Characterization of F-spondin in Japanese flounder (Paralichthys olivaceus) and its role in the nervous system development of teleosts.

F-spondin was originally isolated from the developing embryonic floor plate of vertebrates, secreting numerous kinds of neuron-related molecules. The protein performs a positive function in nervous system development, which is attributed to the high conservation of F-spondin protein, an extracellular matrix (ECM) protein in several species. However, its precise function remains unknown, especially in marine fish. In this study, the F-spondin of Japanese flounder (Paralichthys olivaceus). was cloned, and its expression pattern and structural characteristics were analyzed. The 2421bp-long cDNA ORF of PoF-spondin was obtained and divided into 14 exons spread over 61,496bp of the genomic sequence. Phylogenetic analysis showed that PoF-spondin was actually the ortholog of the human spon1 gene and shared high identities with other teleost spon1a genes. Quantitative RT-PCR analysis showed that PoF-spondin was maternally expressed, and transcripts were present from one-cell stage to hatching stage, peaking at tailbud stage. Tissue distribution analysis indicated that PoF-spondin was detectable mainly in the gonads (especially in the ovary) and the brain. Whole mount in situ hybridization analysis revealed that the PoF-spondin transcription distributed throughout the cleavage of the ball in the early stage and expressed at a high level in the floor plate of the trunk at tailbud and pre-hatching stages. Furthermore, the expression of genes related to nervous system development (spon1b, foxo3b, and foxj1a) was significantly increased after the injection of PoF-spondin into the embryos of wild-type zebrafish. Furthermore, PoF-spondin significantly suppressed the expression of the chordamesoderm marker gene ntl, increased the expression of otx2/krox20, ectoderm mark genes, and left the expression of dorsal mesodermal marker gene gsc unaffected at 50% epiboly stage in zebrafish. In short, our results suggest that PoF-spondin functions in the development of the teleost nervous system.

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