Dynamic Polarization of Rab11a Modulates Crb2a Localization and Impacts Signaling to Regulate Retinal Neurogenesis

Abstract

Interkinetic nuclear migration (IKNM) is the process in which pseudostratified epithelial nuclei oscillate from the apical to basal surface and in phase with the mitotic cycle. In the zebrafish retina, neuroepithelial retinal progenitor cells (RPCs) increase Notch activity with apical movement of the nuclei, and the depth of nuclear migration correlates with the probability that the next cell division will be neurogenic. This study focuses on the mechanisms underlying the relationships between IKNM, cell signaling, and neurogenesis. In particular, we have explored the role IKNM has on endosome biology within RPCs. Through genetic manipulation and live imaging in zebrafish, we find that early (Rab5-positive) and recycling (Rab11a-positive) endosomes polarize in a dynamic fashion within RPCs and with reference to nuclear position. Functional analyses suggest that dynamic polarization of recycling endosomes and their activity within the neuroepithelia modulates the subcellular localization of Crb2a, consequently affecting multiple signaling pathways that impact neurogenesis including Notch, Hippo, and Wnt activities. As nuclear migration is heterogenous and asynchronous among RPCs, Rab11a-affected signaling within the neuroepithelia is modulated in a differential manner, providing mechanistic insight to the correlation of IKNM and selection of RPCs to undergo neurogenesis.

A single-cell guide to retinal development: Cell fate decisions of multipotent retinal progenitors in scRNA-seq

“…These studies have led to numerous hypotheses for candidate gene function in the specification and differentiation of individual retinal cell types, catalyzing the identification of transcription factors Atoh7 ( Math5 ), Crx , and Nrl , for RGC, photoreceptor, and rod photoreceptor specification, respectively ( Brown et al, 2001 ; Chen et al, 1997 ; Mears et al, 2001 ). Furthermore, lineage tracings of individual progenitors have provided insights into the ‘stochastic’ nature of cell fate decisions ( Gomes et al, 2011 ; He et al, 2012 ) and correlated cell features with cell division modes ( Baye and Link, 2007 ; Cayouette and Raff, 2003 ; Clark et al, 2012 , 2021 ; Cohen et al, 2010 ; Kechad et al, 2012 ; Lacomme et al, 2016 ; Malicki, 2004 ). Additionally, the implementation of genetic reporters ( Brzezinski et al, 2011 ; Katoh et al, 2010 ; Rowan and Cepko, 2004 ; Zeng and Sanes, 2017 ) and gain- and/or loss-of-function studies have integrated our understanding of transcription factor landscapes required during development for lineage restriction and proper cell type specification.…”

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

“…A semi-quantitative assay for receptor-mediated endocytosis in zebrafish neuroepithelial tissue Bulk endocytosis has been observed previously in the zebrafish neuroepithelium using the lipophilic dye FM4-64 (Clark et al, 2011), and we have also visualised fluid-phase endocytosis, as described above. However, in order to directly visualize receptor-mediated endocytosis, a specific ligand is required.…”

“…Within these cells, the arrangement of endocytic compartments mirrors that seen in mammalian epithelial cell types. Early endosomal compartments are enriched towards the apical pole, whilst late endosomal compartments are more dispersed throughout the cell body, as previously described (Clark et al, 2011), and visualised live here using EGFP-tagged Rab5c and Rab7, respectively (Fig. S1, see also Fig.…”

“…In zebrafish, the developing brain and its associated ventricular system are easily accessible and can be imaged by microscopy (Fame et al, 2020). In addition, transgenic zebrafish lines to examine the neuroepithelial endocytic pathway have been established and characterised, whilst additional studies have demonstrated the accessibility of the hindbrain ventricle for injection of fluorescent dyes (Clark et al, 2011;Fame et al, 2020;Lowery and Sive, 2005). Here, taking advantage of these features in zebrafish, we set out to establish a semi-quantitative endocytic uptake assay to investigate endocytosis and endocytic trafficking within the developing neuroepithelium.…”

Assessment of endocytic traffic and Ocrl function in the developing zebrafish neuroepithelium

“…Bound ligands internalised into endosomes from the neuroepithelial cell surface can also influence neuroepithelial cell behaviour. The asymmetric segregation of the Notch ligand DeltaD within Rab5 positive SARA endosomes has been shown to be a key neuroepithelial cell fate determinant (Coumailleau et al, 2009; Kressmann et al, 2015; Nerli et al, 2020; Zhao et al, 2021) whilst the positioning of Rab11 recycling endosomes can control developmental signalling within the zebrafish retinal neuroepithelium and Drosophila sensory organ precursor cells (Clark et al, 2020; Emery and Knoblich, 2006; Furthauer and Gonzalez-Gaitan, 2009). Underscoring the importance of understanding how post-Golgi trafficking events regulate neuroepithelial cell function are mutations in multiple trafficking regulators that are linked to CNS abnormalities in mammals.…”