The Function Analysis Of CCSD(Spns2)

S1P(Sphingosine-1-phosphate) is a kind of membrane phospholipids which has significant physiological functions. Besides acting as an intracellular second messenger, S1 P also combines with the S1 P receptor on the cell membrane, and then acts on the downstream signal to produce a series of physiological effects. The studies have shown that Spns2(spinster homologue 2) acts as one of the transporter of S1 P, plays an important role in the migration of zebrafish myocardial precursor cells, and the mutation of Spns2 caused the Zebrafish appearing two hearts. Besides, S1 P also had an important role in the process of migration of immune cells. The circulating S1 P concentration in Spns2-/- mice decreased significantly, resulting in mature T and B lymphocytes cannot migrate to the peripheral circulation and peripheral lymphoid organs.During our experiments, we found that the EGFP homozygous animals of SD-Tg(CAG-EGFP) transgenic rats(green rats) imported from SLC Company in Shizuoka of Japan had abnormal phenotype based eye symptoms, and it also presented recessive heredity. So we inferred that the phenotype of EGFP homozygous transgenic rats may be caused by the EGFP gene insertion mutation in the host cell genome. Because the cornea of the EGFP homozygous transgenic rat lacks the external stimuli perception, the endogenous mutant gene temporarily named for the CCSD(congenital corneal sensory defect). In order to analyze the reason of ocular abnormal symptoms of the EGFP homozygous transgenic(CCSDegfp/egfp) rats, and clarify the functions of CCSD gene, we proceed the following studies:(1) Firstly, we studied the phenotypic analysis of CCSDegfp/egfp rats and the location of CCSD gene in the chromosome. The studies found that: 1) The CCSDegfp/egfp rats in addition to the lack of perception of the cornea responded to the external stimuli, had other abnormal phenotype, including corneal opacity、symblepharon and so on, and the newborn CCSDegfp/egfp rats appeared EOB(eye-open at birth) phenotype. HE staining of the eye paraffin sections showed that CCSDegfp/egfp animals compared to the WT and CCSD+/egfp animal: thickening of the corneal epithelial layer and inflammatory cells in the stroma; the layer of the retinal disorders, particularly the outer nuclear layer. 2) We obtained flanking sequences of CCSD insertion sites in the genome by using nested PCR. The results of the sequence Blast showed that CCSD and Spns2 genes are the same gene. Spns2 gene was located on chromosome 10 of the rat, and EGFP inserted in the first intron of it, thus prevented the transcription. Studies have shown that the insertion mutation of CCSD gene caused the abnormal phenotype of EGFP homozygous transgenic rat- CCSDegfp/egfp, the location study of chromosome found that the EGFP inserted into the endogenous Spns2 gene, causing the deficiency of Spns2 gene expression.Therefore, the CCSD and Spns2 genes were may be the same gene. It indicated that the abnormal phenotype of the CCSDegfp/egfp rats maybe caused by the mutation of Spns2.(2) Our study further compared the phenotype of CCSDegfp/egfp rats with the Spns2-/-mice. First of all, we tested the quantity of immune cells in peripheral blood of CCSDegfp/egfp rats, and found that the number of leukocytes and T、B lymphocytes significantly reduced in its peripheral blood. The phenotype was in accordance with the Spns2-/- mice, and further confirmed that the CCSD and Spns2 were the same gene Secondly, the experiment of the expression vector of Spns2-EGFP fusion protein transfected to He La cells showed that Spns2 clearly expressed on the cell membrane. Finally, we established two anti-Spns2 polyclonal Antibodies: S2(N) and S2(C) by synthesizing N-terminal and C-terminal peptides of Spns2 protein and immunizing rabbits. These two antibodies could identify specifically the Spns2 protein, and some embryonic tissue staining indicated Spns2 expresses in the peripheral blood cells, hepatic cells, and peripheral ganglia.(3) The phenotype effect on Spns2 genetic defect was not clear besides the immune system. Although the EOB phenotype in Spns2-/- mice had been reported, it had not studied the mechanism. so we further studied the molecular mechanism of EOB phenotype by using histological staining、cell culture、immunofluorescence staining and Western blot. During the study we found that: 1) The eyelid margin of CCSDegfp/egfp rats did not move out and fuse like WT and CCSD+/egfp rats; 2) The protein of Spns2 expressed in the leading edge of the WT and CCSD+/egfp rats, but the leading edge of CCSDegfp/egfp rats eyes did not expressed; 3) the deficiency of Spns2 function by reducing the level of EGFR phosphorylation, the intracellular signaling pathways of MEKK1 and c-Jun, caused the EOB phenotype of CCSD+/egfp rats.In summary, our present research found the CCSD and Spns2 are the same gene, and confirmed the previous hypothesis of EGFP gene mutation in the host cell. By studying the EOB phenotype of CCSD+/egfp rats, it was revealed that Spns2 could interfere with cytoskeleton of the skin keratinocyte, and resulted in difficult skin cells emigration. In addition, the study also proved that the CCSDegfp/egfp rats was the good model of Spns2 gene mutation, and provided a very good platform for further studying the function of Spns2 gene.