进行培养。如果一个候选者在萌芽过程中起了作用，那么这个过程就会被打乱，并从由此产生的表型中推断出它的作用。一种新的表达Cre的小鼠品系将被生产出来，以测试经过验证的候选基因在体内的作用。目的2是检验这两个肝脏前体群体是不同前体的假设。命运图谱实验表明，肝脏来自两个不同的细胞群体，它们对肝芽的贡献不同。初步数据表明，这两个种群在分子和形态上彼此不同，也不同于内胚层的其余部分。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图谱。AIM 3旨在测试单个肝芽细胞(也称为肝母细胞)的潜力。间接证据表明，肝母细胞是多能的，既能产生肝细胞，又能产生导管细胞。我们提出了一种遗传标记策略，利用内胚层特异的Creer系和R26R报告基因，在肝芽中产生单个重组事件，打开该细胞及其所有后代中的报告基因。这一追溯的谱系分析将展示肝脏是如何生长的，以及这两种肝细胞类型是否来自共同的肝芽前体。这三个目标的结合将提供有关正常肝脏发育的新信息，这将极大地帮助理解肝脏疾病，并有助于体外诱导肝细胞和肝脏器官发生的研究。 公共卫生相关性：拟议工作的目标是了解在发育过程中导致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年器官做出贡献。实现这些目标将带来对肝脏个体发育和肝脏再生的新见解，将加深我们对肝病问题的理解，为治疗设计提供新的方向，包括从胚胎干细胞生产肝细胞。

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.