始基生殖細胞在斑馬魚胚胎發育過程中需要硫酸乙醯肝素以利遷移和增殖

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

魏可軒(Ke-Hsuan Wei)

指導教授 ： 劉逸軒

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2013年)

https://doi.org/10.6342/NTU.2013.01828

摘要

硫酸乙醯肝素蛋白聚醣 (heparan sulfate proteoglycan, HSPGs) 是由長鏈聚合的硫酸乙醯肝素醣胺聚醣 (Heparan sulfate glycosaminoglycans, HS-GAGs)共價結合核心蛋白所組成，為細胞表面和細胞外基質的主要成分之一。其可與多種因子，例如:生長激素、趨化激素及morphogens等結合，繼而調節細胞內訊息傳遞等行為。早期斑馬魚胚胎發育中，始基生殖細胞 (primordial germ cell, PGC) 於初始發生處依特定路徑遷移至性腺發育的生殖脊。前人研究指出HS-GAGs穩定SDF-1/CXCR4複合體形成，其訊息傳遞亦被證實確能參與始基生殖細胞之遷移。因此，HS-GAGs可能於PGC遷移至生殖脊的過程中扮演重要的角色。
為謹證明HS-GAGs存在於PGC遷移的路徑上，本研究乃進行螢光免疫染色試驗；試驗結果顯示，完整的HS-GAGs存在於PGCs的周圍。 為謹探討HS-GAGs對PGCs遷移的重要性，進一步研究乃針對一細胞期斑馬魚胚進行注射hpse1-nanos-1 3’UTR mRNA，以於PGCs內大量表現特定地乙醯肝素酶 (heparanase1, hpse1)。 鑒於顯微注射試驗開始之前，曾設計的斑馬魚hpse1 (zhpse1)蛋白質序列分析，結果顯示殊與其他多種模式物種諸如：人類、小鼠…等之hpse1頗有高度之同源性，且zhpse1 活性試驗之結果亦顯示吾人選殖的zhpse1確實有水解HS-GAGs的活性。 進一步透過原位雜合試驗 (whole-mount in situ hybridization) 之結果， 顯示HS-GAGs有大量水解導致6 hours postfertilization (hpf) 胚胎 PGCs異常遷移且無法形成規則的四群細胞之情事。因此，吾人乃進一步量化PGCs遷移的距離，結果顯示PGCs確實呈現失序地遷移至異常位置之情事 (p<0.01)。 除此之外，原位雜合試驗結果，顯示HSGAGs大量水解導致8hpf，10hpf和24hpf胚胎內的PGCs數量減少。因此，試驗乃藉由RT-PCR進一步完成量化PGCs特定標誌，結果亦證明在hpse1大量表現之10hpf早期胚中，其PGCs數量確有顯著少於彼等野生型 (wild type) 之胚者。 為謹確認HS-GAGs大量水解導致PGCs數量減少是因為增生能力下降或是細胞凋亡增加，進一步透過DNA 斷裂端標記試驗 (TUNEL assay) 之結果，顯示凋亡的PGCs數量確有顯著多於彼等野生型之胚者。
綜上所述， 鑒於zhpse1在演化過程中頗有高度之同源性，且斑馬魚早期胚中需要HS-GAGs參與以利PGCs遷移和數目增加。

關鍵字

始基生殖細胞 ； 硫酸乙醯肝素醣胺聚醣 ； 細胞遷移 ； 斑馬魚

並列摘要

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.

並列關鍵字

primordial germ cell ； heparan sulfate glycosanimoglycan ； cell migration ； zebrafish

參考文獻

Ara, T., Tokoyoda, K., Sugiyama, T., Egawa, T., Kawabata, K., and Nagasawa, T. (2003). Long-term hematopoietic stem cells require stromal cell-derived factor-1 for colonizing bone marrow during ontogeny. Immunity 19, 257-267.

Barbazuk, W.B., Korf, I., Kadavi, C., Heyen, J., Tate, S., Wun, E., Bedell, J.A., McPherson, J.D., and Johnson, S.L. (2000). The syntenic relationship of the zebrafish and human genomes. Genome research 10, 1351-1358.

Bendel-Stenzel, M.R., Gomperts, M., Anderson, R., Heasman, J., and Wylie, C. (2000). The role of cadherins during primordial germ cell migration and early gonad formation in the mouse. Mechanisms of development 91, 143-152.

Blaser, H., Eisenbeiss, S., Neumann, M., Reichman-Fried, M., Thisse, B., Thisse, C., and Raz, E. (2005). Transition from non-motile behaviour to directed migration during early PGC development in zebrafish. Journal of cell science 118, 4027-4038.

Boldajipour, B., Mahabaleshwar, H., Kardash, E., Reichman-Fried, M., Blaser, H., Minina, S., Wilson, D., Xu, Q., and Raz, E. (2008). Control of chemokine-guided cell migration by ligand sequestration. Cell 132, 463-473.