Intrinsic mechanisms governing retinal progenitor cell biology: Retinal homeobox transcriptional regulation and the function of forkhead transcription factors during eye development

Understanding the development and maintenance of Retinal Progenitor Cells (RPCs) is critical to understanding normal and disease processes within the neural retina. To understand RPC biology, it is important to understand the transcriptional regulation of known intrinsic regulators, and continue to identify new RPC expressed genes to demonstrate their function during eye development. The studies presented in this work address the transcriptional regulation of the Xenopus laevis Rx gene product, Rx2A, and function of two newly recognized RPC genes, FoxO3 and FoxM1.;To further the understanding of transcriptional regulation in RPCs, we characterized the Rx2A promoter in transgenic embryos. Both the distal portion and the proximal portion of the Rx2A promoter are sufficient for expression of a GFP transgene in the developing eyes. We identify a highly conserved element in the distal region of the Rx2A promoter (UCE). Within UCE, an OTX, SOX and POU site act as cis-elements to coordinately specify proper gene expression in the developing eye. We show that the activity of the proximal promoter is dependant on a forkhead-binding element (FBE). In addition, we have shown that the distal region containing the UCE can cooperate with the FBE to maintain robust Rx expression throughout all stages of eye development. The work associated with the transcriptional regulation of Rx furthers our understanding of how this primary retinal transcription factor is regulated. This is applicable to the understanding of RPC development since Rx is one of the first eye field transcription factors expressed in the anterior neural plate as RPCs are specified.;The identification of the FBE within the Rx2A promoter led to further investigation of the involvement of the forkhead family of transcription factors in vertebrate eye development. We present a discussion of current data regarding the expression and function of this family of transcription factors during eye development, and we present the expression pattern of 5 forkhead transcription factors (FoxG1, FoxN2, FoxN4, FoxM1 and FoxP1) in the maturing X. laevis retina. These forkhead gene products have not been previously described in the maturing retina of X. laevis.;We chose to pursue studies of FoxO3 and FoxM1 in the developing neural retina to further the understanding of RPC regulation by transcription factors of the forkhead family. These two factors were chosen for co-current studies for the following reasons: (1) neither gene product had been ascribed a role in developing RPCs, (2) their known functions suggested they act in opposing ways with regards to the cell cycle, and (3) due to their expression patterns both FoxO3 and FoxM1 serve as candidate factors to regulate the Rx2A promoter through the FBE.;To define the role of FoxO3 during vertebrate eye development, we overexpressed FoxO3 RNA in the anterior neural plate of X. leavis embryos. FoxO3 overexpression results in embryos with small eyes. The small eye phenotype is a result of decreased proliferation, induction of apoptosis, and changes in RPCs gene expression. The phenotype can be exacerbated by introducing a threonine to alanine mutation at a conserved PI3K phosphorylation site, which produces a constitutively nuclear form of FoxO3. The changes in gene expression suggest that FoxO3 can function to delay the differentiation of RPCs, although they are properly specified. The data supports our original hypothesis regarding FoxO3 as cell cycle antagonist with the ability to alter the differentiation capacity of RPCs.;To investigate the function of FoxM1 in developing RPCs, we performed loss-of-function studies using morpholino oligonucleotides (MO). FoxM1 knockdown in the anterior neural region of X. laevis results in embryos with slightly smaller eyes and clear defects in retinal lamination. Our data reveals that FoxM1 is not necessary for the specification of RPCs, and suggests that differentiation into the Muller glia, and rod photoreceptor lineages is possible with reduced levels of FoxM1.;The studies presented concern the transcriptional regulation of the Rx gene product, Rx2A, and a role of FoxO3 and FoxM1 during RPC development. Collectively, they represent an advancement in the knowledge regarding two important intrinsic mechanisms governing RPC development: transcriptional regulation and transcription factor function.