Semester

Fall

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Biochemistry

Committee Chair

Peter H Mathers

Abstract

Patterning of the optic vesicle is a crucial step in early vertebrate eye development that organizes uncommitted optic vesicle cells into distinct distal, dorsal, and proximal regions that will give rise to the neural retina, retinal pigment epithelium (RPE), and optic stalk, respectively. Originating from the same sheet of anterior neuroectoderm, uncommitted optic vesicle cells are patterned into neural and non-neural (RPE) retinal domains through the coordinated activities of extrinsic signaling molecules and intrinsic transcription factors. Neural retinal specification is driven by FGF signals emanating from the surface ectoderm and developing lens, while RPE is specified by signals from the extraocular mesenchyme overlying the dorsal optic vesicle, likely a TGF-beta superfamily protein. Similarly, signaling from the optic vesicle has been implicated in directing lens formation from cells in the surface ectoderm. Various experiments have shown that patterning of neural retina and RPE can be altered through the ectopic introduction of signaling molecules, or the manipulation of various developmentally regulated transcription factors. Indeed, in frogs, chicks, and rodents, determination of neural retina and RPE can be interchanged for some time after their initial specification: presumptive RPE cells can transdifferentiate into neural retinal cells, and vice versa, demonstrating the bipotentiality of optic vesicle cells. Specifically, numerous studies demonstrate FGFs, and downstream effectors of FGF signaling, are important mediators of RPE-to-neural retinal transdifferentiation.;Rx is a paired-like homeobox gene that encodes a transcription factor that is expressed in retinal progenitor cells (RPCs) of the developing optic vesicle/cup. As the optic cup forms, Rx expression is restricted to the inner layer of developing optic cup (presumptive neural retina) and is terminated as retinal progenitors exit the cell cycle and differentiate into a neural retinal cell type. Rx expression is maintained through adulthood in Muller glial cells, which have been shown to function like neural retinal stem cells. During embryogenesis, mice homozygous for a targeted Rx-null allele fail to form optic vesicles, demonstrating Rx is intrinsically required for the earliest stage of eye formation---evagination of the optic vesicles. Given the expression of Rx in proliferating retinal progenitors, we hypothesize that following optic vesicle evagination, Rx plays a role in promoting the proliferation of retinal progenitors, which could impact retinal and lens morphogenesis, and is required for the specification and/or maintenance of neural retinal identity.;In this dissertation, the functions of Rx during the early stages of vertebrate ocular development following optic vesicle evagination are ascertained through Rx loss-of-function studies using Cre/loxP conditional inactivation strategies. We demonstrate that inactivation of Rx during the optic vesicle stage, via the Foxg1-Cre animal model, arrests retinal progenitor cell proliferation and prevents neural retinal specification, generating an optic vesicle remnant composed entirely of RPE-fated cells. Further, genetic introduction of an FGF9 transgene, which can promote transdifferentiation of RPE to neural retina, into this optic vesicle-inactivated Rx model is unable to rescue neural retinal formation, suggesting Rx is required during the optic vesicle stage to initiate neural retinal specification. Through the use of the Six3-Cre transgenic animal model, we show that inactivation of Rx during optic cup development also impedes retinal progenitor cell proliferation. Due to delayed onset of Cre expression in the Six3-Cre line compared to Foxg1-Cre, this Rx conditional inactivation model allows for the initiation of optic cup morphogenesis and lens induction, but remains unable to specify neural retinal identity. This model indicates Rx may have roles during optic cup development in RPC proliferation, optic cup morphogenesis and lens induction that are separate from its role in neural retinal specification. Remarkably, introduction of the FGF9 transgene into this optic cup-inactivated Rx model can initiate neural retinal specification and formation, but is unable to maintain neural retinal identity. This compound mutant model suggests delayed Rx inactivation (i.e.- prolonged expression) in the developing optic cup confers competence to undergo FGF-mediated neural retinal specification in Rx-depleted cells. We propose a model in which Rx activity is absolutely required to specify neural retinal cell identity until a temporal or developmental threshold is met during the optic cup stage; after which neural retinal cells can be specified via FGF-signaling, independent of Rx activity.;Overall, the studies contained within this dissertation indicate the Rx gene has functional roles in RPC proliferation, optic cup and lens morphogenesis, and neural retinal specification, and also uncovers a novel role for Rx in determining retinal progenitor cell competence to undergo FGF-mediated RPE-to-neural retinal transdifferentiation.

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