Elucidating the Critical Functions of Yap1 in the Embryonic Development and Regeneration of the Biliary System

项目摘要/摘要 胆汁淤积性肝病是胆道系统的破坏性疾病，约占儿科和外科疾病的45%。 每年10%的成人肝移植。目前还没有靶向治疗方法可用，而且有显著的 未满足的临床需求，以提高患者的存活率。胆管上皮细胞(BECs)具有显著的再生能力 能力，但在严重的胆汁淤积损伤后，肝细胞(肝实质细胞)能够转分化 以修复成人和儿童疾病模型中受损的胆管。因此肝细胞是一种 未开发的水库，用于促进胆道修复。最近，YAP1(YAP1)作为一种 胆管形成和肝再生的关键调节因子。然而，人们对这种机制知之甚少。 在胚胎发育或肝细胞来源的BEC再生过程中，哪种YAP1驱动胆管分化。 我们建立了一种新的Foxa3-Cre YAP1基因敲除(KO)小鼠模型，其中YAP1基因完全缺失 肝祖细胞在胆管谱系诱导前，导致胆管形成失败，严重 胆汁淤积性损伤。最近的阿拉格尔综合征模型显示胆道发育有相似的缺陷。 4个月后，肝细胞来源的胆管大量再生。然而，4个月大的YAP1- 肝功能不全并不能显示肝细胞源性胆管再生。基于这些观察，我们的 最重要的假设是，YAP1对于功能性胆管的形成是不可或缺的，无论其来源如何。 无论是来自胚胎肝脏发育过程中的肝母细胞，还是来自于胚胎肝发育中的转分化肝细胞 严重胆汁淤积性损伤的背景。为了进行调查，我们提出了以下具体目标，这将产生小说 对YAP1信号的机械论洞察，对再生医学和组织的重大影响 旨在促进胆道修复和再生的工程技术。具体目标1：我们将确定 正常肝脏器官发生过程中YAP1活性的个体发生及胆管时间线的探讨 用免疫荧光和三维全肝显微镜观察YAP1 KO小鼠的发育。因此，我们将确定 YAP1缺失导致的胆管三维形态发生的特异性缺陷。具体目标2：基于我们的 初步数据显示YAP1 KO小鼠缺乏肝细胞到BEC的转分化，我们假设 在YAP1阴性的肝细胞中恢复YAP1的表达将选择性地允许这些细胞 对临界胆管肿块进行转分化和修复，以改善持续的胆汁淤积性损伤。我们将使用血统- 追踪以确定在成年YAP1 KO小鼠中是否存在任何肝细胞来源的胆管。到时候我们会的 使用两种互补的方法选择性地将标记的野生型YAP1构建传递给少数人 在YAP1 KO小鼠体内观察肝细胞并追踪其命运以评估其向BECs转分化的能力 和新生的胆管形成。对培训的贡献：本建议结合肝脏的研究培训 病理学，发育生物学，最先进的生物成像和世界级的临床培训， 作为一名学术内科科学家，为富有成效的职业生涯提供坚实的基础。

PROJECT SUMMARY/ABSTRACT Cholestatic liver diseases are devastating illnesses of the biliary system, accounting for ~45% of pediatric and 10% of adult liver transplants annually. Currently no targeted therapies are available, and there is a significant unmet clinical need to improve patient survival. Biliary epithelial cells (BECs) possess significant regenerative capacity, but after severe cholestatic injury hepatocytes (liver parenchymal cells) are able to transdifferentiate into BECs to repair damaged bile ducts in both adult and pediatric models of disease. Hepatocytes are thus an untapped reservoir for promotion of biliary repair. Recently, Yes-associated protein 1 (Yap1) has emerged as a critical regulator of bile duct formation and liver regeneration. However, little is known about the mechanisms by which Yap1 drives biliary differentiation during embryonic development or hepatocyte-derived BEC regeneration. We have developed a novel Foxa3-Cre Yap1 knockout (KO) mouse model in which Yap1 is fully deleted from liver progenitor cells before induction of the biliary lineage, resulting in failure of bile duct formation and severe cholestatic injury. A recent model of Alagille syndrome showing similar defects in biliary development described extensive de novo generation of bile ducts derived from hepatocytes by 4 months. However, 4-month-old Yap1- deficient livers show no evidence of hepatocyte-derived biliary regeneration. Based on these observations, our overarching hypothesis is that Yap1 is indispensable for functional bile duct formation regardless of their origin, whether from hepatoblasts during embryonic liver development or from transdifferentiating hepatocytes in the setting of severe cholestatic injury. To investigate, we propose the following specific aims, which will yield novel mechanistic insights into Yap1 signaling, with significant implications for regenerative medicine and tissue engineering technologies aimed at promoting biliary repair and regeneration. Specific Aim 1: We will determine the ontogeny of Yap1 activity during normal liver organogenesis, and investigate the timeline of bile duct development in Yap1 KO mice using immunofluorescence and 3D whole-liver microscopy. We will thus identify the specific defects in 3D bile duct morphogenesis caused by Yap1 deletion. Specific Aim 2: Based on our preliminary data showing lack of hepatocyte-to-BEC transdifferentiation in Yap1 KO mice, we hypothesize that restoration of Yap1 expression to Yap1-negative hepatocytes will selectively allow these cells to transdifferentiate and restore a critical biliary mass to ameliorate ongoing cholestatic injury. We will use lineage- tracing to determine whether any hepatocyte-derived bile ducts are present in adult Yap1 KO mice. We will then use two complementary approaches to selectively deliver a tagged wild-type Yap1 construct to a small number of hepatocytes in Yap1 KO mice and trace their fate to assess their capacity for transdifferentiation into BECs and de novo bile duct formation. Contribution to Training: This proposal combines research training in liver pathology, developmental biology, and state-of-the-art biological imaging with world-class clinical training, providing a strong foundation for a productive career as an academic physician scientist.