Immunohistochemical localization of GDD1 gene product

Objectives: The human GDD1 (TMEM16E) gene was identified as the causative gene of Gnatho-Diaphyseal Dysplasia (GDD; MIM 166260), an unusual generalized skeletal syndrome with autosomal dominant inheritance. To date, the biochemical function(s) of GDD1 protein and the molecular pathophysiology of GDD1 mutations leading to GDD have not yet been elucidated. Our previous studies demonstrated that the GDD1 gene showed abundant expression in skeletal muscle and bone of human and murine tissues. We suggest that GDD1 plays important roles in muscle/skeletal development and metabolism. In this study, we characterized tissue localization of the murine GDD1 gene products using a GDD1-specific antibody.

Methods: We generated a specific antibody against the GDD1 protein and investigated immunohistochemical localizations of GDD1 in mouse tissues. BDF1 adult mice were perfused through the aortic arch with 4%PFA. Tissue samples were removed and fixed in 4%PFA (Wako) for 24 hours, processed into paraffin-wax, and 5um thick sections were prepared. Immunohistochemical staining was performed using HISTOMOUSE-PLUS kits (Zymed). Primary antibodies were used at the following dilutions: anti-GDD1 at 1:100; anti-GLUT4 at 1:500; anti-Dystrophin at 1:20; anti-Type-X collagen at 1:200; and anti-Type-II collagen at 1:200.

Results: Immunohistochemistry of adult mouse femoral head cartilage revealed GDD1 to be expressed exclusively in hypertrophic chondrocytes of the epiphyseal growth-plate. Expression of GDD1 was also evident in osteoblasts lining mineralized bone surfaces, as well as in articular cartilage cells. Furthermore, immunohistochemical analysis of GDD1 in lower incisor revealed specific expression patterns. GDD1 staining was observed in epithelial and mesenchymal cells during the amelogenesis of the mouse incisors.

Conclusion: Our data suggest diverse cellular role(s) of GDD1 protein in the development of the tooth and musculoskeletal system.