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Thesis
microrna miR-18a regulates neural regeneration in the injured retina
University of West Florida,
Master of Science (MS), University of West Florida
2020
Abstract
In mammals, retinal injuries and photoreceptor degeneration (PD) can damage photoreceptors causing permanent blindness. However, retinal injury in zebrafish (Danio rerio) elicits a rapid stem cell-based response that regenerates photoreceptors and restores vision. Crucial to this are the Müller glia (stem cells) that generate multipotent progenitor cells, which regenerate photoreceptors. Photoreceptor regeneration requires accurate control over the immune response (inflammation), cell cycle exit, and differentiation. Understanding the mechanisms which control photoreceptor regeneration in zebrafish could lead to treatments for blindness that stimulate Müller glia to reestablish vision in humans. Past investigations have demonstrated that microRNAs (miRNAs) are essential regulators of retinal neurogenesis, but lacking is an understanding of the roles of miRNAs in regulating photoreceptor regeneration. Preliminary data with miR-18a mutant fish generated using CRISPR/Cas9 gene-editing indicated that this microRNA regulates photoreceptor regeneration. The objective of this study was to determine if and how miR-18a, following light-induced photoreceptor injury, functions to regulate the cell cycle and/or differentiation in Müller glia and photoreceptor progenitors. In-situ hybridization (ISH) and immunohistochemical (IHC) labeling showed that miR-18a is expressed in microglia, proliferating Müller glia and photoreceptor progenitors, suggesting roles at both early and later stages of the photoreceptor regeneration response. Bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA) immunolabeling experiments showed that, following photoreceptor injury, there are more proliferating cells in miR-18a mutant (CRISPR) retinas compared with x wild-type (WT), demonstrating that miR-18a regulates the cell cycle. Expression of miR-18a in Müller glia and microglia (macrophages) indicates a role in the early regeneration response during which inflammation plays a critical role, and microRNA databases predict that miR- 18a might interact with and regulate several molecules involved in inflammation. These results led to the hypothesis that miR-18a regulates photoreceptor regeneration by regulating inflammation. As an initial test of this hypothesis, ISH was used to show that, in miR- 18a mutants compared with WT, the inflammatory mediator NFkB is expressed for a more extended period of time, indicating that miR-18a might typically function to suppress inflammation. To test this, dexamethasone (corticosteroid) was administered at critical time points to reduce inflammation levels in the miR-18a mutants. By blocking inflammation from days 2 to 6 post-injury, the treatment rescued the phenotype in the mutants, reducing the numbers of proliferating cells to the levels observed in WT fish. Finally, to determine if miR- 18a also regulates photoreceptor differentiation, ISH for rods and cones showed that miR- 18a mutant fish generate photoreceptors slower than wild-type fish but that by 14 days postinjury (dpi) miR-18a mutant fish generate more cones than WT. Together, these results demonstrate that miR-18a regulates the cell cycle and photoreceptor differentiation through molecular control over inflammation.
Files and links (1)
CC BY V4.0, Open Access
Details
- Title
- microrna miR-18a regulates neural regeneration in the injured retina
- Resource Type
- Thesis
- Publisher
- University of West Florida,; Pensacola, Florida :
- Format
- pdf
- Number of pages
- x, 48 leaves
- Copyright
- http://rightsstatements.org/vocab/InC-EDU/1.0/
- Identifiers
- 99380090837906600
- Academic Unit
- Biology
- Language
- English
- Awarding Institution
- University of West Florida; Master of Science (MS)
- Theses and Dissertations
- Master of Science (MS), University of West Florida