Probes for INS

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 mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high fat diet (HFD) and insulinopenia induced by treatment with streptozotocin (STZ), we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by - 28.4±7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06±38.71%, and increased density of SCA1+ (+74.99± 29.19%) but not Runx2 +osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82± 33.05%), senescence gene signature (≈106.66± 34.03%) and LAMIN B1 - senescent cell density (+225.18± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9± 6.2% which was inversely correlated with glucose levels (R 2=0.48, p<0.004) and callus adipose tissue area (R 2=0.3711, p<0.01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycaemia per se and not just insulin levels is detrimental for bone healing.

Although the skeleton is essential for locomotion, endocrine functions, and hematopoiesis, the molecular mechanisms of human skeletal development remain to be elucidated. Here, we introduce an integrative method to model human skeletal development by combining in vitro sclerotome induction from human pluripotent stem cells and in vivo endochondral bone formation by implanting the sclerotome beneath the renal capsules of immunodeficient mice. Histological and scRNA-seq analyses reveal that the induced bones recapitulate endochondral ossification and are composed of human skeletal cells and mouse circulatory cells. The skeletal cell types and their trajectories are similar to those of human embryos. Single-cell multiome analysis reveals dynamic changes in chromatin accessibility associated with multiple transcription factors constituting cell-type-specific gene-regulatory networks (GRNs). We further identify ZEB2, which may regulate the GRNs in human osteogenesis. Collectively, these results identify components of GRNs in human skeletal development and provide a valuable model for its investigation.

The mammalian skull vault is essential to shape the head and protect the brain, but the cellular and molecular events underlying its development remain incompletely understood. Single-cell transcriptomic profiling from early to late mouse embryonic stages provides a detailed atlas of cranial lineages. It distinguishes various populations of progenitors and reveals a high expression of SOXC genes (encoding the SOX4, SOX11, and SOX12 transcription factors) early in development in actively proliferating and myofibroblast-like osteodermal progenitors. SOXC inactivation in these cells causes severe skull and skin underdevelopment due to the limited expansion of cell populations before and upon lineage commitment. SOXC genes enhance the expression of gene signatures conferring dynamic cellular and molecular properties, including actin cytoskeleton assembly, chromatin remodeling, and signaling pathway induction and responsiveness. These findings shed light onto craniogenic mechanisms and SOXC functions and suggest that similar mechanisms could decisively control many developmental, adult, pathological, and regenerative processes.

A non-canonical PRC1 (PRC1.6) prevents precocious meiotic onset. Germ cells alleviate its negative effect by reducing their amount of MAX, a component of PRC1.6, as a prerequisite for their bona fide meiosis. Here, we found that germ cells produced Mga variant mRNA bearing a premature termination codon (PTC) during meiosis as an additional mechanism to impede the function of PRC1.6. The variant mRNA encodes an anomalous MGA protein that lacks the bHLHZ domain and thus functions as a dominant negative regulator of PRC1.6. Notwithstanding the presence of PTC, the Mga variant mRNA are rather stably present in spermatocytes and spermatids due to their intrinsic inefficient background of nonsense-mediated mRNA decay. Thus, our data indicate that meiosis is controlled in a multi-layered manner in which both MAX and MGA, which constitute the core of PRC1.6, are at least used as targets to deteriorate the integrity of the complex to ensure progression of meiosis.