Fig. 6

Localization of miR-409 in CAM samples during the course of gestation by in situ hybridization. NBT/BCIP (blue) was used as the substrate. Magnification: 200X. Bar = 250 μm. Insets represent a higher magnification (400X). a U6 snRNA control probe exhibits nuclear localization. b Scrambled miRNA probe (negative control). c CAM collected at 45d shows strong cytoplasmic signal for miR-409 specifically in chorioallantoic epithelium, vascular endothelium and mesenchymal cells. d and e CAM collected at 4mo and 6mo show a significant reduction in the expression of miR-409. MiR-409 expression was limited to scattered chorionic epithelial cells (arrows [d]), and rare mesenchymal and vascular endothelial cells (* [e]). f CAM collected at 10mo shows scattered, basolateral expression of miR-409 in chorionic epithelial cells (arrows) while no expression was observed in mesenchymal cells. g miR-409 expression was not detected in CAM samples collected postpartum. h The bar graph shows the mean value for each ISH score with standard error bars in CAM during the course of gestation. Mean miR-409 ISH scores between the two independent observers were determined, and mean scores were compared between the different stages of pregnancy