The Zebrafish (Danio Rerio) as a Novel Model to Study Folate-mthfr Interactions During Embryonic Development and Effect(s) on Long-Term Health

Abstract

Abstract Objectives Methylenetetrahydrofolate reductase (MTHFR) catalyzes the synthesis of the bioactive folate form, 5-methyltetrahydrofolate (5MTHF). Common polymorphisms in MTHFR limits the availability of 5MTHF and high intakes of folic acid (FA, synthetic form) may exacerbate these effects. 5MTHF is an alternative supplement to FA, but the interaction of folate form and MTHFR genotype and role in programming metabolic health has not been determined. The zebrafish (Danio rerio) is a proven model system in nutrient and drug discovery studies. The objectives of this study were to: 1) develop a mthfr zebrafish mutant model using CRISPR-Cas9 technology; and 2) identify in zebrafish the relationship between folate form (FA vs 5MTHF) and mthfr genotype on early-life development and metabolic regulation. Methods To assay the function of mthfr in vivo, we used a rapid transient knock-out approach reported to recapitulate germline zebrafish loss-of-function phenotypes. Embryos were co-injected with Cas9 protein and a set of 4 guide RNAs (4gRNA) or Cas9 protein alone (control). Zero to 5 days post-fertilization (dpf), embryos were raised in standard conditions then fed a high cholesterol diet (HCD) up to 15 dpf with a fluorescent cholesteryl BODIPY-C12. The HCD was used to induce obesity as previously reported. Global DNA methylation (5-mC%) was measured at 5 dpf and whole-body and hepatic lipid accumulation and live imaging analyses performed at 15 dpf. Results Compared to control, 4gRNA mthfr zebrafish on an HCD had enlarged liver, greater accumulation of hepatic and whole body lipids and altered lipid transport. They also had 80% lower mthfr mRNA than control zebrafish. Global methylation was ∼15% higher (P = 0.06) in 4gRNA zebrafish suggesting a compensatory dependency on an alternative methyl donor pathway during embryonic development. We are now screening germline mutant carriers and assessing folate and methylation metabolites and the interaction of folate form and genotype. Conclusions The zebrafish mthfr mutant will be a valuable model to examine the mechanisms underlining mthfr-related pathologies and provide a high-throughput in vivo system to ascertain the role of different folate forms on embryonic development and long-term health. Funding Sources CIHR-INMD; EP supported by NSERC-CGS D.