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Semaphorin 3f and post-embryonic regulation of retinal progenitors

Kalifa, Amira1; Hehr, Carrie1; Shewchuk, Katelyn1; Mori-Kreiner, Risa1; Standing, Shaelene1; Halabi, Rami1; McFarlane, Sarah1

Research facility: University of Calgary
Published Jul 07, 2025 on Dryad. https://doi.org/10.5061/dryad.pc866t21r

Data files

Jul 07, 2025 version files 1.64 MB

Abstract

Neural progenitors produce specific cell types that form the circuits of the nervous system. Extrinsic signals regulate both progenitor proliferation and the production of specific neuron types. Where progenitors reside within a progenitor niche determines to which of these signals they are exposed, and thus likely has important consequences on the progeny they produce. Little is known, however, of the signals that determine progenitor location within the niche. Here we show that a member of the Class III family of secreted Semaphorins, Semaphorin3fa (Sema3fa), is required for the orderly arrangement of progenitors with a niche present in the periphery of the larval and adult retina of zebrafish, the ciliary marginal zone (CMZ). We used whole mount and fluorescent slide in situ hybridization to show that CMZ progenitors express mRNAs for various Sema3 receptors, including for nrp2a, nrp2band plxna1. We investigated the function and gene expression of progenitors in wild type fish and in a previously characterized CRISPR/Cas9-generated sema3fa mutant allele (sema3faca304). We found that mutant juvenile fish had a reduced eye size, implicating Sema3fa in the ongoing production of retinal cells by the CMZ. We used EdU labeling, and PCNA and pHH3 immunolabelling to show that larval mutant CMZ progenitors show altered cell cycle parameters. We also performed whole mount in situ hybridization and immunohistochemistry in retinal sections with various mRNA and protein markers of different domains of the CMZ. We found that the spatial organization of functionally distinct progenitors is disrupted. An EdU pulse-chase experiment revealed that the generation of retinal cell types in the appropriate proportions and numbers, as determined by laminar location of cells, was disrupted in the mutant fish. Our data support a model whereby Sema3fa secreted by CMZ progenitors reduces adhesive interactions, which allows for smooth progression of progenitors through the niche, ensuring progenitors receive the correct recipe of extrinsic signals to secure the proper generation of new retinal circuits.

The dataset includes the measured values that formed the averaged data points in the graphs provided in the figures of the manuscript. Each point in the graphs comes from a separate animal (n) and the data is pooled between N=2-4 independent data sets. When areas were measured this was done on retinal sections, either plastic sections of whole mount in situ hybridization samples, or cryostat sections. For cryostat sections, samples were counter-stained with Hoescht to label nuclei. These sections came from the central retina that contained a lens. Two-four images/eye/larva of the central retina, with a lens, were collected. To normalize area measurements we measured the area of the CMZ, as defined by the peripheral edge of the inner plexiform and outer plexiform layers evident either in plastic sections, or as defined by the lack of Hoescht label in cryostat sections.

For the RT-qPCR data the values are from independent data sets, performed on mRNA isolated from 30 surgically isolated eyes. Three technical replicates were performed/data set for each primer set.