In the neural retina (NR), the proliferation of progenitor cells (PCs) and cell differentiation are tightly regulated to ensure the right numbers of each retinal cell type are present in the adult retina. Studies of albino animals (Ilia & Jefferey, 2000) suggest that the retinal pigment epithelium (RPE) may play an important role in the regulation of these processes. In order to better understand the physiological relationship between the NR and the RPE we have examined the role of gap junctions and purinergic receptors in Ca²⁺ signalling in these two tissues.

Retinas (NR and RPE) from embryonic day 4 (E4) to E6 chicks killed by decapitation were dissected in Krebs solution gassed with 95 % O₂ and 5 % CO₂. Using either patch pipettes or sharp microelectrodes, single RPE cells were filled, in control solution or in the presence of the gap junction blocker carbenoxolone, with FITC-Dextran (3000 MW) and Neurobiotin (NB, 256 MW), a combination which marks the injected cell with FITC and any cells coupled to it with NB. Retinas were fixed in 4 % formaldehyde and processed to reveal the NB with Cy3-avidin. Dye-filled cells were imaged on a confocal microscope. Some retinas were subsequently immunostained using an antibody to neuron-specific β-tubulin (TUJ-1) to identify differentiating neurons and re-imaged. Our results show that at E4 RPE cells are coupled via gap junctions both to one another (n = 59 dye fills) and to PCs in the NR (n = 30 dye fills). Coupling to the NR disappears by E6.

Changes in [Ca²⁺]_i in the RPE and NR and the progress of mitosis were simultaneously monitored on the confocal microscope at 36 °C. This was done by first loading retinas with either Oregon Green-BAPTA-AM or fluo-4 AM (10 µM for 1 h) and then, during the imaging process, superfusing them with the vital chromatin dye Hoechst 33342 (2 µM). These experiments show that periodic spontaneous Ca²⁺ signals propagate via gap junctions in both RPE (n = 6 retinae) and NR (n = 8 retinae) and that, albeit rarely, Ca²⁺ signals pass between the two tissues. Ca²⁺ transients propagated as waves that invaded many cells in the RPE while in the NR Ca²⁺ activity was more spatially restricted. The presence of the RPE and purinergic agonists and antagonists had profound effects on the rate of mitosis in the NR that may be mediated by ATP release from the RPE and purinergic receptors present on dividing cells in the NR. Further experiments are required to investigate the role of [Ca ²⁺ ]_i transients and purinergic signalling in the NR and RPE in the regulation of mitosis in the developing retina.

This work was supported by the BBSRC, MRC, Wellcome Trust and the Royal Society.