Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Celllineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER + perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. β-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration.Therefore, quiescent Cxcl12-creER + BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.

Growth plate chondrocytes play central roles in the proper development and growth of endochondral bones. Particularly, a population of chondrocytes in the resting zone expressing parathyroid hormone-related protein (PTHrP) is now recognized as skeletal stem cells, defined by their ability to undergo self-renewal and clonally give rise to columnar chondrocytes in the postnatal growth plate. These chondrocytes also possess the ability to differentiate into a multitude of cell types including osteoblasts and bone marrow stromal cells during skeletal development. Using single-cell transcriptomic approaches and in vivo lineage tracing technology, it is now possible to further elucidate their molecular properties and cellular fate changes. By discovering the fundamental molecular characteristics of these cells, it may be possible to harness their functional characteristics for skeletal growth and regeneration. Here, we discuss our current understanding of the molecular signatures defining growth plate chondrocytes.

Large-scale sequencing efforts have captured a rapidly growing catalogue of genetic variations. However, the accurate establishment of gene variant pathogenicity remains a central challenge in translating personal genomics information to clinical decisions. Interferon Regulatory Factor 6 (IRF6) gene variants are significant genetic contributors to orofacial clefts. Although approximately three hundred IRF6 gene variants have been documented, their effects on protein functions remain difficult to interpret. Here, we demonstrate the protein functions of human IRF6 missense gene variants could be rapidly assessed in detail by their abilities to rescue the irf6 -/- phenotype in zebrafish through variant mRNA microinjections at the one-cell stage. The results revealed many missense variants previously predicted by traditional statistical and computational tools to be loss-of-function and pathogenic retained partial or full protein function and rescued the zebrafish irf6 -/- periderm rupture phenotype. Through mRNA dosage titration and analysis of the Exome Aggregation Consortium (ExAC) database, IRF6 missense variants were grouped by their abilities to rescue at various dosages into three functional categories: wild type function, reduced function, and complete loss-of-function. This sensitive and specific biological assay was able to address the nuanced functional significances of IRF6 missense gene variants and overcome many limitations faced by current statistical and computational tools in assigning variant protein function and pathogenicity. Furthermore, it unlocked the possibility for characterizing yet undiscovered human IRF6 missense gene variants from orofacial cleft patients, and illustrated a generalizable functional genomics paradigm in personalized medicine.