2010 |
Wills, Andrea Elizabeth |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Investigating the Transcriptional Regulation of Liver Specification in Xenopus Tr
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.
|
0.954 |
2011 — 2012 |
Wills, Andrea Elizabeth |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Transcriptional Regulation of Liver Specification in Xenopus Tropicalis
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.
|
0.954 |
2017 — 2018 |
Wills, Andrea Elizabeth |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Defining the Mechanism of Chromatin Accessibility Modifications in Vertebrate Appendage Regeneration @ University of Washington
PROJECT SUMMARY Amphibians are able to undergo scarless healing and regeneration in response to amputation of their appendages. By articulating the cell and molecular basis for this regenerative capacity, we can develop therapeutic strategies to improve regenerative healing in humans. Successful regeneration in amphibians requires the early action of histone deacetylases (HDACs), enzymes that act to condense chromatin and inhibit transcription. However, the genomic and transcriptional targets of HDAC activity are unknown in this context, limiting our ability to form a mechanistic model of epigenetic and transcriptional reprogramming in vertebrate regeneration. It has been difficult to identify these targets because there are few resources for querying chromatin structure in regenerating vertebrate tissue. We have overcome this barrier by using a new assay for transposase accessible chromatin (ATAC-Seq) in Xenopus tropicalis tail regeneration. We have found that thousands of promoter regions are rapidly rendered inaccessible at 6 hours post amputation: the very same time that HDAC activity is required. These regions are later re-opened. Therefore, the central hypothesis of our study is that HDAC activity acts transiently to condense chromatin in these promoter regions by deacetylating specific core histone residues, and is reversed by the later action of histone acetyltransferases (HATs). Here will use our expertise in genomics and functional perturbations of this system to conduct a succinct functional secondary analysis that will test this hypothesis. We will identify the genomic regions that are sensitive to HDAC and HAT activity, the specific residues that are targeted, and the spatiotemporal distribution of histone acetylation in the regenerating tail. Importantly, we will also lay the foundation for future work that will establish how HDAC activity is balanced with other chromatin remodeling activities to reprogram transcription and cell behavior early in regeneration. !
|
1 |
2017 — 2021 |
Wills, Andrea Elizabeth |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Transcriptional Regulatory Mechanisms of Vertebrate Regeneration @ University of Washington
Why do humans fail to regenerate injured central nervous system tissues, when other vertebrates do so readily? In this proposal we take aim at this fundamental question by defining the cell-intrinsic mechanisms that enable spinal cord regeneration in the frog Xenopus tropicalis. Tadpoles of this species are able to regenerate spinal cord tissues and motor function following injury, while adult animals cannot. We will exploit this temporal competence to regenerate in order to understand how regeneration normally proceeds as well as why it might fail. This distinctive biology coupled with the deep set of available tools for functional and genomic analysis makes X. tropicalis a uniquely powerful system for analysis of regeneration. A central goal of spinal cord regeneration research is to identify the cell-intrinsic factors that enable neurogenesis and axon regeneration. Our preliminary analyses in this system have uncovered new insights into these factors and the gene regulatory mechanisms that may form the basis for regenerative competence. First, we have found that tens of thousands of genomic regions shift rapidly to an accessible chromatin conformation, and then unexpectedly to an inaccessible conformation, within the first few hours of regeneration. These rearrangements take place in regions that are heavily enriched for binding sites of FoxO1 and Ascl1, factors that have pioneer activity and critical roles in neural progenitor function. Second, genes specific to differentiated neurons are expressed within hours of amputation, and are surprisingly independent of neural induction and neurogenesis gene activation. Based on these observations, we hypothesize that regenerative competence relies on three features: 1) a robust neural progenitor population, 2) a rapid burst of chromatin remodeling in neural progenitor cells carried out by Ascl1, FoxO1, and other pioneer factors, and 3) activation of neuronal specific genes that allow axonogenesis and neuronal growth in existing differentiated neurons. In this proposal, we will test these predictions by identifying the transcription factors that mediate chromatin remodeling in isolated neural progenitors. We will functionally test the role of Ascl1 and FoxO1 in regeneration using loss-of-function mutants for these factors. We will then identify whether upregulation of axonogenesis genes in regenerating tadpoles represents neuronal repair or neurogenesis, and interrogate whether these genes are upregulated using embryonic gene regulatory elements or regeneration-specific regulatory elements. Finally, we will identify whether regeneration in adult frogs fails due to lack of neural progenitors, failure to initiate chromatin remodeling, or failure to upregulate neuronal morphogenesis genes. By systematically characterizing the events that define regeneration competence in Xenopus, we expect to identify molecular mechanisms that can be targeted for more effective therapeutics in human spinal cord injury patients.
|
1 |