Cell behaviour during optic vesicle morphogenesis in medaka

Vertebrate eye morphogenesis starts with the bilateral evagination of optic vesicles from the forebrain. A failure of evagination leads to the complete absence of eyes. Despite a good knowledge about patterning and retina differentiation, the mechanism underlying evagination remained largely unknown. Studies on optic vesicle morphogenesis and its underlying cellular basis were missing. I therefore used an in vivo imaging approach in medaka fish to investigate the cellular movements and dynamics of early eye formation. Early medaka embryos were not amenable to in vivo imaging due to contractile movements that cause them to rotate in the chorion or in the embedding medium. I first established an in vivo imaging set up for medaka that was the basis for the further 4D analysis of eye morphogenesis. This analysis shows that optic vesicle morphogenesis requires the modulation of the morphogenetic behaviour of anterior neuroectodermal cells within the eye field during late gastrulation, before evagination is evident. While prospective telencephalic cells lateral to the eye field converge towards the midline, retinal precursor cells are retained in their medial movement. Together with the ventrally and laterally directed movement of medial eye field cells, a widened domain in the forebrain is formed that primes evagination. The importance of these movements is demonstrated in eyeless embryos that harbour a mutation in the homeobox transcription factor Rx3 that is specifically expressed in the eye field. Eyeless embryos fail to form optic vesicles. Mutant retinal precursor cells converge towards the midline to the same extent as anteriorly and laterally located telencephalic progenitors and eventually form a neural keel-like structure. The wide domain in the forebrain is absent. Moreover, Rx3 function is required for the modulation of cell shape changes and correct polarization of cells. In wild-type, retinal precursor cells elongate mediolaterally as they move laterally into the growing optic vesicles. In eyeless the cells at the lateral border of the neurula stage eye field adopt a columnar epithelium-like shape reminiscent of the neural tube. They surround medial cells of the neurula stage eye field that remain rounded, indicating a defective polarization of mutant cells. Mosaic analysis showed that optic vesicle evagination can be rescued cell-autonomously by wild-type cells. Detailed 4D time-lapse analysis revealed that the rescue is due to the modulation of medial directed movement and individual cells migrating actively from medial positions laterally in the optic vesicles. This demonstrates that optic vesicle evagination depends on the locally coordinated migration of single cells rather than the movement of a tissue as whole