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.

Summary 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.

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.

Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1a1, Col1a2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.

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.

Autoinflammatory diseases are characterized by dysregulation of the innate immune system, leading to spontaneous inflammation. Pstpip2 cmo mouse strain is a well-characterized model of this class of disorders. Because of the mutation leading to the lack of adaptor protein PSTPIP2, these animals suffer from autoinflammatory chronic multifocal osteomyelitis similar to several human syndromes. Current evidence suggests that it is driven by hyperproduction of IL-1b by neutrophil granulocytes. In this study, we show that in addition to IL-1b, PSTPIP2 also negatively regulates pathways governing reactive oxygen species generation by neutrophil NOX2 NADPH oxidase. Pstpip2 cmo neutrophils display highly elevated superoxide production in response to a range of stimuli. Inactivation of NOX2 NADPH oxidase in Pstpip2 cmo mice did not affect IL-1b levels, and the autoinflammatory process was initiated with similar kinetics. However, the bone destruction was almost completely alleviated, suggesting that dysregulated NADPH oxidase activity is a key factor promoting autoinflammatory bone damage in Pstpip2 cmo mice.

The patterning of repeated structures is a major theme in developmental biology, and the inter-relationship between spacing and size of such structures is an unresolved issue. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a crucial regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development; however, this effect does not occur at the level of gene transcription. Rather, our experimental data, together with computational modeling, indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.