The zebrafish, Danio rerio , as a vertebrate model of ENOSF1 function
Finckbeiner, Steven Michael
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The protein product of the human ENOSF1 (enolase superfamily member 1) gene is an enzyme thought to be involved in the metabolism of S-adenosylmethionine (AdoMet). This study used the zebrafish (Danio rerio) as a vertebrate model of ENOSF1 function during development. Whole mount in situ hybridization (WISH) showed that zebrafish ENOSF1 (drENOSF1) is zygotic and expressed ubiquitously through the first 24 hours post fertilization (hpf). After 24 hours, drENOSF1 becomes restricted to notochord. Modulation of gene dosage in zebrafish can be achieved by injecting mRNA ("knock in") or morpholino antisense oligonucleotides (morpholinos, "knock down") into early cleavage embryos. Embryos injected with drENOSF1-EGFP grew slower than EGFP-injected embryos. drENOSF1-EGFP disappears from the embryos between 48 and 72 hpf, while EGFP remains detectable throughout these time points. This suggests that drENOSF1 is regulated at the level of mRNA or protein stability as well as mRNA expression. Injection of a morpholino targeting ribosome binding of the second ATG (ATG2) in endogenous drENOSF1 resulted in embryos with kinked notochords, shortened anterior-posterior axes, and circulatory edema. Injection of a morpholino targeting normal splicing of exon 10 (e10i10) resulted in embryos with a less severe version of the ATG2 phenotype. RT-PCR of uninjected, standard control morpholino, and e10i10 injected embryos show that e10i10-injected embryos expressed a large proportion of misspliced drENOSF1 transcript. WISH for tissue-specific gene expression showed that embryos injected with both morpholinos have deformed posterior structures, including notochord and pronephros. TUNEL staining revealed increased apoptosis in the peri-notochord region. The TUNEL results are mirrored in WISH for two enzymes needed to synthesize AdoMet: MATIIA increases expression in ATG2-injected embryos, while MATIIB expression decreased. This study represents the first report and characterization of phenotypes associated with animal ENOSF1 knockdown in vivo .