Runt-related transcription factor 2 (Runx2) is involved in the early stage of tooth development. However, only few studies have reported the role of Runx2 in enamel development, which may be attributed to that Runx2 full knockout mice cannot survive after birth. In the present study, we successfully established a Runx2-deficient mouse model using a conditional knockout (cKO) method. We observed a significant reduction in the degree of mineralization and the decreased size of enamel rods in cKO mice. Histological analysis showed the retained enamel proteins in enamel layer at maturation stage in cKO molars. Further analysis by qRT-PCR revealed that the expressions of genes encoding enamel structure proteins, such as amelogenin (AMELX), ameloblastin (AMBN) and enamelin (ENAM), were increased in cKO enamel organs. On the other hand, the expression of kallikrein-related peptidase-4 (KLK4) at the mRNA and protein levels was dramatically decreased from late secretory stage to maturation stage in cKO enamel organs, while the expression of matrix metalloproteinase-20 (MMP-20) was not significantly altered. Finally, immunohistochemistry indicated that the uptake of amelogenins by ameloblasts was significantly decreased in cKO mice. Taken together, Runx2 played critical roles in controlling enamel maturation by increasing synthesis of KLK4 and decreasing synthesis of AMELX, AMBN and ENAM.

Background Mutation in Odontogenesis‐associated phosphoprotein (ODAPH) has been reported to cause recessive hypomineralized amelogenesis imperfecta (AI) in human. However, the exact role of ODAPH in amelogenesis is still unknown. Results ODAPH was identified as a novel constituent of the atypical basal lamina located at the interface between maturation ameloblasts and the enamel by dual immunofluorescence staining of ODAPH and LAMC2. Odaph knockout mice were generated to explore the function of ODAPH in amelogenesis. Odaph−/− mice teeth showed severely attrition and reduced enamel mineralization. Histological analysis showed from transition or early‐maturation stage, ameloblasts were rapidly shortened, lost cell polarity, and exhibited cell pathology. Abundant enamel matrix marked by amelogenin was retained. Temporary cyst‐like structures were formed between flattened epithelial cells and the enamel from maturation stage to eruption. The integrity of the atypical basal lamina was impaired indicated by the reduced diffuse expression of LAMC2 and AMTN. The expression of maturation stage related genes of Amtn, Klk4, Integrinβ6 and Slc24a4 were significantly decreased. Conclusions Our results suggested Odaph played vital roles during amelogenesis by maintaining the integrity of the atypical basal lamina in maturation stage, which may contribute to a better understanding of the pathophysiology of human AI.

As a critical transcription factor, Runx2 plays a fundamental role in the formation and mineralization of bone and tooth (Camilleri & McDonald, 2006;Komori, 2019;Long, 2011). Mutations in the human Runx2 gene cause cleidocranial dysplasia (CCD), an autosomal dominant inheritance characterized by bone and dental defects (Farrow et al., 2018). In CCD patients, dental defects include supernumerary teeth, enamel hypoplasia, the absence of cellular cementum, and abnormally shaped roots (Jaruga et al., 2016;Otto et al., 2002). In addition to its expression in mineralized tissues, Runx2 is also detected in dental follicle cells, cementoblasts, periodontal ligament cells, and junctional epithelium (JE) (Bronckers et al., 2001), indicating that this molecule may play an essential role in the periodontium.Once the enamel completes its mineralization, the ameloblasts are converted into reduced enamel epithelium. With the eruption of teeth, the reduced enamel epithelium fuses with the oral epithelium to form the JE. Amelotin, odontogenic ameloblast-associated protein (ODAM), and secretory calcium-binding phosphoprotein proline-glutamine rich 1 (SCPPPQ1) are expressed in the developmental continuum from enamel epithelium to JE (Moffatt et al., 2006Nishio et al., 2013). Previous studies have suggested an association between the occurrence of enamel defects and periodontal lesions.