Background In our efforts to gain a deeper understanding of retinogenesis we are exploring fundamental regulatory mechanisms (i.e. epigenetic modifications) underlying neuronal differentiation in the developing retina. Furthermore, we are focusing on developmental processes characteristic of retinal photoreceptors (establishment of cell polarity and photoreceptor-bipolar cell synaptogenesis) and of ganglion cell axonal migration (optic disc formation). These are key events in establishing the mature tissue architecture and functional organization of the retina. We have selected candidate molecules/pathways that have been shown in recent literature to be involved in developmental processes in other areas of the nervous system and/or in retinal development in other species. Results 1. Epigenetics regulation of neurogenesis in the retina Chromatin remodeling has attracted researchers attention in recent years as a general intracellular molecular mechanism modulating gene expression and regulating retina cell differentiation. DNA methylation has been suggested to be actively involved in establishing retinal progenitor cell competence (e.g., hypometylation could modulate IRBP gene activation during photoreceptor genesis). However, little is known about the influence of methylation on retinal cell fate. A family of three known DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b, partially cooperate to establish and maintain genomic DNA methylation patterns. To investigate the role of DNA methylation in retinal differentiation retina-specific conditional KO mice have been generated in which Dnmt1, Dnmt3a, or Dnmt3b flanked by loxP sites are ablated at subsequent stages of retinal development by either Six3-Cre, Rx-Cre, or Crx-Cre transgenes. Retinal pathology of Dnmt1 2lox/Rx-Cre mice has been completed. Analysis of retinal development after ablation of Dnmt1 2lox by Six3-Cre is in progress and Dnmt1 2lox/Crx-Cre mice have just recently become available for characterization. 2. Establishment of photoreceptor cell polarity Prickle 1 and 2 are tissue polarity genes that are necessary for the establishment of planar cell polarity (PCP) as well as photoreceptor polarity in Drosophila. We hypothesize that, similar to Drosophila, the two genes play essential roles in initiation and maintenance of photoreceptor polarity in mammals. We confirmed by in situ hybridization and immunohistochemistry that Prickle 1 and 2 are expressed in the developing mouse retina. We have electroporated Prickle 1 and shRNAi against Prickle 1 in vivo in the postnatal retina and are investigating the effects of gain and loss-of-function on photoreceptor polarity. Conditional Prickle 1 and 2 double KO mice are being generated and analysis of their phenotype will complement our in vivo electroporation experiments. 3. Optic disc formation Based on protein structure similarity and on expression pattern in the optic disc region, we hypothesized that Frizzled (Fz)5 and 8 orchestrate Wnt-Frizzled signaling as a redundant back-up system and that their disruption might be involved in the insurgence of coloboma. We have characterized Fz8 expression in the mouse retina using a knock-in beta-gal reporter mouse. A detailed electroretinographic analysis of these mice is currently ongoing. Analysis of Fz5 and Fz8 double KO mice will give insights into the redundancy of these gene functions. 4. Synaptogenesis between photoreceptors and bipolar cells We have established novel gene transfer tools in the laboratory to study gene function and directly image neurogenesis in the rodent retina as a model system, with a goal to understand human retinal circuitry generation. Using in vivo electroporation to deliver RNA-mediated interference (RNAi) constructs to knockdown selected genes, we are investigating the roles of candidates previously shown to be regulated by NRL and are evaluating their involvement in synaptogenesis of rod photoreceptor cells with postsynaptic target cells. We have generated reporter fluorescent tags to identify specific stages of retinal neuronal differentiation. Founders of transgenic mice expressing TdTomato specifically in cones are currently being screened. They will be used for flow sorting experiments and for in vivo imaging. They will be breed with different mouse transgenic lines expressing other fluorochromes in a cell-specific manner. Furthermore, Nrl-Cre transgenic mouse lines have been established driving photoreceptor-specific expression of Cre recombinase. These mice will allow conditional targeted modulation of gene expression in photoreceptors, initiating with their genesis. Significance Our investigations on neuronal differentiation will further our understanding of retinal development. They will also complement our efforts to engineer stem cells into photoreceptors and functionally integrate them into the disrupted architecture of degenerating retinas after transplantation (see project EY000473-01).
