The Role of the Notch Pathway in Bile Duct Development

描述(申请人提供)：遗传性肝内胆管发育障碍是儿科人群发病率和死亡率的主要原因。近年来，在了解肝脏发育的遗传控制方面取得了进展，但对胆管发育的分子调控知之甚少。Jag1基因被鉴定为Alagille综合征(AGS)的致病基因，AGS是一种常染色体显性遗传性疾病，其特征是胆管稀少和其他器官系统的异常，揭示了Jag1和Notch途径在胆管发育中的关键作用。Jag1编码Notch信号通路中的一个配体，在许多器官系统中参与决定细胞命运。在小鼠模型中，Jag1的纯合子靶向破坏会导致早期胚胎死亡，而杂合子突变的小鼠没有表型。我们使用Cre/lox基因打靶系统产生了一只Jag1条件性基因敲除小鼠。这个模型将允许系统地研究Jag1在发育过程中特定时间的多器官系统发育中的作用。初步研究表明，Jag1在原始胆管或导管板细胞中表达，该细胞起源于发育中的小鼠和人类肝脏的肝细胞前体。我们建议通过用肝脏特异的Cre转基因系培育Jag1-loxP小鼠来选择性地消融发育中的肝脏和胆管中的Jag1。利用这个小鼠模型，我们将在没有JAG-1的情况下对胆管板重塑和胆管发育进行详细的研究。此外，我们还将利用实时荧光聚合酶链式反应和微阵列分析来寻找胆管发育过程中Notch通路的独特下游靶点。作为补充策略，我们建议使用一种细胞培养方法，即我们将从胚胎野生型和Jag1缺失的肝脏中获得双潜能肝母细胞系。我们将评估它们对特定刺激的反应，并通过转染激活的Notch来挽救表型

DESCRIPTION (provided by applicant): Inherited disorders of intrahepatic bile duct development are a major cause of morbidity and mortality in the pediatric population. In recent years, advances have been made toward understanding the genetic control of liver development, but relatively little is understood regarding the molecular regulation of bile duct development. Identification of Jag1 as the disease gene for Alagille syndrome (AGS), an autosomal dominant disorder characterized by bile duct paucity along with anomalies in other organ systems, has revealed a crucial role for Jag1 and the Notch pathway in bile duct development. Jag1 encodes a ligand in the Notch signaling pathway, which is involved in cell fate determination in many organ systems. Homozygous targeted disruption of Jag1 in a mouse model causes early embryonic lethality, and the heterozygous mutant mouse has no phenotype. We have generated a Jag1 conditional knockout mouse using the Cre/lox gene targeting system. This model will permit the systematic study of the role of Jag1 in the development of multiple organ systems at specified times during development. Preliminary studies have demonstrated expression of Jag1 in the primitive bile ducts, or ductal plate cells, which originate from hepatocyte precursors in developing mouse and human liver. We propose to selectively ablate Jag1 in the developing liver and bile ducts by breeding the Jag1-loxP mouse with a liver-specific Cre transgenic line. Using this mouse model, we will perform detailed studies of ductal plate remodeling and bile duct development in the absence of Jag 1. In addition, we will employ real time PCR and micro array analysis to identify unique downstream targets of the Notch pathway in bile duct development. As a complementary strategy, we propose to use a cell culture approach in which we will derive bipotential hepatoblast cell lines from embryonic wildtype and Jag1 null livers. We will assess their response to defined stimuli, and rescue the phenotype by transfecting with activated Notch