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

<p>Abstract</p> <p>Background</p> <p>The reduced folate carrier (<it>RFC1</it>) 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 <it>RFC1 </it>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 <it>RFC1</it>^-/-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.</p> <p>Results</p> <p>Affymetrix microarray analysis of the relative gene expression profiles in whole E9.5 <it>RFC1</it>^-/-vs. <it>RFC1</it>^+/+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 <it>RFC1</it>^-/-embryos, while cubilin protein was widely misexpressed.</p> <p>Conclusion</p> <p>Inactivation of <it>RFC1 </it>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.</p

Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex-5

Bilin protein. The level and plane of the sections shown in Figure 6 is illustrated by the line drawn through the embryo shown in the upper left hand corner. The red boxes drawn over the neuroepithelium of embryos shown in and (4× magnification) indicate the orientation of the higher magnification panels (40× magnification) shown in , , , and ; the apical surface (ventricular side) of the neuroepithelial cells is shown on the right of these panels, and the basolateral, or mesenchymal side of the neuroepithelial cells is shown on the left side of the panels. Megalin is expressed exclusively on the apical side of neuroepithelial cells in wildtype embryos (; red arrows), but is expressed on both the apical (; red arrows) and the basolateral side (; purple arrowheads) of the neuroepithelium in mutants. Cubilin expression is also restricted to the apical side of neuroepithelial cells in wildtype embryos (, ; red arrows), but is misexpressed on the basolateral side of the neuroepithelium in nullizygous embryos (, ; purple arrowheads). Staining for cubilin protein is visible in the surface ectoderm of embryos (; black arrows), and discrete staining is also observed in the notochord (, ; yellow arrow). Cubilin expression is highly upregulated in the cranial mesenchyme of embryos (shown in panel ) relative to wildtype littermates, especially in the region surrounding the notochord (; yellow arrow). Staining for cubilin protein is visible in the mesothelial cells of the pericardium of wildtype (; black arrowhead) and mutant embryos (not visible in section shown in ), and although there are a few cells that stain positive for cubilin in the trabeculae of the common ventricular chamber in the wildtype embryo, cubilin expression is dramatically upregulated throughout the myocardium of the mutant (). Normal morphology of the primary head vein/cephalic extension of the anterior cardinal vein (**) and the third branchial arch artery (*) in the wildtype embryo are shown in (6C); comparable structures labeled in the mutant (6D) indicate abnormal vascular morphology. left ventricle.<p><b>Copyright information:</b></p><p>Taken from "Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex"</p><p>http://www.biomedcentral.com/1471-2164/9/156</p><p>BMC Genomics 2008;9():156-156.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2383917.</p><p></p

Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex-2

consisting of a large extracellular region, a single transmembrane domain, and a C-terminal cytoplasmic tail. The extracellular domain of megalin contains four clusters of lipoprotein receptor ligand-binding repeats (blue), growth factor repeats, an EGF repeat, and YWTD spacer regions. The second cluster of ligand-binding repeats has been identified as a common binding site for several ligands including apolipoprotein E (Apo E), apolipoprotein M (Apo M), retinol binding protein (Rbp), and transthyretin (Ttr). Megalin also binds the soluble form of the folate receptor (Folr1), and the morphogen sonic hedgehog (Shh). The cytoplasmic tail of megalin binds Dab2, a cytosolic adapter protein important for megalin-mediated endocytosis, and Dab2 binds and recruits Myo6 to clathrin-coated vesicles. The receptor-associated protein (Lrpap1; RAP) binds both megalin and cubilin. Cubilin is a peripheral membrane receptor comprised of a short amino terminal, eight EGF type domains, and 27 CUB domains (green). The amino-terminal end of cubilin is attached to the extracellular part of amnionless (Amn), and amnionless provides the transmembrane domain necessary for the anchoring and endocytic trafficking of cubilin. Cubilin ligands include transferring (Trf), albumin, hemoglobin, apolipoprotein A1 (ApoA1), and intrinsic factor (IF)-vitamin B.<p><b>Copyright information:</b></p><p>Taken from "Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex"</p><p>http://www.biomedcentral.com/1471-2164/9/156</p><p>BMC Genomics 2008;9():156-156.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2383917.</p><p></p

Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex-4

He apical side of the visceral yolk sac in E9.5 embryos. Cubilin is also highly expressed on the apical side of the visceral yolk sac in E9.5 embryos (). Megalin expression, however, is completely absent from the visceral yolk sac of mutants (). Folr1 protein expression is absent from the apical plasma membrane of the VYS, but demonstrates increased expression in the endothelial cell layer of the yolk sac blood islands ().<p><b>Copyright information:</b></p><p>Taken from "Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex"</p><p>http://www.biomedcentral.com/1471-2164/9/156</p><p>BMC Genomics 2008;9():156-156.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2383917.</p><p></p

Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex-0

T of the genes within each of these gene ontology groups that were differentially expressed following statistical analysis of the microarray data is provided in [Additional file ].<p><b>Copyright information:</b></p><p>Taken from "Microarray analysis of E9.5 reduced folate carrier () knockout embryos reveals altered expression of genes in the cubilin-megalin multiligand endocytic receptor complex"</p><p>http://www.biomedcentral.com/1471-2164/9/156</p><p>BMC Genomics 2008;9():156-156.</p><p>Published online 9 Apr 2008</p><p>PMCID:PMC2383917.</p><p></p