The mature human retina represents a complex neuronal organ composed of various highly differentiated cell types including two distinct classes of photoreceptors, rod and cone cells. These various retina cells develop from precursors in a defined sequential maturation process. Control of this process is of interest in the understanding of normal and abnormal retina development. Analysis, however, has been hampered by the lack of adequate in vitro model systems. Retinoblastoma (RB), an eye tumor in children, can be propagated in vitro and we have shown that RB expresses cone but not rod cell specific genes such as the red/green color photopigment genes and the transducin T omega gene. RB, therefore, represents the only human retina derived cell type grown in vitro with differentiated properties. We propose to isolate transcription controlling elements of the human red and green gene using cultured RB cells as a test system. We will identify pututative promoter-enhancer fragments cloned into chloramphenicol acetyl transferase (CAT) expression vectors and transfect them into RB13 and RB22 cells which express the red/green transcript. Enhancer will be mapped by deletion mutations and their DNA sequence will be determined. Enhancer elements will be used in expression constructs containing various reporter genes to establish transgenic mice. Tissue specific expression of the cis-acting elements will be determined in fetal and mature retina using a beta-galactosidase transgene. The role of an immortalizing oncogene expressed in cone precursor cells will be studied using the adenovirus E1A gene. Growth pattern, cell lineage development and eventual tumor formation will be studied. We will to isolate E1A immortalized retina cells with differentiated properties in culture. Finally, the role of cone precursors in cell lineage development of the fetal retina will be studied by ablation using a Diphteria-Toxin A (DT-A) gene under the control of cis-acting elements from the red or green gene. A strong enhancer element within a 6.0 kb DNA fragment upstream the mRNA start site of the green pigment gene has already been identified using various CAT constructs transfected into RB22 cells, therefore, demonstrating the validity of the proposed approach. This proposal is designed to utilize transcription controlling elements from the red and green pigment gene to establish transgenic mice in which cone cell specific gene expression and cell lineage development of the mammalian retina can be studied.
