Developmental expression of alpha4 protein phosphatase regulatory subunit in tissues affected by Opitz syndrome

Mutations in the Mid1 gene are responsible for X-linked Opitz syndrome, characterized by midline defects of the brain, face, heart, and trachea. The regulatory subunit alpha4 binds protein phosphatase 2A to Mid1 and promotes Mid1 dephosphorylation, in opposition to MAP kinase. To examine the relationship between alpha4 expression and human defects in Opitz syndrome, developmental expression of alpha4 in embryonic day (E) 8-E16 mouse embryos was mapped by immunohistochemistry. At E10, alpha4 was first detected only in the heart. At E11, alpha4 was expressed in epithelium of the mandibular and maxillary arches and in specific subsets of mesenchymal cells within the arches. The fetal heart and brain also highly expressed alpha4. At E16, alpha4 expression broadened to include the heart, brain, skeletal muscle, and tracheal and esophageal epithelium but not smooth muscle. Consistent with immunolocalization in embryos, Western immunoblotting of adult rabbit tissues demonstrated high levels of alpha4 expression in brain, heart, lung, liver, and skeletal muscle with low expression in kidney, uterus, and intestine. Expression in slow type I skeletal muscle was much higher than in fast type II muscle. By using double immunohistochemical staining, alpha4 was coexpressed in mouse skeletal muscle cells containing slow type myosin. Expression of alpha4 overlaps with the tissue defects in Opitz syndrome. The alpha4 gene lies in the linkage interval for FG syndrome, characterized by skeletal muscle and brain defects that coincide closely to the expression pattern of alpha4.