Characterising Photoreceptor Subtypes in the Turquoise Killifish (Nothobranchius furzeri)

Abstract

Purpose : The turquoise killifish (N. furzeri) is an accelerated ageing model that naturally develops age-related retinal disease and vision loss. Photoreceptor subtypes and organisation have not been investigated in turquoise killifish. We assessed the killifish genome for photoreceptor opsin genes to determine the presumptive photoreceptor subtypes and utilised single-cell RNA-sequencing (scRNA-seq) data to determine whether these genes are expressed in photoreceptor cells.

Methods : The turquoise killifish reference genome was assessed for opsin genes. Identified sequences were then BLASTed against the zebrafish genome to assess homology and potentially misannotated genes. ScRNA-seq was performed on killifish retinas isolated from animals at 6 weeks, 12 weeks, and 18 weeks of age. Dissociated cells were captured with the 10x Chromium system (10X Genomics), with library prep and sequencing (Illumina NovaSeq) performed at Genomics Core Leuven. Phylogenetic analysis was performed with representative fish, bird, reptile, amphibian, and mammalian opsin genes.

Results : Genes for the following opsins were identified in the killifish genome: sws1, sws2, rh2, and rho. No annotated red opsin (lws) genes were found, however red opsin-like genes were identified (ENSNFUG00015014616 and ENSNFUG00015014685). Teleost fishes commonly have multiple genes per opsin type, and it is possible that both potential red opsin genes are represented in the killifish retina. BLASTing the coding and predicted protein sequences against the zebrafish genome revealed that both putative killifish lws genes had high identity to zebrafish lws1 and lws2. However, the scRNA-seq dataset showed that only ENSNFUG00015014616 clustered with cones and is likely the true adult killifish red opsin (lws1) gene. Additionally, the identified killifish genes grouped with the same genes in other species via phylogenetic analysis, supporting that the genes identified are correctly named.

Conclusions : The turquoise killifish genome and retinal transcriptome data indicates that they have rods and four types of cone photoreceptors – UV, blue, green, and red light-sensitive cones. It is possible that additional opsin paralogues will be identified in the future and that gene expression differs between development and adulthood. Future studies will utilise in situ hybridisation and immunofluorescence to validate opsin expression in juvenile and adult killifish.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.