Abstract
Purpose :
The zebrafish raifteirí, (raf) model was identified in an N-ethyl-N-nitrosourea mutagenesis screen. raf-/- present with severly compromised visual behavioral. We sought to characterize the raf phenotype and identify the causative gene.
Methods :
Electroretinography measure visual capacity. Light, electron and confocal microscopy analyzed retinal histology. RNA isolated from eyes of 5 days old raf larvae (n=5). The RNA samples were processed using ‘TruSeq Stranded mRNA Sample Preparation’ protocol. The cDNA libraries were sequenced by Illumina Sequencer. Data obtained were processed and ran through the Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) pipeline. Genomic DNA was isolated from 20 pooled, 5 days old raf larval samples and whole genome sequencing (WGS) and bioinformatics analysis performed.
Results :
raf mutants have an average of 0.2 saccades/min OKR response while siblings have an average of 15 saccades/min. Histological analysis of raf-/- retina revealed the photoreceptor layer was thinner and failure to develop the rod and cone outer segments properly. Additionally, gaps were present in the dorsal and ventral side of the lens and vacuoles were observed within the RPE. Immunostaining with rod and cone photoreceptor specific markers indicated that rod photoreceptors were largely absent in the retina and cone photoreceptors were aberrantly formed in the mutants. RNA sequencing (RNAseq) was performed to identify the ‘raf‘ mutant allele and to understand the molecular mechanisms of disease. The MMAPPR pipeline, was ran to call out single nucleotide polymorphisms (SNPs), insertions and deletions (indels). The results were suggestive of the mutant allele being localized to chromosome 23. Approximately 40 potential candidate genes in chromosome 23 have been identified by variant calling and are currently being validated. A total of 118 differentially expressed genes (with a log foldchange of < and ≥ 2 and an adjusted p value of 0.05) were identified in raf samples. WGS sequencing revealed 477,656 SNPs, indels and structural variants inclusive of intronic regions, which are currently being assessed.
Conclusions :
raf mutants are visually impaired and fail to form proper photoreceptor outer segments, indicative of potentially a ciliary mutation. The raf mutant allele is localized to chromosome 23, further analysis needs to be undertaken to identify raf mutant allele.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.