Understanding liver bud emergence, formation and potential

了解肝芽的出现、形成和潜力

• 批准号: 8088116
• 负责人: KIMBERLY D TREMBLAY
• 金额: $ 28.93万
• 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/8088116
• 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）：肝脏是由两种细胞类型组成的重要再生器官：肝细胞和管道细胞。了解这些细胞在再生和发育过程中如何形成对于为众多肝病和损伤开发临床疗法很重要。当前，了解肝脏发育的初始阶段存在很大的差距。该项目的目的是对鼠肝芽的更彻底的了解，鼠肝芽是具有肝脏祖细胞的形态上独特的结构。肝芽使用来自相邻组织的分泌信号，在包括肺，胰腺和甲状腺在内的所有内胚层器官（包括肺，胰腺和甲状腺）的过程中出现。目的1是测试候选信号通路在肝脏萌芽中的作用。为了下调肝脏前体种群中感兴趣的途径，使用了两种使用整个胚胎培养的方法：旨在在局部内胚层中下调候选途径的构造的电穿孔，并直接向培养基添加抑制剂。然后通过发育的肝芽阶段培养操纵的胚胎。如果候选人在萌芽中发挥作用，那么该过程将被中断，并从最终的表型中推断出其作用。将产生一种表达CRE的新型小鼠系，以测试经体内验证的候选物的作用。目的2是检验两个肝前体种群是不同的前体的假设。命运映射实验表明，肝脏源自两个谨慎的细胞种群，对肝芽的贡献有所不同。初步数据表明，这两个群体在分子和形态上彼此之间以及内胚层的其余部分都是不同的。为了评估两个肝前体种群与第三个非肝脏前体之间的分子差异，将产生并比较从单独确认的细胞池中得出的转录曲线。 AIM 3旨在测试单个肝芽细胞的潜力，该细胞也称为肝母细胞。间接证据表明，肝类母细胞是多能的，引起了肝细胞和导管细胞。我们提出了一种遗传标记策略，利用内胚层特异性的CREER系和R26R报告基因在肝芽中产生单个重组事件，该事件打开该细胞中的记者及其所有后代。这种回顾性的谱系分析将证明肝脏的生长以及两种肝细胞类型的生长方式。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的疾病，并有助于旨在在体外诱导肝细胞和肝脏器官发生的研究。 公共卫生相关性：拟议工作的目的是了解在发育过程中引起和支持肝脏规范的分子机制，并探讨早期肝芽细胞如何对成年器官的贡献。实现这些目标将导致对肝脏本体发育和肝脏再生的新见解，这将加深我们对肝病中出问题的理解，提供新的治疗设计方向，包括从胚胎干细胞中产生肝细胞。