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

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

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.This work is supported by the following grants: (I) To J.-R.M.-M.: From the Spanish Ministry of Science, Innovation, and Universities (MICINN): BFU2017-86339P with FEDER funds, MDM-2016-0687 and PY20_00006/Junta de Andalucía. (II) To O.B. Australian Research Council (ARC) Discovery Project (DP190103852). (III) To F.-J.D.-C.: Andalusian Ministry of Health, Equality and Social Policies (PI-0099-2018). (IV) To P.B.: BFU2016-75412-R with FEDER funds; PCIN-2015-176-C02-01/ERA-Net Neuron ImprovVision, and a CBMSO Institutional grant from the Fundación Ramón Areces. (V) To both J.-R.M.-M. and P.B.: BFU2016-81887-REDT, as well as Fundación Ramón Areces-2016 (Supporting L.B.)

Kinase PLK1 regulates the disassembly of the lateral elements and the assembly of the inner centromere during the diakinesis/metaphase I transition in male mouse meiosis

PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes

Genetic control of cell geometry in epithelia: the morphogenesis of the vertebrate optic cup as experimental paradigm

Resumen del póster presentado al 11th Meeting of the Spanish Society for Developmental Biology, celebrado en Girona (España) del 19 al 21 de octubre de 2016.The orchestration of the morphogenetic mechanisms involved in organ formation during embryogenesis and tissue homeostasis entails a precise genetic control of cellular shape. The development of complex organs requires coordination among molecular mechanisms specifying the identity of each cellular domain and downstream effectors. Using available tissue models, only few effector molecules have been identified and the role of many morphogenetic genes has not been explored. Furthermore, little is known about how these effector molecules integrate into broader developmental gene networks. Here we focus on the development of the optic cup as a model to identify key effector genes determining cell and tissue architecture in zebrafish. During eye development an undifferentiated mass of precursor cells from the neural plate, morphologically and molecularly indistinguishable, differentiates into the neural retina (NR) and the retinal-pigmented epithelium (RPE). In the frame of a few hours, the eye specification network bifurcates into mutually exclusive developmental programs for the NR and RPE and each domain displays a distinctive morphology. These differential cell geometries will condition the optic cup invagination. We have isolated these populations by FACS, to perfom an RNAseq analysis of the bifurcating gene networks (GRNs) that establish the identity of these domains. By interrogating eye GRNs, we aim to identify downstream genes operating directly on basic cell morphological properties. This work will contribute to the identification and analysis of novel components of the machinery driving optic cup morphogenesis, many of which will be causative genes for the most common hereditary malformations of the eye.Peer Reviewe

Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis

The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flatteningSpanish AEI (BFU2014-55918-P to FC; BFU2016-75412-R with FEDER support, RED2018-102553-T and PID2019-104186RB-100 to PB), BBVA Foundation (N[16]_BBM_BAS_0078 to FC) and Fundacion Ramon Areces-2016 (to PB)

Image4_Kinase PLK1 regulates the disassembly of the lateral elements and the assembly of the inner centromere during the diakinesis/metaphase I transition in male mouse meiosis.TIF

PLK1 is a serine/threonine kinase with crucial roles during mitosis. However, its involvement during mammalian male meiosis remains largely unexplored. By inhibiting the kinase activity of PLK1 using BI 2536 on organotypic cultures of seminiferous tubules, we found that the disassembly of SYCP3 and HORMAD1 from the lateral elements of the synaptonemal complex during diakinesis is impeded. We also found that the normal recruitment of SYCP3 and HORMAD1 to the inner centromere in prometaphase I spermatocytes did not occur. Additionally, we analyzed the participation of PLK1 in the assembly of the inner centromere by studying its implication in the Bub1-H2AT120ph-dependent recruitment of shugoshin SGO2, and the Haspin-H3T3ph-dependent recruitment of Aurora B/C and Borealin. Our results indicated that both pathways are regulated by PLK1. Altogether, our results demonstrate that PLK1 is a master regulator of the late prophase I/metaphase I transition in mouse spermatocytes.</p