Retinal degeneration diseases pose great health risks. Currently, the effective cures and prevention methods remain elusive. Identification of genes and genetic processes important for the formation and survival of retinal neurons will lead to the development of prevention drugs targeting these genes and processes and to develop approaches to replace the degenerated retinal neurons by transplantation or regeneration from neuronal stem cells. The long-term objective of this proposal is to understand the role of Brn-3 POU-domain transcription factors in retinal neurogenesis and to identify and characterize the roles of their downstream effect genes. The three brn-3 genes, brn-3a, brn-3b and brn-3c, share a highly conserved functional POU-domain and their expression during neurogenesis and in adult is largely overlapping. Targeted mutagenesis studies in mice have shown that deletion of each brn-3 genes lead to unique neuronal phenotypes with the loss of a selected group of neurons. Intriguingly, the uniqueness of knockout phenotypes in each brn-3 mutant closely correlated to its distinctive spatiotemporal expression pattern. In retina, expression of brn-3 genes is mostly overlapping in retinal ganglion cells (RGCs) and the onset of brn-3b expression precedes those of brn-3a and brn-3c. Deletion of brn-3b results in the terminal differentiation failure and apoptosis of approximate 70% of RGCs. Loss of brn-3c has similar effects on a small percentage of RGCs. To further understand the roles of brn-3 genes in retinal neurogenesis and to explore the common molecular mechanisms of brn-3 function, in this application, we will: 1) use the transgenic approach to test the functional equivalence of brn 3 genes. The coding regions of brn-3a and brn-3c will be used to replace brn-3b in the knock-in experiments. The ability of knock-in brn-3 genes to rescue the retinal phenotypes associated with brn-3b knockout will be examined; 2) determine the role of brn-3a in the development of RGCs. Defects in retina of brn-3a-null mice or mice null for brn-3b and brn-3a will be analyzed; 3) identify and characterize in vivo the role of brn-3b downstream genes. We have shown that loss of Brn-3b leads to the diminished RGC expression of transcription factors Gfi-1 and LMO2. Gfi-1 is a zinc-finger protein required for the survival of inner ear hair cells. Recently, we have identified LMO2 as the first transcription factor expressed in the GCL of developing retina in a low-nasal-to-high-temporal gradient. Transgenic approaches will be used to investigate their roles in retinal development, particularly the axon pathfinding and survival of RGCs.
