探討CC趨化素家族基因在斑馬魚體內之表現與功能

Abstract

CC趨化素(chemokine)是一群具有細胞趨化作用的小分子細胞素(cytokine)家族蛋白，因其有兩個位置相鄰的半胱氨酸殘基為共同結構特徵而命名。CC趨化素的主要作用是趨化細胞的遷移，例如有些趨化素能誘導淋巴細胞到淋巴結，或趨化白血球細胞從血液循環到感染及組織損傷部位。目前，關於趨化素的相關研究皆僅侷限於小鼠和人類，在其他非哺乳類的脊椎動物則無足夠證據證明趨化素的存在。為了進一步了解CC趨化素在發育學上扮演的角色，我們以斑馬魚做為研究的動物模式，並成功複製出其與哺乳類動物CCL1、CCL21、CCL25可能具有相同功能之蛋白的基因，再利用原位雜合染色法觀察ccl21及ccl25在胚胎及成魚體內的表現情形。 實驗結果顯示，ccl21能夠表現在斑馬魚的不同組織中，例如七天幼魚的咽囊、腦神經、血管和腸道，以及成魚的脊髓、血管、血球、腎臟和肝臟。在另一方面，ccl25 則只會在胸腺的原基細胞中表現。此外，當我們用morpholino去抑制ccl25的內生性表現之後，發現T前驅細胞就會因此無法進入胸腺。這些結果顯示，CC趨化素確實存在於硬骨魚中，並具有其重要的功能。

CC chemokines, which are distinguished by two adjacent cysteine residues in a conserved position, can be classified by certain spatial and functional characteristics. Many chemokines and their receptors have been previously demonstrated to be implicated in the emigration of leukocytes from the blood stream to inflammation sites. To date, the chemokine system has only been characterized in mouse and human, albeit fragmentary evidence from some non mammalian species suggests that the chemokine system came into existence early in vertebrate evolution. To gain a further understanding of the potential roles of CC chemokines in the development of a representative teleost, here we cloned the zebrafish orthologues of the mammalian ccl1, ccl21, and ccl25 genes, and described expression patterns of ccl21 and ccl25 in early larval and adult fish by whole mount in situ hybridization. Here, we report that the ccl21 transcripts were observed in various tissues such as pharyngeal pouches, pharynx, blood vessel, intestine, and cranial nerve in 7-dpf-larvae, and in spinal cord, blood vessels, blood cells, kidney, and liver in adult fish. On the other hand, expression of ccl25 was only detectable in the presumptive thymic primordium in the pharyngeal region as early as 36 hpf and persisted in later laval stages. Moreover, we found the recruitment of T-lymphoid progenitor cells was significantly abolished by the morpholino-mediated knockdown of ccl25, which suggests ccl25 is indispensable for colonization of thymus. Interestingly, ccl25 was also maternally transcribed in fish oocytes, and was categorized as cortex class mRNA, which localizes to the cortex of the oocyte during oogenesis. Taken together, our results provide the direct evidence to show the existence of CC chemokine orthologues in zebrafish. These temporally and anatomically restricted expression patterns provide basic foundation for the genetic dissection of their function in this model organism.