DEVELOPMENT AND MAINTENANCE OF OUTER SEGMENTS BY ISOLATED CHICK-EMBRYO PHOTORECEPTOR CELLS IN CULTURE

Purpose. To investigate the capacity of isolated chick embryo photorec eptors to develop and maintain outer-segment processes in dissociated cell cultures, in the absence of pigment epithelial and glial cells. M ethods. Cells were obtained from the retinas of embryonic day (ED) 17 chick embryos, after the onset of outer-segment formation in vivo. Aft er a 5- to 12-minute incubation in Ca++- and Mg++-free Hanks' balanced salt solution, neural retinas were freed from other optical tissues, including the pigment epithelium. Retinal cell suspensions were prepar ed by repeated pipeting after mild trypsinization and were grown in se rum-containing medium on a polyornithine-coated substratum. Cell diffe rentiation was evaluated using phase-contrast and transmission electro n microscopes and by autoradiographic analysis of the uptake of putati ve amino acid neurotransmitters, lectin cytochemical analysis, and imm unocytochemical analysis with rod- and cone-specific antibodies. Cells isolated from ED 8 retinas, before the onset of outer-segment formati on in vivo, were also studied. Results. At culture onset, ED 17 cells appeared morphologically undifferentiated and devoid of processes; dif ferentiated features could be detected after 24 to 48 hours in vitro. Photoreceptor cells were the most abundant cell type after 6 days in v itro, followed by nonphotoreceptor multipolar neurons and morphologica lly undifferentiated cells. Autoradiographic analysis showed extensive Na+-dependent uptake of (2,3,4-H-3]gamma-aminobutyric acid in nonphot oreceptor neurons, whereas photoreceptors were labeled predominantly w ith H-3-glutamate. Most of the photoreceptors were labeled with fluore scent peanut lectin and with a sheep polyclonal antibody against bovin e rhodopsin. Subsets of photoreceptors, on the other hand, were immuno reactive with cone- or rod-specific monoclonal antibodies COS-1, OS-2, 50-1B11, or Rho-4D2. Approximately 50% to 65% of the photoreceptors p ositive with these monoclonal antibodies showed a remarkable polarizat ion of immunoreactive materials, which accumulated predominantly, or e ven exclusively, in an outer-segment-like apical process. When viewed on the transmission electron microscope, these outer-segment-like proc esses appeared as distal expansions of the photoreceptor cilium and co ntained disc-like membranous profiles. Outer-segment-like processes al so could be detected using the electron microscope and by immunocytoch emical analysis of cultures of ED 8 retinal cells. Conclusions. After undergoing morphologic dedifferentiation as a result of tissue dissoci ation, isolated retinal photoreceptors, grown in the absence of contac t-mediated cell interactions and of pigment epithelial and glial cells , can regenerate and maintain a highly polarized pattern of structural and molecular organization, including the formation of outer-segment- like processes. The cultures provide an experimental system for the in vestigation of cellular and molecular mechanisms regulating further de velopment and maturation of these photoreceptor structures.