Expression Analysis of Visual Arrestin gene during Ocular Development of Olive Flounder (Paralichthys olivaceus)

Abstract

Olive flounder (Paralichthys olivaceus) is one of the commercial important flatfish species in Korea. The ocular signal transduction pathway is important in newly hatched flounders because it is closely involved in the initial feeding phase thus essential for survival during the juvenile period. However, the study of gene expression during ocular development is incomplete in olive flounder. Therefore we examined the expression analysis of specifically induced genes during the development of the visual system in newly hatched flounders. We searched ocular development-involved gene in the database of expressed sequence tags (ESTs) from olive flounder eye and this gene similar to arrestin with a partial sequence homology. Microscopic observation of retinal formation corresponded with the time of expression of the arrestin gene in the developmental stage. These results suggest that arrestin plays a vital role in the visual signal transduction pathway of the retina during ocular development. The expression of arrestin was strong in the ocular system during the entirety of the development stages. Our findings regarding arrestin have important implications with respect to its biological role and evolution of G-protein coupled receptor (GPCR) signaling in olive flounder. Further studies are required on the GPCR-mediated signaling pathway and to decipher the functional role of arrestin.

Keywords: Arrestin; GPCR signaling; Gene expression; Ocular development; Olive flounder..

Fig. 1

Development of visual system formation of the olive flounder (Paralichythys olivaceus). The experiment was fertilized eggs of olive flounder from the tank, oxygen supply and maintains a temperature of 20°C. Schematic dorsal view of the brain and eyes of a flat fish with key features labeled. (A), (B) Photograph was shown during after hatching for 1 day and 3 days, Magnification: ×400. The following structures can be identified in development olive flounder larvae under dissecting stereomicroscopes: tel, telencephalon; di, diencephalon; with the frontally located epiphysis; tgm, tegmentum; and tectal ventricle; mhb, midbrain-hindbrain boundary. hb, hindbrain; spc, spinalcord; es, eyesac; with lens; rtn, retina. Scale bar, 300 μm.

Fig. 2

The arrestin gene was deduced by bioinformatics analysis for the olive flounder. (A) The amino acid sequence alignment was deduced for the olive flounder arrestin gene. Arrestin sequences are aligned with the several spices. GenBank accession numbers of each species are as follows: Tilapia (Oreochromis niloticus) XP_003452818.1, Zebra Mbuna (Maylandia zebra) XP_004569535.1, Fugu (Takifugu rubripes) XP_003976489.1, Rice fish (Oryzias latipes) NP_001116373.1, Zebrafish (Danio rerio) NP_956853.1. Clone Eyes3 D06 is indicated by Asterisk (*). It is indicates the invariant and conserved residues in Arrestin. The percentages in parentheses indicate the overall amino acid identities. (B) The 2-dimensional protein structure of arrestin protein was predicted in amino acid sequences. Prediction was made by using the TMRPres2D tool of the University of Athens of Greece.

Fig. 3

Expression of arrestin gene induces in the development stage. (A) The experiment was flounder fertilized eggs from the tank, oxygen supply and maintain a temperature of 20°C. Embryo and larva were harvested during hours post fertilization (HPF) and days after hatching (DAH) for the indicated periods. The RNA extracted and analyzed by RT-PCR method. (B) The mRNA expression of arrestin was analyzed by real-time PCR. Different letters over each bar with the standard deviation represent significant differences one group according to unpaired matched comparisons (p<0.05).

Fig. 4

Expression analysis of arrestin gene induces from several tissues in the olive flounder. Experiments are examined with various periods of adult fish. (A) Arrestin gene expression from the segmental tissue (B: brain, M: muscle, F: fin, E: eye, L: liver, S: spleen, K: kidney, G: gill, N: negative) for the olive flounder at 90 days, 10 months and 2 years of age were analyzed using RT-PCR. (B) The mRNA expression of arrestin was analyzed by real-time PCR. Different letters over each bar with the standard deviation represent significant differences one group according to unpaired matched comparisons (p<0.05).