Disruption of the folate pathway in zebrafish causes developmental defects

Marina S Lee,Jenna R Bonner,Erica L Sanchez,Shawn M Burgess,Lawrence C Brody,R Reid Prentice,David J Bernard,Eric T Sause

doi:10.1186/1471-213x-12-12

Abstract

BackgroundFolic acid supplementation reduces the risk of neural tube defects and congenital heart defects. The biological mechanisms through which folate prevents birth defects are not well understood. We explore the use of zebrafish as a model system to investigate the role of folate metabolism during development.ResultsWe first identified zebrafish orthologs of 12 human folate metabolic genes. RT-PCR and in situ analysis indicated maternal transcripts supply the embryo with mRNA so that the embryo has an intact folate pathway. To perturb folate metabolism we exposed zebrafish embryos to methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (Dhfr) an essential enzyme in the folate metabolic pathway. Embryos exposed to high doses of MTX exhibited developmental arrest prior to early segmentation. Lower doses of MTX resulted in embryos with a shortened anterior-posterior axis and cardiac defects: linear heart tubes or incomplete cardiac looping. Inhibition of dhfr mRNA with antisense morpholino oligonucleotides resulted in embryonic lethality. One function of the folate pathway is to provide essential one-carbon units for dTMP synthesis, a rate-limiting step of DNA synthesis. After 24 hours of exposure to high levels of MTX, mutant embryos continue to incorporate the thymidine analog BrdU. However, additional experiments indicate that these embryos have fewer mitotic cells, as assayed with phospho-histone H3 antibodies, and that treated embryos have perturbed cell cycles.ConclusionsOur studies demonstrate that human and zebrafish utilize similar one-carbon pathways. Our data indicate that folate metabolism is essential for early zebrafish development. Zebrafish studies of the folate pathway and its deficiencies could provide insight into the underlying etiology of human birth defects and the natural role of folate in development.

Highlights

Folic acid supplementation reduces the risk of neural tube defects and congenital heart defects
Cloning & RT-PCR Orthologs of eleven enzymes and one transporter involved in folate-one carbon metabolism were identified in zebrafish by BLAST searches, cloned, and characterized (Figure 1 and Table 1)
Most genes identified in this pathway appear to be ‘house keeping’ genes that are maternally loaded, present throughout early embryonic development, and whose expression is predominantly enriched in anterior CNS (Figure 2)

Summary

Introduction

Folic acid supplementation reduces the risk of neural tube defects and congenital heart defects. We explore the use of zebrafish as a model system to investigate the role of folate metabolism during development. Genetic variants in folate pathway genes are associated with increased risk for neural tube defects (NTD) (and other congenital defects) [1], cardiovascular disease [2], cancer [3] and cognitive decline [4]. Women taking supplemental folic acid prior to conception significantly reduce their chance of having an NTD affected pregnancy [5]. The significant advantages of working with zebrafish (external fertilization, large clutch size, and transparency) may render it a useful investigative model for studying the role of folate in development and disease

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