title: Microarray analysis of E9.5 reduced folate carrier (RFC1; Slc19a1) knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex.
abstract: BACKGROUND
  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. 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.
  
  
  RESULTS
  Affymetrix 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 Ingenuitytrade mark 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.
  
  
  CONCLUSIONS
  Inactivation 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.
authors.affiliation: Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Center, Omaha, NE 68198-5455, USA. jvanwaes@unmc.edu
authors.forename: Janee
authors.initials: J
authors.lastname: Gelineau-van Waes
authors.forename: Joyce R
authors.initials: JR
authors.lastname: Maddox
authors.forename: Lynette M
authors.initials: LM
authors.lastname: Smith
authors.forename: Michael
authors.initials: M
authors.lastname: van Waes
authors.forename: Justin
authors.initials: J
authors.lastname: Wilberding
authors.forename: James D
authors.initials: JD
authors.lastname: Eudy
authors.forename: Linda K
authors.initials: LK
authors.lastname: Bauer
authors.forename: Richard H
authors.initials: RH
authors.lastname: Finnell
mesh_terms: D000818:Animals; D004622:Embryo, Mammalian / Q000378:metabolism; D047108:Embryonic Development; D004705:Endocytosis; D005260:Female; D053263:Gene Regulatory Networks; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000737:chemistry / Q000378:metabolism*; D008431:Maternal-Fetal Exchange; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000378:metabolism*; D051818:Replication Protein C / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: C424167:Amn protein, mouse; D026561:Low Density Lipoprotein Receptor-Related Protein-2; C055127:Lrp2 protein, mouse; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C095385:Rfc1 protein, mouse; D014157:Transcription Factors; C029486:intrinsic factor-cobalamin receptor; D051818:Replication Protein C