A major challenge facing cell replacement and regenerative therapies for retinal diseases is generating retinal cells or tissue at the quality and scale necessary to be therapeutically relevant. To meet this challenge, we need to be able to control the growth and developmental potential of progenitor/stem cells such that certain fates can be selected over others and differentiation programs are robust enough to impart desired functions. Key insights into this come from studying how the retina develops using various genetically tractable animal model systems ranging from Drosophila to mouse. In this project, we examine the question of how key developmental transcription factors regulate the neurogenic properties of retinal progenitor cells (RPCs), from the perspectives of promoting neurogenesis, maintaining progenitors in a neurogenic environment, and generating neuronal diversity. In the first aim, we test the hypothesis that the homeodomain protein Vsx2 (formerly Chx10) controls the timing of onset of neurogenesis in the embryonic retina through a cell-autonomous circuit that sets the proper expression levels/activities of a minimum of four other transcription factors in pre-neurogenic RPCs;Mitf, p27Kip1, Pax6, and Sox2. We also test the hypothesis that the acquisition of a dependence on Notch signaling as a mechanism for progenitor cell maintenance is linked to this timing. In the second aim, we address the hypothesis that the homeodomain protein Lhx2 is a key regulator of progenitor cell maintenance, neurogenic output, and progenitor competence during retinal neurogenesis. Using conditional inactivation genetics to bypass Lhx2's early and essential role in optic vesicle patterning and optic cup morphogenesis, we will test the model that Lhx2 maintains RPCs with high neurogenic potential, and by doing so, establishes a dynamic balance with other RPCs such that they persist through the entire interval of retinal development. Second, we will test the model that Lhx2 is required in select RPCs to respond to negative feedback signals from postmitotic, differentiating retinal precursors, and by doing so, limits the production of specific cell types and contributes to the generation of neuronal diversity in the retina. Third, we've identified a critical window during early retinal development Lhx2 is required for RPCs to transition into another competence state necessary for production of later cell types. Only a couple of other factors have been proposed to regulate early versus late competence, and a potential relationship with Lhx2 will be tested. We will also adapt the newly described HA-tagging method with transcriptome profiling to discover new genes regulated by Lhx2 that promote the transition in competence. Completion of these studies will provide new insight into how RPC maintenance and neurogenic potential is controlled during embryogenesis, information that can be applied to stem cell engineering and regenerative medicine.

Public Health Relevance

The eye and its component parts such as the retina are formed through a series of progressive and interdependent processes, and alterations in the timing or robustness of these processes lead to an array of ocular malformations and vision problems. The goal of the proposed research is to understand the underlying molecular and genetic mechanisms driving the capacity of retinal progenitor cells to generate the complement of retinal neurons in the numbers needed for retinal organization and function. The information gained from these studies will provide new knowledge on how the retina develops, on what goes wrong in congenital ocular birth defects, and provides needed information on how to control the growth and neurogenic properties of stem cells and related cell populations for their use in regenerative and cell replacement therapies.

National Institute of Health (NIH)
Research Project (R01)
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Greenwell, Thomas
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University of Utah
Schools of Medicine
Salt Lake City
United States
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Fuhrmann, Sabine; Zou, ChangJiang; Levine, Edward M (2014) Retinal pigment epithelium development, plasticity, and tissue homeostasis. Exp Eye Res 123:141-50
Gordon, Patrick J; Yun, Sanghee; Clark, Anna M et al. (2013) Lhx2 balances progenitor maintenance with neurogenic output and promotes competence state progression in the developing retina. J Neurosci 33:12197-207
Das, Gaurav; Clark, Anna M; Levine, Edward M (2012) Cyclin D1 inactivation extends proliferation and alters histogenesis in the postnatal mouse retina. Dev Dyn 241:941-52
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Das, Gaurav; Choi, Yoon; Sicinski, Piotr et al. (2009) Cyclin D1 fine-tunes the neurogenic output of embryonic retinal progenitor cells. Neural Dev 4:15
Sigulinsky, Crystal L; Green, Eric S; Clark, Anna M et al. (2008) Vsx2/Chx10 ensures the correct timing and magnitude of Hedgehog signaling in the mouse retina. Dev Biol 317:560-75
Barton, Kirston M; Levine, Edward M (2008) Expression patterns and cell cycle profiles of PCNA, MCM6, cyclin D1, cyclin A2, cyclin B1, and phosphorylated histone H3 in the developing mouse retina. Dev Dyn 237:672-82
Clark, Anna M; Yun, Sanghee; Veien, Eric S et al. (2008) Negative regulation of Vsx1 by its paralog Chx10/Vsx2 is conserved in the vertebrate retina. Brain Res 1192:99-113
Defoe, Dennis M; Adams, Lorrie B S; Sun, Jingru et al. (2007) Defects in retinal pigment epithelium cell proliferation and retinal attachment in mutant mice with p27(Kip1) gene ablation. Mol Vis 13:273-86

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