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標題: | 有機滲透壓調節對斑馬魚胚胎循環系統及腦室發育上之影響 The Role of Organic Osmoregulation on the Circulation System and Brain Ventricle Development in Zebrafish Embryos |
作者: | Chun-Lin Ko 柯均霖 |
指導教授: | 劉逸軒(I-Hsuan Liu) |
關鍵字: | 斑馬魚,胚胎發育,有機滲透壓調節,LRRC8A,SWELL1,VRAC,VSOAC, zebrafish development,organic osmoregulation,LRRC8A,SWELL1,VRAC,VSOAC, |
出版年 : | 2017 |
學位: | 碩士 |
摘要: | 目前已知滲透壓之調控途徑乃透過細胞內外之滲透質移動所調控。而滲透壓之調控除了對細胞體積之調節有著重要的貢獻以外,近幾年也有許多研究指出無機離子滲透壓調節機制對於胚胎之腦室型態發生有著密切的影響。然而,過去之研究指出有機滲透調節機制對於滲透壓之調節有著同樣重要的貢獻。因此我們便推測有機滲透調節機制亦參與胚胎發育過程中之型態發生。目前已知之有機滲透調控機制,主要透過體積調節陰離子通道 (volume-regulated anion channel,VRAC) 將牛磺酸 (taurine) 等有機滲透質送至細胞外來達到滲透壓調節之目的。過去本實驗室之研究已證實,若將斑馬魚胚胎中牛磺酸之關鍵合成酵素 (cysteine sulfinic acid decarboxylase) 調降表現時,會引起胚胎循環系統之發育異常,此一結果也間接地支持了有機滲透調節機制同樣影響著胚胎發育的假設。在近幾年之研究已證實,哺乳動物之細胞中 Leucine-rich repeat containing 8A (LRRC8A) 為構成與維持 VRAC 活性不可取代的重要組成蛋白,並因此將其另名為 SWELL1。
為了進一步證實有機滲透壓調節機制在胚胎型態發生中所扮演的角色,我們於斑馬魚之基因庫中找到了兩個可能為 swell1 之基因,並將其命名為 swell1a 與 swell1b。透過反轉錄聚合酶鏈鎖反應 (reverse transcription polymerase chain reaction) 與全胚胎原位雜合染色 (whole-mount in situ hybridization) ,可以發現此二基因於胚齡 0 至 12 hpf (hours post fertilization) 皆會廣泛的表現於整個胚胎體,且於 24 hpf 開始會集中表現於腦室 (ventricle),及近心臟之周邊組織。為解析此二基因於胚胎發育過程之功能,嗎啉基寡核苷酸 (morpholino oligonucleotide,MO) 被設計合成用以調降各基因之表現,結果皆可於 24 hpf 的胚胎中,觀察到腦室以及循環系統之型態發生出現明顯的異常。為了對腦室之發育異常進行定量比較,我們使用 TRITC-dextran 進行腦室成像並對腦室大小進行定量,結果顯示 swell1a 與 swell1b 被調降的胚胎,其腦室空腔顯著小於正常之胚胎。而針對 32 hpf 胚胎進行血管造影的結果亦顯示,swell1a 與 swell1b 受到調降的胚胎,其循環系統及血管網絡之發育顯著出現異常。此外,當 swell1a 與 swell1b 被調降時,胚胎之心跳速率也會顯著小於正常之胚胎。為了確認這些現象的確是因 swell1a 與 swell1b 被調降所導致,swell1a 與 swell1b 之 mRNA 與 MOs 被共同注射於胚胎,結果顯示此二基因之 mRNA 能隨劑量增加而顯著提升正常胚胎之比率。此外,因 VRAC 主要活動特性之一乃藉由輸出牛磺酸來進行滲透壓調節,為了驗證我們所觀察到之胚胎異常表現為VRAC滲透壓調控途徑受到干擾所導致,我們便於 E3 培養水中額外添加牛磺酸 (13mM),以回復胚胎細胞滲透壓之平衡,結果亦發現添加牛磺酸可顯著提升正常胚胎之比率。 綜觀上述之結果,我們的研究指出在斑馬魚基因體中存有兩個對應為哺乳類 LRRC8A 的同源基因,且兩基因皆為母系信息 (maternal message) 基因,並廣泛表現於斑馬魚胚胎發育階段,此二基因似乎功能相似,皆參與有機滲透壓調節機制。而若有其中一基因缺失,可能因總量不足致使有機滲透壓調節機轉受限,將會導致斑馬魚胚胎之腦室及循環系統之型態發育產生異常。 The osmoregulation pathway is so far considered to be regulated by movement of osmolytes inside or outside of a cell. Despite the contribution of osmoregulation to the cell volume regulation, inorganic osmoregulation has also been reported to be attributed to the embryonic cerebral ventricular morphogenesis. Notwithstanding, previous studies has revealed the equivalent significance of organic osmoregulation to inorganic one. Therefore, we presume organic osmoregulation also participates in the morphogenesis of embryonic developmental process. As far as is known, the mechanism of organic osmoregulation is to transfer organic osmolytes such as taurine extracellularly through volume-regulated anion channel (VRAC). In the past, we have demonstrated that knockdown of cysteine sulfinic acid decarboxylase, a key rate limiting enzyme in de novo synthesis of taurine, resulted in aberration of embryonic cardiovascular system in zebrafish, which indirectly supports the hypothesis that organic osmoregulation also plays an important role on embryonic development. In recent years, leucine-rich repeat containing 8A (LRRC8A) has been found in mammals serving as the indispensable component for the volume-regulated anion channel (VRAC) activity and thereby named “SWELL1”. To further confirm the roles of the mechanism of organic osmoregulation on embryonic morphogenesis, two potential swell1 genes were found in zebrafish data base and named swell1a and swell1b. Through reverse transcription polymerase chain reaction and whole-mount in situ hybridization, the RNA expression of two genes could be detected ubiquitously in 0-12 hours-post-fertilization (hpf) embryos and specifically in cerebral ventricles and parenchyma around the heart. To clarify the functions of the two genes in the embryonic development, morpholino oligonucleotides (MO) were used to manipulate gene expression. The result shows that knockdown of either swell1 gene lead to obvious cerebral ventricular and cardiovascular disorder in 24 hpf zebrafish embryos. To quantify the abnormality of the cerebral ventricular development, cerebral ventricles were visualized with TRITC-dextran and the sizes of the visualized cerebral ventricles were quantified for comparison. The outcome demonstrated smaller cerebral ventricles in the groups treated with swell1a or swell1b MO in comparison with normal zebrafish embryos. Angiography at 32 hpf also demonstrated aberrant development of circulatory system and vascular network on swell1a or swell1b knock-down embryos. In addition, swell1a or swell1b knock-down group also showed slower heart beat rate. To further confirm that the developmental aberration was due to the down regulation of swell1a or swell1b, swell1a or swell1b mRNA was co-injected with their corresponding swell1a or swell1b MO and found capable of raise the proportion of normality dose dependently. Furthermore, as one of the features of VRAC is regulate osmotic pressure by releasing taurine, to verify the aberrancy of knock-down embryos is due to disruption of pathway of VRAC osmoregulation, extra taurine (13mM) was added to E3 medium to remain isotonicity in embryonic cells. We found addition of taurine is able to rescue the normality proportion in knock-down embryos. In sum, our study introduces two zebrafish genes corresponding the LRRC8A gene in mammals. They are both maternal massage genes, widely expressing in the embryonic development. They seemly have similar functions in participation of organic osmoregulation. Loss of either gene results in serious cerebral ventricular and cardiovascular disorder due to the limitation of organic osmoregulation. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77928 |
DOI: | 10.6342/NTU201702700 |
全文授權: | 有償授權 |
顯示於系所單位: | 動物科學技術學系 |
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