Identification Of Ly6Superfamily Members With A Special Emphasison Function Of PSCA-like In Zebrafish

A wide range of Ly-6superfamily members have been identified to date in avariety of species. For example, in humans, there exist at least45Ly-6members, andin fruitfly,36new Ly-6members have been documented. Ly-6superfamily proteinsare characterized by a conserved Ly-6/uPAR （LU） domain, which is about70to100amino acids long and contains8to10cysteine residues that form a defineddisulfide-bonding pattern. Ly-6superfamily members are classified into two families:glycophosphatidyl inositol （GPI）-anchored proteins and secreted proteins.Thecysteine positions and the disulfide bonds are well conserved, whereas the otheramino acid sequences are highly variable between these Ly6proteins. Although mostsequences of Ly6proteins are poorly conserved, and even for the LU domain there islow homology besides the conserved cysteines, the LU domains of Ly6proteins sharesimilar spatial structure which looks like a “three-fingers” structure. Ly6membershave been shown to execute diverse function in different biological processes, asexamplified by the immunomodulatory factor CD59, the neuron receptor modifyingfactor LYNX1, and the regulating cell adhesion and migration factor uPAR.In addition,many Ly6genes are associated with some human diseases. For example, SLURP1is alate marker of epidermal differentiation and is absent in Mal de Meleda, geneticvariation in PSCA is linked with susceptibility to diffuse-type gastric cancer andbladder cancer, and inherited complete deficiency of CD59is a cause of paroxysmalnocturnal hemoglobinuria. Moreover, a few Ly-6members have been shown to beinvolved in embryonic development because four Drosophila Ly-6proteins,Boudin,Crooked, Coiled and Crimpled, are required for the formation of septate junction, andzebrafish Lypd6is required for Wnt8-mediated patterning of the mesoderm andneuroectoderm by enhancing Wnt signaling. In spite of these reports, the expressionand function of most Ly6members remains poorly understood.Zebrafish as a powerful animal model can be used for functional study of the role of Ly6genes in embryogenesis. The sequences of members of mammalian Ly-6superfamily known were used as queries against zebrafish database. The resultsrevealed that there were27Ly6members in zebrafish. The bioinformatic analysisshowed that zebrafish Ly6proteins had typical features of Ly6family. All zebrafishLy6proteins contain a signal peptide in N-terminal and one or two LU domains. Onlyfive Ly6proteins were secreted proteins, while all the others were GPI-anchoredproteins. In addition, ten Ly6proteins comprised two LU domains, wheras theremaining Ly6members contained only one LU domain. Zebrafish Ly6genesclustered on the chromosome, in particular, seven Ly6genes which contain two LUdomains clustered on the chromosome17. Whole mount in situ hybridization resultsshowed that zebrafish Ly6genes were specifically expressed in different locationsduring early embryo development, suggesting that zebrafish Ly6genes play multipleroles. We found that a Ly6family gene XP₇06427was expressed in enveloping layer（EVL） at gastrula stage, then expressed in epidermis, pharyngeal arch and pectoral finat later stages; both XP₀02664005and XP₀02664937genes were highly expressedin lateral line system neuromasts; and XP₀03200124and XP₀02665293genes wereexpressed in pigment cell and ionocyte, respectively. Interestingly, a Ly6genePSCA-like was found to be initially expressed in pineal gland at14somites,in dorsalretina at24hpf, and in ventral retina at36hpf; subsequently the expression ofPSCA-like diffused towards the outer retina（ONL） according to the zebrafish“neurogenic wave”. At50hpf, PSCA-like was expressed in the outer retina, and then,PSCA-like was expressed in photoreceptor from3dpf to adult zebrafish.Overexpressed Otx5can induce expression of PSCA-like and conversely knockdownof Otx5inhibited the expression of PSCA-like in pineal gland. In silico analysisshowed that the regulatory sequence of PSCA-like contained multiple binding sites ofOtx5, suggesting that PSCA-like may be directly regulated by Otx5. In addition, theexpression of PSCA-like was not affected by light. Knockdown of PSCA-like bymorpholino oligonucleotides caused the delayed development of retinal photoreceptor.A PSCA-like mutant zebrafish was established using GoldenGate TALEN. In the adultmutant, arrangement of retinal photoreceptors was irregular, photoreceptors assembled abnormally and Gnat2protein was mislocalized in photoreceptor, althoughmutant embryos developed normally. Finally, tight junction OLM was disturbed.Collectively, these results indicate that PSCA-like was necessary for photoreceptordevelopment and maintenance.