Disruption of the folate pathway in zebrafish causes developmental defects

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.

The role of folate metabolism in orofacial development and clefting

“…We saw an overall increase in the severity of the facial phenotypes with concentrations ranging from 110–440 μM of MTX. This was consistent with concentrations used in zebrafish (Lee et al, 2012) (not shown)). Further, we noted considerable variability in facial phenotypes at each concentration (Supp.…”

“…Further, previous work in zebrafish and mouse have shown that decreased DHFR function or loss of a folate binding proteins cause a decrease in cell cycle progression, particularly through S-phase (Lee et al, 2012; Tang et al, 2005). Therefore, we hypothesized that the changes in face shape and size observed with DHFR inhibition in Xenopus could be due to perturbations in cell cycle progression and subsequent decreases in cellular division.…”

“…Such a reduction could at least in part account for the progressively more apparent decrease in the size of the face during the development of MTX-treated embryos and DHFR morphants. Folate deficiency or mutations to folate pathway genes can also result in low birth weight in humans and a smaller embryonic size in model vertebrates, with specific defects in cell cycle progression in zebrafish (Lee et al, 2012; Molloy et al, 2008; Momb et al, 2013; Sun et al, 2011)Lee, 2012). However, decreased proliferation alone could not account for the facial shape changes and lack of differentiation of the jaw muscle and cartilage.…”

“…Single cells were isolated using a protocol adapted from (Lee, 2012). Briefly, 25–30 embryos were isolated in 0.5X calcium free Ringer’s solution (58 mM NaCl, 1.45 mM KCl, 2.5 mM HEPES pH 7.2) and mechanically digested.…”

“…Certainly, changes in folate metabolism have been shown to affect both DNA synthesis and histone methylation during neurulation, heart development, and in diseases such as cancer (Beaudin and Stover, 2009; Momb et al, 2013; Sun et al, 2011; Tang et al, 2005; Wang et al, 2014). Folate deficiency and mutations to genes involved in the folate pathway cause orofacial defects in model vertebrates and humans (Li et al, 2011; Momb et al, 2013; Tang et al, 2005; Wehby and Murray, 2010; Wilcox et al, 2007) (Burgoon et al, 2002; Kao et al, 2014; Lee et al, 2012). However, it is less clear if these results are due to similar processes during orofacial development.…”