Disruption of the folate pathway in zebrafish causes developmental defects
© Lee et al; licensee BioMed Central Ltd. 2012
Received: 17 October 2011
Accepted: 5 April 2012
Published: 5 April 2012
Folic 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.
We 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.
Our 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.
Folate one-carbon metabolism (FOCM) plays an integral role in human development and disease. Genetic variants in folate pathway genes are associated with increased risk for neural tube defects (NTD) (and other congenital defects) , cardiovascular disease , cancer  and cognitive decline . Women taking supplemental folic acid prior to conception significantly reduce their chance of having an NTD affected pregnancy . The mechanism by which folic acid reduces the incidence of NTD’s has not been fully elucidated.
Folate, also known as vitamin B9, is a cofactor for enzymes involved in cellular transmethylation reactions and de novo synthesis of purines and thymidylate . In its reduced form, the folate molecule acts a carrier of single carbon units. The ultimate donors of the methyl groups are serine, glycine, histidine and formate. Methylated folates are cofactors in two steps during inosine synthesis (the precursor of purine bases) and in the synthesis of thymidylate (a precursor necessary for DNA synthesis). The third major cellular reaction using folates as a co-factor is the methionine cycle, whereby the methyl group is added to homocysteine to form methionine. The subsequent product s-adenosyl-methionine is used in the synthesis of neurotransmitters, methylation of phospholipids, DNA methylation, histone methylation and mRNA capping. Folate dependent enzymes are found in the mitochondria, cytoplasm, and nucleus. Folate is a water-soluble vitamin found primarily in leafy vegetables. Due to its protective effect in early pregnancy, folate fortification is mandated in the US with synthetic folate in the form of folic acid added to milled grain products.
We sought to evaluate the zebrafish as a model for studying vertebrate folate and one-carbon metabolism. We aimed to understand the role of FOCM in early organ development. There is little to no information on these pathways in fish. Using bioinformatics we have identified zebrafish orthologs of 12 human folate metabolic genes and characterized their spatial and temporal expression patterns. Chemical and genetic disruption of these pathways demonstrated that the folate pathway is essential for several aspects of zebrafish development, as treated embryos are embryonic lethal. We show cell cycle perturbations as a result of defects in FOCM. We hypothesize these defects are the result of reduced folate substrates for thymidylate synthase, a rate-limiting enzyme in DNA synthesis. 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.
Cloning & RT-PCR
Folate Pathway Protein Homologies
mat1a 75/mat2a 73
Dihydrofolate reductase (Dhfr), a key enzyme in the conversion of dietary folate into the reduced bioactive form that can be utilized by cells (EC 22.214.171.124), is found in the zebrafish genome on chromosome 5. The predicted protein has 61% identity (76% similarity) to the human protein (NP_000782.1) . This transcript is maternally loaded and is present throughout the first 72 hours of development. At 24hpf the transcript is enriched in neural tissues; alar plate midbrain region, CNS, optic tectum, and optic vessels (, Figure 2B).
Methionine adenosyltransferases (MAT) are a family of enzymes that catalyze the synthesis of s-adenosylmethionine from methionine and ATP (EC 126.96.36.199). Folate is a cofactor in the preceding reaction that generates the methionine from homocysteine. Mat1a encodes the liver specific forms of this enzyme, MATI and MATIII . Mat2a is expressed throughout the organism and encodes MATII. The regulatory subunit for MATII is encoded by mat2b. Orthologs of these genes are found in zebrafish. Mat1a is located on chromosome 13 and has 84% identity (94% similarity) to the human protein (NP_000420.1). The transcript for this gene is not expressed until after the onset of gastrulation, by 24 hpf its expression is enriched in the YSL and myotomes (, Figure 2). Mat2b is found on chromosome 21 and has 65% identity (79% similarity) to the human protein (NP_037415.1). This is a maternally loaded transcript and is expressed throughout early zebrafish development. At 24 hpf, its expression is enriched in anterior neural tissues (Figure 2). The zebrafish genome has two paralogs of human Mat2a (NP_005902.1): Mat2aa, with 91% identity (96% similarity) on chromosome 10 and Mat2ab with 89% identity (96% similarity) on chromosome 5. Mat2aa is a maternally expressed gene whose expression decreases as development progresses. Mat2ab is a maternally loaded transcript and is present throughout the early stages of development. Both these transcripts are enriched in myotomes and anterior neural structures at 24 hpf (, Figure 2). The zebrafish genome contains another paralog in the mat family, which we propose to name Mat2a-like (mat2al), located on chromosome 10. This gene has 75% identity to human Mat2a and 73% identity to Mat1a, similar enough to be identified as a methionine adenosyltransferases, but not similar enough to be identified definitively as either one. It has a developmental expression profile, via RTPCR, similar to mat1a, but via in situ hybridization at 3 dpf we have not observed its expression being limited to the primitive liver (data not shown). At 1 dpf, its expression is enriched in the CNS (Figure 2).
Mthfd1 is a cytoplasmic trifunctional enzyme that interconverts methylene THF, methenyl THF, formyl THF, and formate. These folates serve as substrates for downstream reactions that synthesize thymidylate, methionine, and inosine. The protein is partitioned into an N-terminal bifunctional dehydrogenase and cyclohydrase domain (EC 188.8.131.52 and 184.108.40.206), and a C-terminal synthetase domain (EC 220.127.116.11). The mitochondrial protein, Mthfd1L retains a functional synthetase domain. However, while the N-terminal domain is present, mutations in critical residues results in the predicted loss of these enzymatic functions . The mitochondrial dehydrogenase and cyclohydrase reactions are performed by the proteins Mthfd2 and Mthfd2L. Mthfd2 is only found in transformed mammalian cells and embryonic or nondifferentiated tissues  and is essential during embryonic development . Mthfd2L has recently been shown to be the active mitochondrial enzyme in adult tissues . Orthologs of all these human proteins are found in zebrafish. Two paralogs of Mthfd1 (NP_005947.3) are found in zebrafish: mthfd1a has 57% identity (74% similarity) to the human protein and is located on chromosome 20, mthfd1b has 77% identity (88% similarity) and is located on chromosome 17. Mthfd1b is the likely true ortholog of human Mthfd1 as it retains the conserved amino acids in the catalytic domains of this trifunctional enzyme. However, Mthfd1a has a predicted mitochondrial leader sequence (p = 0.9736, ) and has mutations in conserved catalytic residues in the synthetase domain. This transcript is dynamically regulated within the first 72 hpf. Mthfd1b is expressed throughout early development. At 24 hpf, both transcripts are enriched in the CNS, with mthfd1b also expressed in the YSL (, Figure 2). An ortholog of Mthfd1L (NP_056255.2) can be identified in zebrafish, on chromosome 20, with 81% identity (89% similarity) to the synthetase domain. While the zebrafish transcript is predicted to encode a mitochondrial leader sequence, the dehydrogenase/cyclohydrase domain has only 44% identity to the human protein. This transcript is not maternally expressed and is dynamically regulated throughout early development. Its expression is not spatially restricted at 24 hpf (Figure 2B). One ortholog of Mthfd2 (NP_006627.2) is found on chromosome 5 in zebrafish with 80% identity (92% similarity). An Mthfd2L ortholog is located on chromosome on 8, with 67% identity (82% similarity). Both of these transcripts are dynamically expressed throughout the first 72 hours of development, with expression at 24 hpf being enriched in the CNS (Figure 2).
Methylenetetrahydrofolate reductase (Mthfr, EC 18.104.22.168) catalyzes the irreversible reduction of the one carbon donor group on folate to 5-methyl-tetrahydrofolate. This form of folate is the carbon donor in the conversion of homocysteine to methionine. An ortholog of the human protein (NP_ 005948.3) is found in the zebrafish genome on chromosome 8 with 79% identity (89% similarity) (NP_001121727.1). This is also a maternally loaded transcript and is present throughout the early stages of development. At 24 hpf its expression is also enriched in the CNS and myotomes (Figure 2B).
Methionine synthase (Ms, EC 22.214.171.124), encoded by the gene mtr, is the enzyme that catalyzes the conversion of homocysteine to methionine, using 5-methyl-tetrahydrofolate as the methyl group donor and the vitamin cofactor cobalamin (vitamin B12). This enzyme sits at the intersection of the folate pathway and the methionine cycle. Zebrafish has one ortholog of this protein (NP_00245.2) on chromosome 12 with 77% identity (88% similarity). The transcript is maternally loaded and present throughout the first three days of embryonic development. At 24hpf its expression is enriched in the CNS (, Figure 2B).
Thymidylate synthetase (Tyms, EC 126.96.36.199) is an enzyme involved in a rate-limiting step of DNA synthesis: the methylation of dUMP to dTMP. This reaction uses a folate cofactor as the one carbon donor. An ortholog of the human protein (NP_001062.1) is found in the zebrafish genome on chromosome 7 with 82% identity (91% similarity). This is also a maternally loaded transcript and is present throughout the early stages of development. At 24 hpf its expression is also enriched in the CNS (, Figure 2B).
We also investigated the transport protein, Slc25a32 (MFTC), a mitochondrial membrane protein that transports folate into the mitochondrion . Two paralogs of Slc25a32 (NP_110407.2) are found in zebrafish. Slc25a32a is located on chromosome 19 and has 66% identity (81% similarity) to the human protein. This transcript is also maternally expressed and is present throughout early development. At 24 hpf it is expression is not spatially restricted (Figure 2B). Slc25a32b is located on chromosome 16, has 70% identity (82% similarity) to the human protein, and is expressed throughout early embryonic development. Its expression is enriched in anterior neural tissues at 24 hpf (Figure 2B).
In order to investigate the role of the folate pathway in early zebrafish development, we inhibited Dhfr by using either the chemotherapeutic drug methotrexate or antisense morpholino oligonucleotides.
Folinic acid, a folate derivative that can enter the folate metabolic pathway without the enzymatic function of Dhfr, is used during chemotherapy to ameliorate the toxic effects of methotrexate . Embryos pre-treated with folinic acid in addition to 200 μM methotrexate showed fewer severe defects at 2 dpf: less edema, fewer embryos with dorsal curvature and a decrease in cardiac defects (Figure 3B). However, this treatment was unable to completely rescue methotrexate treatment as the embryos died by 5 dpf.
Differentiation and tissue specification
Cell cycle aspects of folate pathway perturbations
Embryos treated with methotrexate are phenotypically abnormal but tissues are normally specified. We hypothesized the defect is in the number of cells in the embryo, due to defects and delays in the cell cycle in response to reduced folate substrate for the enzyme thymidylate synthetase.
Our aim was to identify and characterize components of the folate pathway and to interrogate its role in early zebrafish development. Twelve genes involved in folate metabolism were identified by BLAST searches, cloned, and their expression profiles studied by RT-PCR and RNA in situ hybridization. We also assessed the impact of inhibiting the folate pathway in the developing zebrafish by treatment with the folate antagonist, methotrexate, and by microinjection of antisense morpholino oligonucleotides designed to disrupt Dhfr expression. Inhibition of the folate pathway caused perturbations in the cell cycle, which resulted in embryonic lethality. While it is not surprising that this pathway is essential for development, the mechanism through which this inhibition acts had not been explored. We propose the primary defect in these fish is the lack of folate substrates for Tyms, a rate-limiting step in DNA synthesis, which results in the lack of dTTP pools for DNA synthesis. However, it is also possibile that other methylation reactions, requiring s-adenosylmethionine (a downstream product of folate one- carbon pathway), also play a role in the defects we observed.
We set out to identify and clone 12 orthologs of folate genes in humans. We identified 16 orthologs and paralogs, presumably due to the partial genome duplication that occurred in teleosts . The presence of paralogs can complicate studies that try to establish gene function by looking at single gene knock-out (down) phenotypes. One set of duplicated genes we identified was the mats: mat1a and mat2a. Mat1a encodes the liver specific isoform of the enzyme. Mat2a encodes the globally expressed isoform. Zebrafish has four mat genes. One is clearly mat1a, with 77% identity to the human protein and liver expression at 3 dpf (data not shown). Two genes, mat2aa and mat2ab, appear to be paralogs of human mat2a, with 80% identity to the human protein and without liver restricted expression. The fourth gene, mat2al, has a temporal expression pattern similar to mat1a, but without the liver restricted expression, similar to the mat2as, and relatively low (75% and 73%) identity to the human Mat1a and Mat2a proteins, respectively. This highlights one of the challenges of transferring pathways between species.
Queried members of the folate pathway are generally maternally loaded and ubiquitously expressed (with enrichment in anterior neural tissues), consistent with the idea that they are house-keeping genes involved in an essential cellular process. Inhibition of the pathway with either the folate antagonist methotrexate or by using morpholinos targeting dhfr results in early embryonic lethality. Further study of methotrexate treated embryos indicates defects in the cell cycle, specifically S-phase accumulation and increased apoptosis. By visual morphological criteria and in situ analysis embryos have a normal developmental program through bud stage. Between 12 hpf and 24 hpf development defects arise, as >80% of embryos treated with 400 μM methotrexate die overnight and the surviving embryos are anatomically abnormal. Neural cells condense to form the neural plate around the 2 somites stage (11 hpf) . Most embryos treated with methotrexate do not survive treatment to be queried specifically for neural tube defects. Despite decreased cell numbers due to S-phase delay and concomitant decreased mitosis, the developmental program proceeds and appropriate tissues are specified, although the anterior-posterior axis is severely shortened and the notochord undulates. Convergent/extension defects, resulting in embryos with a shortened A-P axis, are observed in zebrafish expressing rare variants of VANGL1 that are associated with neural tube defects in humans .
One limitation of using zebrafish as a model system for understanding genetic and environmental etiologies of neural tube defects is that the mechanics of zebrafish neurulation differs from that of human and mouse. Zebrafish form a neural plate, which then progresses through a neural keel stage, to a solid neural rod which then cavitates to a neural tube . Zebrafish do not form a neural tube via the bending of the neural plate and fusing of opposing sides. In humans and mice, the failure of these tissues to fuse presents as a neural tube defect, whether in its lethal form of anencephaly or the milder state of spina bifida. However, many of the molecular and genetic aspects of neural tube formation are genetically conserved between humans and fish .
The essential requirement for the folate pathway has been documented in other model organisms ranging from yeast to mice. Of the genes we studied, only dfr1 (dhfr) and cdc21 (tyms) are essential in budding yeast. Mtr −/− mice are embryonic lethal prior to day E9.5 . Mthfd1 −/− mice also are embryonic lethal  as are Mthfd2 −/− mice, by day E13.5, and can be distinguished by their smaller size and pale livers . In contrast Mthfr −/− knock out mice are viable, although with reduced viability, decreased body size, and cerebellar pathology . A small number of reports have investigated folate pathway genes and mutants in zebrafish. Sun et al. reported cardiac defects in zebrafish dhfr morphants. Similar cardiac defects were also reported for zebrafish embryos treated with methotrexate, as well as decreased expression of the cardiac markers (hand2, mef2a, and mef2b) . Neither study reports the early severe defects we observed nor do they interrogate cell cycle properties of affected embryos. Zebrafish tyms mutants were identified in screens looking for genes essential for embryonic  or defective in retinal development . Another zebrafish mutant with ocular defects has been identified as gart, one of two enzymes involved in purine metabolism that uses folate as a cofactor . Our identification and characterization of major components of the folate pathway in zebrafish increases the utility of this model organism in studying the role of folate in normal development and disease.
We provide the first in depth characterization of the folate pathway in zebrafish, by reporting the expression patterns of 16 genes, of which we name 6 genes in ZFIN and provide additional sequence information for 3 genes. We expand upon earlier studies that implicated Dhfr in cardiac development by identifying an earlier defect in zebrafish. Folate starved embryos start to show defects prior to segmentation. We propose this is due to defects in S phase, caused by decreased pools of thymidylate and consequently dTTP, which in turn leads a lengthened cell cycle. As each round of division takes longer, fewer total cells are available in which to form an embryo. We also observed an increase in apoptosis, which we propose to be a consequence of and in response to extended delays in S-phase. Despite the decreased number of cells and decreased size of the entire organism, mutant embryos are able to appropriately specify the neuro-ectodermal and mesodermal tissues that were queried. The typical consequence of folate deficiency is megaloblastic anemia, an enlargement of red blood cells, due to the failure of cellular division of erythrocyte precursors. Our data support the idea that in rapidly dividing hematopoietic or embryonic cells, DNA synthesis is susceptible before purine synthesis and other cellular methylation reactions. Low folate status and hyperhomocysteinemia have been implicated as potentially reversible risk factors for cognitive decline and cardiovascular disease. Our work establishes zebrafish as a model system for studying the folate pathway and demonstrates that zebrafish can serve as a model organism for studying the role of folate in development in order to understand the genetic and environmental etiology underlying some neural tube and congenital heart defects.
The zebrafish, TAB-5 strain (ZIRC, Eugene, OR), were bred and maintained at 28.5°C as described . Animals were staged by hours post-fertilization (hpf) and on morphological criteria. Embryos were treated with Methotrexate USP (GeneraMedix, Liberty Corner, NJ) and/or Leucovorin USP (folinic acid) (Bedford Laboratories, Bedford, OH) within 30 minutes of fertilization in 1X E3. We treated embryos with 5 orders of magnitude more than the minimal chemotherapeutic dose of methotrexate. We used 14 C labeled methotrexate to measure intra-embryonic uptake of the drug. These data suggest that only small amounts of drug permeate the chorion into the embryo (data not shown). Embryos were injected with up to 4 nL of diluted morpholino (Genetools LLC, Philomath, OR) (as determined by drop size in oil) prior to 8-cell stage. Unfertilized embryos were removed between 4–6 hpf and media was changed at 2 dpf. Experimental protocols were reviewed and approved by the NHGRI Animal Use and Care Committee. Morpholino sequences: tp53 GCGCCATTGCTTTGCAAGAATTG , dhfr ATG 5’ ACGGTCTCGCCTTCTTCCCGCCAAG 3’ , dhfr.i3e4 5’ TGTCCTGTGGAGTGACAGAAAATAA 3’, dhfr.MM 5’ ACcGTCTCGgCTTgTTCgCGCCgAAG 3’ (mismatches in lower case).
Orthologs were identified by TBLASTN of the human protein sequence to the zebrafish genome (Ensembl v7) and/or cDNAs. Primers to predicted transcripts were designed to amplify the 5’UTR and CDS. RNA was isolated from 24 hpf embryos using RNA STAT-60 (Amsbio, Abingdon, UK) and transcribed to cDNA (Superscript III, Invitrogen, Carlsbad, CA). Transcripts were amplified (Hotstar Taq, Qiagen, Valencia, CA), cloned into pCRII (Invitrogen, Carlsbad, CA), and sequenced. GenBank accession numbers: JN848834 (mat2aa), JN848835 (mat2ab), JN848836 (mthfr).
RNA was isolated from staged embryos (4 cell through 72 hpf) using RNA STAT-60 (Amsbio, Abingdon, UK) or TRI reagent LS (Sigma-Aldrich, St. Louis, MO). 1 μg of total RNA was transcribed into cDNA (Superscript III SuperMix, Invitrogen, Carlsbad, CA). Primers for RT-PCR were designed to yield 400–600 bp products, biased to the 3’ end of the transcript, and spanned at least one intron - exon boundary ( Additional file 1: Table S1). Multiple overlapping sets of primers were designed to the dhfr transcript to query the efficacy of morpholino induced knock-down ( Additional file 2: Table S2). Aberrant splice products were TOPO-TA cloned (Invitrogen, Carlsbad, CA) and sequenced.
In situ hybridization
In situ probes were designed to be ~500 bp and biased to the 3’UTR. Hybridizations were performed essentially as described .
Embryos were treated with 400 μM methotrexate for 24 hours. 20–40 embryos were processed in duplicate. After treatment, embryos were briefly rinsed in calcium-free Ringers solution (116 mM NaCl, 2.9 mM KCl, 5 mM HEPES pH 7.2), pestled 20 times in calcium-free Ringers, and then passed through 40 μM filter (BD Falcon 352340), rinsed once and resuspended in DNA staining buffer (100 mM Tris pH 7.5, 154 mM NaCl, 1 mM CaCl, 0.5 mM MgCl, 0.2% BSA, 0.1% Nonidet P-40, 250ug/mL Rnase, 20ug/mL propidium iodide) . FACS analysis was performed on FACSCalibur (BD Biosciences) and data analyzed in FlowJo (Ashland, OR).
BrdU and phospho-histone H3 Assays
Phosphohistone-H3 immunochemistry was performed as described . BrdU incorporation was assayed essentially as described  with the following modifications: fixed embryos were permeabilized with methanol and treated with 4 M HCl for 20 minutes. Primary mouse anit-BrdU antibody (clone II5B, Santa Cruz) was used at a 1:250 dilution and secondary AP-conjugated donkey anti-mouse antibody was diluted 1:500. Alkaline phosphatase was developed with NBT/BCIP solution (Roche, Basel, Switzerland).
Folate-one carbon metabolism
Neural tube defects
We thank Stacie Anderson for help with FACS analysis, Darryl Leja for help with figures, Settara Chandrasekharappa for equipment, Amy Singer of the ZFIN Nomenclature Committee for naming help, and Charles River Aquatic Staff for fish care. This research was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health (S.M.B, L.C.B).
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