slc7a6os Gene Plays a Critical Role in Defined Areas of the Developing CNS in Zebrafish

The aim of this study is to shed light on the functional role of slc7a6os, a gene highly conserved in vertebrates. The Danio rerio slc7a6os gene encodes a protein of 326 amino acids with 46% identity to human SLC7A6OS and 14% to Saccharomyces cerevisiae polypeptide Iwr1. Yeast Iwr1 specifically binds RNA pol II, interacts with the basal transcription machinery and regulates the transcription of specific genes. In this study we investigated for the first time the biological role of SLC7A6OS in vertebrates. Zebrafish slc7a6os is a maternal gene that is expressed throughout development, with a prevalent localization in the developing central nervous system (CNS). The gene is also expressed, although at different levels, in various tissues of the adult fish. To determine the functional role of slc7a6os during zebrafish development, we knocked-down the gene by injecting a splice-blocking morpholino. At 24 hpf morphants show morphological defects in the CNS, particularly the interface between hindbrain and midbrain is not well-defined. At 28 hpf the morpholino injected embryos present an altered somite morphology and appear partially or completely immotile. At this stage the midbrain, hindbrain and cerebellum are compromised and not well defined compared with control embryos. The observed alterations persist at later developmental stages. Consistently, the expression pattern of two markers specifically expressed in the developing CNS, pax2a and neurod, is significantly altered in morphants. The co-injection of embryos with synthetic slc7a6os mRNA, rescues the morphant phenotype and restores the wild type expression pattern of pax2a and neurod. Our data suggest that slc7a6os might play a critical role in defined areas of the developing CNS in vertebrates, probably by regulating the expression of key genes.

本研究旨在阐明脊椎动物中高度保守的基因slc7a6os的功能角色。斑马鱼（Danio rerio）的slc7a6os基因编码一条含326个氨基酸的蛋白质，与人源SLC7A6OS的序列同一性为46%，与酿酒酵母（Saccharomyces cerevisiae）的多肽Iwr1的序列同一性为14%。已知酵母Iwr1可特异性结合RNA聚合酶II（RNA pol II），与基础转录机器相互作用并调控特定基因的转录。本研究首次探究了脊椎动物体内SLC7A6OS的生物学功能。斑马鱼slc7a6os属于母源基因，在整个发育周期中均有表达，且在发育中的中枢神经系统（Central Nervous System, CNS）中富集表达。该基因在成鱼的多种组织中亦有表达，但表达水平存在组织特异性差异。为明确slc7a6os在斑马鱼发育过程中的功能，我们通过注射剪接阻断型吗啉代寡核苷酸（morpholino）对该基因进行了敲低。在受精后24小时（hours post fertilization, hpf），吗啉寡核苷酸敲低胚胎（morphants）的中枢神经系统出现形态学缺陷，尤以中脑与后脑的分界模糊为显著特征。受精后28 hpf时，注射吗啉代寡核苷酸的胚胎呈现体节形态异常，且表现出部分或完全运动能力丧失。此阶段，与对照组胚胎相比，敲低胚胎的中脑、后脑及小脑结构受损且分界不清。上述异常表型在后续发育阶段持续存在。与之相符的是，两个特异性在发育中枢神经系统中表达的标记基因pax2a和neurod的表达模式在敲低组胚胎中发生了显著改变。通过共注射合成slc7a6os mRNA可挽救该敲低表型，并恢复pax2a与neurod的野生型表达模式。本研究数据表明，slc7a6os可能在脊椎动物发育中的中枢神经系统特定区域发挥关键作用，其潜在机制可能为调控关键基因的表达。