ZFIN ID: ZDB-FIG-210324-49
Halabi et al., 2021 - Semaphorin 3fa Controls Ocular Vascularization From the Embryo Through to the Adult. Investigative ophthalmology & visual science   62:21 Full text @ Invest. Ophthalmol. Vis. Sci.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Fish:
Anatomical Terms:
Stage Range: Long-pec to Adult
PHENOTYPE:
Fish:
Observed In:
Stage Range: Prim-25 to Day 6

Fig. 1 Sema3fa is expressed by the RPE and inner retina of larval and adult zebrafish. (A–D) sema3fa RNA ISH in whole mount (A) and retinal sections (B–D). ISH signal is detected in the retinal pigment epithelium (RPE) during development (A–C) and into adulthood (D). In larval and adult retina, sema3fa is also expressed strongly in the inner nuclear layer (INL) and CMZ, with expression in the outer nuclear layer (ONL) observed in the adult. (E–G) Expression at 7 dpf of mRNA for the known Sema3fa receptor nrp2b (F,G) but not nrp2a (E) by the endothelial cells of the retinal artery that runs alongside the optic nerve head (yellow arrow) and the choroid vessels lining the back of the eye (black arrows). Note that to minimize the pigment of the RPE, retinal sections were either from larvae treated at 24 hpf with 1-phenyl-2-thiourea (A–C, E–G), or were bleached (D). (H) Chromosomal overview of the sema3fa locus targeted by CRISPR/Cas9 mutagenesis to exon 1 (guide RNA: A1-3) and primers used to identify the mutation. UTR: untranslated region; F: forward primer; R: reverse primer. (I) Schematic representation of WT and premature stop codon mutant proteins. sema3faca304 fish have a 2 bp deletion (dashes) and produce a predicted product of 76 aa. Dashes represent missense amino acids (miss.aa). (J) RT-qPCR of sema3fa mRNA levels in WT and sema3fbca304 embryos at 48 hpf suggest nonsense mediated decay of mRNA transcript (N = 3). Error bar represents standard error of the mean (SEM). (K) Western blot of protein isolated from 5-6 dpf WT and sema3faca304 mutant fish processed with a custom-made antibody against zebrafish Sema3fa. The antibody recognizes a protein of the appropriate size for Sema3fa in WT fish, which is absent in the mutants. Loading control is ß-actin. (L, M) Lateral views of 72 hpf WT (H) and sema3faca304 (I) fish. (N) The ratio of eye width to anterior-posterior body length, measured along the antero-posterior axis, at 36, 48, and 72 hpf, arguing that there are no gross abnormalities in body or eye growth in the sema3fa heterozygote (n = 10) and homozygote (n = 4) mutant embryos as compared to WT (n = 10). Scale bars: A: 100 µm; B: 15 µm; L: 1 mm.

Gene Expression Details
Gene Antibody Fish Conditions Stage Qualifier Anatomy Assay
nrp2a WT standard conditions Days 7-13 retinal neural layer central region ISH
nrp2b WT standard conditions Days 7-13 hyaloid vessel ISH
Days 7-13 optic choroid vascular plexus blood vessel endothelial cell ISH
sema3fa WT standard conditions Long-pec whole organism RTPCR
Protruding-mouth retinal inner nuclear layer ISH
Protruding-mouth retinal pigmented epithelium ISH
Day 5 to Day 6 whole organism WB
Days 7-13 ciliary marginal zone ISH
Days 7-13 retinal inner nuclear layer ISH
Days 7-13 retinal pigmented epithelium ISH
Adult retinal inner nuclear layer ISH
Adult retinal pigmented epithelium ISH
sema3faca304/ca304 standard conditions Long-pec whole organism RTPCR
Day 5 to Day 6 Not Detected whole organism WB
Antibody Labeling Details No data available
Phenotype Details
Fish Conditions Stage Phenotype
sema3faca304/ca304 standard conditions Prim-25 eye normal size, normal
Long-pec eye normal size, normal
Long-pec whole organism sema3fa expression decreased amount, abnormal
Protruding-mouth eye normal size, normal
Day 5 - Day 6 whole organism sema3fa expression absent, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Investigative ophthalmology & visual science for permission to reproduce figures from this article. Please note that this material may be protected by copyright. Full text @ Invest. Ophthalmol. Vis. Sci.