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Fibronectin (FN) is a ubiquitous extracellular matrix glycoprotein essential for the development of various tissues. Mutations in FN cause a unique form of spondylometaphyseal dysplasia, emphasizing its importance in cartilage and bone development. However, the relevance and functional role of FN during skeletal development has remained elusive. To address these aspects, we have generated conditional knockout mouse models targeting the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and both isoforms together in a double knockout (FNdKO). We used these mice to determine the relevance of the two principal FN isoforms in skeletal development from postnatal day one to the adult stage at two months. We identified a distinct topological FN deposition pattern in the mouse limb during different gestational and postnatal skeletal development phases, with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Cartilage-specific cFN emerged as the predominant isoform in the growth plate, whereas circulating pFN remained excluded from the growth plate and confined to the primary and secondary ossification centers. Deleting either isoform independently (cFNKO or pFNKO) yielded only relatively subtle changes in the analyzed skeletal parameters. However, the double knockout of cFN in the growth plate and pFN in the circulation of the FNdKO mice significantly reduced postnatal body weight, body length, and bone length. Micro-CT analysis of the adult bone microarchitecture in FNdKO mice exposed substantial reductions in trabecular bone parameters and bone mineral density. The mice also showed elevated bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice demonstrated changes in the resting, proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and X, decreased apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. Transforming growth factor-β1 and downstream phospho-AKT levels were significantly lower in the FNdKO than in the control mice, revealing a crucial FN-mediated regulatory pathway in chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for chondrogenesis and bone development. Even though cFN and pFN act in different regions of the bone, both FN isoforms are required for the regulation of chondrogenesis, cartilage maturation, trabecular bone formation, and overall skeletal growth., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier B.V.)

In this work, we review the multifaceted connections between osteosarcoma (OS) biology and normal bone development. We summarize and critically analyze existing research, highlighting key areas that merit further exploration. The review addresses several topics in OS biology and their interplay with normal bone development processes, including OS cell of origin, genomics, tumor microenvironment, and metastasis. We examine the potential cellular origins of OS and how their roles in normal bone growth may contribute to OS pathogenesis. We survey the genomic landscape of OS, highlighting the developmental roles of genes frequently altered in OS. We then discuss the OS microenvironment, emphasizing the transformation of the bone niche in OS to facilitate tumor growth and metastasis. The role of stromal and immune cells is examined, including their impact on tumor progression and therapeutic response. We further provide insights into potential development-informed opportunities for novel therapeutic strategies., (Copyright © 2024 Cold Spring Harbor Laboratory Press; all rights reserved.)

Background: Observational studies have shown that childhood obesity is associated with adult bone health but yield inconsistent results. We aimed to explore the potential causal association between body shape and skeletal development., Methods: We used two-sample Mendelian randomization (MR) to estimate causal relationships between body shape from birth to adulthood and skeletal phenotypes, with exposures including placental weight, birth weight, childhood obesity, BMI, lean mass, fat mass, waist circumference, and hip circumference. Independent genetic instruments associated with the exposures at the genome-wide significance level (P < 5 × 10 -8 ) were selected from corresponding large-scale genome-wide association studies. The inverse-variance weighted analysis was chosen as the primary method, and complementary MR analyses included the weighted median, MR-Egger, weighted mode, and simple mode., Results: The MR analysis shows strong evidence that childhood (β = -1.29 × 10 -3 , P = 8.61 × 10 -5 ) and adulthood BMI (β = -1.28 × 10 -3 , P = 1.45 × 10 -10 ) were associated with humerus length. Tibiofemoral angle was negatively associated with childhood BMI (β = -3.60 × 10 -1 , P = 3.00 × 10 -5 ) and adolescent BMI (β = -3.62 × 10 -1 , P = 2.68 × 10 -3 ). In addition, genetically predicted levels of appendicular lean mass (β = 1.16 × 10 -3 , P = 1.49 × 10 -13 ), whole body fat mass (β = 1.66 × 10 -3 , P = 1.35 × 10 -9 ), waist circumference (β = 1.72 × 10 -3 , P = 6.93 × 10 -8 ) and hip circumference (β =1.28 × 10 -3 , P = 4.34 × 10 -6 ) were all associated with tibia length. However, we found no causal association between placental weight, birth weight and bone length/width., Conclusions: This large-scale MR analysis explores changes in growth patterns in the length/width of major bone sites, highlighting the important role of childhood body shape in bone development and providing insights into factors that may drive bone maturation., Competing Interests: Declaration of competing interest All authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bone formation is tightly modulated by genetically encoded molecular proteins that interact to regulate cellular differentiation and secretion of bony matrix. Many transcription factors are known to coordinate these events by controlling gene transcription within networks. However, not all factors involved are known. Here, we identified a novel function for Zinc Finger Homeobox 3 (Zfhx3), a gene encoding a transcription factor, as a regulator of bone metabolism. We knocked out Zfhx3 conditionally in mice in either chondrocytes or osteoblasts and characterized their bones by micro-CT in 12-week-old mice. We observed a negative effect in linear bone growth in both knockout mice but reduced bone mass only in mice with Zfhx3 deleted in osteoblasts. Loss of Zfhx3 expression in osteoblasts affected trabecular bone mass in femurs and vertebrae in both sexes but influenced cortical bone volume fraction only in females. Moreover, transcriptional analysis of femoral bones in osteoblast Zfhx3 conditional knockout mice revealed a reduced expression of osteoblast genes, and histological evaluation of trabecular bones suggests that Zfhx3 causes changes in bone formation and not resorption. The loss of Zfhx3 causes reductions in trabecular bone area and osteoid volume, but no changes in the expression of osteoclast differentiation markers or number of TRAP stained osteoclasts. These studies introduce Zfhx3 as a relevant factor toward understanding gene regulatory networks that control bone formation and development of peak bone mass., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Splicing and endoplasmic reticulum (ER)-proteostasis are two key processes that ultimately regulate the functional proteins that are produced by a cell. However, the extent to which these processes interact remains poorly understood. Here, we identify SNRPB and other components of the Sm-ring, as targets of the unfolded protein response and novel regulators of export from the ER. Mechanistically, The Sm-ring regulates the splicing of components of the ER export machinery, including Sec16A, a component of ER exit sites. Loss of function of SNRPB is causally linked to cerebro-costo-mandibular syndrome (CCMS), a genetic disease characterized by bone defects. We show that heterozygous deletion of SNRPB in mice resulted in bone defects reminiscent of CCMS and that knockdown of SNRPB delays the trafficking of type-I collagen. Silencing SNRPB inhibited osteogenesis in vitro, which could be rescued by overexpression of Sec16A. This rescue indicates that the role of SNRPB in osteogenesis is linked to its effects on ER-export. Finally, we show that SNRPB is a target for the unfolded protein response, which supports a mechanistic link between the spliceosome and ER-proteostasis. Our work highlights components of the Sm-ring as a novel node in the proteostasis network, shedding light on CCMS pathophysiology., (© 2024. The Author(s).)

Intermuscular bones, which are present in numerous economically significant fish species, have a negative impact on the development of aquaculture. The Asb15b gene, primarily expressed in skeletal muscle, plays a crucial role in regulating protein turnover and the development of muscle fibers. It stimulates protein synthesis and controls the differentiation of muscle fibers. In this study, we employed CRISPR/Cas9 technology to generate homozygous zebrafish strains with 7 bp and 49 bp deletions in the Asb15b gene. Subsequent analyses using skeleton staining demonstrated a substantial reduction in the number of intermuscular bones in adult Asb15b -/- -7 bp and Asb15b -/- -49 bp mutants compared to the wild-type zebrafish, with decreases of 30 % (P < 0.001) and 40 % (P < 0.0001), respectively. Histological experiments further revealed that the diameter and number of muscle fibers in adult Asb15b -/- mutants did not exhibit significant changes when compared to wild-type zebrafish. Moreover, qRT-PCR experiments demonstrated significant differences in the expression of bmp6 and runx2b genes, which are key regulators of intermuscular bone development, during different stages of intermuscular bone development in Asb15b -/- mutants. This study strongly suggests that the Asb15b gene plays a crucial role in regulating intermuscular bone development in fish and lays the groundwork for further exploration of the role of the Asb15b gene in zebrafish intermuscular bone development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

Translation of mRNA to protein is tightly regulated by transfer RNAs (tRNAs), which are subject to various chemical modifications that maintain structure, stability, and function. Deficiency of tRNA N7-methylguanosine (m7G) modification in patients causes a type of primordial dwarfism, but the underlying mechanism remains unknown. Here we report that the loss of m7G rewires cellular metabolism, leading to the pathogenesis of primordial dwarfism. Conditional deletion of the catalytic enzyme Mettl1 or missense mutation of the scaffold protein Wdr4 severely impaired endochondral bone formation and bone mass accrual. Mechanistically, Mettl1 knockout decreased abundance of m7G-modified tRNAs and inhibited translation of mRNAs relating to cytoskeleton and Rho GTPase signaling. Meanwhile, Mettl1 knockout enhanced cellular energy metabolism despite incompetent proliferation and osteogenic commitment. Further exploration revealed that impairment of Rho GTPase signaling upregulated the level of branched-chain amino acid transaminase 1 (BCAT1) that rewired cell metabolism and restricted intracellular α-ketoglutarate (αKG). Supplementation of αKG ameliorated the skeletal defect of Mettl1-deficient mice. In addition to the selective translation of metabolism-related mRNAs, we further revealed that Mettl1 knockout globally regulated translation via integrated stress response (ISR) and mammalian target of rapamycin complex 1 (mTORC1) signaling. Restoring translation by targeting either ISR or mTORC1 aggravated bone defects of Mettl1-deficient mice. Overall, our study unveils a critical role of m7G tRNA modification in bone development by regulation of cellular metabolism and indicates suspension of translation initiation as a quality control mechanism in response to tRNA dysregulation.

This study investigated the differences in bone growth and turnover and calcium (Ca) and phosphorus (P) uptake among three different breeds of growing-finishing pigs. Ninety healthy Duroc, Xiangcun black (XCB), and Taoyuan black (TYB) pigs (30 pigs per breed) at 35 day-old (D) with the average body weight (BW) of their respective breed were assigned and raised to 185 D. The results showed that Duroc pigs had higher bone weight and length than the XCB and TYB pigs at 80, 125, and 185 D and the bone index at 185 D ( p  < 0.05). Duroc pigs had higher bone mineral densities (femur and tibia) compared with the other two breeds at 80 D and 125 D, whereas TYB pigs had higher mineral content and bone breaking load (rib) compared with the other two breeds at 185 D ( p  < 0.05). The bone morphogenetic protein-2 and osteocalcin concentrations were higher, and TRACP5b concentration was lower in serum of TYB pigs at 125 D ( p  < 0.05). Meanwhile, 1,25-dihydroxyvitamin D 3 , parathyroid hormone, thyroxine, and fibroblast growth factor 23 concentrations were higher in serum of TYB pigs at 185 D ( p  < 0.05). The TYB pigs had higher apparent total tract digestibility of P at 80 D and 185 D and bone Ca and P contents at 185 D in comparison to the Duroc pigs ( p  < 0.05). Furthermore, gene expressions related to renal uptake of Ca and P differed among the three breeds of pigs. Collectively, Duroc pigs have higher bone growth, whereas TYB pigs have a higher potential for mineral deposition caused by more active Ca uptake.

RNA-binding proteins (RBPs), which regulate gene expression through post-transcriptional modifications of RNAs, play a role in diverse biological processes that include bone cell development and bone tissue formation. RBP dysregulation may result in aberrant bone homeostasis and contribute to various bone diseases. The function of RBPs in bone physiology and pathophysiology and the underlying molecular mechanisms have been extensively studied in recent years. This article provides a review of such studies, highlighting the potential of RBPs as pivotal targets for therapeutic intervention.

Stem cells remain in a quiescent state for long-term maintenance and preservation of potency; this process requires fine-tuning regulatory mechanisms. In this study, we identified the epigenetic landscape along the developmental trajectory of skeletal stem cells (SSCs) in skeletogenesis governed by a key regulator, Ptip (also known as Paxip1, Pax interaction with transcription-activation domain protein-1). Our results showed that Ptip is required for maintaining the quiescence and potency of SSCs, and loss of Ptip in type II collagen (Col2) + progenitors causes abnormal activation and differentiation of SSCs, impaired growth plate morphogenesis, and long bone dysplasia. We also found that Ptip suppressed the glycolysis of SSCs through downregulation of phosphoglycerate kinase 1 (Pgk1) by repressing histone H3 lysine 27 acetylation (H3K27ac) at the promoter region. Notably, inhibition of glycolysis improved the function of SSCs despite Ptip deficiency. To the best of our knowledge, this is the first study to establish an epigenetic framework based on Ptip, which safeguards skeletal stem cell quiescence and potency through metabolic control. This framework is expected to improve SSC-based treatments of bone developmental disorders., (Copyright © 2024 Science China Press. Published by Elsevier B.V. All rights reserved.)

Family with sequence similarity 20 member C (FAM20C) is a Golgi casein kinase that phosphorylates extracellularly-secreted regulatory proteins involved in bone development and mineralization, but its specific role in bone development is still largely unknown. In this study, to examine the specific mechanisms that FAM20C influences bone development, we cross-bred Osx-Cre with FAM20C flox/flox mice to establish a Osx-Cre; FAM20C flox/flox knockout (oKO) mouse model; FAM20C was KO in pre-osteoblasts. oKO development was examined at 1-10 weeks, in which compared to control FAM20C flox/flox , they had lower body weights and bone tissue mineralization. Furthermore, oKO had lower bone volume fractions, thickness, and trabecular numbers, along with higher degrees of trabecular separation. These mice also had decreased femoral metaphyseal cartilage proliferation layer, along with thickened hypertrophic layer and increased apoptotic cell counts. Transcriptomic analysis found that differentially-expressed genes in oKO were concentrated in the osteoclast differentiation pathway, in line with increased osteoclast presence. Additionally, up-regulation of osteoclast-related, and down-regulation of osteogenesis-related genes, were identified, in which the most up-regulated genes were signal regulatory protein β-1 family (Sirpb1a-c) and mitogen-activated protein kinase 13. Overall, FAM20C KO in pre-osteoblasts leads to abnormal long bone development, likely due to subsequent up-regulation of osteoclast differentiation-associated genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The transcription factor SRY-related HMG box 9 (Sox9) is essential for chondrogenesis. Mutations in and around SOX9 cause campomelic dysplasia (CD) characterized by skeletal malformations. Although the function of Sox9 in this context is well studied, the mechanisms that regulate Sox9 expression in chondrocytes remain to be elucidated. Here, we have used genome-wide profiling to identify 2 Sox9 enhancers located in a proximal breakpoint cluster responsible for CD. Enhancer activity of E308 (located 308 kb 5' upstream) and E160 (located 160 kb 5' upstream) correlated with Sox9 expression levels, and both enhancers showed a synergistic effect in vitro. While single deletions in mice had no apparent effect, simultaneous deletion of both E308 and E160 caused a dwarf phenotype, concomitant with a reduction of Sox9 expression in chondrocytes. Moreover, bone morphogenetic protein 2-dependent chondrocyte differentiation of limb bud mesenchymal cells was severely attenuated in E308/E160 deletion mice. Finally, we found that an open chromatin region upstream of the Sox9 gene was reorganized in the E308/E160 deletion mice to partially compensate for the loss of E308 and E160. In conclusion, our findings reveal a mechanism of Sox9 gene regulation in chondrocytes that might aid in our understanding of the pathophysiology of skeletal disorders.

Chondrocyte differentiation controls skeleton development and stature. Here we provide a comprehensive map of chondrocyte-specific enhancers and show that they provide a mechanistic framework through which non-coding genetic variants can influence skeletal development and human stature. Working with fetal chondrocytes isolated from mice bearing a Col2a1 fluorescent regulatory sensor, we identify 780 genes and 2'704 putative enhancers specifically active in chondrocytes using a combination of RNA-seq, ATAC-seq and H3K27ac ChIP-seq. Most of these enhancers (74%) show pan-chondrogenic activity, with smaller populations being restricted to limb (18%) or trunk (8%) chondrocytes only. Notably, genetic variations overlapping these enhancers better explain height differences than those overlapping non-chondrogenic enhancers. Finally, targeted deletions of identified enhancers at the Fgfr3, Col2a1, Hhip and, Nkx3-2 loci confirm their role in regulating cognate genes. This enhancer map provides a framework for understanding how genes and non-coding variations influence bone development and diseases., (© 2024. The Author(s).)

Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as "junk DNA". Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies., (© 2024. The Author(s).)

Runt-related transcription factor (RUNX1) is a transcription factor closely involved in hematopoiesis. RUNX 1 gene mutation plays an essential pathogenic role in the initiation and development of hematological tumors, especially in acute myeloid leukemia. Recent studies have shown that RUNX1 is also involved in the regulation of bone development and the pathological progression of bone-related diseases. RUNX1 promotes the differentiation of mesenchymal stem cells into chondrocytes and osteoblasts and modulates the maturation and extracellular matrix formation of chondrocytes. The expression of RUNX1 in mesenchymal stem cells, chondrocytes, and osteoblasts is of great significance for maintaining normal bone development and the mass and quality of bones. RUNX1 also inhibits the differentiation and bone resorptive activities of osteoclasts, which may be influenced by sexual dimorphism. In addition, RUNX1 deficiency contributes to the pathogenesis of osteoarthritis, delayed fracture healing, and osteoporosis, which was revealed by the RUNX1 conditional knockout modeling in mice. However, the roles of RUNX1 in regulating the hypertrophic differentiation of chondrocytes, the sexual dimorphism of activities of osteoclasts, as well as bone loss in diabetes mellitus, senescence, infection, chronic inflammation, etc, are still not fully understood. This review provides a systematic summary of the research progress concerning RUNX1 in the field of bone biology, offering new ideas for using RUNX1 as a potential target for bone related diseases, especially osteoarthritis, delayed fracture healing, and osteoporosis., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Board of Journal of Sichuan University (Medical Sciences).)

Estrogen receptor α (ESR1) is involved in E2 signaling and plays a major role in postmenopausal bone loss. However, the molecular network underlying ESR1 has not been explored. We used systems genetics and bioinformatics to identify important genes associated with Esr1 in postmenopausal bone loss. We identified ~2300 Esr1-coexpressed genes in female BXD bone femur, functional analysis of which revealed 'osteoblast signaling' as the most enriched pathway. PPI network led to the identification of 25 'female bone candidates'. The gene-regulatory analysis revealed RUNX2 as a key TF. ANKRD1 and RUNX2 were significantly different between osteoporosis patients and healthy controls. Sp7, Col1a1 and Pth1r correlated with multiple femur bone phenotypes in BXD mice. miR-3121-3p targeted Csf1, Ankrd1, Sp7 and Runx2. β-estradiol treatment markedly increased the expression of these candidates in mouse osteoblast. Our study revealed that Esr1-correlated genes Ankrd1, Runx2, Csf1 and Sp7 may play important roles in female bone development., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Inc.)

Background: Presenilin 1 (PSEN1) is one of the genes linked to the prevalence of early onset Alzheimer's disease. In mice, inactivation of Psen1 leads to developmental defects, including vertebral malformation and neural development. However, little is known about the role of PSEN1 during the development in other species., Objective: To investigate the role of PSEN1 in vertebral development and the pathogenic mechanism of neurodegeneration using a pig model., Methods: CRISPR/Cas9 system was used to generate pigs with different mutations flanking exon 9 of PSEN1, including those with a deleted exon 9 (Δexon9). Vertebral malformations in PSEN1 mutant pigs were examined by X-ray, micro-CT and micro-MRI. Neuronal cells from the brains of PSEN1 mutant pigs were analyzed by immunoflourescence, followed by image analysis including morphometric evaluation via image J and 3D reconstruction., Results: Pigs with a PSEN1 null mutation (Δexon9-12) died shortly after birth and had significant axial skeletal defects, whereas pigs carrying at least one Δexon9 allele developed normally and remained healthy. Effects of the null mutation on abnormal skeletal development were also observed in fetuses at day 40 of gestation. Abnormal distribution of astrocytes and microglia in the brain was detected in two PSEN1 mutant pigs examined compared to age-matched control pigs. The founder pigs were bred to establish and age PSEN1ΔE9/+ pigs to study their relevance to clinical Alzheimer's diseases., Conclusions: PSEN1 has a critical role for normal vertebral development and PSEN1 mutant pigs serves as novel resources to study Alzheimer's disease.

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor activated canonically by numerous cytokines and other factors, with significant roles in immunity, immune diseases, and cancer. It has also been implicated in several human skeletal disorders, with loss-of-function (LOF) mutations associated with aberrant skeletal development. To gain further insights, two zebrafish STAT3 lines were investigated: a complete LOF knockout (KO) mutant and a partial LOF mutant with the transactivation domain truncated (ΔTAD). Consistent with other studies, the KO mutants were smaller, with reduced length in early embryos exacerbated by a decreased growth rate from 5 days postfertilization (dpf). They displayed skeletal deformities that approached 80% incidence by 30 dpf, with a significant reduction in early bone but not cartilage formation. Further analysis additionally identified considerable abrogation of caudal fin regeneration, concomitant with a paucity of infiltrating macrophages and neutrophils, which may be responsible for this. Most of these phenotypes were also observed in the ΔTAD mutants, indicating that loss of canonical STAT3 signaling was the likely cause. However, the impacts on early bone formation and regeneration were muted in the ΔTAD mutant, suggesting the potential involvement of noncanonical functions in these processes.

Primary cilia are non-motile, microtubule-based sensory organelle present in most vertebrate cells with a fundamental role in the modulation of organismal development, morphogenesis, and repair. Here we focus on the role of primary cilia in embryonic and postnatal skeletal development. We examine evidence supporting its involvement in physiochemical and developmental signaling that regulates proliferation, patterning, differentiation and homeostasis of osteoblasts, chondrocytes, and their progenitor cells in the skeleton. We discuss how signaling effectors in mechanotransduction and bone development, such as Hedgehog, Wnt, Fibroblast growth factor and second messenger pathways operate at least in part at the primary cilium. The relevance of primary cilia in bone formation and maintenance is underscored by a growing list of rare genetic skeletal ciliopathies. We collate these findings and summarize the current understanding of molecular factors and mechanisms governing primary ciliogenesis and ciliary function in skeletal development and disease., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Genetic variants of the transcription factor SIX1 and its co-factor EYA1 underlie 50% of Branchio-oto-renal syndrome (BOR) cases. BOR is characterized by craniofacial defects, including malformed middle ear ossicles leading to conductive hearing loss. In this work, we expand our knowledge of the Six1 gene regulatory network by using a Six1-null mouse line to assess gene expression profiles of E10.5 mandibular arches, which give rise to the neural crest (NC)-derived middle ear ossicles and lower jaw, via bulk RNA sequencing., Results: Our transcriptomic analysis led to the identification of 808 differentially expressed genes that are related to translation, NC cell differentiation, osteogenesis, and chondrogenesis including components of the WNT signaling pathway. As WNT signaling is a known contributor to bone development, we demonstrated that SIX1 is required for expression of the WNT antagonist Frzb in the mandibular arch, and determined that SIX1 expression results in repression of WNT signaling., Conclusion: Our results clarify the mechanisms by which SIX1 regulates the development of NC-derived craniofacial elements that are altered in SIX1-associated disorders. In addition, this work identifies novel genes that could be causative to this birth defect and establishes a link between SIX1 and WNT signaling during patterning of NC cells., (© 2023 American Association for Anatomy.)

This study is the first to investigate the process of osteoclast (OCL) differentiation, its potential functions, and the associated mRNA and signalling pathways in embryonic palatal bone. Our findings suggest that OCLs are involved in bone remodelling, bone marrow cavity formation, and blood vessel formation in embryonic palatal bone. We observed TRAP-positive OCLs at embryonic day 16.5 (E16.5), E17.5, and E18.5 at the palatal process of the palate (PPP) and posterior and anterior parts of the palatal process of the maxilla (PPMXP and PPMXA, respectively), with OCL differentiation starting 2 days prior to TRAP positivity. By comparing the key periods of OCL differentiation between PPMX and PPP (E14.5, E15.5, and E16.5) using RNA-seq data of the palates, we found that the PI3K-AKT and MAPK signalling pathways were sequentially enriched, which may play critical roles in OCL survival and differentiation. Csf1r, Tnfrsff11a, Ctsk, Fos, Tyrobp, Fcgr3, and Spi1 were significantly upregulated, while Pik3r3, Tgfbr1, and Mapk3k7 were significantly downregulated, in both PPMX and PPP. Interestingly, Tnfrsff11b was upregulated in PPMX but downregulated in PPP, which may regulate the timing of OCL appearance. These results contribute to the limited knowledge regarding mRNA-specific steps in OCL differentiation in the embryonic palatal bone., (© 2023. Springer Nature Limited.)

Endochondral ossification requires proper control of chondrocyte proliferation, differentiation, survival, and organization. Here we show that knockout of α-parvin, an integrin-associated focal adhesion protein, from murine limbs causes defects in endochondral ossification and dwarfism. The mutant long bones were shorter but wider, and the growth plates became disorganized, especially in the proliferative zone. With two-photon time-lapse imaging of bone explant culture, we provide direct evidence showing that α-parvin regulates chondrocyte rotation, a process essential for chondrocytes to form columnar structure. Furthermore, loss of α-parvin increased binucleation, elevated cell death, and caused dilation of the resting zones of mature growth plates. Single-cell RNA-seq analyses revealed alterations of transcriptome in all three zones (i.e., resting, proliferative, and hypertrophic zones) of the growth plates. Our results demonstrate a crucial role of α-parvin in long bone development and shed light on the cellular mechanism through which α-parvin regulates the longitudinal growth of long bones., (© 2023. West China School of Stomatology Sichuan University.)

Insulin-like growth factor-I (IGF-I) levels, which are reduced by age, and cortical bone dimensions are major determinants of fracture risk in elderly subjects. Inactivation of liver-derived circulating IGF-I results in reduced periosteal bone expansion in young and older mice. In mice with lifelong depletion of IGF-I in osteoblast lineage cells, the long bones display reduced cortical bone width. However, it has not previously been investigated whether inducible inactivation of IGF-I locally in bone in adult/old mice affects the bone phenotype. Adult tamoxifen-inducible inactivation of IGF-I using a CAGG-CreER mouse model (inducible IGF-IKO mice) substantially reduced IGF-I expression in bone (-55%) but not in liver. Serum IGF-I and body weight were unchanged. We used this inducible mouse model to assess the effect of local IGF-I on the skeleton in adult male mice, avoiding confounding developmental effects. After tamoxifen-induced inactivation of the IGF-I gene at 9 months of age, the skeletal phenotype was determined at 14 months of age. Computed tomography analyses of tibia revealed that the mid-diaphyseal cortical periosteal and endosteal circumferences and calculated bone strength parameters were decreased in inducible IGF-IKO mice compared with controls. Furthermore, 3-point bending showed reduced tibia cortical bone stiffness in inducible IGF-IKO mice. In contrast, the tibia and vertebral trabecular bone volume fraction was unchanged. In conclusion, inactivation of IGF-I in cortical bone with unchanged liver-derived IGF-I in older male mice resulted in reduced radial growth of cortical bone. This suggests that not only circulating IGF-I but also locally derived IGF-I regulates the cortical bone phenotype in older mice., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Introduction: Both the calvarial and the cortical bones develop through intramembranous ossification, yet they have very different structures and functions. The calvaria enables the rapid while protected growth of the brain, whereas the cortical bone takes part in locomotion. Both types of bones undergo extensive modeling during embryonic and post-natal growth, while bone remodeling is the most dominant process in adults. Their shared formation mechanism and their highly distinct functions raise the fundamental question of how similar or diverse the molecular pathways that act in each bone type are., Methods: To answer this question, we aimed to compare the transcriptomes of calvaria and cortices from 21-day old mice by bulk RNA-Seq analysis., Results: The results revealed clear differences in expression levels of genes related to bone pathologies, craniosynostosis, mechanical loading and bone-relevant signaling pathways like WNT and IHH, emphasizing the functional differences between these bones. We further discussed the less expected candidate genes and gene sets in the context of bone. Finally, we compared differences between juvenile and mature bone, highlighting commonalities and dissimilarities of gene expression between calvaria and cortices during post-natal bone growth and adult bone remodeling., Discussion: Altogether, this study revealed significant differences between the transcriptome of calvaria and cortical bones in juvenile female mice, highlighting the most important pathway mediators for the development and function of two different bone types that originate both through intramembranous ossification., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Janssen, Kalev-Altman, Shalit, Sela-Donenfeld and Monsonego-Ornan.)

The enthesis is a transitional tissue between tendon and bone that matures postnatally. The development and maturation of the enthesis involve cellular processes likened to an arrested growth plate. In this study, we explored the role of fibroblast growth factor 9 (Fgf9), a known regulator of chondrogenesis and vascularization during bone development, on the structure and function of the postnatal enthesis. First, we confirmed spatial expression of Fgf9 in the tendon and enthesis using in situ hybridization. We then used Cre-lox recombinase to conditionally knockout Fgf9 in mouse tendon and enthesis (Scx-Cre) and characterized enthesis morphology as well as mechanical properties in Fgf9 ScxCre and wild-type (WT) entheses. Fgf9 ScxCre mice had smaller calcaneal and humeral apophyses, thinner cortical bone at the attachment, increased cellularity, and reduced failure load in mature entheses compared to WT littermates. During postnatal development, we found reduced chondrocyte hypertrophy and disrupted type X collagen (Col X) in Fgf9 ScxCre entheses. These findings support that tendon-derived Fgf9 is important for functional development of the enthesis, including its postnatal mineralization. Our findings suggest the potential role of FGF signaling during enthesis development., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Growth differentiation factor 11 (GDF11) and GDF8 (MSTN) are closely related TGF-β family proteins that interact with nearly identical signaling receptors and antagonists. However, GDF11 appears to activate SMAD2/3 more potently than GDF8 in vitro and in vivo. The ligands possess divergent structural properties, whereby substituting unique GDF11 amino acids into GDF8 enhanced the activity of the resulting chimeric GDF8. We investigated potentially distinct endogenous activities of GDF11 and GDF8 in vivo by genetically modifying their mature signaling domains. Full recoding of GDF8 to that of GDF11 yielded mice lacking GDF8, with GDF11 levels ∼50-fold higher than normal, and exhibiting modestly decreased muscle mass, with no apparent negative impacts on health or survival. Substitution of two specific amino acids in the fingertip region of GDF11 with the corresponding GDF8 residues resulted in prenatal axial skeletal transformations, consistent with Gdf11 -deficient mice, without apparent perturbation of skeletal or cardiac muscle development or homeostasis. These experiments uncover distinctive features between the GDF11 and GDF8 mature domains in vivo and identify a specific requirement for GDF11 in early-stage skeletal development., (© 2023 Lian et al.)

Intermuscular bones (IBs), which are little, bony spicules in muscle, are embedded in lower teleosts' myosepta. Despite the importance of studying IB development in freshwater aquaculture species, the genes associated with IB development need to be further explored. In the present study, we identified four stages of IB development in barbel steed (Hemibarbus labeo), namely stage 1: IBs have not emerged, stage 2: a few small IBs have emerged in the tail, stage 3: longer IBs gradually emerged in the tail and stage 4: all of the IBs in the tail are mature and long, via Alizarin red staining. Subsequently, we used the HiseqXTen platform to sequence and de novo assemble the transcriptome of epaxial muscle (between 35th and 40th myomere) of barbel steed at 29 days (stage 1) and 42 days (stage 3) after hatching. A total of 190,814 unigenes were obtained with an average length and N50 of 648 bp and 1027 bp, respectively. We found 2174 differentially expressed genes (DEGs) between stages 1 and 3, of which 378 and 1796 were up- and down-regulated, respectively. Functional enrichment analysis showed that several DEGs functioned in ossification, positive regulation of osteoblast differentiation, osteoblast differentiation, and BMP signaling pathway, and were further enriched in signal pathway, including osteoclast differentiation, TGF-β signaling pathway, cytokine-cytokine receptor interaction, Jak-STAT signaling pathway, and other KEEG pathways. In conclusion, we identified genes that may be related to IB development, such as kazal type serine peptidase inhibitor domain 1 (KAZALD1), extracellular matrix protein 1 (ECM1), tetranectin, bone morphogenetic protein 1 (bmp1), acid phosphatase 5 (ACP5), collagen type XI alpha 1 chain (COL11A1), matrix metallopeptidase 9 (MMP9), pannexin-3 (PANX3), sp7 transcription factor (Sp7), and c-x-c motif chemokine ligand 8 (CXCL8), by comparing the transcriptomes of epaxial muscle before and after IB ossification. This study provided a theoretical basis for identifying the molecular mechanisms underlying IB development in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Skeletal shape and mechanical properties define, to a large extent, vertebrate morphology and physical capacities. During development, skeletal morphogenesis results from dynamic communications between chondrocytes, osteoblasts, osteoclasts, and other cellular components of the skeleton. Later in life, skeletal integrity depends on the regulatory cascades that assure the equilibrium between bone formation and resorption. Finally, during aging, skeletal catabolism prevails over anabolism resulting in progressive skeletal degradation. These cellular processes depend on the transcriptional cascades that control cell division and differentiation in each cell type. Most Distal-less ( Dlx ) homeobox transcription factors are directly involved in determining the proliferation and differentiation of chondrocytes and osteoblasts and, indirectly, of osteoclasts. While the involvement of Dlx genes in the regulation of skeletal formation has been well-analyzed thanks to several mutant mouse models, the role of these genes in the maintenance of bone integrity has been only partially studied. The importance of Dlx genes for adult bone tissues is evidenced by their central role in the regulatory pathways involving Osx/Sp 7 and Runx2 , the two major master genes of osteogenesis. Dlx genes appear to be involved in several bone pathologies including, for example, osteoporosis. Indeed, at least five large-scale GWAS studies which aimed to detect loci associated with human bone mineral density (BMD) have identified a known DLX5/6 regulatory region within chromosome 7q21.3 in proximity of SEM1/FLJ42280/DSS1 coding sequences, suggesting that DLX5/6 expression is critical in determining healthy BMD. This review aims to summarize the major findings concerning the involvement of Dlx genes in skeletal development and homeostasis and their involvement in skeletal aging and pathology.

Objectives: Idiopathic short stature (ISS), a disorder of unknown cause, accounts for approximately 80% of the clinical diagnoses of children with short stature. Exosomal circular RNA in plasma has been implicated in various disease processes. However, the role of exosome-derived circRNA in ISS has not been elucidated yet., Methods: Plasma exosomes of ISS and normal children were cocultured with human chondrocytes. Microarray analysis and RT-PCR identified the differential expression of circRNA in exosomes between ISS and normal children. Hsa&#95;circ&#95;0063476 was upregulated or downregulated in human chondrocytes. Subsequently, overexpression rats of hsa&#95;circ&#95;0063476 was constructed via adenoviral vector to further validate the role of hsa&#95;circ&#95;0063476 on longitudinal bone growth via in vivo experiment., Results: The plasma exosome of ISS children suppressed the expression of markers of chondrocyte hypertrophy and endochondral ossification. Subsequently, upregulation of hsa&#95;circ&#95;0063476 in ISS exosome was identified. In vitro experiments demonstrated that chondrocyte proliferation, cell cycle and endochondral ossification were suppressed, and apoptosis was increased following hsa&#95;circ&#95;0063476 overexpression in human chondrocytes. Conversely, silencing hsa&#95;circ&#95;0063476 in human chondrocytes can show opposite outcomes. Our study further revealed hsa&#95;circ&#95;0063476 overexpression in vitro can enhance chondrocyte apoptosis and inhibit the expression of markers of chondrocyte proliferation and endochondral ossification via miR-518c-3p/DDX6 axis. Additionally, the rats with hsa&#95;circ&#95;0063476 overexpression showed a short stature phenotype., Conclusions: The authors identified a novel pathogenesis in ISS that exosome-derived hsa&#95;circ&#95;0063476 retards the expression of markers of endochondral ossification and impairs longitudinal bone growth via miR-518c-3p/DDX6 axis, which may provide a unique therapeutic avenue for ISS., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Segmental overgrowth of the skeletal muscles with bone involvement in body extremities, predominantly affecting the upper limb, is an extremely rare condition with only 40-50 affected children described clinically. The molecular pathogenesis of this disorder remains largely unclear except for the identification of a somatic PIK3CA mutation in each of the six patients genetically tested, all restricted to upper limbs in the literature. This study aimed to further characterize the molecular defects for patients affected with segmental overgrowth of the skeletal muscles by analyzing a 9-gene panel selected from the PI3K/AKT/mTOR pathway and genes associated with other related conditions. Nineteen unrelated patients were chosen for this study, comprising ten upper limb (nine unilateral and one bilateral) and nine lower limb (eight unilateral and one bilateral) cases with variable bone involvement. In each case, an activating PIK3CA mutation (p.E110del, p.N345K, p.E542K, p.E545K, p.H1047R, or p.H1047L) was identified in the affected muscle tissue with variant allele frequencies ranging from 13.88 to 30.43%, while no mutation was detected in the paired peripheral blood sample, indicating somatic mosaicism. All detected mutations were limited to PIK3CA and were previously reported in other overgrowth syndromes currently categorized under the PIK3CA-Related Overgrowth Spectrum (PROS). Our study provides strong molecular evidence that isolated segmental overgrowth of the skeletal muscle with bone involvement is a subtype of PROS. Our findings expand the PROS clinical presentations with a newly molecularly classified condition and can provide guidance in clinical and molecular diagnosis and treatment for patients with this condition., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Mutation is the basis of phenotypic changes and serves as the source of natural selection. The development of limbs has been the milestone in vertebrate evolution. Several limb and bone-related genes were verified experimentally, but other indirect and regulatory factors of limb development remained untested, especially very few cases were observed in natural environment. We report a naturally born serpentized pig without hindlimbs. Whole genome sequencing followed by comparative genomic analysis revealed multiple interesting patterns on the handicapped pig-specific mutations. Although the bone-related genes are not directly subjected to mutations, other regulatory factors such as the RNA deaminase genes Adar are damaged in the handicapped pig, leading to the abolished A-to-I deamination in many functional, conserved genes as well as the bone-related genes. This is a precious case that the limbless phenotype is observed in naturally born non-model organisms. Our study broadened the generality of the limbless phenotype across mammals and extended the regulation of hindlimb development to other non-bone-related genes. Our knowledge of limb and bone-related mutations and regulation would also contribute to human genetics., (© 2021. Institute of Plant Genetics, Polish Academy of Sciences, Poznan.)

Background: The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9., Results: We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9 null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT., Conclusion: Taken together, we discovered that Fgf9 may play an influential role in muscle-bone cross-talk during embryonic and postnatal development., (© 2021 American Association for Anatomy.)

Skeletal deformities are typical AD-HIES manifestations, which are mainly caused by heterozygous and loss-of-function mutations in Signal transducer and activator of transcription 3 (STAT3). However, the mechanism is still unclear and the treatment strategy is limited. Herein, we reported that the mice with Stat3 deletion in osteoblasts, but not in osteoclasts, induced AD-HIES-like skeletal defects, including craniofacial malformation, osteoporosis, and spontaneous bone fracture. Mechanistic analyses revealed that STAT3 in cooperation with Msh homeobox 1(MSX1) drove osteoblast differentiation by promoting Distal-less homeobox 5(Dlx5) transcription. Furthermore, pharmacological activation of STAT3 partially rescued skeletal deformities in heterozygous knockout mice, while inhibition of STAT3 aggravated bone loss. Taken together, these data show that STAT3 is critical for modulating skeletal development and maintaining bone homeostasis through STAT3-indcued osteogenesis and suggest it may be a potential target for treatments., (© 2021. The Author(s).)

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group ( n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

We performed genome-wide association study meta-analysis to identify genetic determinants of skeletal age (SA) deviating in multiple growth disorders. The joint meta-analysis (N = 4557) in two multiethnic cohorts of school-aged children identified one locus, CYP11B1 (expression confined to the adrenal gland), robustly associated with SA (rs6471570-A; β = 0.14; P = 6.2 × 10 -12 ). rs6410 (a synonymous variant in the first exon of CYP11B1 in high LD with rs6471570), was prioritized for functional follow-up being second most significant and the one closest to the first intron-exon boundary. In 208 adrenal RNA-seq samples from GTEx, C-allele of rs6410 was associated with intron 3 retention (P = 8.11 × 10 -40 ), exon 4 inclusion (P = 4.29 × 10 -34 ), and decreased exon 3 and 5 splicing (P = 7.85 × 10 -43 ), replicated using RT-PCR in 15 adrenal samples. As CYP11B1 encodes 11-β-hydroxylase, involved in adrenal glucocorticoid and mineralocorticoid biosynthesis, our findings highlight the role of adrenal steroidogenesis in SA in healthy children, suggesting alternative splicing as a likely underlying mechanism., (© 2021. The Author(s).)

Different cellular mechanisms contribute to osteocyte development. And while critical roles for members of the zinc finger protein SNAI family (SNAIs) have been discussed in cancer-related models, there are few reviews summarizing their importance for chondrocyte-to-osteocyte development. To help fill this gap, we review the roles of SNAIs in the development of mature osteocytes from chondrocytes, including the regulation of chondro- and osteogenesis through different signaling pathways and in programmed cell death. We also discuss how epigenetic factors-including DNA methylation, histone methylation and acetylation, and noncoding RNAs-contribute differently to both chondrocyte and osteocyte development. To better grasp the important roles of SNAIs in bone development, we also review genotype-phenotype correlations in different animal models. We end with comments about the possible importance of the SNAI family in cartilage/bone development and the potential applications for therapeutic goals., (© 2021 New York Academy of Sciences.)

Bone remodeling is a dynamic process between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Disrupted bone remodeling is a key factor in postmenopausal osteoporosis, a metabolic disorder characterized by deteriorated bone microarchitecture and increased risk of fracture. Recent studies have shown that piwi-binding RNA (piRNA) is involved in the pathogenesis of certain diseases at the post-transcriptional level. Here, we analyzed piRNA-63049 (piR-63049), which may play an essential role in bone remodeling. The expression of piR-63049 significantly increased in both bone tissues and plasma of osteoporotic rats and postmenopausal osteoporotic patients. Overexpressing piR-63049 could inhibit the osteoblastogenesis of bone marrow stromal cells (BMSCs) while knocking down piR-63049 could promote the osteoblastogenesis of BMSCs through the Wnt2b/β-catenin signaling pathway. Moreover, knocking-down piR-63049 (piR-63049-antagonist) in vivo could attenuate the bone loss in ovariectomized rats by promoting bone formation. Taken together, the current study shows that piR-63049 inhibits bone formation through the Wnt2b/β-catenin signaling pathway. This novel piRNA may be a potential target to increase bone formation in bone loss disorders such as postmenopausal osteoporosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here, we identify SLC1A5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show SLC1A5 acts cell autonomously to regulate protein synthesis and osteoblast differentiation. SLC1A5 provides both glutamine and asparagine which are essential for osteoblast differentiation. Mechanistically, glutamine and to a lesser extent asparagine support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development., Competing Interests: DS, YY, LS, GZ, CK No competing interests declared, (© 2021, Sharma et al.)

Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlh f/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlh f/f mice from homozygous parents (Dmp1Cre.Pthlh f/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlh f/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlh f/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlh f/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlh f/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR)., (© 2021 American Society for Bone and Mineral Research (ASBMR).)

Osteoporosis is a systemic bone metabolism disease that often causes complications, such as fractures, and increases the risk of death. The nucleotide-binding oligomerization domain-like-receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates the maturation and secretion of Caspase-1 dependent proinflammatory cytokines interleukin (IL)-1β and IL-18, mediates inflammation, and induces pyroptosis. The chronic inflammatory microenvironment induced by aging or estrogen deficiency activates the NLRP3 inflammasome, promotes inflammatory factor production, and enhances the inflammatory response. We summarize the related research and demonstrate that the NLRP3 inflammasome plays a vital role in the pathogenesis of osteoporosis by affecting the differentiation of osteoblasts and osteoclasts. IL-1β and IL-18 can accelerate osteoclast differentiation by expanding inflammatory response, and can also inhibit the expression of osteogenic related proteins or transcription factors. In vivo and in vitro experiments showed that the overexpression of NLRP3 protein was closely related to aggravated bone resorption and osteogenesis deficiency. In addition, abnormal activation of NLRP3 inflammasome can not only produce inflammation, but also lead to pyroptosis and dysfunction of osteoblasts by upregulating the expression of Caspase-1 and gasdermin D (GSDMD). In conclusion, NLRP3 inflammasome overall not only accelerates bone resorption, but also inhibits bone formation, thus increasing the risk of osteoporosis. Thus, this review highlights the recent studies on the function of NLRP3 inflammasome in osteoporosis, provides information on new strategies for managing osteoporosis, and investigates the ideal therapeutic target to treat osteoporosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, An, Yang, Liu, Guan, Ma and Tang.)

Bone fragility is a pathological condition caused by altered homeostasis of the mineralized bone mass with deterioration of the microarchitecture of the bone tissue, which results in a reduction of bone strength and an increased risk of fracture, even in the absence of high-impact trauma. The most common cause of bone fragility is primary osteoporosis in the elderly. However, bone fragility can manifest at any age, within the context of a wide spectrum of congenital rare bone metabolic diseases in which the inherited genetic defect alters correct bone modeling and remodeling at different points and aspects of bone synthesis and/or bone resorption, leading to defective bone tissue highly prone to long bone bowing, stress fractures and pseudofractures, and/or fragility fractures. To date, over 100 different Mendelian-inherited metabolic bone disorders have been identified and included in the OMIM database, associated with germinal heterozygote, compound heterozygote, or homozygote mutations, affecting over 80 different genes involved in the regulation of bone and mineral metabolism. This manuscript reviews clinical bone phenotypes, and the associated bone fragility in rare congenital metabolic bone disorders, following a disease taxonomic classification based on deranged bone metabolic activity.

Signal transducer and activator of transcription 3 (STAT-3) gain-of-function (GOF) syndrome is an early-onset monogenic inborn error of immunity characterized by multi-organ autoimmune disorders, growth failure and lymphoproliferation. We describe that STAT-3 GOF syndrome may be presented with hypogammaglobulinemia and recurrent severe upper and lower respiratory tract infections. In addition, the patient had lymphoproliferation, short stature and interstitial lung disease. Chest computerized tomography examinations showed mild bronchiectasis with areas of non-fibrosing alveolar-interstitial disease and maldevelopment of bilateral first ribs. Using Sanger sequencing, we revealed a novel c.508G>C, p.D170H STAT-3 variant affecting the coiled coil domain of STAT-3. Functional studies confirmed that p.D170H was a GOF variant, as shown by increased phosphorylated STAT-3 (pSTAT-3) and STAT-3 transcriptional activity. Our observation suggests that STAT-3 GOF syndrome can manifest in early childhood with hypogammaglobulinemia and recurrent severe respiratory tract infections. We suggest that patients with lymphoproliferation, hypogammaglobulinemia and severe recurrent infections should be screened for STAT-3 variants, even if autoimmune manifestations are missing., (© 2021 The Authors. Clinical & Experimental Immunology published by John Wiley & Sons Ltd on behalf of British Society for Immunology.)

The adaptation and diversity of animals to the extreme environments of the Qinghai-Tibet Plateau (QTP) are typical materials to study adaptive evolution. The recently discovered Jinchuan yak population has many individuals with multiple ribs. However, little is known about this yak's origin, evolution, and the genetic mechanisms that formed its unique multirib trait. Here, we report a valuable population genome resource of the Jinchuan yak by resequencing the whole genome of 150 individuals. Population genetic polymorphism and structure analysis reveal that Jinchuan yak can be differentiated as a unique and original yak population among the domestic yak. Combined with geological change, the Jinchuan yak's evolutionary origin is speculated to be about 6290 years ago, which may be related to the unique geographical environment of the eastern edge of the QTP during this period. Compared with other domestic yaks, this new population has 280 positively selected genes. The genes related to skeletal function hold a considerable and remarkable proportion, suggesting that the specific skeletal characteristics have been enhanced in the adaptive evolution of Jinchuan yak in the extreme plateau environment. The genome-wide association study has revealed that TUBA8 and TUBA4A, the genes that regulate the cytoskeleton, are potential genes associated with the multirib trait. Our findings provide a basis to further understand the generation mechanism of the adaptive evolution of this new population in high-altitude extreme environments and the multivertebrate trait of domestic animals., (© 2020 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

We previously showed that conditional disruption of the Phd2 gene in chondrocytes led to a massive increase in long bone trabecular bone mass. Loss of Phd2 gene expression or inhibition of PHD2 activity by a specific inhibitor resulted in a several-fold compensatory increase in Phd3 expression in chondrocytes. To determine if expression of PHD3 plays a role in endochondral bone formation, we conditionally disrupted the Phd3 gene in chondrocytes by crossing Phd3 floxed ( Phd3 flox/flox ) mice with Col2α1-Cre mice. Loss of Phd3 expression in the chondrocytes of Cre + ; Phd3 flox/flox conditional knockout (cKO) mice was confirmed by real time PCR. At 16 weeks of age, neither body weight nor body length was significantly different in the Phd3 cKO mice compared to Cre - ; Phd3 flox/flox wild-type (WT) mice. Areal BMD measurements of total body as well as femur, tibia, and lumbar skeletal sites were not significantly different between the cKO and WT mice at 16 weeks of age. Micro-CT measurements revealed significant gender differences in the trabecular bone volume adjusted for tissue volume at the secondary spongiosa of the femur and the tibia for both genotypes, but no genotype difference was found for any of the trabecular bone measurements of either the femur or the tibia. Trabecular bone volume of distal femur epiphysis was not different between cKO and WT mice. Histology analyses revealed Phd3 cKO mice exhibited a comparable chondrocyte differentiation and proliferation, as evidenced by no changes in cartilage thickness and area in the cKO mice as compared to WT littermates. Consistent with the in vivo data, lentiviral shRNA-mediated knockdown of Phd3 expression in chondrocytes did not affect the expression of markers of chondrocyte differentiation ( Col2 , Col10 , Acan , Sox9 ). Our study found that Phd2 but not Phd3 expressed in chondrocytes regulates endochondral bone formation, and the compensatory increase in Phd3 expression in the chondrocytes of Phd2 cKO mice is not the cause for increased trabecular bone mass in Phd2 cKO mice.

The transcription factor Nkx3.2 (Bapx1) is an important chondrocyte maturation inhibitor. Previous Nkx3.2 knockdown and overexpression studies in non-mammalian gnathostomes have focused on its role in primary jaw joint development, while the function of this gene in broader skeletal development is not fully described. We generated a mutant allele of nkx3.2 in zebrafish with CRISPR/Cas9 and applied a range of techniques to characterize skeletal phenotypes at developmental stages from larva to adult, revealing loss of the jaw joint, fusions in bones of the occiput, morphological changes in the Weberian apparatus, and the loss or deformation of bony elements derived from basiventral cartilages of the vertebrae. Axial phenotypes are reminiscent of Nkx3.2 knockout in mammals, suggesting that the function of this gene in axial skeletal development is ancestral to osteichthyans. Our results highlight the broad role of nkx3.2 in zebrafish skeletal development and its context-specific functions in different skeletal elements., Competing Interests: The authors have declared that no competing interests exist.

Cortical bone structure is a crucial determinant of bone strength, yet for many years studies of novel genes and cell signalling pathways regulating bone strength have focused on the control of trabecular bone mass. Here we focus on mechanisms responsible for cortical bone development, growth, and degeneration, and describe some recently described genetic-driven modifications in humans and mice that reveal how these processes may be controlled. We start with embryonic osteogenesis of preliminary bone structures preceding the cortex and describe how this structure consolidates then matures to a dense, vascularised cortex containing an increasing proportion of lamellar bone. These processes include modelling-induced, and load-dependent, asymmetric cortical expansion, which enables the cortex's transition from a highly porous woven structure to a consolidated and thickened highly mineralised lamellar bone structure, infiltrated by vascular channels. Sex-specific differences emerge during this process. With aging, the process of consolidation reverses: cortical pores enlarge, leading to greater cortical porosity, trabecularisation and loss of bone strength. Each process requires co-ordination between bone formation, bone mineralisation, vascularisation, and bone resorption, with a need for locational-, spatial- and cell-specific signalling pathways to mediate this co-ordination. We will discuss these processes, and a number of cell-signalling pathways identified in both murine and human genetic studies to regulate cortical bone mass, including signalling through gp130, STAT3, PTHR1, WNT16, NOTCH, NOTUM and sFRP4., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Growth disorders in the orofacial bone development process may lead to orofacial deformities. The balance between bone matrix formation by mesenchymal lineage osteoblasts and bone resorption by osteoclasts is vital for orofacial bone development. Although the mechanisms of orofacial mesenchymal stem cells (OMSCs) in orofacial bone development have been studied intensively, the communication between OMSCs and osteoclasts remains largely unclear. Methods: We used a neural crest cell-specific knockout mouse model to investigate orofacial bone development in GATA-binding protein 4 (GATA4) morphants. We investigated the underlying mechanisms of OMSCs-derived exosomes (OMExos) on osteoclastogenesis and bone resorption activity in vitro . miRNAs were extracted from OMExos, and differences in miRNA abundances were determined using an Affymetrix miRNA array. Luciferase reporter assays were used to validate the binding between GATA4 and miR-206-3p in OMSCs and to confirm the putative binding of miR-206-3p and its target genes in OMSCs and osteoclasts. The regulatory mechanism of the GATA4-miR-206-3p axis in OMSC osteogenic differentiation and osteoclastogenesis was examined in vitro and in vivo . Results: Wnt1-Cre;Gata4 fl/fl mice (cKO) not only presented inhibited bone formation but also showed active bone resorption. Osteoclasts cocultured in vitro with cKO OMSCs presented an increased capacity for osteoclastogenesis, which was exosome-dependent. Affymetrix miRNA array analysis showed that miR-206-3p was downregulated in exosomes from shGATA4 OMSCs. Moreover, the transcriptional activity of miR-206-3p was directly regulated by GATA4 in OMSCs. We further demonstrated that miR-206-3p played a key role in the regulation of orofacial bone development by directly targeting bone morphogenetic protein-3 (Bmp3) and nuclear factor of activated T -cells, cytoplasmic 1 (NFATc1). OMExos and agomiR-206-3p enhanced bone mass in Wnt1-cre;Gata4 fl/fl mice by augmenting trabecular bone structure and decreasing osteoclast numbers. Conclusion: Our findings confirm that miR-206-3p is an important downstream factor of GATA4 that regulates the functions of OMSCs and osteoclasts. These results demonstrate the efficiency of OMExos and microRNA agomirs in promoting bone regeneration, which provide an ideal therapeutic tool for orofacial bone deformities in the future., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The molecular mechanism of bone metastasis in breast cancer is largely unknown. Herein, we aimed to identify the key genes and long non-coding RNAs (lncRNAs) related to the bone metastasis of breast cancer using a bioinformatics approach. We screened differentially expressed genes and lncRNAs between normal breast and breast cancer bone metastasis samples using the GSE66206 dataset from the Gene Expression Omnibus. We also constructed a differentially expressed lncRNA-mRNA interaction network and analyzed the node degrees to identify the driving genes. After finding potential pathogenic modules of breast cancer bone metastasis, we identified breast cancer bone metastasis-related modules and functional enrichment analysis of the genes and lncRNAs in the modules. Based on the above analysis, we constructed a differentially expressed lncRNA-mRNA network related to bone metastasis in breast cancer and identified core driver genes, including BNIP3 and the lncRNA RP11-317-J19.1. The role of core driver genes and lncRNAs in the network implies their biological functions in regulating bone development and remodeling. Thus, targeting the core driver genes and lncRNAs in the network may be a promising therapeutic strategy to manage bone metastasis.

Osteoporosis is an increasing burden on public health as the world-wide population ages and effective therapeutics are severely needed. Two pathways with high potential for osteoporosis treatment are the retinoic acid (RA) and endocannabinoid system (ECS) signaling pathways. We sought to elucidate the roles that these pathways play in bone development and maturation. Here, we use chemical treatments to modulate the RA and ECS pathways at distinct early, intermediate, and late times bone development in zebrafish. We further assessed osteoclast activity later in zebrafish and medaka. Finally, by combining sub-optimal doses of AR and ECS modulators, we show that enhancing RA signaling or reducing the ECS promote bone formation and decrease osteoclast abundance and activity. These data demonstrate that RA signaling and the ECS can be combined as sub-optimal doses to influence bone growth and may be key targets for potential therapeutics., (Copyright © 2021 Elsevier B.V. All rights reserved.)