University of Kentucky UKnowledge Theses and Dissertations--Biology Biology 2016 ROLE OF HAIRY-RELATED (HER) GENES DURING VERTEBRATE RETINAL DEVELOPMENT AND REGENERATION Stephen G. Wilson University of Kentucky, [email protected] Digital Object Identifier: http://dx.doi.org/10.13023/ETD.2016.249 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Wilson, Stephen G., "ROLE OF HAIRY-RELATED (HER) GENES DURING VERTEBRATE RETINAL DEVELOPMENT AND REGENERATION" (2016). Theses and Dissertations--Biology. 36. https://uknowledge.uky.edu/biology_etds/36 This Doctoral Dissertation is brought to you for free and open access by the Biology at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Biology by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Stephen G. Wilson, Student Dr. Ann C. Morris, Major Professor Dr. David F. Westneat, Director of Graduate Studies ROLE OF HAIRY-RELATED (HER) GENES DURING VERTEBRATE RETINAL DEVELOPMENT AND REGENERATION __________________________ DISSERTATION __________________________ A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Stephen G. Wilson Lexington, Kentucky Director: Ann C. Morris, Associate Professor of Biology Lexington, Kentucky 2016 Copyright © Stephen G. Wilson 2016 ABSTRACT OF DISSERTATION ROLE OF HAIRY-RELATED (HER) GENES DURING VERTEBRATE RETINAL DEVELOPMENT AND REGENERATION Development and regeneration of the vertebrate eye are the result of complex interactions of regulatory networks and spatiotemporally controlled gene expression events. During embryonic retinal development, the coordination of cell signaling and transcriptional regulation allows for a relatively homogenous sheet of neuroepithelial cells to proliferate and differentiate in-to a multilayered, light sensitive retinal tissue. Following injury, the retinas of many cold-blooded vertebrates, such as the zebrafish, undergo a proliferative response that results not only in new retinal cells of the correct type in the correct location, but also functional integration of these cells and restoration of vision. In order for embryonic retinal neurogenesis to proceed correctly, systems must be in place that restrict subsets of progenitor cells from differentiation. Pools of actively proliferating retinal progenitor cells are maintained to fill the needs of developmental processes and normal growth of the retina. In addition, subsets of radial glia in the retina retain the ability to de-differentiate into proliferating progenitor cells to meet the demands of the regenerating retina. All of these processes rely on the tight coordination of extrinsic and intrinsic cues, as well as regulation of gene expression by transcription factors. Although a considerable amount of work has been conducted to identify key regulators of retinal de velopment and regeneration, many gene regulatory networks which include both master signaling pathways as well as individual transcription factors remain poorly characterized. Some of these factors implicated in retinal development and regeneration are members of the Hairy/Enhancer of Split (Hes) superfamily of genes, including the Hairy-related (Her) factors Her4 and Her9. Her transcription factors are basic-helix-loop-helix-orange (bHLH-O) transcription factors that bind to palindromic E- and N-box canonical sequences in the promoters of target genes. Her factors have been previously shown to play roles in a diverse array of developmental and neurogenic processes, including neural tube closure, floor plate development, somitogenesis, and development of various components of the central nervous system as well as the cranial sensory placodes. The roles of her4 and her9 in retinogenesis, however, remain undefined. To determine the possible roles of her4 and her9 factors in the retina, I characterized the expression patterns of these factors during developmental retinal neurogenesis and/or regeneration, examined loss of function phenotypes, and identified signaling pathways that modulate expression of these factors. Chapter 1 of this dissertation provides an overview of vertebrate retina and retinal development, the known functions of her4 in other tissues, and the Notch-Delta signaling pathway. Chapter 2 provides evidence that her4 is a primary effector of the Notch pathway during retinal development, and examines the role of her4 expressing cells during regeneration of the mature zebrafish retina within the context of both chronic and acute photoreceptor damage paradigms. In addition, generation and validation of the transgenic her4:Kaede zebrafish which was used to identify the lineage of her4-expressing cells is described. Characterization of her9 during retinal development, identification of the retinoic acid signaling pathway as a regulator of her9 expression in the retina, and the role her9 plays during retinal vasculogenesis are discussed in Chapter 3. Chapter 4 discusses the generation of her9 knock-out zebrafish lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterization of mutant phenotypes in mosaic her9 mutant F0 fish. In addition, in Chapter 4 I also discuss the screening processes used to identify and characterize genetic lesions in the her9 allele and establish various lines that stably transmit deleterious her9 alleles in the germline, and provide preliminary data of the her9 mutant phenotype. Finally, in Chapter 5 I discuss conclusions from the data generated from this dissertation, additional studies that would ii expand upon this work, and the implications of these results on the broader understanding of retinal development and regeneration. My dissertation incorporates reverse genetic analysis in zebrafish, biochemical analysis, transgenesis, and various molecular approaches to help better understand the roles of her4 and her9 during retinal neurogenesis. Moreover, these studies may also contribute to a better understanding of retinal development, and disease pathogenesis. It is my hope that this work could also ultimately contribute, even if in some small way, to the goal of enabling human patients who have suffered from vision loss a means by which a damaged retina could be regenerated and functional vision restored. KEYWORDS: Retina, Development, Regeneration, Vasculogenesis, Notch-Delta, Retinoic Acid, her4, her9, Zebrafish, CRISPR/Cas9 Stephen G. Wilson Student’s Signature April 25, 2016 Date iii ROLE OF HAIRY-RELATED (HER) GENES DURING VERTEBRATE RETINAL DEVELOPMENT AND REGENERATION By Stephen G. Wilson Ann C. Morris Director of Dissertation David F. Westneat Director of Graduate Studies May 24, 2016 Date iv For HDW ffffffffff v ACKNOWLEDGEMENTS “Things are only impossible until they are not” – Captain Jean-Luc Picard I love the simplicity and implication of one of Captain Picard’s more famous quotes. Truly, things are only impossible until they are not, and I find this to be especially relevant to the field of biology. I find it absolutely amazing that in the span of roughly one hundred years, our understanding of the concept of a gene went from mathematical abstractions of heritable traits to now being able to precisely describe the molecular interactions of nucleic acids and proteins within context of larger networks that lead to fundamental biological principles. I think that one of the things that really drove me into this field is the idea that it is now possible to not only understand but to fix or even improve various aspects of our own biology. I strongly believe that what we now consider to be impossible will indeed be possible in the near future, and we will all live long and prosper. First and foremost, I want to acknowledge my amazing wife, Hilary. I don’t think that any of this would have been remotely possible without her encouragement, love,