FOLATE-RESPONSIVE DYSGENESIS

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The objective of the proposed studies is to demonstrate the central role of the folate receptor (FR) in regulation of transfer of maternal folates across the placenta and of normal embryogenesis. Abnormal regulation of FR may be established as a cause for dysgenesis of the developing embryo, in particular the production of neural tube defects and abnormalities of neural crest cells. Since these cells undergo bursts of proliferation, the FR-regulated transfer of folate to the placenta is essential to their development. Five specific aims are proposed. First, the PI will determine whether the expression of FR in mouse neural crest cells correlate with bursts of cellular proliferation by a) demonstrating effects of arrested proliferation in conjunction with antifolate methotrexate at 12-hour intervals; and b) correlating these results with changes in FR expression by immunohistochemistry and in situ hybridization. Second, the PI will determine whether in vivo folate deficiency induces up regulation of FR in placenta and tissues on gestation day 17 using folate-deficient or folate-replete diets. Approaches to FR expression in the placenta and tissues will include Northern and Western blots to study transcriptional and post-translational events as well as nuclear run-on studies of transcription of FR. Folate deficiency will be monitored by folate and homocysteine measurements. Fetal tissues will be evaluated by immunohistochemistry and in situ hybridization. Third, a series of experiments are proposed to determine whether sustained quenching of placental FR during maternal folate deficiency using antisense FR will adversely affect placental proliferation and result in fetal growth retardation. These studies will use liposomes engineered to deliver antisense FR cDNA incorporated in a placenta specific promoter at two time points, gestational days 8 and 16. Fourthly, his group will determine whether the interaction of a specific human 43-kDa trans-factor liver protein with the cis-element in the 5'-UTR of FR is identical in the mouse model. These studies will involve isolation and purification of the trans-factor protein from mouse placenta and characterization of its function using specific antibody. They will extend previous observations that homocysteine (Hcy) is a mediator of the trans - cis interaction. These experiments will use both gel shift assays with placental slices and large (500-100 mM) concentrations of Hcy. Fifthly, the group will measure the interaction of the cis and trans elements in ex vivo mouse fetal culture. This will be achieved by a complex strategy of molecular cloning of the mouse trans factor and determining concordance of its expression with FR in mouse fetal tissues. Also they will evaluate the effect of down-regulation of FR expression by antisense oligonucleotides to both the cis and the trans elements.