Celf 1 regulation of dmrt 2 a is required for somite symmetry and left-right patterning during zebrafish development

INTRODUCTION Transcriptional control has important roles in generating a properly organized body during vertebrate development (Naiche et al., 2005; Schier and Talbot, 2005; Shivdasani, 2002). However, levels of particular transcripts are highly variable in space and time during development because of the stochastic nature and the spatiotemporal regulation of transcription (Blake et al., 2003; Elowitz et al., 2002). Embryos must therefore have systems that precisely control the amounts of proteins, to regulate multiple developmental processes at post-transcriptional levels (Gebauer and Hentze, 2004). RNA-binding proteins (RBPs) are known to provide such a mechanism. For instance, an RBP named Cugbp Elav-like family member 1 (Celf1) controls gene expression at multiple post-transcriptional levels, including alternative splicing, deadenylation and mRNA decay, and fine-tunes the abundance of proteins that are synthesized from its target mRNAs (Barreau et al., 2006; Vlasova et al., 2008). Celf1 target mRNAs have been identified by a yeast three-hybrid screen, a SELEX (systematic evolution of ligands by exponential enrichment) approach, an in vitro binding assay and RNA immunoprecipitation assays (Lee et al., 2010; Marquis et al., 2006; Mori et al., 2008; Rattenbacher et al., 2010; Suzuki et al., 2002; Takahashi et al., 2000). In HeLa cells, Celf1 binds to >600 shortlived mRNAs that are involved in cell growth, motility and apoptosis (Rattenbacher et al., 2010), suggesting that Celf1 coordinately regulates protein expression from multiple mRNAs. Analyses of the conserved sequences among these mRNAs reveal that UG-rich elements or UGU repeats, such as UGUUUGUUUGU and UGUGUGUGUGU, are the binding sequences of Celf1 (Rattenbacher et al., 2010); these sequences are similar to target elements identified by other methods in human, mouse and frog (Lee et al., 2010; Marquis et al., 2006; Mori et al., 2008; Takahashi et al., 2000). Celf1 is broadly expressed in early vertebrate embryos, whereas Celf1 expression is restricted to specific regions, such as eyes and somites (Gautier-Courteille et al., 2004; Hashimoto et al., 2006; Kress et al., 2007), suggesting a role(s) for Celf1 in the regulation of developmental processes. However, loss of Celf1 function in either frog or mouse embryos has only minor effects on their development; knockdown of the Xenopus Celf1 ortholog EDENBP results in only a defect of somite segmentation (GautierCourteille et al., 2004). Some Celf1-knockout mice are viable to the adult stage, although they display growth retardation (Kress et al., 2007). These mild effects on development may be explained by redundant actions among Celfs and/or by high abundance of proteins synthesized from Celf1 target mRNAs. Therefore, the physiological roles for Celf1 in the regulation of vertebrate development remain largely unclear. A possible strategy to explore such a role for Celf1 is to deplete proteins synthesized from Celf1 target mRNAs. We thus overexpressed celf1 in zebrafish embryos to intensify Celf1mediated post-transcriptional regulation. This manipulation resulted in failures of bilateral symmetric somitogenesis and leftright (LR) asymmetric patterning, defects which are similar to the phenotype induced by knockdown of a single gene named doublesex and mab-3 related transcription factor 2a (dmrt2a, formerly terra) (Saude et al., 2005). We also found that Celf1 downregulates dmrt2a expression by binding to the 3 UTR of dmrt2a mRNA. Furthermore, blocking the interaction between Celf1 and dmrt2a mRNA reduced the effects of celf1 overexpression on both somite symmetry and LR asymmetric patterning. Our results therefore identify a crucial role of the specific Celf1-mRNA pair in generating a properly organized body during vertebrate development, and provide a powerful tool for analyzing the roles of specific RBP-mRNA pairs in various experimental settings. 1Gene Regulation Research, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Nara 630-0101, Japan. 2Cooperative Research Division, Nara Institute of Science and Technology, 8916-5 Takayama, Nara 630-0101, Japan.