Primordial germ cells require heparan sulfate to properly migrate and multiply during early embryonic development in the zebrafish Daino rerio

• Main Authors: Ke-Hsuan Wei, 魏可軒
• Other Authors: I-Hsuan Liu
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/37754369467624708039

碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 101 === Heparan sulfate glycosaminoglycan (HS-GAG) is a linear polysaccharide ubiquitously residing on the animal cell surface and the extracellular matrix usually in the form of heparan sulfate proteoglycans. It binds many proteins, such as various chemokines, growth factors and morphogens, and in turn modulates intracellular signal transduction. After specified, zebrafish primordial germ cells (PGCs) migrate following specific routes from their sites of origin to gonadal ridge during early embryonic development. Previous studies showed that HS-GAGs participate in the SDF-1/CXCR4 signaling which plays a key role in the guidance of PGCs migration. Therefore, HS-GAGs may play important role in the migration of PGCs into gonadal ridges. To assess the existence of HS-GAGs in the migrating PGCs, whole-mount immunohistochemistry revealed that cleaved HS-GAGs were found in PGCs whereas intact HS-GAGs were presented in the vicinity of PGCs. To study the potential role of HS-GAG in zebrafish PGCs migration, we overexpressed recombinant hpse1 specifically in PGCs by injecting hpse1-nanos-1 3’UTR mRNA in l-cell stage embryos of zebrafish. In silico analysis, indicated that zebrafish hpse1 is highly conserved throughout vertebrates, while dot blot analysis demonstrated the HS-GAGs degrading activity of the hpse1 we cloned. Whole-mount in situ hybridization showed that PGCs mismigrated and barely formed four clusters at 6 hours postfertilization (hpf). We further calculated the migration distance showed that PGCs disorderly migrate to different position (p<0.01). In addition, observation under the fluorescent microscopy suggested that PGCs in Tg(Kop:EGFP-F-nanos1-3’UTR) transgenic line fail to increase in number after injecting hpse1-nanos-1 3’UTR mRNA in 8hpf, 10hpf and 24 hpf embryos. Quantitative RT-PCR indicated that overexpressing recombinant hpse1 in 10 hpf embryos resulted in the significantly decreased expression level of PGCs marker gene compared to the wild type embryos (p<0.05). TUNEL assay and quantitative analysis showed that overexpressing recombinant hpse1 in 10 hpf embryos resulted in the significantly increased percentage of apoptotic PGCs (p<0.001). These results suggested that zebrafish hpse1 is conserved throughout evolution and indicated that PGCs migration and increasing in number requires HS-GAGs.