Osteoarthritis (OA) is a progressive joint disease habitually linked to ageing, characterized by the gradual breakdown of cartilage leading to pain and reduced mobility. Historically viewed as mainly a "wear and tear" condition, new insights suggest that OA may be part of an evolutionary, age-related biological process rather than mainly driven by mechanical damage.

This conceptual paper discusses the model of antagonistic pleiotropy that proposes that certain genes beneficial early in life may contribute to diseases in the context of OA.

Findings indicate that OA is connected to biological and not to chronological age supporting the idea that OA is not merely a wear and tear process. Chondrocyte hypertrophy, essential in endochondral bone formation at a (pre)reproductive age, is stimulated by a displaced and wrongly timed endochondral ossification quasi-program in age-related OA. Age-related chondrocyte hypertrophic differentiation in articular cartilage is likely driven by loss of loading-induced TGF-β signaling.

Comprehending OA within this evolutionary and biological frame provides a solid alternative to the theory of "wear and tear", offering insights into further understanding, prevention and disease management.

Hedgehog (HH) signaling, transduced at the primary cilium, plays a crucial role in limb longitudinal growth and digit formation, but its involvement in thumb development has been underestimated. This study identifies Merlin (Nf2) as a critical regulator of limb development, modulating the ciliary trafficking of the HH receptor, Smoothened. Merlin is predominantly expressed in limb buds and growth plates. Conditional knockout of Merlin in limb mesenchyme in mice results in dwarfism, brachydactyly, and thumb hypoplasia, with transcriptomic profiling and molecular analyses revealing disrupted HH signaling. Mechanistically, Merlin interacts with ARF6 to regulate the ciliary transport of Smoothened via RAB11+ vesicles. Importantly, pharmacological enhancement of HH signaling significantly corrected the limb defects caused by Merlin deletion. These findings highlight the essential role of Merlin in regulating longitudinal limb growth and thumb morphogenesis via primary cilium-HH signaling, suggesting potential therapeutic strategies for related limb dysplasias.

Indian hedgehog (IHH) gene codes an important signal molecule mediating chondrogenesis and bone development in chickens, which are key factors that affect body weight and several other significant economic traits. The aim of this study was to construct an IHH knockout cell model using CRISPR-associated protein 9 (CRISPR/Cas9) technology to further analyze the function of IHH. TA cloning was used to screen the single-guide RNA (sgRNA1) [45 %] and sgRNA3 (30.8 %) with the highest targeting efficiency. Monoclonal cells were selected by flow cytometry for TA cloning sequencing to construct the IHH knockout cell model. Quantitative PCR (qPCR) was used to detect the changes in downstream gene expression levels after IHH knockout. TA cloning sequencing results showed that the IHH knockout cell model was successfully constructed, and two mutation types were generated with a 100 % mutation rate. In addition, qPCR results revealed that the expression of patched 1 (PTCH1), smoothened, frizzled class receptor (Smo), glioma-associated oncogene homolog 1 (Gli1), glioma-associated oncogene homolog 2 (Gli2), and osteopontin (OPN) was significantly lower in the IHH knockout group, while that of type II collagen (Col Ⅱ) was significantly higher. These results lay a theoretical foundation for the successful application of knockout technology in poultry functional genomics research and provide a stable knockout cell line model for further study of chicken IHH gene function.

Low back pain (LBP), one of the most common health problems, is the leading cause of disability globally. Intervertebral disc degeneration (IDD) accounts for most LBP. However, the molecular mechanism underlying IDD remains unclear, and the existing treatment strategy for IDD is still limited. A growing body of evidences suggest that the Hedgehog (HH) pathway plays an essential role in the formation, maintenance, and degeneration of intervertebral discs (IVDs), with Sonic HH (SHH) being primarily involved in the development and maturation of the IVDs and a strong link between Indian HH(IHH) and disc calcification. This review provides an overview of the role of the HH signaling pathway in the developmental maturation and degeneration of IVDs and suggests potential therapeutic targets for IDD that may interfere with HH signaling.

Axolotl digits offer an experimentally versatile model for studying complex tissue regeneration. Here, we provide a comprehensive morphological and molecular characterization of digit regeneration, revealing both conserved features and notable divergences from classical limb regeneration. Digit blastemas progress through similar morphological stages, are nerve-dependent, contain key regenerative cell populations, and express many canonical morphogens and mitogens. However, they exhibit minimal expression of the A-P patterning genes Shh, Fgf8, and Grem1; suggesting distal outgrowth and patterning occur independently of these signals. Joint regenerative fidelity varies significantly across digits and cannot be explained by differences in nerve supply, cell proliferation, or differential expression of any patterning genes assessed in this study. Furthermore, functional experiments reveal Hedgehog signaling is essential for interphalangeal joint regeneration, but activation alone is insufficient to improve fidelity in less robust digits. This system combines experimental accessibility with intrinsic variation in regenerative outcomes, making it an ideal platform to identify critical determinants of successful tissue regeneration and refine models of appendage patterning.

Porcupine (PORCN) is a membrane-bound protein of the endoplasmic reticulum, which modifies Wnt proteins by adding palmitoleic acid. This modification is essential for Wnt ligand secretion. Patients with mutated PORCN display various skeletal abnormalities likely stemming from disrupted Wnt signaling pathways during the chondrocyte differentiation. To uncover the mechanism of PORCN action during chondrogenesis, we used 2 different PORCN inhibitors, C59 and LGK974, in several model systems, including micromasses, 3D cell cultures, long bone tissue cultures, and zebrafish animal model. PORCN inhibitors enhanced cartilaginous extracellular matrix (ECM) production and accelerated chondrocyte differentiation, which resulted in the earlier induction of cellular hypertrophy as well as cartilaginous mass expansion in micromass cultures and cartilaginous organoids. In addition, both PORCN inhibitors expanded the hypertrophic zone and reduced the proliferative zone in the growth plate. This led to a significant increase in cartilaginous tissue and ultimately resulted in the elongation of tibias in the mouse organ cultures. Also, LGK974 treatment of Danio rerio embryos induced expansion of craniofacial cartilage width together with the shortening of the body axis, which was consistent with a phenomenon occurring upon inhibition of non-canonical Wnt signaling. By combining PORCN inhibition with exogenous Wnt proteins activating either canonical/β-catenin (WNT3a) or non-canonical (WNT5a) signaling, we propose that the key mechanism mediating pro-chondrogenic effects of PORCN inhibition is the removal of canonical ligands that prevent chondrocyte differentiation. In summary, our results provide evidence of the distinct role of PORCN in both the early and late stages of cartilage development. Further, our data demonstrate that PORCN inhibitors can be used in the experimental and clinical strategies that need to trigger chondrocyte differentiation and/or cartilage outgrowth.

The SH2 domain-containing protein tyrosine phosphatase 2 (SHP2, also known as PTP2C), encoded by PTPN11, is ubiquitously expressed and has context-specific effects. It promotes RAS/MAPK signaling downstream of receptor tyrosine kinases, cytokine receptors, and extracellular matrix proteins, and was shown in various lineages to modulate cell survival, proliferation, differentiation, and migration. Over the past decade, PTPN11 inactivation in chondrocytes was found to cause metachondromatosis, a rare disorder characterized by multiple enchondromas and osteochondroma-like lesions. Moreover, SHP2 inhibition was found to mitigate osteoarthritis pathogenesis in mice, and abundant but incomplete evidence suggests that SHP2 is crucial for cartilage development and adult homeostasis, during which its expression and activity are tightly regulated transcriptionally and posttranslationally, and by varying sets of functional partners. Fully uncovering SHP2 actions and regulation in chondrocytes is thus fundamental to understanding the mechanisms underlying both rare and common cartilage diseases and to designing effective disease treatments. We here review current knowledge, highlight recent discoveries and controversies, and propose new research directions to answer remaining questions.

The mandibular condylar cartilage (MCC) is a dual-function component of the temporomandibular joint (TMJ), acting as both articular cartilage for jaw movement and growth cartilage for vertical growth of the mandibular condyle. Parathyroid hormone-related protein (PTHrP) plays a critical role in orchestrating chondrogenesis in the long bone, and its importance is also highlighted in both MCC development and TMJ function. Here, we discuss the role of PTHrP in the development, growth and diseases of the MCC. PTHrP is a key morphogen in the MCC that regulates chondrogenesis by promoting chondrocyte proliferation and preventing premature hypertrophic differentiation. Exclusively expressed in the superficial layer, PTHrP diffuses across the MCC and targets chondrocytes in deeper layers, regulating transcription factors such as RUNX2 and SOX9. PTHrP regulates chondrocyte differentiation through two main pathways: the PTHrP-PTH1R signalling pathway, which suppresses hypertrophy and the PTHrP-Ihh negative feedback loop, which balances proliferation and hypertrophy. In the postnatal murine MCC, PTHrP levels are high early on and decrease after the onset of mastication around P21. Altered mechanical environments, such as those therapeutically induced as mandibular advancement, increase PTHrP expression, promoting chondrocyte proliferation and delaying hypertrophy. PTHrP also plays a dual role in adult TMJ diseases, particularly in osteoarthritis (OA); PTHrP expression transiently increases during the early stages of TMJ-OA to promote cell proliferation, but its eventual decrease contributes to the progression of the disease. This highlights the complex role of PTHrP in maintaining MCC homeostasis and its potential involvement in TMJ-OA pathology. The MCC combines the characteristics of growth and articular cartilage and functions distinctively in three phases: development before occlusion, growth after the occlusion is established, and maintenance after the growth is complete. While PTHrP plays a multifaceted role in all phases, further research is needed to fully understand how it regulates MCC development, growth and diseases.

Chronic pancreatitis (CP) is a fibro-inflammatory disease of the pancreas with no specific cure. Research highlighting the pathogenesis and especially the therapeutic aspect remains limited. Aberrant activation of developmental pathways in adults has been implicated in several diseases. Hedgehog pathway is a notable embryonic signaling pathway, known to promote fibrosis of various organs when overactivated. The aim of this study is to explore the role of the hedgehog pathway in the progression of CP and evaluate its inhibition as a novel therapeutic strategy against CP. CP was induced in mice by repeated injections of l-arginine or caerulein in two separate models. Mice were administered with the FDA-approved pharmacological hedgehog pathway inhibitor, vismodegib during or after establishing the disease condition to inhibit hedgehog signaling. Various parameters of CP were analyzed to determine the effect of hedgehog pathway inhibition on the severity and progression of the disease. Our study shows that hedgehog signaling was overactivated during CP and its inhibition was effective in improving the histopathological parameters associated with CP. Vismodegib administration not only halted the progression of CP but was also able to resolve already-established fibrosis. In addition, inhibition of hedgehog signaling resulted in the reversal of pancreatic stellate cell activation ex vivo. Findings from our study justify conducting clinical trials using vismodegib against CP and, thus, could lead to the development of a novel therapeutic strategy for the treatment of CP.NEW & NOTEWORTHY Hedgehog signaling is activated in human and experimental models of CP. Inhibition of hedgehog signaling using an FDA-approved inhibitor, vismodegib, leads to the resolution of fibrosis and improves CP. This study has immense and immediate translational benefits.

MicroRNAs (miRNAs) are critical regulators of the skeleton. In the growth plate, these small non-coding RNAs modulate gene networks that drive key stages of chondrogenesis, including proliferation, differentiation, extracellular matrix synthesis and hypertrophy. These processes are orchestrated through the interaction of pivotal pathways including parathyroid hormone-related protein (PTHrP), Indian hedgehog (IHH), and bone morphogenetic protein (BMP) signaling. This review highlights the miRNA-mRNA target networks essential for chondrocyte differentiation. Many miRNAs are differentially expressed in resting, proliferating and hypertrophic cartilage zones. Moreover, differential enrichment of specific miRNAs in matrix vesicles is also observed, providing means for chondrocytes to influence the function and differentiation of their neighbors by via matrix vesicle protein and RNA cargo. Notably, miR-1 and miR-140 emerge as critical modulators of chondrocyte proliferation and hypertrophy by regulating multiple signaling pathways, many of them downstream from their mutual target Hdac4. Demonstration that a human gain-of-function mutation in miR-140 causes skeletal dysplasia underscores the clinical relevance of understanding miRNA-mediated regulation. Further, miRNAs such as miR-26b have emerged as markers for skeletal disorders such as idiopathic short stature, showcasing the translational relevance of miRNAs in skeletal health. This review also highlights some miRNA-based therapeutic strategies, including innovative delivery systems that could target chondrocytes via cartilage affinity peptides, and potential applications related to treatment of physeal bony bridge formation in growing children. By synthesizing current research, this review offers a nuanced understanding of miRNA functions in growth plate biology and their broader implications for skeletal health. It underscores the translational potential of miRNA-based therapies in addressing skeletal disorders and aims to inspire further investigations in this rapidly evolving field.

Developmental dysplasia of the hip (DDH) is a developmental disorder that has long-term chronic pain and limited hip joint mobility. The aim of the current study is to understand the specific chondrocyte composition involved in DDH development, identify effective biomarkers for DDH prediction, and elucidate the gene regulatory elements driving DDH progression.

In this study, we performed an integrated analysis combining single-cell RNA sequencing and single-cell assay for transposase-accessible chromatin sequencing to investigate the molecular programs and epigenetic changes governing human DDH pathogenesis. Validation of marker genes for distinct chondrocyte populations was performed via immunohistochemical assays, alongside characterization of regulatory elements specific to DDH.

Our analysis identified seven molecularly distinct chondrocyte populations in DDH cartilage, including a novel inflammatory chondrocyte population with unique molecular signatures. Furthermore, we reconstructed the differentiation trajectory of chondrocytes, shedding light on their roles in DDH pathogenesis. Integrative analyses of transcriptomic and chromatin accessibility profiles highlighted shared regulatory features and transcriptional programs among chondrocyte subtypes, with several regulatory elements linked to DDH progression. Immunohistochemical validation corroborated the presence of key marker genes in distinct chondrocyte subsets.

Our findings contribute to clarifying the cellular heterogeneity of DDH and offer insights into potential early diagnostic and therapeutic strategies for this condition.

Ectodysplasin-A (EDA) has been reported to be involved in mouse condylar development, but the specific functions of EDA in maintaining homeostasis of the temporomandibular joint (TMJ) remain unclear. This study aimed to explore the underlying roles and related mechanisms of EDA in temporomandibular joint osteoarthritis (TMJOA).

The TMJOA mouse model was established by unilateral discectomy and the alteration of EDA expression was detected. EDA knockout male mice and their wild-type male littermates were used to clarify the effect of EDA on TMJOA. Mouse condylar chondrocytes were extracted to explore the potential mechanisms. The effects of local injection of supplementary EDA on condyles were also evaluated morphologically and histologically.

The expression of EDA was downregulated in condylar cartilage after TMJOA modeling. EDA deficiency aggravated degeneration and inflammation of condylar cartilage in TMJOA mice. In vitro studies demonstrated that EDA deficiency upregulated the expression of inflammatory cytokines, while supplementary EDA exhibited anticatabolic and anti-inflammatory effects on tumor necrosis factor-α (TNFα)-treated mouse condylar chondrocytes. Mechanistically, EDA deficiency effectively activated activating transcription factor 4 (ATF4) to upregulate Indian hedgehog (Ihh) signaling pathway and thereby aggravated the inflammation. Inhibition of ATF4 resulted in blocking of Ihh signaling. The selective pharmacological inhibition of Ihh signaling attenuated TNF-α-induced chondrocyte destruction and the release of inflammatory cytokines. Furthermore, intra-articular application of EDA significantly alleviated the osteoarthritic cartilage destruction after discectomy.

EDA deficiency aggravated TMJOA by modulating ATF4/Ihh pathway, which confirmed the essential role of EDA in maintaining TMJ cartilage homeostasis and its potential application in TMJOA treatment.

Endochondral ossification generates most of the load-bearing bones, recapitulating it in human cells remains a challenge. Here, we report generation of SOX9+ sclerotomal progenitors (scl-progenitors), a mesenchymal precursor at the pre-condensation stage, from human pluripotent stem cells and development of osteochondral induction methods for these cells. Upon lineage-specific induction, SOX9+ scl-progenitors have not only generated articular cartilage but have also undergone spontaneous condensation, cartilaginous anlagen formation, chondrocyte hypertrophy, vascular invasion, and finally bone formation with stroma, thereby recapitulating key stages during endochondral ossification. Moreover, self-organized growth plate-like structures have also been induced using SOX9+ scl-progenitor-derived fusion constructs with chondro- and osteo-spheroids, exhibiting molecular and cellular similarities to the primary growth plates. Furthermore, we have identified ITGA9 as a specific surface marker for reporter-independent isolation of SOX9+ scl-progenitors and established a culture system to support their expansion. Our work highlights SOX9+ scl-progenitors as a promising tool for modeling human skeletal development and bone/cartilage bioengineering.

Chondrodysplasias with multiple dislocations are rare skeletal disorders characterized by hyperlaxity, joint dislocations, and growth retardation. Chondrodysplasias with multiple dislocations have been linked to pathogenic variants in genes encoding proteins involved in the proteoglycan (PG) biosynthesis. In this study, by exome sequencing analysis, we identified a homozygous nonsense variant (NM_001297654.2: c.1825C>T, p.Arg609*) in the discoidin domain receptor 1 (DDR1) gene in a patient presenting joint dislocations, hyperlaxity, and cerebellar hypoplasia. Functional studies revealed decreased PG production in patient fibroblasts. We further demonstrated that DDR1 inhibition impaired the Indian Hedgehog signaling pathway in chondrocytes, decreased differentiation and mineralization in osteoblasts, and disrupted p38 MAPK signaling in both cell types. Additionally, we showed that DDR1 inhibition affected the noncanonical WNT signaling pathway in human skeletal cells and decreased PG production in chondrocytes. These findings suggest that DDR1 is a new gene involved in the group of chondrodysplasias with multiple dislocations and highlights its essential role in human skeletal and brain development.

Longitudinal skeletal growth takes place in the cartilaginous growth plates. While growth plates are found at either end of conventional long bones, they occur at a variety of locations in the mammalian skeleton. For example, the metacarpals and metatarsals (MT) in the hands and feet form only a single growth plate at one end, and the pisiform in the wrist is the only carpal bone to contain a growth plate. We take advantage of this natural anatomical variation to test which components of the PTHrP/Ihh feedback loop, a fundamental regulator of chondrocyte differentiation, are specific to growth plate function.

Parathyroid hormone-like hormone (Pthlh), the gene that transcribes parathyroid hormone-related peptide (PTHrP), is expressed in the reserve zone of the growth plate-forming end of the MT. At the opposite end, the absence of a PTHrP+ reserve zone results in premature chondrocyte differentiation and Indian hedgehog (Ihh) expression. Pthlh is expressed in the reserve zone of the developing pisiform, confirming the existence of a true growth plate.

A pool of PTHrP+ reserve zone chondrocytes is a defining characteristic of growth plates, and its patterning may be key to evolved differences in growth plate location in the mammalian skeleton.

Chondrocytes in articular cartilage can secrete extracellular matrix to maintain cartilage homeostasis. It is well known that articular cartilage chondrocytes are sensitive to mechanical loading and that mechanical stimuli can be translated to biological processes. This study provides deep insight into the impact of mechanical loading on chondrocytes via single-cell RNA sequencing (scRNA-seq). Five cartilage tissue samples from the high-loading region of medial cartilage from the upper tibia (the TL group) and six cartilage tissue samples from the low-loading region of lateral cartilage from the upper tibia (the TN group) were obtained from six donors and subjected to scRNA-seq. TL and TN cartilage tissues from another donor were subjected to immunohistochemical staining. In total, 132,685 cells were analyzed and assigned to 11 cell types. The functions, developmental relationships and interactions of these cell types were determined, and gene transcription data were also evaluated. In addition, differentially expressed genes between the TL and TN groups and their functions were identified. The hub genes for the TL group were identified as GAPDH, FN1, VEGFA, LDHA, SOD1, CTGF, DCN, SERPINE1, ENO1 and CAV1, whereas the hub genes for the TN group included ACTB, CD44, MMP2, COL1A1, COL1A2, SPP1, CTGF, MYC, CCL2, and IGF1. The different enrichment terms indicated that physiological mechanical loading may induce reactive oxygen species accumulation and thus cause ferroptosis in chondrocytes.

The Hedgehog (Hh) signaling pathway is critical for regulating cell growth, survival, fate determination, and the overall patterning of both vertebrate and invertebrate body plans. Aberrations in Hh signaling are associated with congenital abnormalities and tumorigenesis. In vertebrates, Hh signaling depends uniquely on primary cilia, microtubule-based organelles that extend from the cell surface. Over the last 2 decades, studies have demonstrated that key molecules regulating Hh signaling dynamically accumulate in primary cilia via intraflagellar transport systems. Moreover, through the primary cilia, extracellular signals are converted to stabilize GLI2 and GLI3 that are transcription factors that play a central role in regulating Hh signaling at the post-translational modification level. Recent in vivo and anatomical studies have uncovered crucial molecules that facilitate the conversion of extracellular signals into the intracellular stabilization of GLI2/GLI3 via primary cilia, emphasizing their essential roles in tissue development and tumorigenesis. This review explores the regulatory mechanisms of GLI2/GLI3 with a focus on mammalian tissue development.

The skeleton is an important organ in the human body. Bone defects caused by trauma, inflammation, tumors, and other reasons can impact the quality of life of patients. Although the skeleton has a certain ability to repair itself, the current most effective method is still autologous bone transplantation due to factors such as blood supply and defect size. Modern medicine is attempting to overcome these limitations through cell therapy, with mesenchymal stem cells (MSCs) playing a crucial role. MSCs can be extracted from different tissues, and their differentiation potential varies depending on the source. Various cells and cell secretions can influence this process. This article, based on previous research, reviews the effects of macrophages, endothelial cells (ECs), nerve cells, periodontal cells, and even some bacteria on MSC osteogenic differentiation, aiming to provide a reference for multicell coculture strategies related to osteogenesis.

Mesenchymal stem cells (MSCs) are crucial for skeletal development, homeostasis, and repair, primarily through their differentiation into osteoblasts and other skeletal lineage cells. Key signaling pathways, including Wnt, TGF-β/BMP, PTH, Hedgehog, and IGF, act as critical regulators of MSC osteogenesis, playing pivotal roles in maintaining bone homeostasis and facilitating regeneration. These pathways interact in distinct ways at various stages of bone development, mineralization, and remodeling. This review provides an overview of the molecular mechanisms by which these pathways regulate MSC osteogenesis, their influence on bone tissue formation, and their implications in bone diseases and therapeutic strategies. Additionally, we explore the potential applications of these pathways in bone tissue engineering, with a particular focus on promoting the use of MSCs as seed cells for bone defect repair. Ultimately, this review aims to highlight potential avenues for advancing bone biology research, treating bone disorders, and enhancing regenerative medicine.

Osteoarthritis (OA) is a common joint disease characterized by pain and functional impairment, which severely impacts the quality of life of middle-aged and elderly individuals. During normal bone development, chondrocyte hypertrophy is a natural physiological process. However, in the progression of OA, chondrocyte hypertrophy becomes one of its key pathological features. Although there is no definitive evidence to date confirming that chondrocyte hypertrophy is the direct cause of OA, substantial experimental data indicate that it plays an important role in the disease's pathogenesis. In this review, we first explore the mechanisms underlying chondrocyte hypertrophy in OA and offer new insights. We then propose strategies for inhibiting chondrocyte hypertrophy from the perspectives of targeting signaling pathways and tissue engineering, ultimately envisioning the future prospects of OA treatment.

The initial interzone cells for synovial joints originate from chondrocytes, but such critical transition is minimally understood. With single-cell RNA sequencing (scRNA-seq) of murine embryonic knee joint primordia, we discovered that heightened expression of glycolysis genes characterized developing interzone cells when compared to flanking chondrocytes. Conditional deletion of the glucose transporters Glut1 and/or Glut3, in either the incipient pre-skeletal mesenchyme with Prx1Cre or in chondrocytes with Col2Cre, disrupted interzone formation dose-dependently. In contrast, deletion of Glut1/3 in established interzone cells with Gdf5Cre did not have similar severe disruption of joint development. scRNA-seq revealed that Glut1/3 deletion by Prx1Cre impeded Tgfβ signaling in the developing interzone cells. Direct elimination of Tgfβ signaling with Prx1Cre partially phenocopied the deletion of Glut1/3 in impairing interzone formation. Tgfβ stimulated glycolysis in chondrocytes via activation of mTOR and Hif1α in vitro. The data support that the essential conversion of chondrocytes to interzone cells requires a transient elevation of glycolysis partly dependent on Tgfβ signaling.

The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre-hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA-seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre-hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre-hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP-seq and ATAC-seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre-hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates.

Hedgehog and canonical Wnt signaling pathways and the transcription factors Runx2 and Sp7 are essential for osteoblast differentiation. Ihh is necessary for the commitment of perichondrial mesenchymal cells to Runx2+ osteoprogenitors and for the formation of the bone collar and primary spongiosa. Runx2 is needed for osteoblast differentiation during both endochondral and intramembranous ossification. It regulates the commitment of mesenchymal cells to osteoblast-lineage cells and their proliferation by inducing the expression of Hedgehog, Fgf, Wnt, Pthlh signaling pathway genes, and Dlx5. The Runx2-induced expression of Fgfr2 and Fgfr3 is important for the proliferation of osteoblast-lineage cells. Runx2 induces Sp7 expression and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Runx2, Sp7, and canonical Wnt signaling induce the differentiation of preosteoblasts into osteoblasts. Canonical Wnt signaling, but not Sp7, enhances the proliferation of osteoblast-lineage cells. In mature osteoblasts, Runx2 plays an important role in the expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap/Bglap2. The canonical Wnt signaling pathway is also crucial for bone formation by mature osteoblasts. Sp7 is needed for osteocytes to acquire a sufficient number of processes and a reduction in these processes results in osteocyte apoptosis and cortical porosity.

Bone tissue provides structural support for our bodies, with the inner bone marrow (BM) acting as a hematopoietic organ. Within the BM tissue, two types of stem cells play crucial roles: mesenchymal stem cells (MSCs) (or skeletal stem cells) and hematopoietic stem cells (HSCs). These stem cells are intricately connected, where BM-MSCs give rise to bone-forming osteoblasts and serve as essential components in the BM microenvironment for sustaining HSCs. Despite the mid-20th century proposal of BM-MSCs, their in vivo identification remained elusive owing to a lack of tools for analyzing stemness, specifically self-renewal and multipotency. To address this challenge, Cre/loxP-based cell lineage tracing analyses are being employed. This technology facilitated the in vivo labeling of specific cells, enabling the tracking of their lineage, determining their stemness, and providing a deeper understanding of the in vivo dynamics governing stem cell populations responsible for maintaining hard tissues. This review delves into cell lineage tracing studies conducted using commonly employed genetically modified mice expressing Cre under the influence of LepR, Gli1, and Axin2 genes. These studies focus on research fields spanning long bones and oral/maxillofacial hard tissues, offering insights into the in vivo dynamics of stem cell populations crucial for hard tissue homeostasis.

Copy number variation (CNV) is a crucial component of genetic diversity in the genome, serving as the foundation for the genetic architecture and phenotypic variability of complex traits. In this study, we examined CNVs in the Danzhou (DZ) chicken, an indigenous breed exclusive to Hainan Province, China. By employing whole-genome resequencing data from 200 DZ chickens, we conducted a comprehensive genome-wide analysis of CNVs using CNVpytor and performed CNV-based genome-wide association studies (GWAS) on 6 body size traits, including body slope length (BSL), keel length (KeL), tibial length (TiL), tibial circumference (TiC), chest width (ChW), and chest depth (ChD) utilizing linear mixed model methods considering a genomic relationship matrix. We identified a total of 144,265 autosomal CNVs among the 200 individuals, comprising 67,818 deletions and 76,447 duplications. After merging these variants together, we obtained 4,824 distinct copy number variant regions, which accounted for approximately 20% of the chicken autosomal genome. Furthermore, we discovered several significantly associated CNV segments with body size traits located proximal to genes such as IHH, WNT6, WNT10A, LPR4, FZD2, WNT7B, and GNAS that have been extensively implicated in skeletal development and growth processes. These findings enhance our understanding of CNVs in chickens and their potential impact on body size traits by revealing candidate genes involved in the regulation of these traits. This establishes a solid framework for future studies and may prove particularly beneficial for exploring genetic structural variation in chickens.

Mitochondrial Permeability Transition Pore (MPTP) and its key positive regulator, Cyclophilin D (CypD), control activity of cell oxidative metabolism important for differentiation of stem cells of various lineages including osteogenic lineage. Our previous work (Sautchuk et al., 2022) showed that CypD gene, Ppif, is transcriptionally repressed during osteogenic differentiation by regulatory Smad transcription factors in BMP canonical pathway, a major driver of osteoblast (OB) differentiation. Such a repression favors closure of the MPTP, priming OBs to higher usage of mitochondrial oxidative metabolism. The physiological role of CypD/MPTP regulation was demonstrated by its inverse correlation with BMP signaling in aging and bone fracture healing in addition to the negative effect of CypD gain-of-function (GOF) on bone maintenance. Here we show evidence that CypD GOF also negatively affects bone development and growth as well as fracture healing in adult mice. Developing craniofacial and long bones presented with delayed ossification and decreased growth rate, respectively, whereas in fracture, bony callus volume was diminished. Given that Genome Wide Association Studies showed that PPIF locus is associated with both body height and bone mineral density, our new data provide functional evidence for the role of PPIF gene product, CypD, and thus MPTP in bone growth and repair.

Chondrocyte hypertrophy is a potential target for osteoarthritis (OA) treatment, with Indian hedgehog (IHH), glioma-associated oncogene homolog (GLI), and hypoxia-inducible factor-2α (HIF-2α) being closely associated with chondrocyte hypertrophy during OA progression. Whereas IHH can modulate chondrocyte hypertrophy, interference with IHH signalling has not achieved the anticipated therapeutic effects and poses safety concerns, necessitating further clarification of the specific mechanisms by which IHH affects articular cartilage degeneration. Inhibition of the HIF-2α overexpression in cartilage slows the progression of early OA, but the mechanisms underlying HIF-2α accumulation in OA cartilage remain unclear. The aim of this study was to determine the function of Ihh, as well as its downstream factors, in chondrocytes, based on an early osteoarthritis (OA) mouse model and in vitro chondrocyte model.

Investigated the expression levels and locations of IHH-GLI-1 pathway in normal and early degenerated human cartilage, comparing them with HIF-2α and its downstream factors. RT-qPCR, Western blotting, Crystal violet staining, and EdU assays were used to evaluate the pecific regulatory mechanisms of the IHH-GLI-1-HIF-2α signalling axis in normal chondrocytes and in chondrocytes under inflammatory conditions. Validated the impact of IHH on early cartilage degeneration and the relationship between the IHH-GLI-1 pathway and the expression levels and expression locations of HIF-2α and its downstream factors in Col2a1-CreERT2;Ihhfl/fl mice.

In early-stage degenerative joint cartilage, the GLI-1 pathway in hypertrophic chondrocytes exhibited similar changes in location and levels to HIF-2α and its downstream factor vascular endothelial growth factor (VEGF). In vitro, IHH-GLI-1-HIF-2α signalling activation in chondrocytes under physiological hypoxic conditions inhibited chondrocyte proliferation. In chondrocytes stimulated by inflammatory environments, IHH inhibited the degradation of HIF-2α via the GLI-1 pathway, thereby promoting HIF-2α protein expression. Elevated HIF-2α expression further enhanced intracellular IHH-GLI-1 levels, generating a positive feedback loop to collectively regulate the expression of downstream hypertrophic factors and matrix-degradation factors. In vivo, conditional Ihh knockout in mouse chondrocytes downregulated Hif-2α protein expression in early degenerative cartilage tissue and affected the expression of downstream Vegf and hypertrophic factors.

During OA progression, the IHH-GLI-1-HIF-2α axis mainly operates within hypertrophic chondrocytes, exacerbating cartilage degeneration by regulating hypertrophic chondrocyte functions, cartilage matrix degradation, and microvascular invasion.

This study identifies the IHH-GLI-1-HIF-2α signalling axis and reveals its potential as a therapeutic target for OA.

Thoracic ossification of the ligamentum flavum (TOLF) is characterized by ectopic ossification of the ligamentum flavum in the thoracic spine and is considered the main cause of thoracic spinal stenosis and spinal cord disease. Osteoblast specific transcription factor Osterix (Osx) is required for bone formation, and there is no bone formation or ossification without Osx. Surgical intervention is recognized as the only effective method for TOLF treatment with set of complications. However, underlying mechanisms of TOLF are not well understood. This paper summarizes the pathogenesis of TOLF. Some relevant factors have been discussed, such as mechanical stress, genetic susceptibility genes, endocrine and trace element metabolism abnormalities, which may associate with TOLF. More recent studies using proteomics technology and RNA sequencing approach have discovered that some new factors participate in TOLF by upregulation of Osx gene expression including inflammatory factors. TOLF is a unique disease involving multiple factors. On the other hand, studies on TOLF pathogenic mechanism may provide new ideas for finding possible upstream regulatory factors of Osx and further developing novel drugs to stimulate new bone formation to treat osteoporosis.

Despite the functional importance of the vertebral skeleton, little is known about how individual vertebrae elongate or achieve disproportionate lengths as in the giraffe neck. Rodent tails are an abundantly diverse and more tractable system to understand mechanisms of vertebral growth and proportion. In many rodents, disproportionately long mid-tail vertebrae form a 'crescendo-decrescendo' of lengths in the tail series. In bipedal jerboas, these vertebrae grow exceptionally long such that the adult tail is 1.5x the length of a mouse tail, relative to body length, with four fewer vertebrae. How do vertebrae with the same regional identity elongate differently from their neighbors to establish and diversify adult proportion? Here, we find that vertebral lengths are largely determined by differences in growth cartilage height and the number of cells progressing through endochondral ossification. Hypertrophic chondrocyte size, a major contributor to differential elongation in mammal limb bones, differs only in the longest jerboa mid-tail vertebrae where they are exceptionally large. To uncover candidate molecular mechanisms of disproportionate vertebral growth, we performed intersectional RNA-Seq of mouse and jerboa tail vertebrae with similar and disproportionate elongation rates. Many regulators of posterior axial identity and endochondral elongation are disproportionately differentially expressed in jerboa vertebrae. Among these, the inhibitory natriuretic peptide receptor C (NPR3) appears in multiple studies of rodent and human skeletal proportion suggesting it refines local growth rates broadly in the skeleton and broadly in mammals. Consistent with this hypothesis, NPR3 loss of function mice have abnormal tail and limb proportions. Therefore, in addition to genetic components of the complex process of vertebral evolution, these studies reveal fundamental mechanisms of skeletal growth and proportion.

The human skeleton is a multifunctional organ made up of multiple cell types working in concert to maintain bone and mineral homeostasis and to perform critical mechanical and endocrine functions. From the beginning steps of chondrogenesis that prefigures most of the skeleton, to the rapid bone accrual during skeletal growth, followed by bone remodeling of the mature skeleton, cell differentiation is integral to skeletal health. While growth factors and nuclear proteins that influence skeletal cell differentiation have been extensively studied, the role of cellular metabolism is just beginning to be uncovered. Besides energy production, metabolic pathways have been shown to exert epigenetic regulation via key metabolites to influence cell fate in both cancerous and normal tissues. In this review, we will assess the role of growth factors and transcription factors in reprogramming cellular metabolism to meet the energetic and biosynthetic needs of chondrocytes, osteoblasts, or osteoclasts. We will also summarize the emerging evidence linking metabolic changes to epigenetic modifications during skeletal cell differentiation.

The role of rare non-coding variation in complex human phenotypes is still largely unknown. To elucidate the impact of rare variants in regulatory elements, we performed a whole-genome sequencing association analysis for height using 333,100 individuals from three datasets: UK Biobank (N = 200,003), TOPMed (N = 87,652) and All of Us (N = 45,445). We performed rare ( < 0.1% minor-allele-frequency) single-variant and aggregate testing of non-coding variants in regulatory regions based on proximal-regulatory, intergenic-regulatory and deep-intronic annotation. We observed 29 independent variants associated with height at P <  6 × 10 - 10 after conditioning on previously reported variants, with effect sizes ranging from -7cm to +4.7 cm. We also identified and replicated non-coding aggregate-based associations proximal to HMGA1 containing variants associated with a 5 cm taller height and of highly-conserved variants in MIR497HG on chromosome 17. We have developed an approach for identifying non-coding rare variants in regulatory regions with large effects from whole-genome sequencing data associated with complex traits.

Endometrial Cancer (EC) is one of the most common gynecological malignancies. Despite its prevalence, molecular pathways, such as the Sonic Hedgehog (SHH) pathway, have not been extensively studied in the context of EC. This study aims to explore the clinical implications of SHH expression in EC, potentially uncovering new insights into the disease's pathogenesis and offering valuable insights for therapeutic strategies in EC. We utilized data from The Cancer Genome Atlas (TCGA) to divide the dataset into 'High SHH' and 'Low SHH' groups based on a gene signature score derived from SHH pathway-related genes. We explored the clinical and tumor characteristics of these groups, focusing on key cancer hallmarks, including stemness, proliferation, cytolytic activity, tumor micro-environment, and genomic instability. 'High SHH' tumors emerged as a distinct category with favorable clinical and molecular features. These tumors exhibited lower proliferation rates, reduced angiogenesis, and diminished genomic instability, indicating a controlled and less aggressive tumor growth pattern. Moreover, 'High SHH' tumors displayed lower stemness, highlighting a less invasive phenotype. The immune micro-environment in 'High SHH' tumors was enriched with immune cell types, such as macrophage M0, monocytes, B cells, CD8 T cells, CD4 T cells, follicular helper T cells, and natural killer cells. This immune enrichment, coupled with higher cytolytic activity, suggested an improved anti-tumor immune response. Our study sheds light on the clinical significance of Sonic signaling in EC. 'High SHH' tumors exhibit a unique molecular and clinical profile associated with favorable cancer hallmarks, lower grades, and better survival. These findings underscore the potential utility of SHH expression as a robust prognostic biomarker, offering valuable insights for tailored therapeutic strategies in EC. Understanding the SHH pathway's role in EC contributes to our growing knowledge of this cancer and may pave the way for more effective treatment strategies in the future.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Heterotopic ossification (HO) is a pathological process that generates ectopic bone in soft tissues. Hedgehog signaling (Hh signaling) is a signaling pathway that plays an important role in embryonic development and involves three ligands: sonic hedgehog (Shh), Indian hedgehog (Ihh) and desert hedgehog (Dhh). Hh signaling also has an important role in skeletal development. This paper discusses the effects of Hh signaling on the process of HO formation and describes several signaling molecules that are involved in Hh-mediated processes: parathyroid Hormone-Related Protein (PTHrP) and Fkbp10 mediate the expression of Hh during chondrogenesic differentiation. Extracellular signal-regulated kinase (ERK), GNAs and Yes-Associated Protein (YAP) interact with Hh signaling to play a role in osteogenic differentiation. Runt-Related Transcription Factor 2 (Runx2), Mohawk gene (Mkx) and bone morphogenetic protein (BMP) mediate Hh signaling during both chondrogenic and osteogenic differentiation. This paper also discusses possible therapeutic options for HO, lists several Hh inhibitors and explores whether they could serve as emerging targets for the treatment of HO.

Animals use a small number of morphogens to pattern tissues, but it is unclear how evolution modulates morphogen signaling range to match tissues of varying sizes. Here, we used single-molecule imaging in reconstituted morphogen gradients and in tissue explants to determine that Hedgehog diffused extracellularly as a monomer, and rapidly transitioned between membrane-confined and -unconfined states. Unexpectedly, the vertebrate-specific protein SCUBE1 expanded Hedgehog gradients by accelerating the transition rates between states without affecting the relative abundance of molecules in each state. This observation could not be explained under existing models of morphogen diffusion. Instead, we developed a topology-limited diffusion model in which cell-cell gaps create diffusion barriers, which morphogens can only overcome by passing through a membrane-unconfined state. Under this model, SCUBE1 promoted Hedgehog secretion and diffusion by allowing it to transiently overcome diffusion barriers. This multiscale understanding of morphogen gradient formation unified prior models and identified knobs that nature can use to tune morphogen gradient sizes across tissues and organisms.

Insulin-like growth factor (IGF) signaling is required for proper growth and skeletal development in vertebrates. Consequently, its dysregulation may lead to abnormalities of growth or skeletal structures. IGF is involved in the regulation of cell proliferation and differentiation of chondrocytes. However, the availability of bioactive IGF may be controlled by antagonizing IGF binding proteins (IGFBPs) in the circulation and tissues. As the metalloproteinase PAPP-A specifically cleaves members of the IGFBP family, we hypothesized that PAPP-A activity liberates bioactive IGF in cartilage. In PAPP-A knockout mice, the femur length was reduced and the mice showed a disorganized columnar organization of growth plate chondrocytes. Similarly, zebrafish lacking pappaa showed reduced length of Meckel's cartilage and disorganized chondrocytes, reminiscent of the mouse knockout phenotype. Expression of chondrocyte differentiation markers (sox9a, ihha, and col10a1) was markedly affected in Meckel's cartilage of pappaa knockout zebrafish, indicating that differentiation of chondrocytes was compromised. Additionally, the zebrafish pappaa knockout phenotype was mimicked by pharmacological inhibition of IGF signaling, and it could be rescued by treatment with exogenous recombinant IGF-I. In conclusion, our data suggests that IGF activity in the growing cartilage, and hence IGF signaling in chondrocytes, requires the presence of PAPP-A. The absence of PAPP-A causes aberrant chondrocyte organization and compromised growth in both mice and zebrafish.

The rapid development of modern society has led to an increasing severity in the generation of new pollutants and the significant emission of old pollutants, exerting considerable pressure on the ecological environment and posing a serious threat to both biological survival and human health. The skeletal system, as a vital supportive structure and functional unit in organisms, is pivotal in maintaining body shape, safeguarding internal organs, storing minerals, and facilitating blood cell production. Although previous studies have uncovered the toxic effects of pollutants on vertebrate skeletal systems, there is a lack of comprehensive literature reviews in this field. Hence, this paper systematically summarizes the toxic effects and mechanisms of environmental pollutants on the skeletons of vertebrates based on the evolutionary context from fish to mammals. Our findings reveal that current research mainly focuses on fish and mammals, and the identified impact mechanisms mainly involve the regulation of bone signaling pathways, oxidative stress response, endocrine system disorders, and immune system dysfunction. This study aims to provide a comprehensive and systematic understanding of research on skeletal toxicity, while also promoting further research and development in related fields.

Skeletal dysplasias are group of rare genetic diseases resulting from mutations in genes encoding structural proteins of the cartilage extracellular matrix (ECM), signaling molecules, transcription factors, epigenetic modifiers, and several intracellular proteins. Cell division, organelle maintenance, and intracellular transport are all orchestrated by the cytoskeleton-associated proteins, and intracellular processes affected through microtubule-associated movement are important for the function of skeletal cells. Among microtubule-associated motor proteins, kinesins in particular have been shown to play a key role in cell cycle dynamics, including chromosome segregation, mitotic spindle formation, and ciliogenesis, in addition to cargo trafficking, receptor recycling, and endocytosis. Recent studies highlight the fundamental role of kinesins in embryonic development and morphogenesis and have shown that mutations in kinesin genes lead to several skeletal dysplasias. However, many questions concerning the specific functions of kinesins and their adaptor molecules as well as specific molecular mechanisms in which the kinesin proteins are involved during skeletal development remain unanswered. Here we present a review of the skeletal dysplasias resulting from defects in kinesins and discuss the involvement of kinesin proteins in the molecular mechanisms that are active during skeletal development.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.