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AFFYMETRIX ANALYSIS OF E9.5 RFC MOUSE KO EMBRYOS REVEALS ALTERED EXPR’N OF GENES IN THE CUBILIN-MEGALIN COMPLEX

ABSTRACT: 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 embryo lethality, 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. Affymetrix microarray analysis and quantitative RT-PCR validation of the relative gene expression profiles in E9.5 RFC1-/- vs. RFC1+/+ embryos indicates a dramatic downregulation of multiple genes involved in erythropoiesis, and upregulation of several genes that form the cubilin-megalin multiligand endocytic receptor complex. Megalin protein expression disappears from the visceral yolk sac of RFC1-/- embryos, and cubilin protein is widely misexpressed. 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, vitamin B12, and other nutrients, lipids and morphogens required for normal embryogenesis. Comparison of RFC KO, wildtype normal embryos vs. RFC KO, nullizygous affected embryos Keywords: reduced folate carrier knockout, folate receptor, cubilin, megalin, embryos, gene expression, neural tube defect, chorioallantoic fusion

ORGANISM(S): Mus musculus

PROVIDER: GSE10659 | GEO | 2008/03/17

SECONDARY ACCESSION(S): PRJNA107627

REPOSITORIES: GEO

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Project description:The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. To further understand the role of Notch signaling during endothelial differentiation, this study uses both an in vivo gain-of-function and in vivo loss-of-function approach. At embryonic day 9.5, embryos with activated Notch1 signaling in the endothelia display a variety of growth and cardiovascular defects, and die soon after E10.5. Most notably, the extra-embryonic vasculature of the yolk sac displays remodeling differentiation defects. In the wild-type yolk sac, the primary vascular network has begun to reorganize, forming the large primary vessels and the smaller capillaries. In the activated Notch1 embryos remodeling is defective; the vasculature have an enlarged surface with decreased inter-vessel space. Embryos with ablated Notch signaling also display growth and vascular defects at E9.5 similar to the activated Notch1 embryos, however they exhibit a lack of vascular remodeling in the yolk sac, retaining the simple vascular plexus seen at E8.5. These results indicate that Notch signaling plays a critical role in the remodeling of the vasculature in the early embryo, particularly in the extra embryonic region. A conditional transgenic system was used in this study to activate Notch signaling. The ubiquitous ROSA26Notch transgene with a Neo/stop cassette flanked by loxP sites, followed by the N1-ICD cDNA, was recombined with a Tie2-CRE mouse, resulting in the removal of the STOP cassette and the subsequent activation of the Notch1-intracellular domain. This allowed for the overexpression and expansion of Notch signaling in all endothelial cells. Male Tie2-Cre mice were mated with female ROSA26Notch mice and resulting embryos were dissected at embryonic day 9.5. To ablate Notch signaling, Tie2-Cre mice were used in a two generation cross to obtain Tie2-Cre; Rbpj flox/flox embryos. These embryos lack RBPJ binding activity in the endothelia. In both instances embryos were used for genotyping and the yolk sac were separated and used to isolate total RNA with an RNeasy mini kit. The RNA was analyzed with the Mouse Genome 430A Array from Affymetrix. Samples were performed in duplicate, and RNA from wild type yolk sac tissues was compared to activated Notch and RBPJ loss-of-function yolk sac tissues.

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AFFYMETRIX ANALYSIS OF E9.5 RFC MOUSE KO EMBRYOS REVEALS ALTERED EXPR’N OF GENES IN THE CUBILIN-MEGALIN COMPLEX

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