Semaphorin 3f and post-embryonic regulation of retinal progenitors

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.