Molecular characterization and expression pattern of taurine transporter in zebrafish during embryogenesis

Abstract

Taurine and its transporter (TauT) are expressed in preimplantation embryos, but their role in embryogenesis is not known. To investigate the role of TauT during embryonic development, we cloned and functionally characterized the zebrafish TauT. The zebrafish TauT cDNA codes for a protein of 625 amino acids which is highly homologous to mammalian TauT. When expressed in mammalian cells, zebrafish TauT mediates taurine uptake in a Na⁺/Cl⁻-dependent manner with a Na⁺:Cl⁻:taurine stoichiometry of 2:1:1. In the zebrafish embryo, taurine and TauT mRNA are present during early cleavage stages, indicating that both the transporter and its substrate are maternally derived. During embryogenesis, zygotic expression of TauT mRNA is evident in the retina, brain, heart, kidney, and blood vessels. Knockdown of TauT by antisense morpholino oligonucleotides leads to cell death in the central nervous system and increased mortality. These findings suggest a specific role for TauT during development in vertebrates.

Introduction

Taurine is an abundant amino acid present in all vertebrates. Intracellular taurine concentrations can range up to 50 mM (Huxtable, 1992). In most vertebrates, taurine is synthesized in the liver and then taken up actively by other tissues (Tappaz, 2004). Uptake of taurine in mammalian cells is mediated by an efficient transport system in the plasma membrane which utilizes transmembrane gradients of Na⁺ and Cl⁻ as the driving force. The transporter (TauT or SLC6A6) responsible for the uptake has been cloned and characterized in several animal species (Chen et al., 2004). Taurine, along with glycine, is essential for the generation of conjugated bile acids (Huxtable, 1992). It also functions as an osmolyte (Pasantes-Morales et al., 1998) and is involved in the covalent modification of the wobble position of anticodons in specific mitochondrial tRNAs (Suzuki et al., 2002, Kirino et al., 2004). Taurine also protects cells against various types of injury (Huxtable, 1992). Lastly, there is evidence for taurine in mediating trophic effects, antioxidant function, regulation of ion channels and intracellular calcium levels, and protein phosphorylation (Huxtable, 1992).

Animal studies have provided evidence for the biological importance of taurine in vivo. Cats cannot synthesize taurine and therefore depend on dietary sources of taurine (Knopf et al., 1978). Cats fed a taurine-free diet become taurine-deficient with profound clinical consequences that include retinal degeneration and cardiomyopathy (Pion et al., 1987, Hayes and Trautwein, 1989). Rodents synthesize taurine, and it is difficult to create taurine deficiency in these animals. However, genetic deletion of the taurine transporter gene (taut) in mice causes taurine deficiency (Heller-Stilb et al., 2002). The primary phenotype of taut^−/− mice is retinal degeneration and skeletal myopathy, without cardiac involvement (Heller-Stilb et al., 2002, Warskulat et al., 2004, vom Dahl et al., 2000). In humans, taurine deficiency occurs as a consequence of prolonged parenteral nutrition in premature infants, and this is associated with retinal dysfunction (Sturman and Chesney, 1995).

Taurine is also critical for normal embryonic development. Female-derived reproductive tract fluids in several different animal species, including humans, contain high levels of taurine (Leese et al., 1979, Borland et al., 1980, Casslen, 1987). Taurine is present in both oocytes and embryos where it functions as an osmolyte (Schultz et al., 1981, Dumoulin et al., 1992, Dumoulin et al., 1997, Devreker et al., 1999). Consistent with maternally provided taurine, preimplantation mouse embryos express high levels of taurine transporter activity (Van Winkle et al., 1994). Despite the convincing evidence for the occurrence of taurine and taurine transporter in female reproductive fluids, oocytes, and early embryos, an essential role for this amino acid in embryogenesis has only been suggestive. Homozygous TauT null (taut^−/−) mice are viable but growth retarded (Heller-Stilb et al., 2002, Warskulat et al., 2004). Despite the observations that homozygous TauT knockout mice are viable, taurine may still have a role during embryogenesis. Null mice are generated by mating male and female heterozygotes (Heller-Stilb et al., 2002). Heterozygous females do synthesize taurine and plasma levels of taurine are not markedly reduced in these animals, even during pregnancy. Although homozygous embryos and the embryo-derived placenta do not express TauT, other transporters may mediate the transfer of taurine across the placenta. For example, particular isoforms of GABA transporters can transport taurine (Sivakami et al., 1992) and these isoforms could facilitate transplacental transfer of taurine to the developing taut^−/− embryo. Furthermore, heterozygous females are likely to provide significant amounts of taurine as well as TauT mRNA in eggs during oogenesis and this is independent of the germline genotype.

Zebrafish is used widely as a model system to study various aspects of embryonic development. There are several advantages in this model system. This includes transparency of the embryos, rapid development, and external fertilization. In addition, targeted genes can be silenced effectively with the injection of morpholino nucleotides into one-cell stage embryos, and the influence of resultant gene silencing on the development of the embryo can be monitored. Taurine has been detected in the nervous system of zebrafish, including the retina (Marc and Cameron, 2001, Sakata et al., 2003), but there is no information available on the transporter responsible for cellular uptake of taurine in this animal species. This prompted us to use zebrafish embryo as the model system with three primary goals: (a) to characterize the zebrafish taurine transporter at the molecular and functional level, (b) to monitor the expression pattern of the transporter during various stages of embryonic development, and (c) to determine the role of the transporter in embryogenesis.