斑馬魚低溫適應之機制

Abstract

摘要 有別於對低溫敏感的哺乳動物，外溫動物在體溫降低時仍能存活。研究外溫動物能夠在低溫環境下存活、生長以及生殖的原因，一直是生物學家探討的研究主題。我們將斑馬魚以短期及長期低溫處理，並以生物晶片的方法比較低溫處理與常溫下鰓組織的基因表現相。結果顯示與離子調節相關的基因在低溫下大量被誘導。進一步分析低溫對於鰓離子調節及酸鹼平衡的影響，發現在低溫環境下，斑馬魚鰓上離子細胞內的運輸蛋白及與離子細胞分化相關的轉錄因子，皆大量表現，顯示斑馬魚能夠主動調節生理反應，以應付在低溫環境下離子流失及酸鹼平衡的問題。以鈣離子調節為例，在低溫環境下，上皮鈣離子通道(ECaC)的基因表現大量增加，同時伴隨著鈣離子流入速率增加，表示在低溫適應過程中，這是一種補償的生理反應。在以Phospho-histone H3偵測細胞增生與以TUNEL偵測細胞凋亡的實驗中發現，在低溫處理下，除了細胞分裂速率降低之外，細胞凋亡的情形也降低，顯示斑馬魚面對低溫環境時，可以藉由產生更多的離子細胞以維持鰓組織的功能，藉以適應低溫環境。 我們進一步比較斑馬魚鰓組織及腦組織對低溫的反應。主成分分析法(PCA)的結果顯示，斑馬魚鰓組織在長期及短期低溫處理採取不同的策略，相較於鰓組織，腦部則相對穩定。結合統計、聚類分析及SNEA分析結果發現，在低溫的環境下，鰓組織表現較高的細胞活性及緊迫反應，而腦組織則表現較高的細胞保護相關基因。我們進一步研究在低溫適應中，鰓組織與腦組織之間的交互作用。以生物晶片的方法，我們發現一個與isotocin神經發育、分化相關的轉錄因子Orthopedia的基因表現受到低溫的誘導，進而發現一群與isotocin神經分化相關的基因在低溫環境下皆大量表現。而在注射Orthopedia反義核酸以弱化Orthopedia蛋白質表現後，與離子細胞相關的基因、離子細胞的數目及離子吸收功能皆受到抑制。接著我們以cDNA cloning的方法選殖了二型isotocin的接受器，並測量這二型接受器在鰓上的基因表現，結果發現在低溫的環境下，這二型isotocin接受器的基因表現量均大量增加。這些結果顯示下視丘神經性荷爾蒙isotocin在離子及滲透壓調節機制上，扮演著重要的角色。綜上所述，本研究除了篩選出在斑馬魚鰓及腦組織中受到低溫調節的基因，更提供了分子生理的證據說明鰓及腦組織在低溫適應中的交互作用。

Abstract Ectothermic vertebrates are different from mammals that are sensitive to hypothermia and they have to maintain core temperature for survival. Why and how ectothermic animals can survive, grow and reproduce in low temperature have been for a long time a scientifically challenging and important inquiry to biologists. We used a microarray to profile the gill transcriptome in zebrafish (Danio rerio) after exposure to low temperature. Adult zebrafish were acclimated to a low temperature of 12 °C for 1 day only (1-d) and up to 30 days (30-d), and the gill transcriptome was compared to wild types by oligonucleotide microarray hybridization. The gill transcriptome profiles revealed that ionoregulation-related genes were highly upregulated in cold-acclimated zebrafish. This paved way to investigate the role of ionoregulatory genes in zebrafish gills during cold acclimation. Cold acclimation caused upregulation of genes that are essential for ionocyte specification, differentiation, ionoregulation, acid/base balance, and increased cell number among cells expressing these genes. For instance, epithelial Ca2+ channel (ECaC), an ionoregulatory gene mRNA expression was increased in parallel with the level of Ca2+ influx, revealing a functional compensation after long-term acclimation to cold. Phospho-histone H3 and TUNEL staining showed that the cell turnover rate was retarded in cold-acclimated gills. These results suggest that gills may sustain their functions by yielding mature ionocytes from preexisting undifferentiated progenitors in low-temperature environments. We further adopt a transcript screening approach to compare the cold responses in zebrafish gills and brain. Principle component analysis of the gene expression profiles indicated that gills developed different strategies depending on the durations of cold exposure while brain remained more stable. Based on ANOVA, clustering analysis, and sub-network enrichment analysis (SNEA), gill exhibits higher cell activities and 3 stress responses while brain activates more genes related to cellular protection during cold acclimation. We also extended the study on the interactions between these two organs during cold acclimation. A transcription factor gene, orthopedia (otp), which is associated with isotocin neuronal development, was stimulated by cold in the zebrafish brain. A group of isotocin-related genes were also stimulated by cold in the zebrafish brain. Otp knockdown decreased the mRNA expressions of these ionocyte-related genes, the numbers of ionocytes, and ion uptake functions. Moreover, two isotocin receptor isoforms were cloned, and the mRNA expressions of these two genes were found to be upregulated in zebrafish gills during cold acclimation. These data suggest that the hypothalamic neurohormone, isotocin, plays some roles in the control pathways of iono/osmoregulatory mechanisms in zebrafish. Taken together, the present study not only identify cold induced genes in zebrafish gill and brain but also provide molecular evidences to elucidate the interactions between these two organs during cold acclimation.