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SOX8 was linked in a genome-wide association study to human height heritability, but roles in chondrocytes for this close relative of the master chondrogenic transcription factor SOX9 remain unknown. We undertook here to fill this knowledge gap. High-throughput assays demonstrate expression of human SOX8 and mouse Sox8 in growth plate cartilage. In situ assays show that Sox8 is expressed at a similar level as Sox9 in reserve and early columnar chondrocytes and turned off when Sox9 expression peaks in late columnar and prehypertrophic chondrocytes. Sox8-/- mice and Sox8fl/flPrx1Cre and Sox9fl/+Prx1Cre mice (inactivation in limb skeletal cells) have a normal or near normal skeletal size. In contrast, juvenile and adult Sox8fl/flSox9fl/+Prx- 1Cre compound mutants exhibit a 15 to 20% shortening of long bones. Their growth plate reserve chondrocytes progress slowly toward the columnar stage, as witnessed by a delay in down-regulating Pthlh expression, in packing in columns and in elevating their proliferation rate. SOX8 or SOX9 overexpression in chondrocytes reveals not only that SOX8 can promote growth plate cell proliferation and differentiation, even upon inactivation of endogenous Sox9, but also that it is more efficient than SOX9, possibly due to greater protein stability. Altogether, these findings uncover a major role for SOX8 and SOX9 in promoting skeletal growth by stimulating commitment of growth plate reserve chondrocytes to actively proliferating columnar cells. Further, by showing that SOX8 is more chondrogenic than SOX9, they suggest that SOX8 could be preferred over SOX9 in therapies to promote cartilage formation or regeneration in developmental and degenerative cartilage diseases. [ABSTRACT FROM AUTHOR]

Objectives: Fibroblast-like synoviocytes (FLS) and articular chondrocytes (AC) derive from a common pool of embryonic precursor cells. They are currently believed to engage in largely distinct differentiation programs to build synovium and articular cartilage and maintain healthy tissues throughout life. We tested this hypothesis by deeply characterizing and comparing their transcriptomic attributes., Methods: We profiled the transcriptomes of freshly isolated AC, synovium, primary FLS, and dermal fibroblasts from healthy adult humans using bulk RNA sequencing assays and downloaded published single-cell RNA sequencing data from freshly isolated human FLS. We integrated all data to define cell-specific signatures and validated findings with quantitative reverse transcription PCR of human samples and RNA hybridization of mouse joint sections., Results: We identified 212 AC and 168 FLS markers on the basis of exclusive or enriched expression in either cell and 294 AC/FLS markers on the basis of similar expression in both cells. AC markers included joint-specific and pan-cartilaginous genes. FLS and AC/FLS markers featured 37 and 55 joint-specific genes, respectively, and 131 and 239 pan-fibroblastic genes, respectively. These signatures included many previously unrecognized markers with potentially important joint-specific roles. AC/FLS markers overlapped in their expression patterns among all FLS and AC subpopulations, suggesting that they fulfill joint-specific properties in all, rather than in discrete, AC and FLS subpopulations., Conclusion: This study broadens knowledge and identifies a prominent overlap of the human adult AC and FLS transcriptomic signatures. It also provides data resources to help further decipher mechanisms underlying joint homeostasis and degeneration and to improve the quality control of tissues engineered for regenerative treatments., (© 2021 The Authors. ACR Open Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology.)

The RNA in situ hybridization assay is essential in many studies to evaluate gene expression in vivo. It consists of generating tissue sections and subsequently hybridizing these sections with RNA probes. Keeping RNA intact is a challenge while harvesting tissue samples, processing through embedding, sectioning them, and conditioning the sections for hybridization. These challenges are particularly strong for adult skeletal tissues due to their copious, dense, and mineralized extracellular matrices. Here, we describe a method optimized to successfully hybridize RNA species, even of low abundance, in adult mouse bone and cartilage samples. This method involves tissue fixation with paraformaldehyde, demineralization with Morse's solution and paraffin embedding, all of which can be completed in 4 days. Sections are then generated and hybridized using a 1-day standard protocol. Sections prepared using this method are compatible with immunostaining and standard staining procedures for skeletal tissues.

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo-synthesized genomic DNA. 5-bromo-2'-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2'-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.