重新審視硬骨魚鰓的離子調節機制：活體功能分析的新進展

Abstract

離子調節器官的分子與生理功能分析在魚類生理學研究是至關重要的。硬骨魚的鰓富含離子細胞，是最主要之離子調節器官，負責運輸鹽類及排除體內多餘的酸和含氮廢物。由於成魚鰓的結構複雜，且鰓腔外有鰓蓋阻隔，過去並沒有任何方法可以直接、活體測定鰓的功能，而先前離子調節之活體功能分析多著重於皮膚表皮(胚胎、仔魚時期之離子調節器官)，並推測皮膚表皮的離子調節機制與鰓相似。然而，在缺乏鰓上活體功能分析的情況下，這樣的推論或許並不合適。本研究旨在重新審視硬骨魚鰓上的離子調節機制，不僅希望在鰓上建立活體電生理測定平台來探討相關運輸機制，更預期透過單細胞轉錄體分析找到未知且重要的離子運輸蛋白或調控因子，進而活體鑑定其功能。 第一章主要將掃描式離子選擇電極技術(SIET)應用到斑馬魚的鰓上，建立活體電生理測定平台。藉由此技術，首次證明了鰓上排氫、排銨與鈉吸收的功能，也意外發現斑馬魚四對鰓的功能強弱不同。此外，搭配各類抑制劑處理，證實了離子細胞中氫離子幫浦(VHA)、鈉氫交換蛋白(NHE)、鈉氯共轉運蛋白(NCC)的功能，奠定了斑馬魚鰓上排酸與鈉吸收的路徑。第二章主要透過單細胞轉錄體分析，於斑馬魚和印度青鱂魚鰓上分別發掘到新的運輸蛋白(SLC26a11)和轉錄因子(ERRγ2)專一地表現在離子細胞上。在酸適應後之成年斑馬魚中，以vivo-morpholino弱化其SLC26a11的表現會降低其鰓的排氫能力，說明SLC26a11參與斑馬魚的酸適應機制。而透過morpholino弱化青鱂魚胚胎ERRγ2的表現會減弱其離子細胞吸收鈉、氯離子的能力，以及NCC1之表現，說明ERRγ2能藉由調控運輸蛋白的表現來協助青鱂魚在淡水的適應。第三章主要進一步探討第一章在斑馬魚四對鰓功能差異的發現。藉由表現量分析、細胞計數與外部型質測量，發現四對鰓存在離子調節的功能性分功，其第一、二對鰓主要負責排氫、排銨的功能，第三、四對鰓則主要負責鈉離子的吸收，而在面對酸逆境時，四對鰓與排酸相關的運輸蛋白表現皆會上升至一致，這說明四對鰓皆具有很好的可塑性，更暗示其存在不同的離子調節機制。綜合以上，本研究不僅為魚類生理學提供一個全新的平台去測定鰓的功能，更闡述了鰓上複雜的離子調節機制與深入探討的重要性。

Molecular and physiological analyses in ionoregulatory organs are essential for fish physiology study. Teleost gills rich in ionocytes are the most important ionoregulatory organs for salt transport and excretion of acid and nitrogenous waste. Owing to adult gills exhibiting a complex structure and the opercular covering outside of the gill cavity, there were no direct methods for in vivo functional assays in the gills. Previous in vivo evidence on ion-transporting function was mostly obtained in the skin (ionoregulatory organ in embryonic/larval stages), and it was speculated that adult gills and embryonic/larval skin share similar ionoregulatory mechanisms. However, without in vivo functional evidence from the gills, this presumption is too premature. The present study aims to revisit branchial ionoregulatory mechanisms in teleosts. Not just to establish an in vivo electrophysiological platform on the gills and investigate branchial functions, the present study expects to explore novel transporters or regulators essential for ion regulation using single-cell transcriptome analysis, and further identifies their functional roles. In Chapter 1, the scanning ion-selective electrode technique (SIET) was applied to zebrafish gills for establishing a platform for in vivo electrophysiological detection. For the first time, the present study proved the functions of H+ excretion, NH4+ excretion, and Na+ uptake. By treating with different inhibitors, the functions of several transporters, vacuolar-type H+ ATPase (VHA), Na+/H+ exchanger (NHE), and Na+-Cl- co-transporter (NCC) were further proven, which consolidated the pathways for acid excretion and Na+ uptake in zebrafish gills. In Chapter2, a novel transporter (SLC26a11) and transcription factor (estrogen-related receptor γ2, ERRγ2) were explored using single-cell transcriptome analysis, which are specifically expressed in gill ionocytes of zebrafish and Indian medaka, respectively. Knockdown of SLC26a11 (by vivo-morpholino) decreased branchial H+ excretion in acid-acclimated zebrafish, suggesting that SLC26a11 is involved in acid acclimation mechanisms in adult zebrafish. Knockdown of ERRγ2 in medaka embryos reduced Na+ and Cl- uptake capacities of ionocytes, and also down-regulated the expression of NCC1, suggesting that ERRγ2 controls the expression of specific transporters for FW acclimation. In Chapter 3, the functional differences between four gill arches (discovered in Chapter 1) were further investigated. By expression analysis, cell counting, morphological measurement, the present study demonstrated a functional division between gill arches. The 1st – 2nd gill arches are major for H+ and NH4+ excretion, and the 3rd – 4th gill arches for Na+ uptake. When encountering acidifying environments, expression of the transporters related to acid excretion within four gill arches would be elevated and maintained at similar levels, which means the four gill arches possessing a high plasticity and also implies distinct ionoregulatory mechanisms. Taken together, the present study not only provides a new detecting platform for branchial functions in fish physiology, but also elucidates the complexity of ionoregulatory mechanisms in the gills and the importance of further investigation.