The Role of FAM20B-catalyzed Proteoglycans in Tooth Development

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Project summary/Abstract Supernumerary teeth can cause a broad range of dental complications. As extra teeth are formed on existing dentition, unraveling the molecular mechanism of supernumerary tooth formation will not only help develop the therapeutic strategy for this disease but also provide insights into tooth regeneration. Despite the significant progress in understanding the regulatory role of morphogens, growth factors, and transcriptional factors in supernumerary tooth formation, little is known about the role of extracellular components such as proteoglycans in this pathological process. Our recent studies show that inactivation of dental epithelial Fam20B, a newly discovered xylose kinase essential for glycosaminoglycan (GAG) assembly, leads to supernumerary incisors in mice. Our pilot study reveals that GAG deficiency in the dental epithelium leads to ectopic activation of WNT signaling, and that an ectopic Sox2 expression is located in the same area, which normally should disappear from this site after E14.5. Our in vitro study shows that GAGs on certain FAM20B-catalyzed proteoglycans suppress WNT signaling but facilitate Wise- mediated inhibition on WNT. Conversely, administering WNT inhibitor to the mutant embryos rescued the tooth phenotype in some cases. These data led us to form our central hypothesis that certain FAM20B- catalyzed proteoglycans regulate tooth renewal by mediating the stem cell renewal via negative regulation on WNT signaling in the dental epithelium. To test this hypothesis, we propose the following three specific aims: (1) To determine if FAM20B-catalyzed proteoglycans mediate tooth renewal via negative regulation on WNT signaling, and if GAG-mediated Wise inhibition on WNT underlies the supernumerary tooth formation. We will perform "rescue" experiments by overexpressing DKK1 or Wise in the dental epithelium to inhibit the overactivated WNT signaling in K14Cre/+;Fam20Bfl/fl mice. (2) To determine whether FAM20B- catalyzed proteoglycans regulate tooth renewal by mediating the stem cell renewal in the dental epithelium. We will perform lineage tracing and inducible knockout experiments to determine the role of stem cell renewal in GAG deficiency-caused supernumerary teeth, and if the FAM20B-catalyzed PGs mediate stem cell renewal in the dental epithelium in a cell-autonomous manner. (3) To identify the FAM20B-catalyzed proteoglycans responsible for the supernumerary tooth formation. We will determine the expression pattern of FAM20B-catalyzed proteoglycans in tooth and identify those negatively regulating WNT signaling. The biological function of candidate proteoglycans will be determined by gene knockdown and organ culture methods. The completion of this study will advance our understanding about the molecular mechanism underlying supernumerary tooth formation and help in laying the groundwork for tooth regeneration. As FAM20B and proteoglycans are extensively expressed in many tissues, the knowledge gained from this study may also provide valuable insights into the proteoglycan-mediated signaling in other tissues.