Figure 6
Cubilin and megalin protein expression in E9.5 embryos. Immunohistochemical staining was performed on transverse sections through E9.5 RFC1+/+ and RFC1-/- embryos in order to examine the relative expression and distribution of megalin and cubilin 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 6C and 6D (4× magnification) indicate the orientation of the higher magnification panels (40× magnification) shown in 6A, 6B, 6E, and 6G; 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 RFC1 wildtype embryos (6A; red arrows), but is expressed on both the apical (6B; red arrows) and the basolateral side (6B; purple arrowheads) of the neuroepithelium in RFC1 mutants. Cubilin expression is also restricted to the apical side of neuroepithelial cells in RFC1 wildtype embryos (6C, 6E; red arrows), but is misexpressed on the basolateral side of the neuroepithelium in RFC1 nullizygous embryos (6D, 6G; purple arrowheads). Staining for cubilin protein is visible in the surface ectoderm of RFC1+/+ embryos (6C; black arrows), and discrete staining is also observed in the notochord (6C, 6F; yellow arrow). Cubilin expression is highly upregulated in the cranial mesenchyme of RFC1-/- embryos (shown in panel 6D) relative to wildtype littermates, especially in the region surrounding the notochord (6H; yellow arrow). Staining for cubilin protein is visible in the mesothelial cells of the pericardium of RFC1 wildtype (6I; black arrowhead) and RFC1 mutant embryos (not visible in section shown in 6J), 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 RFC1 mutant (6J). Normal morphology of the primary head vein/cephalic extension of the anterior cardinal vein (**) and the third branchial arch artery (*) in the RFC1 wildtype embryo are shown in (6C); comparable structures labeled in the RFC1 mutant (6D) indicate abnormal vascular morphology. LV: left ventricle.