Knockout of sws2a and sws2b in Medaka (Oryzias latipes) Reveals Their Roles in Regulating Vision-Guided Behavior and Eye Development

Abstract

The medaka (Oryzias latipes) is an excellent vertebrate model for studying the development of the retina. Its genome database is complete, and the number of opsin genes is relatively small compared to zebrafish. Short wavelength sensitive 2 (sws2), a G-protein-coupled receptor expressed in the retina, has been lost in mammals, but its role in eye development in fish is still poorly understood. In this study, we established a sws2a and sws2b knockout medaka model by CRISPR/Cas9 technology. We discovered that medaka sws2a and sws2b are mainly expressed in the eyes and may be regulated by growth differentiation factor 6a (gdf6a). Compared with the WT, sws2a^-/- and sws2b^-/- mutant larvae displayed an increase in swimming speed during the changes from light to dark. We also observed that sws2a^-/- and sws2b^-/- larvae both swam faster than WT in the first 10 s of the 2 min light period. The enhanced vision-guided behavior in sws2a^-/- and sws2b^-/- medaka larvae may be related to the upregulation of phototransduction-related genes. Additionally, we also found that sws2b affects the expression of eye development genes, while sws2a is unaffected. Together, these findings indicate that sws2a and sws2b knockouts increase vision-guided behavior and phototransduction, but on the other hand, sws2b plays an important role in regulating eye development genes. This study provides data for further understanding of the role of sws2a and sws2b in medaka retina development.

Keywords: behavior; eye development; medaka; sws2a; sws2b.

Figure 1

Conservation analysis and expression characterization of sws2a and sws2b in medaka. (A) Sequence alignment of amino acids in sws2a and sws2b (n = 3). (B) Differences of sws2a and sws2b mRNA expression in different tissues of medaka adult. (C) Expression of sws2a and sws2b in larva and adults (n = 6).

Figure 2

Generation and characterization of sws2a and sws2b mutant medaka. (A,B) Design target sites of sws2a and sws2b based on CRISPR/Cas9 technology. Exons are represented by boxes, and single-guide RNAs (sgRNAs) are labeled with blue color in exon. The sgRNA sequences are highlighted in red, and the −4 bp and −274 bp deletions are indicated by sequencing validation. (C,D) Illustration of deduced protein structure of WT and sws2a and sws2b mutants. These numbers represent amino acid positions from the initiation codon. (E,F) The sws2a and sws2b protein tertiary structure prediction of WT and sws2a and sws2b mutants.

Figure 3

The mRNA levels of sws2a and sws2b, and the transcription factors gdf6a and foxq2 in the larval eyes at 6 dph. (A) sws2a. (B) sws2b. (C) gdf6a. (D) foxq2. All data are expressed as the mean ± SEM (n = 6). Vertical bars not sharing the same letter are significantly different (p < 0.05).

Figure 4

Analysis of feeding and the behavioral tests among WT, sws2a^−/−, and sws2b^−/− medaka at the larval stage. (A) Relative levels of ingested Artemia shown in the digestive tract (n = 60). (B) Total length of medaka from 1 to 10 days from first feeding (n = 15/group). (C) The statistical analysis of swimming speed during photoperiod stimulation period in WT, sws2a^−/−, and sws2b^−/− larvae. (D) The swimming speed during the last 2 min dark and the first 10 s of the 2 min light period (* p < 0.05 by Student’s t-test). Vertical bars not sharing the same letter are significantly different (p < 0.05).

Figure 5

The transcriptional levels of opsin- and phototransduction-related genes in the larval eyes at 6 dph. (A) Genes involved in opsin (sws1, rh2-a, rh2-b, rh2-c, lws, gnat2, arr3b, rho, and gnat1). (B) Genes involved in phototransduction (gnb3a, gnb3b, grk7a, and grk7b, and pde6c). The data are represented by mean ± SEM (n = 6). Vertical bars not sharing the same letter are significantly different (p < 0.05).

Figure 6

The results of the qRT-PCR analysis of eye development genes in larval eyes at 6 dph. (A) six3a. (B) sin3b. (C) six6. (D) six7. (E) pax6a. (F) pax6b. All data are expressed as the mean ± SEM (n = 6). Vertical bars not sharing the same letter are significantly different (p < 0.05).