Autoimmune amelogenesis imperfecta in patients with APS-1 and coeliac disease.

Ameloblasts are specialized epithelial cells in the jaw that have an indispensable role in tooth enamel formation-amelogenesis1. Amelogenesis depends on multiple ameloblast-derived proteins that function as a scaffold for hydroxyapatite crystals. The loss of function of ameloblast-derived proteins results in a group of rare congenital disorders called amelogenesis imperfecta2. Defects in enamel formation are also found in patients with autoimmune polyglandular syndrome type-1 (APS-1), caused by AIRE deficiency3,4, and in patients diagnosed with coeliac disease5-7. However, the underlying mechanisms remain unclear. Here we show that the vast majority of patients with APS-1 and coeliac disease develop autoantibodies (mostly of the IgA isotype) against ameloblast-specific proteins, the expression of which is induced by AIRE in the thymus. This in turn results in a breakdown of central tolerance, and subsequent generation of corresponding autoantibodies that interfere with enamel formation. However, in coeliac disease, the generation of such autoantibodies seems to be driven by a breakdown of peripheral tolerance to intestinal antigens that are also expressed in enamel tissue. Both conditions are examples of a previously unidentified type of IgA-dependent autoimmune disorder that we collectively name autoimmune amelogenesis imperfecta.

Intrinsically disordered proteins drive enamel formation via an evolutionarily conserved self-assembly motif.

The formation of mineralized tissues is governed by extracellular matrix proteins that assemble into a 3D organic matrix directing the deposition of hydroxyapatite. Although the formation of bones and dentin depends on the self-assembly of type I collagen via the Gly-X-Y motif, the molecular mechanism by which enamel matrix proteins (EMPs) assemble into the organic matrix remains poorly understood. Here we identified a Y/F-x-x-Y/L/F-x-Y/F motif, evolutionarily conserved from the first tetrapods to man, that is crucial for higher order structure self-assembly of the key intrinsically disordered EMPs, ameloblastin and amelogenin. Using targeted mutations in mice and high-resolution imaging, we show that impairment of ameloblastin self-assembly causes disorganization of the enamel organic matrix and yields enamel with disordered hydroxyapatite crystallites. These findings define a paradigm for the molecular mechanism by which the EMPs self-assemble into supramolecular structures and demonstrate that this process is crucial for organization of the organic matrix and formation of properly structured enamel.

Skeletal dysmorphology and mineralization defects in Fgf20 KO mice.

Introduction: Fibroblast growth factor 20 (Fgf20), a member of the Fgf9 subfamily, was identified as an important regulator of bone differentiation and homeostasis processes. However, the role of Fgf20 in bone physiology has not been approached yet. Here we present a comprehensive bone phenotype analysis of mice with functional ablation of Fgf20. Methods: The study conducts an extensive analysis of Fgf20 knockout mice compared to controls, incorporating microCT scanning, volumetric analysis, Fgf9 subfamily expression and stimulation experiment and histological evaluation. Results: The bone phenotype could be detected especially in the area of​ the lumbar and caudal part of the spine and in fingers. Regarding the spine, Fgf20-/- mice exhibited adhesions of the transverse process of the sixth lumbar vertebra to the pelvis as well as malformations in the distal part of their tails. Preaxial polydactyly and polysyndactyly in varying degrees of severity were also detected. High resolution microCT analysis of distal femurs and the fourth lumbar vertebra showed significant differences in structure and mineralization in both cortical and trabecular bone. These findings were histologically validated and may be associated with the expression of Fgf20 in chondrocytes and their progenitors. Moreover, histological sections demonstrated increased bone tissue formation, disruption of Fgf20-/- femur cartilage, and cellular-level alterations, particularly in osteoclasts. We also observed molar dysmorphology, including root taurodontism, and described variations in mineralization and dentin thickness. Discussion: Our analysis provides evidence that Fgf20, together with other members of the Fgf9 subfamily, plays a crucial regulatory role in skeletal development and bone homeostasis.

The dynamics of supernumerary tooth development are differentially regulated by Sprouty genes.

In mice, a toothless diastema separates the single incisor from the three molars in each dental quadrant. In the prospective diastema of the embryo, small rudimentary buds are found that are presumed to be rudiments of suppressed teeth. A supernumerary tooth occurs in the diastema of adult mice carrying mutations in either Spry2 or Spry4. In the case of Spry2 mutants, the origin of the supernumerary tooth involves the revitalization of a rudimentary tooth bud (called R2), whereas its origin in the Spry4 mutants is not known. In addition to R2, another rudimentary primordium (called MS) arises more anteriorly in the prospective diastema. We investigated the participation of both rudiments (MS and R2) in supernumerary tooth development in Spry2 and Spry4 mutants by comparing morphogenesis, proliferation, apoptosis, size and Shh expression in the dental epithelium of MS and R2 rudiments. Increased proliferation and decreased apoptosis were found in MS and R2 at embryonic day (ED) 12.5 and 13.5 in Spry2(-/-) embryos. Apoptosis was also decreased in both rudiments in Spry4(-/-) embryos, but the proliferation was lower (similar to WT mice), and supernumerary tooth development was accelerated, exhibiting a cap stage by ED13.5. Compared to Spry2(-/-) mice, a high number of Spry4(-/-) supernumerary tooth primordia degenerated after ED13.5, resulting in a low percentage of supernumerary teeth in adults. We propose that Sprouty genes were implicated during evolution in reduction of the cheek teeth in Muridae, and their deletion can reveal ancestral stages of murine dental evolution.

Early evolution of enamel matrix proteins is reflected by pleiotropy of physiological functions.

Highly specialized enamel matrix proteins (EMPs) are predominantly expressed in odontogenic tissues and diverged from common ancestral gene. They are crucial for the maturation of enamel and its extreme complexity in multiple independent lineages. However, divergence of EMPs occured already before the true enamel evolved and their conservancy in toothless species suggests that non-canonical functions are still under natural selection. To elucidate this hypothesis, we carried out an unbiased, comprehensive phenotyping and employed data from the International Mouse Phenotyping Consortium to show functional pleiotropy of amelogenin, ameloblastin, amelotin, and enamelin, genes, i.e. in sensory function, skeletal morphology, cardiovascular function, metabolism, immune system screen, behavior, reproduction, and respiratory function. Mice in all KO mutant lines, i.e. amelogenin KO, ameloblastin KO, amelotin KO, and enamelin KO, as well as mice from the lineage with monomeric form of ameloblastin were affected in multiple physiological systems. Evolutionary conserved motifs and functional pleiotropy support the hypothesis of role of EMPs as general physiological regulators. These findings illustrate how their non-canonical function can still effect the fitness of modern species by an example of influence of amelogenin and ameloblastin on the bone physiology.

Multi-Level Approach for Comprehensive Enamel Phenotyping.

Enamel is the hardest tissue in mammalian organisms and is the layer covering the tooth. It consists of hydroxyapatite (HAP) crystallites, which mineralize on a protein scaffold known as the enamel matrix. Enamel matrix assembly is a very complex process mediated by enamel matrix proteins (EMPs). Altered HAP deposition or disintegration of the protein scaffold can cause enamel defects. Various methods have been established for enamel phenotyping, including MicroCT scanning with various resolutions from 9 µm for in vivo imaging to 1.5 µm for ex vivo imaging. With increasing resolution, we can see not only the enamel layer itself but also a detailed map of mineralization. To study enamel microstructure, we combine the MicroCT analysis with scanning electron microscopy (SEM), which enables us to perform element analyses such as calcium-carbon ratio. However, the methods mentioned above only show the result-already formed enamel. Stimulated emission depletion (STED) microscopy provides extra information about protein structure in the form of EMP localization and position before enamel mineralization. A combination of all these methods allows analyzing the same sample on multiple levels-starting with the live animal being scanned harmlessly and quickly, followed by sacrifice and high-resolution MicroCT scans requiring no special sample preparation. The biggest advantage is that samples remain in perfect condition for SEM or STED microscopic analysis. © 2022 Wiley Periodicals LLC. Basic Protocol 1: In vivo MicroCT scanning of mouse Basic Protocol 2: Ex vivo HR-MicroCT of the teeth Basic Protocol 3: SEM for teeth microstructure Basic Protocol 4: Stimulated emission depletion (STED) microscopy.

Letting the 'cat' out of the bag: pouch young development of the extinct Tasmanian tiger revealed by X-ray computed tomography.

The Tasmanian tiger or thylacine (Thylacinus cynocephalus) was an iconic Australian marsupial predator that was hunted to extinction in the early 1900s. Despite sharing striking similarities with canids, they failed to evolve many of the specialized anatomical features that characterize carnivorous placental mammals. These evolutionary limitations are thought to arise from functional constraints associated with the marsupial mode of reproduction, in which otherwise highly altricial young use their well-developed forelimbs to climb to the pouch and mouth to suckle. Here we present the first three-dimensional digital developmental series of the thylacine throughout its pouch life using X-ray computed tomography on all known ethanol-preserved specimens. Based on detailed skeletal measurements, we refine the species growth curve to improve age estimates for the individuals. Comparison of allometric growth trends in the appendicular skeleton (fore- and hindlimbs) with that of other placental and marsupial mammals revealed that despite their unique adult morphologies, thylacines retained a generalized early marsupial ontogeny. Our approach also revealed mislabelled specimens that possessed large epipubic bones (vestigial in thylacine) and differing vertebral numbers. All of our generated CT models are publicly available, preserving their developmental morphology and providing a novel digital resource for future studies of this unique marsupial.

Migration of Founder Epithelial Cells Drives Proper Molar Tooth Positioning and Morphogenesis.

The proper positioning of organs during development is essential, yet little is known about the regulation of this process in mammals. Using murine tooth development as a model, we have found that cell migration plays a central role in positioning of the organ primordium. By combining lineage tracing, genetic cell ablation, and confocal live imaging, we identified a migratory population of Fgf8-expressing epithelial cells in the embryonic mandible. These Fgf8-expressing progenitors furnish the epithelial cells required for tooth development, and the progenitor population migrates toward a Shh-expressing region in the mandible, where the tooth placode will initiate. Inhibition of Fgf and Shh signaling disrupted the oriented migration of cells, leading to a failure of tooth development. These results demonstrate the importance of intraepithelial cell migration in proper positioning of an initiating organ.

Enamel microarchitecture of a tribosphenic molar.

The tribosphenic molar is a dental apomorphy of mammals and the molar type from which all derived types originated. Its enamel coat is expected to be ancestral: a thin, evenly distributed layer of radial prismatic enamel. In the bat Myotis myotis, we reinvestigated the 3D architecture of the dental enamel using serial sectioning combined with scanning electron microscopy analyses, biometrics of enamel prisms and crystallites, and X-ray diffraction. We found distinct heterotopies in enamel thickness (thick enamel on the convex sides of the crests, thin on the concave ones), angularity of enamel prisms, and in distribution of particular enamel types (prismatic, interprismatic, aprismatic) and demonstrated structural relations of these heterotopies to the cusp and crest organization of the tribosphenic molar. X-ray diffraction demonstrated that the crystallites composing the enamel are actually the aggregates of much smaller primary crystallites. The differences among particular enamel types in degree of crystallite aggregation and the variation in structural microstrain of the primary crystallites (depending upon the duration and the mechanical context of mineralization) represent factors not fully understood as yet that may contribute to the complexity of enamel microarchitecture in a significant way.