鈣聯蛋白(calnexin)弱化抑制斑馬魚側線發育

側線是兩生類與魚類特有的器官，用以感知外界水流的方向與速率.此器官是由側線神經母細胞所組成，其中包含了纖毛細胞與支持細胞。為了研究可能參與在側線發育的候選基因，我們利用斑馬魚資料庫網站(ZFIN)搜尋在側線神經母細胞所表現的基因,並發現有一群鈣離子結合蛋白特定表現在這個位置。鈣離子是一個熟知的二級傳訊者,並參與許多細胞活動包括細胞黏合與移動。其中我們對calreticulin(鈣網蛋白) 和 calnexin(鈣聯蛋白) 特別感興趣. 鈣網蛋白和鈣聯蛋白為內質網中伴隨蛋白(chaperon)之一，其主要功能在於幫助新生成醣蛋白的疊合與品質管制。加上其可與鈣離子結合之特性，鈣聯蛋白可影響多種細胞活性如內質網壓力反應，細胞凋亡，細胞黏著及胞器生成等。而此等細胞活性為動物早期胚胎發育所必需，因此我們認為鈣網蛋白和鈣聯蛋白可能在側線發育過程扮演一重要之角色，並選用模式生物斑馬魚研究其功能。斑馬魚的鈣網蛋白和鈣聯蛋白基因與人類的基因具有高度相似度，藉由全固定原位雜合我們發現兩基因的mRNA廣泛表現於早期斑馬魚胚中，而在受精後36小時開始表現在側線神經母細胞初始細胞中，爾後更專一地表現在側線神經母細胞。藉由顯微注射反義吗啉寡聚核苷酸（morpholino oligonucleotides, MO）降低鈣網蛋白和鈣聯蛋白在斑馬魚胚之表現量,我們發現隨著鈣聯蛋白MO劑量的提高，利用各種染劑檢査側線神經母細胞叢、每單位內的神經母細胞細胞及纖毛，我們發現其數量均有逐漸減少的情形，而這些缺陷可以藉由同時注入體外合成的鈣聯蛋白mRNA 所部分回復。但這樣的情形在減少鈣網蛋白表現量減弱的斑馬魚中並沒有明顯的效果。再者以原位雜合觀察纖毛細胞的記號基因eya1與支持細胞的記號基因claudinb亦顯示兩者皆因鈣聯蛋白之弱化而降低，此等結果顯示鈣聯蛋白可能參與側線纖毛細胞與支持細胞形成的過程。而SDF1a/CXCR7已知為引導神經母細胞細胞初始細胞移動的訊息，然 SDF1a mRNA之表現卻因鈣聯蛋白之弱化而片段化，其顯示鈣聯蛋白可能經由影響SDF1a/CXCR7訊息來調節斑馬魚側線細胞之形成及發育過程。

關鍵字

側線；鈣聯蛋白；鈣網蛋白；側線神經母細胞

並列摘要

Lateral line is a mechanosensory system in fish and amphibian to detect local water flow and pressure. This system comprises a series of neuromast clusters, which are formed by sensory hair cells and support cells. To further investigate candidate genes which may be involved in lateral line development, we screened genes which are expressed in the lateral line of zebrafish by examining available expression patterns in the Zebrafish Information Network (ZFIN, http://zfin.org). We found a group of calcium binding proteins expressed specifically at the lateral line system. Calcium is a well known second messenger that exerts many cellular activities, including cell adhesion and migration, via its binding proteins. At these candidate genes we are particularly interested in calreticulin and calnexin. These genes are an endoplasmic reticulum (ER) calcium binding protein and known chaperon to ensure proper folding and quality control of newly synthesized glycoproteins that allow it to participate in cellular activities like ER stress, apoptosis, cell adhesion and organogenesis. Those cellular activities are essential for embryogenesis. Therefore, we hypothesized that calnexin may play a role during lateral line development. We have identified zebrafish calreticulin and calnexin with high similarity to their human homologs. Whole-mount in situ hybridization (WISH) and RT-PCR analyses revealed that calreticulin and calnexin mRNAs are ubiquitously expressed during early embryogenesis in zebrafish. WISH analysis further showed that the expression domain of calreticulin and calnexin was first observed at the migrating primordium of lateral line at 36 h post fertilization and the calreticulin and calnexin mRNAs were specifically localized to the neuromasts. Knockdown of calnexin by antisense morphonino oligonucleotides (MOs) resulted in a dose-dependent reduction in numbers of neuromast clusters, neuromasts per cluster and hair cells in lateral line that could be rescued by over-expressing respective mRNAs. WISH analysis by neuromast maker eya1and neuromast support cell marker claudinb, both showed reduced cluster numbers in calnexin morphants. It indicated that calnexin may be involved in both neuromast and support cell deposition. But these phenotypes were not obvious in the calreticulin morphants. Furthermore, the abolition of calnexin expression resulted in fragmented SDF1a expression, which implies that loss of calnexin might interfere with the SDF/CXCR7 cue for neuromast primodium migration. These results suggest that calnexin is essential for neuromast formation during lateral line development.

並列關鍵字

lateral line ； neuromast ； calreticulin ； calnexin ； promodium ； deposition

參考文獻

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國際替代計量

鈣聯蛋白(calnexin)弱化抑制斑馬魚側線發育

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