Multiphoton imaging of chick retinal development in relation to gap junctional communication

Neural progenitor cells in the developing retina extend processes that stretch from the basal vitread surface to the apical ventricular surface. During the cell cycle, the nucleus undergoes interkinetic nuclear migration (INM), moving in a vitread direction during G1, passing through S-phase at its peak and then, on entering G2, returning towards the ventricular surface where it enters M-phase and divides. We have previously shown that individual saltatory movements of the nucleus correlate with transient changes in cytosolic calcium concentration within these progenitor cells and that these events spread to neighbouring progenitors through connexin43 (Cx43) gap junction channels, thereby coordinating the migration of coupled clusters of cells. Disrupting coupling with pharmacological agents, Cx43-specific antisense oligodeoxynucleotides (asODNs) or dominant negative Cx43 (dnCx43) inhibits the sharing of calcium events, reducing the number that each cell experiences and significantly slowing INM. We have developed protocols for imaging migrating progenitor cells by confocal microscopy over relatively short periods, and by multiphoton microscopy over more extended periods that include complete cell cycles. We find that perturbing gap junctional communication not only slows the INM of progenitor cells but also apparently prevents them from changing direction at critical phases of the cell cycle. It also disrupts the migration of young neurons to their appropriate layers after terminal division and leads to their ectopic differentiation. The ability to perform extended time-lapse imaging over 3D volumes in living retina using multiphoton microscopy should now allow fundamental mechanisms governing development of the retinal neuroepithelium to be probed in detail.