Investigating the transcriptional regulation of liver specification in Xenopus tr

描述(由申请人提供)：从多能前体细胞中确定确定的细胞命运需要随着时间的推移协调整合细胞自主和非自主线索。就肝脏而言，肝细胞的命运被认为是通过一系列的承诺过程获得的：细胞首先通过细胞外Nodal信号导向内胚层命运，然后通过特定转录因子如Gata4和FoxA1的表达获得肝脏功能，然后通过细胞外信号的作用被限制发育到其他前内胚层器官，包括来自横隔的BMP信号和来自心脏中胚层的成纤维细胞生长因子信号。这项研究的目的是回答肝脏命运规范中的三个悬而未决的问题：Nodal信号的哪些直接靶点在建立肝脏功能中起作用；FoxA1的直接靶点是什么；BMP和FGF调节肝脏诱导的体内机制是什么？这些问题解决了我们对转录层次的理解上的差距，肝脏发育的已知因素通过这些转录层次发挥作用，以及肝脏发育的每个阶段是如何机械地与下一个阶段联系在一起的。直接回答这些问题需要在早期发育阶段使用大量胚胎，这是羊膜模型系统的局限性，以前许多关于肝脏发育的研究都是在羊膜模型系统中进行的。因此，这项拟议的研究将在热带爪蛙身上进行，它拥有大量易于操作的胚胎，非常适合快速筛选和生化研究。拟议的项目将生成一个代表Nodal信号靶标的原位杂交表达模式数据库，该数据库将向社区提供。拟议的项目还将需要培训和发展最近开发的生化和高通量测序方法方面的专门知识，特别是修改现有的染色质免疫沉淀和高通量测序(CHIP-SEQ)方案，用于带有FoxA1抗体的热带血吸虫胚胎。最后，该项目将促进在热带X线虫进行的基础研究与将发现应用于人类胚胎干细胞定向分化为肝脏命运之间的合作。这些问题的答案将阐明我们对肝脏器官发生的理解，并将应用于开发从胚胎干细胞获得肝细胞的新方案，这是最终治疗肝病的主要目标。 公共卫生相关性：了解在正常胚胎发育期间细胞从多能命运转变为肝脏命运所发生的步骤，已经并将继续直接为控制人类胚胎干细胞向肝细胞分化的努力提供信息。胚胎干细胞或其他非肝细胞来源的肝细胞对肝病患者来说是一种潜在的肝移植替代疗法。此外，对肝脏发育阶段的机械性理解将有助于深入了解一些肝脏疾病的遗传或发育起源，这些疾病是由于肝脏发育计划执行不当而引起的，以及如何诊断或治疗这些疾病。

DESCRIPTION (provided by applicant): The establishment of a committed cell fate from a pluripotent precursor cell requires the coordinated integration of cell autonomous and non-autonomous cues through time. In the case of the liver, hepatic cell fate is thought to be acquired through a sequential process of commitment: cells are first directed to an endoderm fate by extracellular Nodal signaling, then acquire liver competence through the expression of specific transcription factors such as Gata4 and FoxA1, then are restricted from developing into other anterior endoderm organs by the action of extracellular signals including BMP signals from the septum transversum and FGF signals from the cardiac mesoderm. The goal of this research is to answer three unresolved questions in liver fate specification: which of the direct targets of Nodal signaling act in establishing liver competence; what are the direct targets of FoxA1; and what is the in vivo mechanism by which BMP and FGF regulate hepatic induction? These questions address gaps in our understanding of the transcriptional hierarchy through which known factors in liver development act, and of how each stage of liver development is mechanistically linked to the next. Answering these questions directly requires large numbers of embryos at early developmental stages, a limitation of amniote model systems where many previous studies on liver development have been conducted. Therefore, the proposed research will be carried out in the frog Xenopus tropicalis, which has large numbers of readily manipulated embryos ideal for rapid screening and biochemical investigations. The proposed project will generate a database of in situ hybridization expression patterns representing Nodal signaling targets, which will be made available to the community. The proposed project will also entail training and the development of expertise in recently-developed biochemical and high-throughput sequencing methodologies, specifically including the adaptation of existing protocols for chromatin immunoprecipitation and high throughput sequencing (ChIP-Seq) for use in X. tropicalis embryos with FoxA1 antibodies. Finally, this project will foster collaborations between basic research carried out in X. tropicalis and applications of the findings to directed differentiation of human embryonic stem cells to liver fates. The answers to these questions will elaborate our understanding of liver organogenesis, and will have applications to the development of new protocols for deriving hepatocytes from embryonic stem cells, a major goal for the eventual treatment of liver disease. PUBLIC HEALTH RELEVANCE: Understanding the steps that occur as a cell changes from a pluripotent fate to a liver fate during normal embryological development has and will continue to directly inform efforts to control the differentiation of human embryonic stem cells to liver cells. Liver cells derived from embryonic stem cells or other non-liver cell types are a potential therapeutic alternative to liver transplant for patients suffering from liver diseases. Further, a mechanistic understanding of the stages of liver development will provide insight into the genetic or developmental origins of some liver diseases arising from improper execution of the liver development program, and how they could be diagnosed or treated.