Abstract
BackgroundThe reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Targeted inactivation of the murine RFC1 gene results in post-implantation embryolethality, but daily folic acid supplementation of pregnant dams prolongs survival of homozygous embryos until mid-gestation. At E10.5 RFC1-/- embryos are developmentally delayed relative to wildtype littermates, have multiple malformations, including neural tube defects, and die due to failure of chorioallantoic fusion. The mesoderm is sparse and disorganized, and there is a marked absence of erythrocytes in yolk sac blood islands. The identification of alterations in gene expression and signaling pathways involved in the observed dysmorphology following inactivation of RFC1-mediated folate transport are the focus of this investigation.ResultsAffymetrix microarray analysis of the relative gene expression profiles in whole E9.5 RFC1-/- vs. RFC1+/+ embryos identified 200 known genes that were differentially expressed. Major ontology groups included transcription factors (13.04%), and genes involved in transport functions (ion, lipid, carbohydrate) (11.37%). Genes that code for receptors, ligands and interacting proteins in the cubilin-megalin multiligand endocytic receptor complex accounted for 9.36% of the total, followed closely by several genes involved in hematopoiesis (8.03%). The most highly significant gene network identified by Ingenuity™ Pathway analysis included 12 genes in the cubilin-megalin multiligand endocytic receptor complex. Altered expression of these genes was validated by quantitative RT-PCR, and immunohistochemical analysis demonstrated that megalin protein expression disappeared from the visceral yolk sac of RFC1-/- embryos, while cubilin protein was widely misexpressed.ConclusionInactivation of RFC1 impacts the expression of several ligands and interacting proteins in the cubilin-amnionless-megalin complex that are involved in the maternal-fetal transport of folate and other nutrients, lipids and morphogens such as sonic hedgehog (Shh) and retinoids that play critical roles in normal embryogenesis.
Highlights
The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells
The 12 known genes in our data set that have been identified as cell surface receptors, ligands, or interacting proteins in the cubilin-megalin multiligand endocytic receptor complex are listed in Table 1, and the complete set of 200 known genes that were differentially expressed following statistical analysis of the microarray data are provided [see Additional file 1]
The fold-change represents the value for the comparison of level of gene expression in the RFC1-/- embryos relative to the RFC1+/+ embryos. (The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus [18] and are accessible to the public through GEO Series accession number GSE10659)
Summary
The reduced folate carrier (RFC1) is an integral membrane protein and facilitative anion exchanger that mediates delivery of 5-methyltetrahydrofolate into mammalian cells. Adequate maternal-fetal transport of folate is necessary for normal embryogenesis. Folate and vitamin B12 are essential vitamins derived from various food sources that play an important role in erythropoiesis, DNA biosynthesis, and embryogenesis. Nutritional deficiencies or genetic variations that impact folate and/or vitamin B12 homeostasis may result in megaloblastic anemia [1], and failure of maternal-fetal transport of these nutrients has been shown to adversely impact normal embryogenesis [2,3,4,5]. Tetrahydrofolate, the reduced form of folate, functions as an important co-factor for donating or accepting methyl groups. Adequate levels of folate for the incorporation of single carbon groups into purine, or methylation of deoxyuridylate to form thymidylate, are necessary for DNA biosynthesis. According to the "folate-trap hypothesis", cobalamin deficiency results in 'trapping' of tetrahydrofolate, producing a functional folate deficiency that leads to impaired erythropoiesis and DNA biosynthesis [7]
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