Gene Expression and Functional Analysis of Cysteine Sulfinic Acid Decarboxylase during Embryonic Development of Zebrafish
Date Issued
2011
Date
2011
Author(s)
Chang, Yen-Chia
Abstract
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.
Subjects
CSAD
taurine
zebrafish
embryogenesis
heart
Type
thesis
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