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 a..., Methods
Animals: Larvae were used from the respective in-crosses of wild type (WT) Tupfel Long Fin (TL) fish, sema3faca304/ca304 zebrafish, and for the latter a corresponding WT line generated from the same clutch that gave rise to the specific sema3faca304/ca304 fish line (Halabi et al., 2021). For whole mount in situ hybridization, pigment synthesis was inhibited by adding 1-phenyl-2-thiourea (PTU) to the E3 solution at 12-24 hpf.
Eye Size Measurements: Live sema3faca304 and WT siblings were imaged from a dorsal orientation at 10 days post fertilization (dpf). Separate sets of one-month old fish were imaged from a lateral orientation. The areas of the pigmented eyes of larvae (dorsal view) and juveniles (lateral view) were measured in a masked fashion and normalized to the nose to swim bladder length and the size of the head (measured to the back of the gills), respectively.
Sema3fa Overexpression: We engineered a sema3fa heat shock construct, hsp70:sema3fa:p3E-MTpA in pdestTol2CG2 wi..., # Dryad dataset

Dataset DOI: [10.5061/dryad.pc866t21r](10.5061/dryad.pc866t21r)

## Description of the data and file structure

**Figure 2 (Data):** Comparison of WT and *sema3faca304 *zebrafish eyes and CMZ

Figure 2E - Size of eyes normalized to body length (nose to posterior swim bladder). Measurements of the area of the eye of live 10-11 dpf larvae from a dorsal view. Eye area was normalized to the size of the larva. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).

Figure 2F - Size of eyes, measured from a lateral view, normalized to the head size (nose to front of gills) at 1 month. Data is pooled from 2 independent data sets. Raw data (Excel) and averaged data (Prism).

Figure 2I-K - Cumulative EdU labelling at 72 hpf for 1, 2,4,6 and 12 hours;12 um frozen transverse 72 hpf retinal sections immunolabeled for PCNA and EdU and Hoescht counter-stained. Area of PCNA expression (I) and area of intense PCNA (J) expression represented as a percen...,