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.

Public Health Relevance

Tadpoles of the frog Xenopus tropicalis are able to regenerate central nervous system tissues, while adult frogs cannot, making this a powerful model organism in which to discover the factors that govern regeneration competence. We have recently discovered that tadpole regeneration proceeds through rapid remodeling of chromatin upstream of gene expression, and in this proposal we will probe the events both upstream and downstream of this remodeling to discover the transcription factors and gene regulatory elements that trigger neurogenesis in regenerating tadpoles but are missing in adult frogs. By identifying the critical set of factors that define regenerative competence in Xenopus, we can gain insight into how to construct therapies that will better improve regenerative competence in human patients with spinal cord injuries.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
Research Project (R01)
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Neurogenesis and Cell Fate Study Section (NCF)
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Jakeman, Lyn B
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University of Washington
Schools of Medicine
United States
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Arbach, Hannah E; Harland-Dunaway, Marcus; Chang, Jessica K et al. (2018) Extreme nuclear branching in healthy epidermal cells of the Xenopus tail fin. J Cell Sci 131: