Celf1 regulation of dmrt2a is required for somite symmetry and left-right patterning during zebrafish development

Abstract

SUMMARYRNA-binding proteins (RBPs) bind to numerous and diverse mRNAs to control gene expression post-transcriptionally, although the in vivo functions of specific RBP-mRNA interactions remain largely unknown. Here, we show that an RBP named Cugbp, Elav-like family member 1 (Celf1) controls expression of a gene named doublesex and mab-3 related transcription factor 2a (dmrt2a), which is essential for somite symmetry and left-right patterning during zebrafish development. Celf1 promotes dmrt2a mRNA decay by binding to UGU repeats in the 3ЈUTR of dmrt2a mRNA such that celf1 overexpression reduces the amount of dmrt2a mRNA, leading to asymmetric somitogenesis and laterality defects. Furthermore, blocking the Celf1-dmrt2a mRNA interaction by a target protector morpholino alleviates failures in somite symmetry and left-right patterning that are caused by celf1 overexpression. Our results therefore demonstrate that Celf1-dependent fine-tuning of dmrt2a expression is essential for generating bilateral symmetry of somites and left-right asymmetric patterning during zebrafish development.

Celf1 Is Required for Formation of Endoderm-Derived Organs in Zebrafish

“…In addition, we recently showed that celf1 is required for somite symmetry and cardiac laterality [13]. In the course of our investigation of other laterality organs such as liver and pancreas, we find an unexpected role for celf1 in zebrafish organogenesis; celf1 is essential for the formation of endoderm-derived organs.…”

“…Since cardiac laterality was altered in celf1 knockdown (KD) embryos [13], we reasoned that the lateralities of endoderm-derived organs such as liver, pancreas and gut are also altered in celf1 KD embryos. To test this, we injected celf1 morpholinos ( celf1 -MOs) into transgenic line Tg [ sox17:GFP ] [14], whose GFP is expressed in endoderm cells and dorsal forerunner cells, and observed the formation of endoderm-derived organs in celf1 KD embryos.…”

“…Instead, celf1 expression is restricted to specific regions such as eyes and pectoral fins at later stages [13,18,19]. We therefore reasoned that celf1 regulates endoderm formation during gastrulation and secondarily affects the formation of endoderm-derived organs in later embryos.…”

“…Since celf1 is essential for the growth and migration of endoderm cells, we looked for possible targets by undertaking a search for genes involved in endoderm proliferation and migration. Because either UG rich sequences or UGU repeats are existed in 3′ UTR of many mRNAs that encode cell cycle and migration regulators [5] and because flanking U-rich or UA-rich elements to UG/UGU sequences also affects the binding affinity of Celf1 [13], we thought that a sequence containing U/UA-rich elements and at least four UGU repeats within 35 bp would be a strong candidate for the Celf1-binding site. We thus selected gata5 ( gata-binding protein 5 ) and cdc42 as potential targets of Celf1 for the following reasons (Figure 5A).…”

“…To test whether Celf1 binds to gata5 and cdc42 mRNAs in vivo , we performed RIP assays by using Celf1 antiserum. Although Celf1 did not bind to cyclinA1 ( ccna1 ) mRNA (negative control) [13], Celf1 associated with gata5 and cdc42 mRNAs (Figure 5B). To investigate whether Celf1 affects the expression of gata5 and cdc42 , we performed qPCR analyses in control and celf1 KD embryos.…”