斑馬魚cx34.4基因之鑑定與功能分析

連接蛋白（connexin; CX）可在相鄰細胞間形成間隙連接通道 (gap junction)，此通道具有親水性、低選擇性和低電阻等特點，允許離子、水分子及相對分子質量小於分子量1000 Da 在細胞間自由流動，是細胞間完成電子、化學信息交換的重要通道。因此間隙連接在組織和器官中具有重要的調控功能。連接蛋白基因的轉錄、轉譯、修飾、組裝、轉運等過程中出了問題，都會影響細胞間的訊息傳遞。近年來在很多不同 CX 的研究中，也了解到 CX 基因的突變在人類造成許多疾病的發生。先前實驗室針對斑馬魚 cx34.4 基因進行研究。如以半定量 RT-PCR 觀察成魚器官 cx34.4 基因表現，發現表現量最高為皮膚，其次為內耳。從全胚胎原位雜交實驗中發現，內耳的 cx34.4 表達並不明顯，反而在肌肉表現是明顯的。因此為更進一步瞭解 cx34.4 基因在斑馬魚早期胚胎發育時期的表現及功能性的角色。本研究我們利用全胚胎原位雜交的方式觀察基因在胚胎發育時期表現情形，實驗結果顯示 24~96hpf 發現在肌肉、心臟，胸鰭，有訊號的表現。進一步我們將全胚胎原位雜交的胚胎 72hpf、96hpf 做石蠟切片，結果可看到zfcx34.4表現在斑馬魚骨骼肌當中的慢肌。藉由 WISH 和石蠟切片的研究，我們證明了 cx34.4 明顯表現在 24-96hpf 班馬魚的慢肌上。同時本研究也想瞭解 cx34.4 在斑馬魚體內的生理功能，我們利用反股寡核酸 (Antisense Morpholino Oligonucleotides , MO) 顯微注射抑制 cx34.4 基因的功能。觀察其外表形可發現胚胎體軸有嚴重的彎曲、體軸縮短、卵黃形狀異常以及有心臟水腫的現象，而死亡率會隨著 MO 劑量增加而增強。為了確認 MO 對 cx34.4 基因的抑制是專一性的，我們建構了帶有紅色螢光蛋白的 cx34.4 表現質體(cx34.4-TaqRFP)，藉由統計螢光表現率來探討 MO 的專一性，結果發現 cx34.4 的 MO 可以有效的抑制紅色螢光表現，因此我們確認 cx34.4 MO 可以很專一性的 knockdown cx34.4 的基因表現。綜合以上實驗的結果，cx34.4 基因 MO 會專一性抑制cx34.4 後，基因被抑制後會造成肌肉的發育異常和身體扭曲。因此，我們推測 cx34.4 在早期胚胎特別是在慢肌的發育扮演著一個重要角色。

Connexins (Cxs) are homologous four transmembrane domain proteins that are the major components of gap junctions. These gap junctions facilitate direct intercellular communication between adjacient cells, allowing for the transmission of both electrical and chemical signals. Previous studies have showed that the expression pattern of zebrafish cx34.4 was similar to human CX30.3 (GJB4) in HeLa cell model. However, phylogenetic analysis also showed zebrafish cx34.3 gene shared 62% similarity with human CX30.3 gene. During embryogenesis, zebrafish cx34.4 gene was expressed from 1.5 hours post-fertilization (hpf) to 5 days post-fertilization (dpf), especially from 12hpf to 48hpf. The temporal experiment has been conducted in whole larvae from 24 to 48 hpf, in which whole-mount in situ hybridization (WISH) signals were apparently observed in the slow muscle. Morpholino knockdown studies have showed that the cx34.4 morphants displayed a shortened, curved anterior-posterior axis with more severe heart edema phenotype. In this study, we conducted the previous studies to investigate the physiological functions of cx34.4 in the zebrafish embryonic development. Zebrafish cx34.4 transcripts were revealed significantly in slow muscle of the 72-96 hpf embryos and tparaffin-section tissues as previous results. Meanwhile, RT-PCR analysis of the muscle tissues of the 24-96 hpf embryos were consistent with WISH signals. In addition, we established the fusion protein construct Cx34.3-TagRFP that was injected into embryos in the presence or absence of cx34.3-targeting morpholinos. We have found that cx34.3-targeting MO was able to specifically knockdown the expression of TagRFP-tagged Cx34.3 fusion proteins in a dose-dependent manner. Based on above results, zebrafish cx34.4 gene may play an important role in the early embryonic development, especially in the slow muscle.