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    • br STAR Methods br Acknowledgments Anthony Koleske is thanke

    2022-07-04


    STAR★Methods
    Acknowledgments Anthony Koleske is thanked for helpful discussions and for providing p190RhoGAP-A cDNA. Anatoly Kiyatkin and Mark Lemmon are thanked for use of the BioTek Synergy 2 plate reader for the MANT assays. Leena Kuruvilla is thanked for assistance with the thermal shift assays. David Calderwood is thanked for the H-Ras cDNA. Albert Chan is thanked for technical help with XDS. ByungHak Ha is thanked for technical input. Staff at beamline 24-ID-E (NE-CAT-E) at the Advanced Photon Source, Argonne National Laboratory are thanked. This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by NIH grant P41GM103403. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract no. DE-AC02-06CH11357. NIH grants R01NS085078, R01GM102262, and S10OD018007 funded the research.
    Introduction Development of teeth as epithelial appendages is a complex process regulated by inductive interaction between the epithelium and the underlying mesenchymal cells. The earliest event of tooth development is the thickening of the epithelium (the primary dental lamina), followed by condensation of the mesenchymal (R,S)-Anatabine mg [1], [2]. The development of the tooth crown advances through various stages defined by the morphology of the epithelium (bud, cap, and bell) and is followed by the formation of the root. The transition from the bud to the cap stage is a critical step in tooth morphogenesis. Signals from the enamel knot, an early epithelial signaling center, regulate growth and determine the site of epithelial folds that correspond directly with the cusp pattern of the mature tooth [3]. During the cap and bell stages, the size and shape of the tooth crown become apparent by the differentiation of cells into ameloblasts and odontoblasts that secrete the mineralizing matrices of the enamel and dentin, respectively. In the bell stage, the dental epithelium (enamel organ) segregates into four distinct cell types: inner enamel epithelial cells (IEEs), outer dental epithelial cells (OEEs), stratum intermedium (SI), and stellate reticulum (SR). The IEEs eventually differentiate into ameloblasts [4]. In the subsequent transitional stage from crown to root formation, the central core of the epithelium (SI and SR) disappears, leaving only a double layer of IEEs and OEEs called Hertwig's epithelial root sheath (HERS). It directs root growth and gives rise to a fenestrated network of epithelial cells which covers the root, known as the epithelial cell rests of Malassesz (ERM) [4]. The differentiation of epithelial cells into functional ameloblasts comprises several steps of morphological and functional changes. In the proliferation stage, the low columnar IEEs actively proliferate to form the basic shape of the tooth. Then, in differentiation stage, IEEs grow into columnar cells (preameloblasts) with more protein synthesizing organelles. The distal ends of the preameloblasts are flat, and the enamel matrix secreted is called rodless enamel matrix. In the secretory stage, the cells (secretory ameloblasts) lengthen, polarize, and form conical projections called Tome's process and deposit enamel in the form of rods. In transitional stage, when enamel reaches its full thickness, the height of ameloblasts decrease and protein synthesizing organelles are drastically reduced (transitional stage ameloblasts). The number of the ameloblasts is reduced by apoptosis in this stage. In the maturation stage, the ameloblasts modulate and transport specific ions necessary for the simultaneous deposition of minerals, and at the same time they also degrade enamel proteins and resorb the degraded proteins and water. The ameloblasts initiate a series of repetitive morphological change at the enamel surface, in which tight junction and deep membrane infoldings periodically appear (ruffle-ended ameloblasts [RA]), then disappear for short intervals (smooth ended ameloblasts [SA]) from distal end of the cells. In the regressive stage, the ameloblasts (reduced enamel epithelium) lose their differentiation and become short cuboidal cell, which is indistinguishable from other layers of the enamel organ. Reduced enamel epithelium remains on the surface of formed enamel until the tooth erupts. After crown morphogenesis, the boundary where IEEs and OEEs meet, referred to as the cervical loop, ceases to differentiate into ameloblasts and forms HERS with OEEs to induce root formation [4], [5], [6], [7].