The role of Runx2 and Sp7 in ameloblasts differentiation and postnatal enamel synthesis

Abstract

In mammals, the mineralized tissues include bone, dentin, enamel, and cementum. Runx2 and its downstream target Sp7 are essential transcription factors for the osteoblast differentiation and formation of bone tissue. However, transcription factors that regulate ameloblast differentiation and enamel secretion are less understood. To uncover the role of Runx2 and Sp7 in ameloblasts differentiation and enamel synthesis, we established a colony of Runx2 and Sp7 floxed mice. Both genes were deleted specifically in ameloblasts using K14-Cre transgenic mice. The successful deletion of Runx2 and Sp7 genes in the epithelium, including ameloblasts, was confirmed using Td-Tomato and GFP reporter, respectively. Histological analysis of newborn littermates with Runx2 or Sp7 deletion showed comparable numbers and positions of both incisors and molars. Gene expression and histologic analysis revealed that Runx2 or Sp7 deficient ameloblast surprisingly progress through normal differentiation. However, combined deletion of Runx2 and Sp7 genes (DKO), resulted in an enlarged and rounded tooth with significant disruptions in cusp development. Both histologic and gene expression analysis revealed an accelerated ameloblast and odontoblast maturation in newborn DKO mice. Surprisingly, enamel matrix and mineralized dentin was present in the DKO molars. Interestingly large number of different sized nodules of acellular matrix were noted in the pulp, and in between ameloblast and odontoblast layers of DKO mice. To investigate postnatal tooth development prior to the eruption, histological and molecular analysis were performed on 10-days old (PN10) littermates. Unlike the newborn stage, a significant 2.5-fold increase in the enamel matrix was observed in the molars and incisors of Runx2 deficient mice. Comparable expression of Amelogenin, Enamelin and Amelotin genes indicates increase enamel matrix in Runx2 mutant is not linked to increase synthesis. Unlike the cuboidal mature ameloblast in wild-type molars, Runx2 mutant showed only elongated ameloblast that are reminiscent of secretory stage. A marked reduction in the levels of KLK4, strongly suggests a failure in degradation of enamel matrix. Unlike the wild-type littermates, the enamel matrix in 10-day old Sp7 mutants was increased by 2-fold. However, the number of elongated secretory ameloblast and the cuboidal mature ameloblast were comparable among wild-type and Sp7 mutant molars. Consistent with the individual deficiency of Sp7 and Runx2, the DKO mice showed a 4- fold increase in the amount of enamel matrix at the cusp and 6-fold increase in the cervical area of molars. Comparable levels of Amelogenin, Enamelin, and Amelotin in DKO mice indicate that the increase in enamel matrix is not associated with increased synthesis. Surprisingly, DKO molars showed only mature cuboidal ameloblasts, even in the cervical area where majority of the cells should be long columnar secretory ameloblasts. A notable decrease in KLK4 expression, strongly suggests a failure in the degradation of the enamel matrix in DKO mice. To examine enamel synthesis following tooth eruption, 1-month-old mice were subjected to µCT, histology, and qPCR analysis. Runx2 deletion in ameloblasts led to a chalky white enamel in both molars and incisors. Gross appearance of 1-month old molar and incisor showed a significant loss of enamel with marked attrition at the cusp and incisal edge. To quantify changes in the enamel, µCT analysis was performed on the mandibular incisors. Compared to the WT, Runx2 littermates showed a significant decrease in the density and a 75% reduction in overall enamel volume. Interestingly, the expression of Amelogenin, Amelotin, and KLK4 showed a significant reduction in the 1-month-old Runx2 mutant incisor but not in the molars. The gross appearance of enamel in the 1-month-old incisor and molar of Sp7 mutants was translucent and indistinguishable from the wild-type littermates. Furthermore, µCT analysis revealed comparable enamel volume and density in the Sp7-deficient incisors. A similar expression levels of ameloblast marker genes between WT and Sp7 littermates suggests that enamel impairments noted at the pre-eruption stage are resolved after tooth eruption. The molars and incisors of 1-month old DKO mice exhibited a chalky white appearance with pronounced loss of enamel and attrition at the cusp and incisal edge. The µCT analysis confirmed either a patchy presence or near loss of enamel. Quantification from four littermates showed marked reduction in volume and density of enamel in the DKO mice. Interestingly, the body weight of DKO mice was 50% less than WT littermates. This decrease in body weight is likely due to poor food intake by DKO mice due to tooth sensitivity from enamel loss. Furthermore, an increased mineralized surface that initiated from the cervical loop was evident in the DKO incisor. The assessment of dynamic synthesis by double calcein labeling demonstrated higher rate of dentin formation in the DKO mice. Collectively, our data revealed that Runx2 and Sp7 control the removal of enamel matrix through transcriptional regulation of KLK4 enzyme. Combined deficiency of Runx2 and Sp7 accelerate ameloblast differentiation and postnatal enamel synthesis.