Gene Expression and Functional Analysis of Cysteine Sulfinic Acid Decarboxylase during Embryonic Development of Zebrafish

半胱亞磺酸脫羧酶 (cysteine sulfinic acid decarboxylase, CSAD; EC 4.1.1.29) 為牛磺酸 (taurine) 生合成路徑中，速率決定步驟之關鍵酵素，早期於大鼠和小鼠之肝臟組織中曾被選殖並報導。牛磺酸廣泛存在於脊椎動物體中，其生成的路徑在許多組織均可發生。牛磺酸參與許多生理功能，例如：合成膽鹽、維持細胞滲透壓、參與脂質代謝以及減緩氧化壓力。另外，在胚胎發育的過程中牛磺酸是個不可或缺的營養分，但其確切的功能仍待釐清。卵生動物的胚胎可自卵黃攝取或是自行生合成牛磺酸，而抑制斑馬魚胚之牛磺酸運送蛋白 (taurine transporter) 的表現僅會增加中樞神經系統的細胞凋亡，因此在斑馬魚發育過程中，各組織或許都能自行生合成牛磺酸。 為探討胚胎發育過程中牛磺酸生合成的重要性，首先我們選殖斑馬魚的 CSAD (zCSAD) 並且分析其與不同物種蛋白質序列的異同，結果顯示其與各模式物種 (小鼠和大鼠) 之 CSAD 有高度的同源性。不同於人類 CSAD 之外泌特性，根據訊號胜肽預測及細胞轉染的結果顯示 zCSAD 為非外泌性蛋白質。利用 RT-PCR 及原位雜合試驗 (whole-mount in situ hybridization) ，發現 zCSAD 可能為母系遺傳基因，且於各胚胎發育時期和中胚層組織，如中腎管、脊索及心臟生成等區域皆有不同程度之表現。顯微注射反義序列核酸 (morpholino, MOs) 至斑馬魚胚抑制 zCSAD 表達，會造成牛磺酸濃度降低、增加胚胎早期死亡率、提高心臟生成區域和尾巴之細胞死亡率、尾部發育異常、心包水腫及心管彎曲異常。利用共同注射 zCSAD MOs 與 mRNA 或培養液中添加牛磺酸皆能夠降低抑制 zCSAD 所造成之發育異常和死亡率。為釐清其中之分子機制，利用 real-time PCR 檢測抑制 zCSAD 轉譯之斑馬魚胚的心臟血管生成基因、細胞凋亡基因和 unfolded protein response regulator 基因，發現其表現量皆有些微增加，但統計上沒有顯著差異。 綜合上述， CSAD 在不同脊椎動物物種間，具有高度保留性。而斑馬魚胚胎體內牛磺酸之平衡維持，主要透過 CSAD 路徑自行生成，且牛磺酸對於細胞存活以及心臟正常之發育過程，皆扮演相當重要的角色。Cysteine sulfinic acid decarboxylase (CSAD; EC 4.1.1.29) is the rate-limiting enzyme in the biosynthesis of taurine, 2-aminoethane sulfonic acid. CSAD is also named sulfinoalanine decarboxylase and cysteine-sulfinate decarboxylase, and has been cloned and characterized in liver of rat and mice. Taurine can be found abundantly in blood cells, muscles and brain of vertebrates and its biosynthesis was detected in various extrahepatic tissues. There are numbers of positive effects of taurine on physiological processes such as bile salt synthesis, osmoregulation, lipid metabolism and oxidative stress inhibition. Taurine is also critical for normal embryonic development, but its exact role in embryogenesis remains to be elucidated. The acquisition of taurine in embryos depends on de novo synthesis and placental/yolk transfer. Knockdown of taurine transporter induced only slightly increased apoptosis in central nervous system during zebrafish development. It implies that de novo synthesis may play a more dominant role during zebrafish embryogenesis. To investigate the roles of de novo synthesis of taurine during embryonic development, zebrafish CSAD (zCSAD) was cloned and the cDNA encodes for a protein of 482 amino acids with its sequence highly homologous to mammalian CSAD and conserved throughout evolution. The zCSAD was only detected in the cell lysate but not in the medium of HEK293T cells transiently transfected by zCSAD, suggesting that zCSAD is not a secreted protein. Semi-quantitative RT-PCR detected zCSAD mRNA as early as 0 hour post fertilization (hpf) indicating the existence of maternal zCSAD message. Whole-mount in situ hybridization demonstrated that zCSAD was expressed in yolk syncytial layer and various mesoderm tissues such as pronephric duct, notochord and cardiogenic field during early embryogenesis. Knockdown of zCSAD by morpholino oligos (MOs) reduced taurine level in embryos and increased early mortality, elevated cell death in cardiogenic region and tail, pericardial edema and malformation of tail. Coinjecting zCSAD MOs and mRNA could partially rescue the cardiac phenotypes, indicating that zCSAD is important for heart development. On the other hand, when embryos were incubated in 13 mM taurine supplement buffer after injecting MOs, taurine treatment could diminish the mortality and cardiac phenotypes at 72 hpf, suggesting that the heart malformation caused by zCSAD knockdown was due to taurine deficiency. To further investigate the molecular mechanism by which taurine regulates these phenotypes, real-time PCR was performed to detect mRNA expression of heart formation marker (NK2 transcription factor related 5, Nkx2.5), hematopoiesis markers (runt-related transcription factor 1, runx1; kinase insert domain receptor like, KDRL), apoptosis markers (BCL2-antagonist of cell death; caspase 3) and unfolded protein response regulator genes (activating transcription factor 6, ATF6; X-box binding protein-1 spliced form, XBP-1S) in zCSAD knockdown embryos and the results showed a trend of numerical increase without significance. In conclusion, knockdown of zCSAD reduced taurine biosynthesis in zebrafish embryos and resulted in increasing cell death and malformations of heart. These findings indicated that taurine de novo synthesis via CSAD plays an important role in cardiac development and as a cell survival factor in zebrafish embryos