Rh蛋白在斑馬魚胚胎皮膚的功能
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2013
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Rh蛋白是在脊椎動物中發現的氣體通道蛋白，被認為具有運輸氨以及二氧化碳的能力。在魚類中，鰓(成魚)以及皮膚(胚胎仔魚)都是主要用來呼吸的器官，但是目前仍不確定是由何種特定細胞來執行排氨以及二氧化碳的功能，也尚未清楚Rh蛋白在其中扮演的角色。在我的研究中，我將利用斑馬魚胚胎，證明Rh蛋白參與皮膚氨以及二氧化碳運輸的功能。
在第一章的研究中，我以螢光免疫染色證明Rhcg1表現在富含氫幫浦細胞(HR cell)的頂端細胞膜上。利用SIET分析仔魚體表細胞的排氨功能後發現，HR cell比它型表皮細胞具有更強的排氨能力，而此排氨能力也隨抑制Rhcg1的表現而顯著降低。我也發現HR cell在高氨下仍可維持排氨作用，但若是抑制氫幫浦(H+-ATPase)或Rhcg1的表現則會使得HR cell失去高氨下的排氨能力，顯示H+-ATPase以及Rhcg1是HR cell執行主動排氨的關鍵分子。
我在第二章要探討排氨以及鈉離子吸收的運輸機制。透過高氨環境抑制排氨將使得鈉離子吸收能力降低。而增加鈉離子的吸收後則使排氨量增加，顯示氨與鈉離子的運輸息息相關。抑制了Rhcg1以及鈉氫交換蛋白(Na+/H+ exchanger, NHE3b)的表現後發現排氨與吸鈉量皆降低。抑制這兩蛋白也影響了體內鈉離子的含量，顯示Rhcg1以及NHE3b是魚類進行排氨依賴性的鈉離子吸收機制的重要蛋白。
於第三章我將分析另一Rh蛋白Rhbg在仔魚皮膚上的分布與功能。利用原位雜交以及免疫螢光染色我證明Rhbg表現在皮膚keratinocyte頂端與底側端的細胞膜上。與抑制Rhcg1相比，抑制了Rhbg的表現會造成更嚴重的排氨能力失調，顯示Rhbg對於排氨的影響更大。然而，Rhbg的抑制將造成Rhcg1的表現增加以及HR cell排氨能力的提升，這些現象說明補償性的排氨機制是藉由HR cell來調節。
在最後的章節中，我分析了仔魚皮膚Rh蛋白與二氧化碳運輸的相關性。研究發現利用高氨環境會抑制二氧化碳的排放，而高碳酸水也會降低氨的排放量，顯示二氧化碳與氨可能透過同一路徑排放。抑制了Rhbg蛋白會顯著降低二氧化碳排放量，但抑制Rhcg1則不會造成此現象。本實驗也利用H+探針測量表皮二氧化碳的水合(產生H+)與碳酸的水解(減少H+)，藉以分析細胞膜對於二氧化碳的通透性。在高碳酸水的浸泡實驗中，抑制Rhbg將減少體表鹼化的程度，說明較少的二氧化碳通過表皮。這些數據證實Rhbg是魚類排放二氧化碳的重要路徑。
Rhesus glycoproteins (Rh proteins) are the gas channels in vertebrates, and were suggested to conduct ammonia and CO2. In fish, adult gill and larva skin are the organs responsible for gas transport, but the specific cell types for ammonia or CO2 excretion were not identified yet. It is also unclear whether Rh proteins are involved in fish gas excretion? In present study, I use zebrafish larval as animal model to demonstrate the gas excretion function of Rh proteins in skin epithelium. In chapter 1, I used SIET to examine the ammonia excretion ability of skin epithelial cells. The subcellular localization of Rhcg1 was also demonstrated by immunohistochemistry. Results showed that Rhcg1 was distributed in the apical membrane of HR cell. The HR cells exhibited a stronger ammonia efflux than other cell types (keratinocytes and non-HR ionocytes) and the efflux was significant reduced after transcriptional knockdown of Rhcg1 by Rhcg1-antisense morpholino (MO). Under high ammonia environment, HR cell was able to exhibit ammonia efflux, suggesting that HR cell actively excrete ammonia. In chapter 2, an ammonium-dependant Na+ uptake mechanism was demonstrated. The inhibition of ammonia excretion caused a significant decrease of Na+ uptake. Induce of Na+ uptake by high Na+ environment increase ammonia excretion, indicating a linkage of Na+ uptake to ammonia. Knockdown of Rhcg1 or Na+/H+ exchanger (NHE3b) impaired both ammonia excretion and Na+ uptake. The larvae Na+ content was also reduced in the MO-injected larvae. These results suggested that Rhcg1 and NHE3b are the key molecules in this Na+ uptake mechanism. In chapter 3, I analyzed the expression and function of Rhbg in the skin of zebrafish larvae. The results of in situ hybridization and immunohistochemistry showed that Rhbg were expressed at the apical and basolateral membrane of keratinocytes. Knockdown of Rhbg inhibited the ammonia efflux in keratinocytes and caused a severe deficient in total ammonia excretion. On the other hand, Rhbg MO induced the expression of Rhcg1 and elevated the NH4+ efflux in HR cells, suggesting a compensatory effect was occurred. The involvement of Rh proteins in CO2 excretion was showed in chapter 4. In this chapter, CO2 excretion was inhibited in the larvae treated with high ammonia environment. On the other hand, environmental hypercapnia inhibit ammonia excretion as well, suggesting that CO2 shared common pathway of ammonia. The knockdown of Rhbg but not Rhcg1 impaired the CO2 excretion, revealing that Rhbg is important for CO2 transport. I used H+ probe to analyze the hydration/dehydration of surface CO2/HCO3- and determined the membrane permeability to CO2. While exposing to hypercapnia, the change of surface pH was reduced in Rhbg MO-injected larvae, indicating that Rhbg regulated the CO2 permeability.
Rhesus glycoproteins (Rh proteins) are the gas channels in vertebrates, and were suggested to conduct ammonia and CO2. In fish, adult gill and larva skin are the organs responsible for gas transport, but the specific cell types for ammonia or CO2 excretion were not identified yet. It is also unclear whether Rh proteins are involved in fish gas excretion? In present study, I use zebrafish larval as animal model to demonstrate the gas excretion function of Rh proteins in skin epithelium. In chapter 1, I used SIET to examine the ammonia excretion ability of skin epithelial cells. The subcellular localization of Rhcg1 was also demonstrated by immunohistochemistry. Results showed that Rhcg1 was distributed in the apical membrane of HR cell. The HR cells exhibited a stronger ammonia efflux than other cell types (keratinocytes and non-HR ionocytes) and the efflux was significant reduced after transcriptional knockdown of Rhcg1 by Rhcg1-antisense morpholino (MO). Under high ammonia environment, HR cell was able to exhibit ammonia efflux, suggesting that HR cell actively excrete ammonia. In chapter 2, an ammonium-dependant Na+ uptake mechanism was demonstrated. The inhibition of ammonia excretion caused a significant decrease of Na+ uptake. Induce of Na+ uptake by high Na+ environment increase ammonia excretion, indicating a linkage of Na+ uptake to ammonia. Knockdown of Rhcg1 or Na+/H+ exchanger (NHE3b) impaired both ammonia excretion and Na+ uptake. The larvae Na+ content was also reduced in the MO-injected larvae. These results suggested that Rhcg1 and NHE3b are the key molecules in this Na+ uptake mechanism. In chapter 3, I analyzed the expression and function of Rhbg in the skin of zebrafish larvae. The results of in situ hybridization and immunohistochemistry showed that Rhbg were expressed at the apical and basolateral membrane of keratinocytes. Knockdown of Rhbg inhibited the ammonia efflux in keratinocytes and caused a severe deficient in total ammonia excretion. On the other hand, Rhbg MO induced the expression of Rhcg1 and elevated the NH4+ efflux in HR cells, suggesting a compensatory effect was occurred. The involvement of Rh proteins in CO2 excretion was showed in chapter 4. In this chapter, CO2 excretion was inhibited in the larvae treated with high ammonia environment. On the other hand, environmental hypercapnia inhibit ammonia excretion as well, suggesting that CO2 shared common pathway of ammonia. The knockdown of Rhbg but not Rhcg1 impaired the CO2 excretion, revealing that Rhbg is important for CO2 transport. I used H+ probe to analyze the hydration/dehydration of surface CO2/HCO3- and determined the membrane permeability to CO2. While exposing to hypercapnia, the change of surface pH was reduced in Rhbg MO-injected larvae, indicating that Rhbg regulated the CO2 permeability.
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斑馬魚, 氨, Rh蛋白, 二氧化碳, 皮膚, 氫幫浦蛋白, Zebrafish, Ammonia, Rh protein, CO2, skin, H+-ATPase