Commitment of cells to form a retina occurs during early embryogenesis as the neural plate is regionalized into specific domains. Once specified to become retina, the precursor cells proliferate as stem cells until signaled to stop dividing and differentiate. Retinal stem cells are multipotent, giving rise to all of the cells of the pigmented and neural retina, and are capable of participating in retinal regeneration in lower vertebrates. We have chosen to study genes likely to control retinal stem cell fate in an effort to determine the molecular mechanisms that control the establishment and proliferation of the retina. Several homeobox-containing trasncriptional regulatory genes are important for the proper formation of the eye during embryonic development, including Pax6, Chx10 and Lhx2. A new family of retinal homeobox genes, Rx, is expressed in a spatial and temporal pattern consistent with a role in retinal stem cell specification and proliferation. The function of the Rx gene is crucial for proper eye formation, as overexpression causes hyperproliferation of the neural retina and retinal pigment epithelium, and targeted deletion of the Rx gene in the mouse eliminates optic vesicle formation and results in the birth of eyeless mouse pups. Our proposed studies are aimed at identifying the mechanisms by which Rx regulates early mammalian eye development. We have designed three strategies in different experimental systems to study the role of Rx during retinal formation, proliferation and differentiation. Using gene expression studies in mice carrying different ocular mutations, we will investigate whether Rx is necessary for the initial specification of retinal cell fate. An analysis of later Rx function during retinal stem cell proliferation and differentiation will be performed using molecular techniques in primary mouse retinal cultures and human retinoblastoma cells. Finally, we propose to screen DNA samples from anophthalmic patients for mutations in the human Rx gene. These experiments will elucidate the role of the Rx homeobox gene in human eye development. The proposed studies have important implications for our basic understanding of ocular organogenesis, and may contribute to medical treatment strategies for retinoblastoma, retinal degeneration and anophthalmia.