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.
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.
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