Morphogenesis of the zebrafish retinal pigment epithelium and its involvement in optic cup formation

Abstract

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-06-2019Esta tesis tiene embargado el acceso al texto completo hasta el 28-12-2020Understanding the processes that govern the acquisition of organ shape during development is a main scientific goal, for which the eye has attracted notable attention. The eye primordium forms through the folding of a bi-layered structure, the optic vesicle, giving rise to the optic cup. This event occurs concomitantly with the differentiation of two main cell populations: the retinal pigmented epithelium (RPE) and the neural retina. While becoming specified, both cell types undergo extensive morphogenetic changes that have been proposed to act as driving forces for optic cup folding. This idea has been verified for the neural retina but not for the RPE. Using the zebrafish as a model, in this thesis we have studied in detail the changes that RPE cells undergo and asked if these are required for optic cup folding. To this end, we used a zebrafish line–based on an enhancer of the bhlhe40 gene specifically expressed in the RPE–that allowed the early and specific visualization of RPE cells in vivo. Combining this tool with time-lapse analysis, we demonstrate that RPE specification occurs in a small group of cells located in the dorsal optic vesicle, which then extend to cover the whole surface of the eye. This expansion is largely linked to a dramatic cell shape conversion from a pseudostratified epithelium to a monolayer of flat and then squamous cells, on which cell proliferation has little influence. Indeed, RPE cells reduce their proliferation rate during this morphogenetic change, and this reduction is critical because forced maintenance of cell proliferation impairs morphogenesis. The notable surface increase of the RPE as a whole is instead concomitant with the reduction of the apico-basal axis of individual cells and the expansion of their surface area, so that cells undergo an apparent “stretching”. Supporting this view, individual cell volume is conserved and there is only a minimal increase in the overall RPE volume. Both myosin II activity and microtubule dynamics are required for RPE cell flattening, and this event, in turn, actively contributes to optic cup folding. Our results suggest a model, based on analogies with other epithelia, in which myosin II could confer stiffness to RPE cells whereas changes in microtubule orientation could be instrumental for cell rotation, both making an elongated flat epithelium in a short time. Time-course RNAseq analysis of the gene regulatory network behind early RPE development indicates that RPE specification occurs very early, making the RPE rapidly diverging from the neural retina. Notably, among these genes there are transcription factors and cytoskeletal proteins that could increase RPE stiffness. The bhlhe40 gene itself was found among the up-regulated genes. However, here we show that its function seems dispensable for eye morphogenesis. All in all, this study shows that RPE cell flattening is a cell autonomous process promoted by cytoskeleton dynamics, which contributes to drive the folding of the zebrafish optic vesicle into a cup. It also provides initial cues of its genetic regulation.This thesis was supported by The Spanish Ministry of Science, Innovation and Universities (BFU2016-75412-R and BFU2014-55918-P), from Fundación BBVA (IN[16]_BBM_BAS_0078) and Fundación Ramón Areces. Institutional grants from the Fundación Ramón Areces and Banco de Santander to the CBMSO are also acknowledged