The long-term goal of this project is to elucidate the molecular mechanisms underlying photoreceptor cell fate specification in the vertebrate retina. Our recent studies suggest that the genetic program that drives photoreceptor cell fate specification involves neuroD and neurogenin2 (ngn2). The current project addresses four specific questions: (1) Are neuroD and ngn2 expressed at the right place during the right time to be intrinsic factors participating in photoreceptor neurogenesis? Double labeling will be used to determine whether cells expressing ngn2, or neuroD, are proliferating, postmitotic, or both. (2) What is the role of ngn2 in photoreceptor cell fate determination? Does ngn2 specify photoreceptor fate exclusively, or does it also lead to the genesis of other types of retinal cells? Both gain- and loss-of-function analyses will be used to study the function of ngn2, using retroviral-driven misexpression of ngn2 and an active repression construct in the developing chick retina. Retinal pigment epithelial (RPE) cell cultures derived from day 6 chick embryos will be used to examine whether ngn2 can selectively induce RPE transdifferentiation towards a photoreceptor fate. (3) Is neuroD required for photoreceptor production? In other words, will the suppression of neuroD expression and the repression of NeuroD protein function result in a photoreceptor deficit in an otherwise normal retina? Antisense oligonucleotides and Engrailed-mediated active repression will be used to suppress neuroD expression and NeuroD protein function, respectively. Retinal neurogenesis will be analyzed to determine whether attenuation of neuroD expression and function will produce photoreceptor-deficient retinas. (4) Is neuroD sufficient to induce the entire program of photoreceptor differentiation when ectopically expressed in RPE cells that will be provided with an environment for differentiation within the subretinal space of a chick embryo? Transdifferentiating cells will be placed into the subretinal space of chick embryos whose retinal neurogenesis has been manipulated to produce fewer photoreceptor cells. The developmental potential of the microinjected cells will be assessed at the molecular and cellular levels to determine whether these cells can develop into bona fide photoreceptors. These four studies promise new insight into the genetic control of photoreceptor fate specification. Furthermore, they may have clinical implications: neuroD, in conjunction with other factors, may be able to transform cultured RPE cells into photoreceptor cells as a source of cells for photoreceptor replacement therapies.