Taurine homeostasis requires de novo synthesis via cysteine sulfinic acid decarboxylase during zebrafish early embryogenesis

Abstract

Cysteine sulfinic acid decarboxylase (Csad) is the rate-limiting enzyme in the de novo biosynthesis of taurine. There are a number of physiological roles of taurine, such as bile salt synthesis, osmoregulation, lipid metabolism, and oxidative stress inhibition. To investigate the role of de novo synthesis of taurine during embryonic development, zebrafish csad was cloned and functionally analyzed. Semi-quantitative RT-PCR showed that csad transcripts are maternally deposited, while whole-mount in situ hybridization demonstrated that csad is expressed in yolk syncytial layer and various embryonic tissues such as notochord, brain, retina, pronephric duct, liver, and pancreas. Knockdown of csad significantly reduced the embryonic taurine level, and the affected embryos had increased early mortality and cardiac anomalies. mRNA coinjection and taurine supplementation rescued the cardiac phenotypes suggesting that taurine originating from the de novo synthesis pathway plays a role in cardiac development. Our findings indicated that the de novo synthesis pathway via Csad plays a critical role in taurine homeostasis and cardiac development in zebrafish early embryos.

Taurine Biosynthesis in a Fish Liver Cell Line (ZFL) Adapted to a Serum-Free Medium

“…Cellular CDO concentrations are tightly regulated by the rate of proteasomal degradation as controlled by polyubiquitination [57,58,59]. CDO concentrations can change up to 45-fold [60], multiplied by the potential 10-fold difference in catalytic efficiency [61], for a total potential change in CDO activity of up to 450-fold [40]. CSAD, a pyridoxal-5′-phosphate (PLP)-dependent enzyme, is the rate-limiting enzyme for taurine biosynthesis in mouse [62].…”

“…To achieve this goal, we have successfully adapted an adult zebrafish liver cell line (ZFL) to serum-free synthetic medium (UltraMEM™-ITES), allowing us to control taurine levels. Unlike many carnivorous fish, the omnivorous zebrafish has a complete taurine biosynthetic pathway and is not dependent on taurine in the diet, being able to convert methionine via cysteine into taurine [40]. The usefulness of ZFL cells adapted to serum-free medium was demonstrated by an investigation of the effect of taurine on growth rate, expression of the genes involved in taurine biosynthesis and transport, as well as the relationship between taurine and methionine levels.…”

Sterol O-Acyltransferase 2 Contributes to the Yolk Cholesterol Trafficking during Zebrafish Embryogenesis

“…The system was maintained at 28.5°C with a light/dark cycle of 14/10 h, and the fish were fed with live adult brine shrimp twice a day [21]. Embryos were collected after spontaneous spawning, allowed to develop and staged by hour-postfertilization (hpf) at 28.5°C using morphological criteria [22].…”

“…To express Soat enzymes, the coding sequences of soats were subcloned into the pT7-IRES2-DsRed or pT7-IRES2-eGFP vectors [21] with EcoR I to generate pSoat1-IRES2-DsRed, pSoat1-IRES2-eGFP, pSoat2-IRES2-DsRed or pSoat2-IRES2-eGFP, respectively. These plasmids allow the cistronic expression of a fluorescent protein (eGFP or DsRed) together with the genes of interest and hence the expression could be confirmed under a fluorescent microscope.…”

“…To demonstrate the spatial expressions of zebrafish soats during embryonic development, whole-mount in situ hybridization was performed as described previously [21]. Briefly, a 300~400 bp of sense and antisense digoxigenin-labeled riboprobes for soat2 were synthesized by in vitro transcription using T7 polymerase with the T7 RNA polymerase promoter introduced at the 5’-end of the probe sequences by performing nested PCR.…”