Molecular Physiological Study on Na+ uptake/acid-base regulation mechanisms in Japanese medaka (Oryzias latipes)

廣鹽性的硬骨魚類主要藉由調節其滲透壓的平衡來面對環境中的鹽度、離子濃度，及pH值變化。在先前的研究中，成魚的鰓部以及胚胎時期的表皮中，有一群特化的離子細胞，稱為富含粒線體細胞(Mitochondria-rich cells, MR cells)，負責離子平衡機制。在斑馬魚的研究中發現，不同型態的離子細胞具有不同的運輸蛋白，以進行不同的離子之運輸；並且在胚胎發育過程中有轉錄因子會參與離子細胞的分化。然而，通道蛋白的調節功能，以及運輸機制的研究中還有許多未明瞭的議題。本篇的研究以日本種稻田魚做為模式來探討鈉吸收及酸鹼平衡機制。 鈉氫交換蛋白-2/3(Na+/H+ Exchanger 2/3, NHE2/3)和氫離子幫浦(H+-ATPase, HA)等運輸蛋白以及離子細胞相關轉錄因子(forkhead box I 3, FOXI3; glial cell missing 2, GCM2)的序列已選殖。且經由原位雜交與免疫染色結果得知，可利用鈉鉀幫浦(Na+/K+-ATPase)標定稻田魚所有的離子細胞，這些離子細胞分為兩群，一群表現氫離子幫浦、另一群表現鈉氫交換蛋白。利用即時定量聚合Euryhaline teleosts have to cope with the osmotic and ionic gradients of aquatic environments with diverse salinities, ion compositions, and pH values. Previous studies suggested that mitochondria-rich (MR) cells are specialized ionocytes, which are the main site responsible for ion regulation mechanisms in fish gills and embryonic skin. However, there are still many unclear issues of how transporters are functioning in fish ion regulation mechanism. The present study used Japanese medaka as a model to examine the roles of the related ion transporters in fish Na+ uptake and acid/base balance mechanisms. hree ion transporters (Na+/H+ exchanger 2/3, NHE2/3; and V-type H+-ATPase and two transcription factors (forkhead box transcription factor I 3, FOXI3; and glial cell missing 2, GCM2) related to ionocyte differentiation have been successfully cloned from Japanese medaka. Using double in situ hybridization/immunocytochemistry, Na+/K+-ATPase (NKA) and NHE were colocalized in MR cells, H+-ATPase (HA) was localized in a part of MR cells. In qRT-PCR experiments of adult medaka gills, after acclimation to acidic freshwater slc9a2 (NHE2), slc9a3 (NHE3) and atp6v1a (H+-ATPase) were up-regulated, while gcm2 was down-regulated; on the other hand, slc9a3, atp6v1a, and foxi3 were up-regulated during acclimation to low sodium water. aken together, NHE and HA may play some roles in sodium uptake/acid-base regulation pathways in medaka, and the 2 transcriptional factors, foxi3 and gcm2, may participate in the ionocyte differentiation pathway.中文摘要........................................................................................................................1bstract..........................................................................................................................2ntroduction..................................................................................................................4echanisms of sodium uptake and acid-base regulation in freshwater fish..................4omparisons of the ionocytes between species.............................................................5edaka as a model to study sodium uptake and acid-base regulation mechanisms......7ims of studies...............................................................................................................8aterials and Methods..............................................................................................10xperimental animals...................................................................................................10cclimation experiments..............................................................................................10mbryos collection..............................................................................................11reparation of total RNA..............................................................................................11everse transcription-RCR (RT-PCR) analysis............................................................12olecular cloning and sequencing analysis.................................................................13ranslational knockdown with antisense morpholino oligonucleotides (MO) ...........13uantitative reverse transcription-PCR (qRT-PCR)................................................... 14hole-mount in situ hybridization..............................................................................14hole-mount immunocytochemistry...........................................................................16tatistical analysis........................................................................................................17esults.........................................................................................................................18on transporters..........................................................................................................18. Protein expression patterns of ion transporters on medaka embryos......................18. mRNA expression patterns of ion transporters on medaka embryos.......................19. Triple labeling of NKA, HA protein and slc9a3 mRNA on the medaka gill...........20. Phylogenetic analysis of medaka genes..................................................................20. Density of NHE3 cells in embryo acclimated to different Na+ concentration and acidic waters.................................................................................................................21. Effects of environmental pH and Na+ concentration on NHE2/3 and H+-ATPase mRNA expression in medaka gills...............................................................................21ifferentiation related transcriptional factors........................................................22. Expression of foxi3 and gcm2 on medaka embryos................................................22. The effects of Loss-of function experiments of foxi3 and gcm2 on NKA+ cells….22. Effects of environmental pH and Na+ concentration on foxi3 and gcm2 mRNA expression in medaka gills...........................................................................................23iscussion....................................................................................................................24eferences....................................................................................................................30ables............................................................................................................................34igures..........................................................................................................................3