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

阐明 Yap1 在胆道系统胚胎发育和再生中的关键功能

基本信息

批准号：
10132724
负责人：
Laura Maria Molina
金额：
$ 5.1万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10132724
关键词：
3-Dimensional Accounting Adult Affect Age-Months Alagille Syndrome Biliary Biological Birth Cell Fraction Cell Proliferation Cell Survival Cells Child Childhood Cholestasis Clinical Data Defect Dependovirus Development Developmental Biology Disease model Duct (organ) structure Embryo Embryonic Development Epithelial Cells Etiology Event Experimental Designs Failure Fibrosis Foundations Future Generations Goals Growth Hepatic Hepatocyte Image Immunofluorescence Immunologic Impairment Inherited Injections Intrahepatic bile duct Knockout Mice Learning Life Life Expectancy Liver Liver Regeneration Manuscripts Medical Mentorship Microscopy Modeling Molecular Morbidity - disease rate Morphogenesis Mus Natural regeneration Organogenesis Pathology Patients Phenotype Physicians Process Proteins Publications Quality of life Regenerative Medicine Regenerative capacity Research Training Role Savings Scientist Serotyping Signal Transduction Sleeping Beauty Structure System Tail Techniques Technology TimeLine Tissue Engineering Tissues Training Veins base bile duct biliary tract career cell regeneration cell type cholestatic injury cholestatic liver disease hepatocyte injury improved insight intrahepatic liver development liver imaging liver transplantation mortality mouse model notch protein novel novel therapeutics oval cell polarized cell postnatal prevent progenitor regeneration potential regenerative therapy regenerative tissue repaired restoration stem cells targeted treatment transdifferentiation

项目摘要

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.

项目概要/摘要胆汁淤积性肝病是胆道系统的毁灭性疾病，约占儿童和成人患者的 45% 每年 10% 的成人肝移植。目前尚无可用的靶向治疗方法，且存在显着的提高患者生存率的未满足的临床需求。胆道上皮细胞（BEC）具有显着的再生能力能力，但严重胆汁淤积损伤后肝细胞（肝实质细胞）能够转分化进入 BEC 以修复成人和儿童疾病模型中受损的胆管。因此，肝细胞是未开发的促进胆道修复的储库。最近，Yes 相关蛋白 1 (Yap1) 已成为一种胆管形成和肝再生的关键调节因子。然而，人们对其机制知之甚少 Yap1 在胚胎发育或肝细胞源性 BEC 再生过程中驱动胆管分化。我们开发了一种新型 Foxa3-Cre Yap1 敲除 (KO) 小鼠模型，其中 Yap1 被完全删除肝祖细胞在诱导胆管谱系之前被破坏，导致胆管形成失败并严重胆汁淤积性损伤。最近的 Alagille 综合征模型显示出类似的胆道发育缺陷 4个月时，来自肝细胞的胆管广泛从头生成。然而，4个月大的Yap1- 缺陷肝脏没有显示肝细胞来源的胆道再生的证据。根据这些观察，我们总体假设是 Yap1 对于功能性胆管的形成是不可或缺的，无论其起源如何，无论是来自胚胎肝脏发育过程中的成肝细胞，还是来自肝细胞的转分化严重胆汁淤积性损伤的情况。为了进行调查，我们提出以下具体目标，这将产生新颖的结果对 Yap1 信号传导的机制见解，对再生医学和组织具有重要意义旨在促进胆道修复和再生的工程技术。具体目标 1：我们将确定正常肝器官发生过程中 Yap1 活性的个体发育，并研究胆管的时间轴使用免疫荧光和 3D 全肝显微镜观察 Yap1 KO 小鼠的发育情况。我们将因此确定 Yap1 缺失引起的 3D 胆管形态发生的特定缺陷。具体目标 2：基于我们初步数据显示 Yap1 KO 小鼠缺乏肝细胞向 BEC 的转分化，我们假设恢复 Yap1 阴性肝细胞的 Yap1 表达将选择性地允许这些细胞转分化并恢复关键的胆汁质量，以改善持续的胆汁淤积性损伤。我们将使用血统- 追踪以确定成年 Yap1 KO 小鼠中是否存在任何肝细胞来源的胆管。我们随后将使用两种互补的方法选择性地将标记的野生型 Yap1 构建体传递给少数人观察 Yap1 KO 小鼠的肝细胞并追踪其命运以评估其转分化为 BEC 的能力和胆管从头形成。对培训的贡献：该提案结合了肝脏研究培训病理学、发育生物学和最先进的生物成像以及世界一流的临床培训，为学术医师科学家的富有成效的职业生涯提供坚实的基础。