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LASER CAPTURE MICRODISSECTION AND GENE ARRAY ANALYSIS OF PALATAL EPITHELIAL AND MESENCHYMAL CELLS EXPOSED TO TCDD
Abbott, B D. AND J E. Schmid. LASER CAPTURE MICRODISSECTION AND GENE ARRAY ANALYSIS OF PALATAL EPITHELIAL AND MESENCHYMAL CELLS EXPOSED TO TCDD. Presented at Teratology Society Meeting, Vancouver, BC, CANADA, June 26 - July 01, 2004.
Palatal shelves from embryos exposed on gestation day (GD) 12 to either retinoic acid (RA) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) contact but fail to fuse. It is of interest to know if diverse agents that induce clefting via the same etiology also activate the same biochemical response pathways. This study evaluates gene expression in palatal epithelial (epi) and mesenchymal (mes) cells after exposure to TCDD. As palatogenesis involves interactions between epi and mes cells, this study also compares gene expression in epi and mes. C57BL/6J pregnant mice were dosed with either corn oil or 24 g TCDD/kg body weight on GD 12. Fetal heads were collected on GD14 from 5 control and 5 treated litters and stored frozen. Frozen sections were prepared and laser capture microdissection (LCM, Arcturus PixCell II) was used to collect medial epi cells from the unfused, secondary palatal shelves and mes from the zone adjacent to the epi. Epi cells from 4 fetuses per litter were pooled, and mes cells were similarly pooled. Total RNA was prepared (Arcturus PicoPure Kit), RNA quality confirmed via Agilent Bioanalyzer and RNA was amplified two rounds using the Arcturus RiboAmp Kit. Radiolabeled cDNA probes were prepared using random primers and 33P-dCTP. The probe was hybridized to Panorama Mouse Cytokine Gene Arrays (SigmaGenosys), images acquired and analyzed (Phoretix Array3 Software). Data sets were log transformed, normalized and statistically analyzed. Of the 526 genes on the arrays, 54 were detected as expressed in epi and/or mes. Follistatin and WNT-4 were expressed only in epi and EphA3 only in mes. CIS2, NT-4 were higher in epi, while LPS, TGF 2, and others showed higher expression in mes. TCDD exposure affected the expression of TGF 2, CIS2, TrkC, Duffy, TIMP-2, and Midkine in epi and/or mes. This project is on-going and will compare gene expression patterns between TCDD and RA.
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