FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.

BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.

Detection and Quantitation of Label-Retaining Cells in Mouse Incisors using a 3D Reconstruction Approach after Tissue Clearing.

The murine incisor is an organ that grows continuously throughout the lifespan of the mouse. The epithelial and mesenchymal stem cells residing in the proximal tissues of incisors give rise to progeny that will differentiate into ameloblasts, odontoblasts, and pulp fibroblasts. These cells are crucial in supporting the sustained turnover of incisor tissues, making the murine incisor an excellent model for studying the homeostasis of adult stem cells. Stem cells are believed to contain long-living quiescent cells that can be labeled by nucleotide analogs such as 5-ethynyl-2´-deoxyuridine (EdU). The cells retain this label over time and are accordingly named label-retaining cells (LRCs). Approaches for visualizing LRCs in vivo provide a robust tool for monitoring stem cell homeostasis. In this study, we described a method for visualizing and analyzing LRCs. Our innovative approach features LRCs in mouse incisors after tissue clearing and whole-mount EdU staining followed by confocal microscopy and a 3-dimensional (3D) reconstruction with the imaging software. This method enables 3D imaging acquisition and non-biased quantitation compared to traditional LRCs analysis on sectioned slides.

The phosphorylation of serine55 in enamelin is essential for murine amelogenesis.

Amelogenesis imperfecta (AI) is an inherited developmental enamel defect affecting tooth masticatory function, esthetic appearance, and the well-being of patients. As one of the major enamel matrix proteins (EMPs), enamelin (ENAM) has three serines located in Ser-x-Glu (S-x-E) motifs, which are potential phosphorylation sites for the Golgi casein kinase FAM20C. Defects in FAM20C have similarly been associated with AI. In our previous study of EnamRgsc514 mice, the Glu57 in the S55-X56-E57 motif was mutated into Gly, which was expected to cause a phosphorylation failure of Ser55 because Ser55 cannot be recognized by FAM20C. The severe enamel defects in ENAMRgsc514 mice reminiscent of Enam-knockout mouse enamel suggested a potentially important role of Ser55 phosphorylation in ENAM function. However, the enamel defects and ENAM dysfunction may also be attributed to distinct physicochemical differences between Glu57 and Gly57. To clarify the significance of Ser55 phosphorylation to ENAM function, we generated two lines of Enam knock-in mice using CRISPR-Cas9 method to eliminate or mimic the phosphorylation state of Ser55 by substituting it with Ala55 or Asp55 (designated as S55A or S55D), respectively. The teeth of 6-day or 4-week-old mice were subjected to histology, micro-CT, SEM, TEM, immunohistochemistry, and mass spectrometry analyses to characterize the morphological, microstructural and proteomic changes in ameloblasts, enamel matrix and enamel rods. Our results showed that the enamel formation and EMP expression in S55D heterozygotes (Het) were less disturbed than those in S55A heterozygotes, while both homozygotes (Homo) had no mature enamel formation. Proteomic analysis revealed alterations of enamel matrix biosynthetic and mineralization processes in S55A Hets. Our present findings indicate that Asp55 substitution partially mimics the phosphorylation state of Ser55 in ENAM. Ser55 phosphorylation is essential for ENAM function during amelogenesis.