Transient knockdown and overexpression reveal a developmental role for the zebrafish enosf1b gene.

Steve Finckbeiner,Raman Sood,Pin-Joe Ko,Bruce Dolnick,Janice Sufrin,Paul Liu,Blake Carrington,Kenneth Gross

doi:10.1186/2045-3701-1-32

Abstract

BackgroundDespite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s). Results of previous studies suggest involvement of the β splice form of ENOSF1 in breast and colon cancers. This study used the zebrafish (Danio rerio) as a vertebrate model of ENOSF1β function.ResultsWhole mount in situ hybridization (WISH) showed that zebrafish ENOSF1β (enosf1b) is zygotic and expressed ubiquitously through the first 24 hours post fertilization (hpf). After 24 hpf, enosf1b expression is restricted to the notochord. Embryos injected with enosf1b-EGFP mRNA grew slower than EGFP mRNA-injected embryos but caught up to the EGFP-injected embryos by 48 hpf. Embryos injected with ATG or exon 10 enosf1b mRNA-targeting morpholinos had kinked notochords, shortened anterior-posterior axes, and circulatory edema. WISH for ntl or pax2a expression showed that embryos injected with either morpholino have deformed notochord and pronephros. TUNEL staining revealed increased apoptosis in the peri-notochord region.ConclusionsThis study is the first report of ENOSF1 function in a vertebrate and shows that ENOSF1 is required for embryonic development. Increased apoptosis following enosf1b knockdown suggests a potential survival advantage for increased ENOSF1β expression in human cancers.

Highlights

Despite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s)
Ensembl databases followed by MUSCLE alignment and phylogenetic tree construction reveal that the ENOSF1b gene was likely present in the last common ancestor of cephalochordates (Ciona sp, Figure 1) and vertebrates and lost in different vertebrate classes
Expression of enosf1b was manipulated during zebrafish development by injecting enosf1b-EGFP mRNA or antisense morpholino oligonucleotides into 1-2 cell stage embryos

Summary

Introduction

Despite detailed in vivo knowledge of glycolytic enolases and many bacterial non-enolase members of the superfamily, little is known about the in vivo function of vertebrate non-enolase enolase superfamily members (ENOSF1s). This study used the zebrafish (Danio rerio) as a vertebrate model of ENOSF1b function. Sequence information and computational techniques can be used to group proteins into evolutionarily meaningful families and larger superfamilies. Dayhoff defined protein families as groups of proteins with high (>50%) sequence identity [1]. Members of protein superfamilies have lower sequence identities, but statistically significant pairwise alignment scores. Both protein families and protein superfamilies are thought to be monophyletic [1,2,3,4]. The enolase superfamily (ENOSF), named after the enolase of glycolysis, is used as a model of protein superfamily evolution [4,7]. Members of the enolase superfamily share a common ENOSF fold and catalyze a common half reaction: they all abstract protons adjacent to carboxyl groups from a

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