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.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.

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.