- Research article
- Open Access
Reduced-folate carrier (RFC) is expressed in placenta and yolk sac, as well as in cells of the developing forebrain, hindbrain, neural tube, craniofacial region, eye, limb buds and heart
© Maddox et al; licensee BioMed Central Ltd. 2003
- Received: 29 May 2003
- Accepted: 29 July 2003
- Published: 29 July 2003
Folate is essential for cellular proliferation and tissue regeneration. As mammalian cells cannot synthesize folates de novo, tightly regulated cellular uptake processes have evolved to sustain sufficient levels of intracellular tetrahydrofolate cofactors to support biosynthesis of purines, pyrimidines, and some amino acids (serine, methionine). Though reduced-folate carrier (RFC) is one of the major proteins mediating folate transport, knowledge of the developmental expression of RFC is lacking. We utilized in situ hybridization and immunolocalization to determine the developmental distribution of RFC message and protein, respectively.
In the mouse, RFC transcripts and protein are expressed in the E10.0 placenta and yolk sac. In the E9.0 to E11.5 mouse embryo RFC is widely detectable, with intense signal localized to cell populations in the neural tube, craniofacial region, limb buds and heart. During early development, RFC is expressed throughout the eye, but by E12.5, RFC protein becomes localized to the retinal pigment epithelium (RPE).
Clinical studies show a statistical decrease in the number of neural tube defects, craniofacial abnormalities, cardiovascular defects and limb abnormalities detected in offspring of female patients given supplementary folate during pregnancy. The mechanism, however, by which folate supplementation ameliorates the occurrence of developmental defects is unclear. The present work demonstrates that RFC is present in placenta and yolk sac and provides the first evidence that it is expressed in the neural tube, craniofacial region, limb buds and heart during organogenesis. These findings suggest that rapidly dividing cells in the developing neural tube, craniofacial region, limb buds and heart may be particularly susceptible to folate deficiency.
- Retinal Pigment Epithelium
- Neural Tube
- Optic Vesicle
- Trophoblastic Giant Cell
- Renal Tubular Epithelium
Folates are highly lipophobic, bivalent anions that can only minimally traverse biological membranes by simple diffusion . Because they pass so inefficiently through biological membranes, supply of folates through mammalian cell plasma membranes must occur by a mediated process . Reduced-folate carrier (RFC; additionally known as RFC-1, FOLT, RFT-1 or SLC19A1) (Mr 60 kDa) is a typical transport protein with 12 membrane-spanning domains. RFC preferentially transports reduced folates, such as N5-methytetrahydrofolate (MTF), the most common form of circulating folate. In adult tissues, RFC is expressed in the brush-border membrane of jejunum, ileum, duodenum and colon and in the basolateral membrane of renal tubular epithelium, hepatocytes, choroid plexus  and the retinal pigment epithelium (RPE) of the eye .
The importance of RFC during embryogenesis has been demonstrated by the use of targeted gene deletion. Mice with targeted deletions of RFC die at an early embryonic age . Thus, although it is evident the RFC is needed for normal embryonic development, the tissue-specific requirements for RFC during embryogenesis remain largely a mystery. In the present study we offer the first developmental analysis of the embryonic tissue Distribution of RFC message and protein in mouse placenta and developing embryo.
Expression of RFC mRNA and protein in E10.0 mouse uterine sections
Expression of mRNA encoding RFC in whole-mount embryos from E9.0 to E11.5
Immunohistochemical detection of RFC protein at E9.0 through E10.5
In trans-uterine sections of E10.0 pregnant mouse uterus, we found that RFC mRNA and protein were widely distributed throughout the placenta. While studies by Wang et al.  suggested that RFC expression in adult tissues is confined to specialized cell types such as the brush-border membrane of the jejunum, ileum, duodenum, and colon or the basolateral membranes of renal tubular epithelium, hepatocytes and choroid plexus, more recent studies suggest a more ubiquitous expression in adult tissues . In accordance with these recent findings, in situ hybridization and immunolocalization analysis of whole-mount embryos revealed that RFC mRNA and protein were widely present. At E9.0 the signal was most intense in the optic vesicles, forebrain, mandible, heart, somites and tail bud. By E10.5 RFC was also detectable in the neural tube, limb buds and nasal pits. In the eye RFC mRNA and protein were expressed ubiquitously through E12.0. By E12.5, However, the RFC protein was no longer present throughout the developing eye; instead it had segregated to the apical plasma membrane of the RPE, its adult location in the eye .
This study has important clinical implications concerning the mechanism by which the beneficial effects of folate supplementation during pregnancy are derived. Many studies have shown a statistically significant decrease in the number of neural tube defects (NTD's) suffered in offspring of female patients who were given supplemental vitamins containing folate during pregnancy [7–9]. To a lesser extent, folate supplementation has also been shown to decrease the incidence of craniofacial abnormalities [10–12], cardiovascular defects [13, 14] and limb abnormalities  in humans. This work demonstrates that RFC mRNA and protein are present in rapidly dividing cell populations of the mouse embryo during early organogenesis of these structures.
The pattern of RFC mRNA and protein expression is developmentally regulated. While RFC expression is most strongly present in specialized cell-types in adult mice, during development RFC mRNA and protein are expressed in the placenta, yolk sac and throughout the embryo, with higher levels of expression being confined to cell populations in the neural tube, forebrain, hindbrain, craniofacial region, eye, limb buds, heart, somites and tail. Mutations in the RFC gene or deficient maternal folate intake may lead to developmental defects by decreasing proliferation rates in the cell populations determined by this study to express high levels of RFC during development.
ICR male and female mice were purchased from Harlan Sprague Dawley (Indianapolis, IN). Timed pregnancies were obtained by checking mating plugs and the morning a plug was detected was defined as E0.5. Pregnant female ICR mice were sacrificed by cervical dislocation, the uterine horns opened immediately and the embryos collected in cold diethyl pyrocarbonate (DEPC)-treated (0.01 M) PBS. Care and use of mice adhered to the principles set forth in DHEW Publication NIH 80–23 (Guiding Principles in the care and use of animals).
Whole-mount in situ hybridization
The sense and antisense digoxigenin (DIG)-labeled cRNA probes for mouse RFC transcripts were prepared using a DIG RNA labeling kit (Boehringer Mannheim, Indianapolis, IN) following the manufacturer's protocol. The cDNA used to generate cRNA probes was obtained by reverse transcriptase polymerase chain reaction (RTPCR) performed on total eye RNA using the primers 5'-AGCGATAAGCCTACAGGATGG-AGACCTA-3' (sense) and 5'-CTGCAGGCTCAGCGGACCTTGGCT-3' (antisense) which amplified a 618-bp fragment between bases 1558 and 2175 of the published RFC sequence (Genbank accession number NM031196) . The amplified product was cloned into pGEM-T vector in an orientation producing sense probe by T7 RNA polymerase-mediated transcription and antisense probe by SP6 RNA polymerase-mediated transcription. The identity of the amplified products was confirmed by nucleotide sequencing and in situ hybridization was performed as previously described . Specimen were photographed without clearing using a Zeiss Axioskop microscope or a Zeiss Stemi-2000 C dissecting scope equipped with a Spot camera.
Immunohistochemistry on cryosections was performed as previously described . Whole embryos were processed for immunohistochemistry by fixation in 4% PFA for 2 h at 4°C followed by rinsing in PBS. Embryos were dehydrated through a graded ethanol series and the tissues were re-fixed with methanol:dimethylsulfoxide (DMSO) (4:1) overnight at room temperature followed by methanol:DMSO:30% H2O2 (4:1:1) at room temperature for 4 h. Embryos were rehydrated through reverse ethanol series. Nonspecific binding was blocked by incubating the embryos in PBSTMD (2% skim milk powder, 1% DMSO in PBS containing 0.1% Tween 20). The RFC-1 anti-peptide antibody was raised against the peptide sequence RPKRSLFFNRDDRGRC, corresponding to residues 205–220 of human RFC-1 and was used at a 1:500 dilution [3, 18]. The specificity of this antibody has been described . Control experiments were carried out by incubating the antibody with the RPKRSLFFNRDDRGRC peptide at a concentration of 2 μg/ml for 30 minutes prior to beginning the experiment. Incubations were carried out in PBSTMD overnight at 4°C. The antibody solution was removed and embryos were rinsed twice with PBS following which they were washed 4 × 1 h in PBST. Embryos were blocked with PBSTMD for 4 h, the secondary horse-radish peroxidase (HRP)-conjugated antibodies were added in PBSTMD (1:100) and the embryos were incubated overnight at 4°C. Embryos were rinsed 2× with PBS and washed for 4 × 1 h in PBST before being incubated for 1 h in 500 μl DAB solution (1 mg/ml in PBST). 500 μl of 0.03% H2O2 was added and 3–5 minutes were allowed for the color to develop before the embryos were rinsed in PBS and photographed using a Zeiss Axioskop microscope equipped with a Spot camera.
We would like to thank Sue Johnson for help in the preparation of this manuscript. We would like to thank Dr. Pamela Martin for technical advice. This work was supported by National Institutes of Health Grants EY 12830 and HD 37150-01.
- Yang CH, Sirotnak FM, Dembo M: Interaction between anions and the reduced folate/methotrexate transport system in L1210 cell plasma membrane vesicles: directional symmetry and anion specificity for differential mobility of loaded and unloaded carrier. J Membr Biol. 1984, 79: 285-292.View ArticlePubMedGoogle Scholar
- Wang Y, Zhao R, Russell RG, Goldman ID: Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis. Biochemica et Biophysica Acta. 2001, 1513: 49-54. 10.1016/S0005-2736(01)00340-6.View ArticleGoogle Scholar
- Chancy CD, Kekuda R, Huang W, Prasad PD, Kuhnel J, Sirotnak FM, Roon P, Ganapathy V, Smith SB: Expression and differential polarization of the reduced-folate carrier-1 and the folate receptor α in mammalian retinal pigment epithelium. J Biol Chem. 2000, 275: 20676-20684. 10.1074/jbc.M002328200.View ArticlePubMedGoogle Scholar
- Zhao R, Russell RG, Wang Y, Liu L, Gao F, Kneitz B, Edelmann W, Goldman ID: Rescue of embryonic lethality in reduced folate carrier-deficient mice by maternal folic acid supplementation reveals early neonatal failure of hematopoietic organs. J Biol Chem. 2001, 276: 10224-10228.View ArticlePubMedGoogle Scholar
- Smith SB, Huang W, Chancy C, Ganapathy V: Regulation of the reduced folate carrier by nitric oxide in cultured human retinal pigment epithelial cells. Invest Biochem Biophys Res Commun. 1999, 257: 279-283. 10.1006/bbrc.1999.0452.View ArticleGoogle Scholar
- Whetstine JR, Flatley RM, Matherly LH: The human reduced folate carrier gene is ubiquitously and differentially expressed in normal human tissues: identification of seven non-coding exons and characterization of a novel promoter. Biochem J. 2002, 367: 629-40. 10.1042/BJ20020512.PubMed CentralView ArticlePubMedGoogle Scholar
- Czeizel AE, Dudas I: Prevention of the first occurrence of neural-tube defects by periconceptual vitamin supplementation. New Engl J Med. 1992, 327: 1832-1835.View ArticlePubMedGoogle Scholar
- Berry RJ, Li Z, Erickson JD, Li S, Moore CA, Wang H, Mulinare J, Zhao P, Wong LC, Gindler J, Hong S, Correa A: Prevention of neural-tube defects with folic acid in China. N Engl J Med. 1999, 341: 1485-1490. 10.1056/NEJM199911113412001.View ArticlePubMedGoogle Scholar
- Ray JG, Meier C, Vermeulen MJ, Boss S, Wyatt PR, Cole DEC: Association of neural tube defects and folic acid food fortification in Canada. Lancet. 2002, 360: 2047-2048. 10.1016/S0140-6736(02)11994-5.View ArticlePubMedGoogle Scholar
- Shaw GM, Lammer EJ, Wasserman CR, O'Malley CD, Tolarova MM: Risks of orofacial clefts in children born to women using multivitamins containing folic acid periconceptionally. Lancet. 1995, 346: 393-396. 10.1016/S0140-6736(95)92778-6.View ArticlePubMedGoogle Scholar
- Tolarova M, Harris J: Reduced recurrence of orofacial clefts after periconceptual supplementation with high-dose folic acid and multivitamins. Teratology. 1995, 51: 71-78.View ArticlePubMedGoogle Scholar
- Itikala PR, Watkins ML, Mulinare J, Moore CA, Liu Y: Maternal multivitamin use and orofacial clefts in offspring. Teratology. 2001, 63: 79-86. 10.1002/1096-9926(200102)63:2<79::AID-TERA1013>3.3.CO;2-V.View ArticlePubMedGoogle Scholar
- Czeizel AE: Prevention of congenital abnormalities by periconceptual multivitamin supplementation. Br Med J. 1993, 306: 1645-1648.View ArticleGoogle Scholar
- Botto LD, Khoury MJ, Mulinare J, Erickson JD: Periconceptional multivitamin use and the occurrence of conotruncal heart defects: results from a population-based, case-control study. Pediatrics. 1996, 98: 911-917.PubMedGoogle Scholar
- Czeizel AE: Limb-reduction defects and folic acid supplementation. Lancet. 1995, 345: 932-10.1016/S0140-6736(95)90052-7.View ArticlePubMedGoogle Scholar
- Brigle KE, Spinella MJ, Sierra EE, Goldman ID: Characterization of a mutation in the reduced folate carrier in a transport defective L1210 murine leukemia cell line. J Biol Chem. 1995, 270: 22974-22979. 10.1074/jbc.270.14.7842.View ArticlePubMedGoogle Scholar
- Maddox DM, Condie BG: Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development. BMC Dev Biol. 2001, 1: 1-10.1186/1471-213X-1-1.PubMed CentralView ArticlePubMedGoogle Scholar
- Naggar H, Ola MS, Moore P, Huang W, Bridges CC, Ganapathy V, Smith SB: Downregulation of the reduced-folate carrier by glucose in cultured retinal pigment epithelial cells and in streptozotocin-induced diabetic mice. Invest Ophthalmol Vis Sci. 2002, 43: 556-563.PubMedGoogle Scholar
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