With increasing age of the population, countries across the globe are facing a substantial increase in osteoporotic fractures. Genetic association signals for fractures have been reported at the RSPO3 locus, but the causal gene and the underlying mechanism are unknown. Here we show that the fracture reducing allele at the RSPO3 locus associate with increased RSPO3 expression both at the mRNA and protein levels, increased trabecular bone mineral density and reduced risk mainly of distal forearm fractures in humans. We also demonstrate that RSPO3 is expressed in osteoprogenitor cells and osteoblasts and that osteoblast-derived RSPO3 is the principal source of RSPO3 in bone and an important regulator of vertebral trabecular bone mass and bone strength in adult mice. Mechanistic studies revealed that RSPO3 in a cell-autonomous manner increases osteoblast proliferation and differentiation. In conclusion, RSPO3 regulates vertebral trabecular bone mass and bone strength in mice and fracture risk in humans.
The interaction between signaling pathways is a central question in the study of organogenesis. Using the developing murine tongue as a model, we uncovered unknown relationships between Sonic hedgehog (SHH) and retinoic acid (RA) signaling. Genetic loss of SHH signaling leads to enhanced RA activity subsequent to loss of SHH-dependent expression of Cyp26a1 and Cyp26c1. This causes a cell identity switch, prompting the epithelium of the tongue to form heterotopic minor salivary glands and to overproduce oversized taste buds. At developmental stages during which Wnt10b expression normally ceases and Shh becomes confined to taste bud cells, loss of SHH inputs causes the lingual epithelium to undergo an ectopic and anachronic expression of Shh and Wnt10b in the basal layer, specifying de novo taste placode induction. Surprisingly, in the absence of SHH signaling, lingual epithelial cells adopted a Merkel cell fate, but this was not caused by enhanced RA signaling. We show that RA promotes, whereas SHH, acting strictly within the lingual epithelium, inhibits taste placode and lingual gland formation by thwarting RA activity. These findings reveal key functions for SHH and RA in cell fate specification in the lingual epithelium and aid in deciphering the molecular mechanisms that assign cell identity.
Carbonic anhydrases (CAs) play fundamental roles in several physiological events, and emerging evidence points at their involvement in an array of disorders, including cancer. The expression of CAs in the different cells of teeth is unknown, let alone their expression patterns during odontogenesis. As a first step towards understanding the role of CAs during odontogenesis, we used immunohistochemistry, histochemistry and in situ hybridization to reveal hitherto unknown dynamic distribution patterns of eight CAs in mice. The most salient findings include expression of CAII/Car2 not only in maturation-stage ameloblasts (MA) but also in the papillary layer, dental papilla mesenchyme, odontoblasts and the epithelial rests of Malassez. We uncovered that the latter form lace-like networks around incisors; hitherto these have been known to occur only in molars. All CAs studied were produced by MA, however CAIV, CAIX and CARPXI proteins were distinctly enriched in the ruffled membrane of the ruffled MA but exhibited a homogeneous distribution in smooth-ended MA. While CAIV, CAVI/Car6, CAIX, CARPXI and CAXIV were produced by all odontoblasts, CAIII distribution displayed a striking asymmetry, in that it was virtually confined to odontoblasts in the root of molars and root analog of incisors. Remarkably, from initiation until near completion of odontogenesis and in several other tissues, CAXIII localized mainly in intracellular punctae/vesicles that we show to overlap with LAMP-1- and LAMP-2-positive vesicles, suggesting that CAXIII localizes within lysosomes. We showed that expression of CAs in developing teeth is not confined to cells involved in biomineralization, pointing at their participation in other biological events. Finally, we uncovered novel sites of CA expression, including the developing brain and eye, the olfactory epithelium, melanoblasts, tongue, notochord, nucleus pulposus and sebaceous glands. Our study provides important information for future single or multiple gene targeting strategies aiming at deciphering the function of CAs during odontogenesis.
The aim of this study was to analyse the hitherto largely unknown expression patterns of some specific cellular and extracellular molecules during palate and nasal cavity development. We showed that epithelia of the developing palate and the vomerine epithelium express similar sets of structural proteins. With the exception of keratin 15, which becomes barely detectable in the elevated palatal shelves, nearly all of these proteins become upregulated at the presumptive areas of fusion and in the adhering epithelia of the palate and nasal septum. In vivo and in vitro analyses indicated that reduction in the amount of keratin 15 protein is independent of Tgfbeta-Alk5 signalling. Foxa1 expression also highlighted the regionalization of the palatal and nasal epithelia. Owing to the lack of reliable markers of the palatal periderm, the fate of peridermal cells has been controversial. We identified LewisX/stage-specific embryonic antigen-1 as a specific peridermal marker, and showed that numerous peridermal cells remain trapped in the medial epithelial seam (MES). The fate of these cells is probably apoptosis together with the rest of the MES cells, as we provided strong evidence for this event. Heparan sulphate, chondroitin-6-sulphate, and versican displayed dynamically changing distribution patterns. The hitherto-unknown innervation pattern of the developing palate was revealed. These findings may be of value for unravelling the pathogenesis of palatal clefting.
Deciphering how signaling pathways interact during development is necessary for understanding the etiopathogenesis of congenital malformations and disease. In several embryonic structures, components of the Hedgehog and retinoic acid pathways, two potent players in development and disease are expressed and operate in the same or adjacent tissues and cells. Yet whether and, if so, how these pathways interact during organogenesis is, to a large extent, unclear. Using genetic and experimental approaches in the mouse, we show that during development of ontogenetically different organs, including the tail, genital tubercle, and secondary palate, Sonic hedgehog (SHH) loss-of-function causes anomalies phenocopying those induced by enhanced retinoic acid signaling and that SHH is required to prevent supraphysiological activation of retinoic signaling through maintenance and reinforcement of expression of the Cyp26 genes. Furthermore, in other tissues and organs, disruptions of the Hedgehog or the retinoic acid pathways during development generate similar phenotypes. These findings reveal that rigidly calibrated Hedgehog and retinoic acid activities are required for normal organogenesis and tissue patterning.
Background Bone loss is often observed adjacent to inflammatory processes. The WNT signaling pathways have been implicated as novel regulators of both immune responses and bone metabolism. WNT16 is important for cortical bone mass by inhibiting osteoclast differentiation, and we have here investigated the regulation of WNT16 by several members of the pro-inflammatory gp130 cytokine family. Methods The expression and regulation of Wnt16 in primary murine cells were studied by qPCR, scRNAseq and in situ hybridization. Signaling pathways were studied by siRNA silencing. The importance of oncostatin M (OSM)-induced WNT16 expression for osteoclastogenesis was studied in cells from Wnt16 -deficient and wild-type mice. Results We found that IL-6/sIL-6R and OSM induce the expression of Wnt16 in primary mouse calvarial osteoblasts, with OSM being the most robust stimulator. The induction of Wnt16 by OSM was dependent on gp130 and OSM receptor (OSMR), and downstream signaling by the SHC1/STAT3 pathway, but independent of ERK. Stimulation of the calvarial cells with OSM resulted in enhanced numbers of mature, oversized osteoclasts when cells were isolated from Wnt16 deficient mice compared to cells from wild-type mice. OSM did not affect Wnt16 mRNA expression in bone marrow cell cultures, explained by the finding that Wnt16 and Osmr are expressed in distinctly different cells in bone marrow, nor was osteoclast differentiation different in OSM-stimulated bone marrow cell cultures isolated from Wnt16 −/- or wild-type mice. Furthermore, we found that Wnt16 expression is substantially lower in cells from bone marrow compared to calvarial osteoblasts. Conclusion These findings demonstrate that OSM is a robust stimulator of Wnt16 mRNA in calvarial osteoblasts and that WNT16 acts as a negative feedback regulator of OSM-induced osteoclast formation in the calvarial bone cells, but not in the bone marrow.
Osteoporosis is a common skeletal disease, with increased risk of fractures. Currently available osteoporosis treatments reduce the risk of vertebral fractures, mainly dependent on trabecular bone, whereas the effect on non-vertebral fractures, mainly dependent on cortical bone, is less pronounced. WNT signaling is a crucial regulator of bone homeostasis, and the activity of WNTs is inhibited by NOTUM, a secreted WNT lipase. We previously demonstrated that conditional inactivation of NOTUM in all osteoblast lineage cells increases the cortical but not the trabecular bone mass. The aim of the present study was to determine if NOTUM increasing cortical bone is derived from osteoblast precursors/early osteoblasts or from osteocytes/late osteoblasts. First, we demonstrated Notum mRNA expression in Dmp1-expressing osteocytes and late osteoblasts in cortical bone using in situ hybridization. We then developed a mouse model with inactivation of NOTUM in Dmp1 expressing osteocytes and late osteoblasts (Dmp1-creNotumflox/flox mice). We observed that the Dmp1-creNotumflox/flox mice displayed a substantial reduction of Notum mRNA in cortical bone, resulting in increased cortical bone mass and decreased cortical porosity in femur, but no change in trabecular bone volume fraction (BV/TV) in femur or in the lumbar vertebrae L5 in Dmp1-creNotumflox/flox mice as compared to control mice. In conclusion, osteocytes and late osteoblasts are the principal source of NOTUM in cortical bone, and NOTUM derived from osteocytes/late osteoblasts reduces cortical bone mass. These findings demonstrate that inhibition of osteocyte/late osteoblast-derived NOTUM might be an interesting pharmacological target to increase cortical bone mass and reduce non-vertebral fracture risk.
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