The Role of the Notch Pathway in Bile Duct Development

描述（由申请方提供）：遗传性肝内胆管发育障碍是儿科人群发病和死亡的主要原因。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了解相对较少。Alagille综合征（AGS）是一种常染色体显性遗传疾病，其特征是胆管缺乏沿着其他器官系统的异常，Jag 1作为疾病基因的鉴定揭示了Jag 1和Notch通路在胆管发育中的关键作用。Jag 1编码Notch信号通路中的配体，其参与许多器官系统中的细胞命运决定。在小鼠模型中Jag 1的纯合靶向破坏导致早期胚胎致死，而杂合突变小鼠没有表型。利用Cre/lox基因打靶系统构建了Jag 1基因条件性敲除小鼠。该模型将允许系统研究Jag 1在发育过程中特定时间在多器官系统发育中的作用。初步研究表明，Jag 1在原始胆管或胆管板细胞中表达，这些细胞来源于发育中的小鼠和人类肝脏中的肝细胞前体。我们建议选择性地消融Jag 1在发展中的肝脏和胆管的Jag 1-loxP小鼠与肝脏特异性的Cre转基因系育种。使用这种小鼠模型，我们将进行详细的研究导管板重塑和胆管发育的Jag 1的情况下。此外，我们将采用真实的时间PCR和微阵列分析，以确定独特的下游目标的Notch途径在胆管发育。作为一种补充策略，我们建议使用一种细胞培养方法，我们将从胚胎野生型和Jag 1基因缺失的肝脏中获得双能成肝细胞系。我们将评估它们对特定刺激的反应，并通过激活Notch的免疫抑制来挽救表型

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