Epigenetic mechanisms of osteoarthritis risk in human skeletal development.

The epigenome, including the methylation of cytosine bases at CG dinucleotides, is intrinsically linked to transcriptional regulation. The tight regulation of gene expression during skeletal development is essential, with ∼1/500 individuals born with skeletal abnormalities. Furthermore, increasing evidence is emerging to link age-associated complex genetic musculoskeletal diseases, including osteoarthritis (OA), to developmental factors including joint shape. Multiple studies have shown a functional role for DNA methylation in the genetic mechanisms of OA risk using articular cartilage samples taken from aged patients. Despite this, our knowledge of temporal changes to the methylome during human cartilage development has been limited. We quantified DNA methylation at ∼700,000 individual CpGs across the epigenome of developing human articular cartilage in 72 samples ranging from 7-21 post-conception weeks, a time period that includes cavitation of the developing knee joint. We identified significant changes in 8% of all CpGs, and >9400 developmental differentially methylated regions (dDMRs). The largest hypermethylated dDMRs mapped to transcriptional regulators of early skeletal patterning including MEIS1 and IRX1 . Conversely, the largest hypomethylated dDMRs mapped to genes encoding extracellular matrix proteins including SPON2 and TNXB and were enriched in chondrocyte enhancers. Significant correlations were identified between the expression of these genes and methylation within the hypomethylated dDMRs. We further identified 811 CpGs at which significant dimorphism was present between the male and female samples, with the majority (68%) being hypermethylated in female samples. Following imputation, we captured the genotype of these samples at >5 million variants and performed epigenome-wide methylation quantitative trait locus (mQTL) analysis. Colocalization analysis identified 26 loci at which genetic variants exhibited shared impacts upon methylation and OA genetic risk. This included loci which have been previously reported to harbour OA-mQTLs (including GDF5 and ALDH1A2 ), yet the majority (73%) were novel (including those mapping to CHST3, FGF1 and TEAD1 ). To our knowledge, this is the first extensive study of DNA methylation across human articular cartilage development. We identify considerable methylomic plasticity within the development of knee cartilage and report active epigenomic mediators of OA risk operating in prenatal joint tissues.

Activin A marks a novel progenitor cell population during fracture healing and reveals a therapeutic strategy.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-ß superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.

DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells.

Upon stimulation by extrinsic stimuli, stem cells initiate a programme that enables differentiation or self-renewal. Disruption of the stem state exit has catastrophic consequences for embryogenesis and can lead to cancer. While some elements of this stem state switch are known, major regulatory mechanisms remain unclear. Here we show that this switch involves a global increase in splicing efficiency coordinated by DNA methyltransferase 3α (DNMT3A), an enzyme typically involved in DNA methylation. Proper activation of murine and human embryonic and haematopoietic stem cells depends on messenger RNA processing, influenced by DNMT3A in response to stimuli. DNMT3A coordinates splicing through recruitment of the core spliceosome protein SF3B1 to RNA polymerase and mRNA. Importantly, the DNA methylation function of DNMT3A is not required and loss of DNMT3A leads to impaired splicing during stem cell turnover. Finally, we identify the spliceosome as a potential therapeutic target in DNMT3A-mutated leukaemias. Together, our results reveal a modality through which DNMT3A and the spliceosome govern exit from the stem state towards differentiation.

Molecular diagnosis and novel genes and phenotypes in a pediatric thoracic insufficiency cohort.

Thoracic insufficiency syndromes are a genetically and phenotypically heterogeneous group of disorders characterized by congenital abnormalities or progressive deformation of the chest wall and/or vertebrae that result in restrictive lung disease and compromised respiratory capacity. We performed whole exome sequencing on a cohort of 42 children with thoracic insufficiency to elucidate the underlying molecular etiologies of syndromic and non-syndromic thoracic insufficiency and predict extra-skeletal manifestations and disease progression. Molecular diagnosis was established in 24/42 probands (57%), with 18/24 (75%) probands having definitive diagnoses as defined by laboratory and clinical criteria and 6/24 (25%) probands having strong candidate genes. Gene identified in cohort patients most commonly encoded components of the primary cilium, connective tissue, and extracellular matrix. A novel association between KIF7 and USP9X variants and thoracic insufficiency was identified. We report and expand the genetic and phenotypic spectrum of a cohort of children with thoracic insufficiency, reinforce the prevalence of extra-skeletal manifestations in thoracic insufficiency syndromes, and expand the phenotype of KIF7 and USP9X-related disease to include thoracic insufficiency.

Impaired glucose metabolism underlies articular cartilage degeneration in osteoarthritis.

Osteoarthritis (OA) is the leading joint disease characterized by cartilage destruction and loss of mobility. Accumulating evidence indicates that the incidence and severity of OA increases with diabetes, implicating systemic glucose metabolism in joint health. However, a definitive link between cellular metabolism in articular cartilage and OA pathogenesis is not yet established. Here, we report that in mice surgically induced to develop knee OA through destabilization of medial meniscus (DMM), expression of the main glucose transporter Glut1 is notably reduced in joint cartilage. Inducible deletion of Glut1 specifically in the Prg4-expressing articular cartilage accelerates cartilage loss in DMM-induced OA. Conversely, forced expression of Glut1 protects against cartilage destruction following DMM. Moreover, in mice with type I diabetes, both Glut1 expression and the rate of glycolysis are diminished in the articular cartilage, and the diabetic mice exhibit more severe cartilage destruction than their nondiabetic counterparts following DMM. The results provide proof of concept that boosting glucose metabolism in articular chondrocytes may ameliorate cartilage degeneration in OA.

Primary Cilia Direct Murine Articular Cartilage Tidemark Patterning Through Hedgehog Signaling and Ambulatory Load.

Articular cartilage (AC) is essential for body movement but is highly susceptible to degenerative diseases and has poor self-repair capacity. To improve current subpar regenerative treatments, developmental mechanisms of AC should be clarified and, specifically, how its postnatal multizone organization is acquired. Primary cilia are cell surface organelles crucial for mammalian tissue morphogenesis. Although their importance for chondrocyte function is appreciated, their specific roles in postnatal AC morphogenesis remain unclear. To explore these mechanisms, we used a murine conditional loss-of-function approach (Ift88-flox) targeting joint-lineage progenitors (Gdf5Cre) and monitored postnatal knee AC development. Joint formation and growth up to juvenile stages were largely unaffected. However, mature AC (aged 2 months) exhibited disorganized extracellular matrix, decreased aggrecan and collagen II due to reduced gene expression (not increased catabolism), and marked reduction of AC modulus by 30%-50%. In addition, and unexpectedly, we discovered that tidemark patterning was severely disrupted, as was hedgehog signaling, and exhibited specificity based on regional load-bearing functions of AC. Interestingly, Prg4 expression was markedly increased in highly loaded sites in mutants. Together, our data provide evidence that primary cilia orchestrate postnatal AC morphogenesis including tidemark topography, zonal matrix composition, and ambulation load responses. © 2022 American Society for Bone and Mineral Research (ASBMR).

Hox11 expression characterizes developing zeugopod synovial joints and is coupled to postnatal articular cartilage morphogenesis into functional zones in mice.

Previous studies on mouse embryo limbs have established that interzone mesenchymal progenitor cells emerging at each prescribed joint site give rise to joint tissues over fetal time. These incipient tissues undergo structural maturation and morphogenesis postnatally, but underlying mechanisms of regulation remain unknown. Hox11 genes dictate overall zeugopod musculoskeletal patterning and skeletal element identities during development. Here we asked where these master regulators are expressed in developing limb joints and whether they are maintained during postnatal zeugopod joint morphogenesis. We found that Hoxa11 was predominantly expressed and restricted to incipient wrist and ankle joints in E13.5 mouse embryos, and became apparent in medial and central regions of knees by E14.5, though remaining continuously dormant in elbow joints. Closer examination revealed that Hoxa11 initially characterized interzone and neighboring cells and was then restricted to nascent articular cartilage, intra joint ligaments and structures such as meniscal horns over prenatal time. Postnatally, articular cartilage progresses from a nondescript cell-rich, matrix-poor tissue to a highly structured, thick, zonal and mechanically competent tissue with chondrocyte columns over time, most evident at sites such as the tibial plateau. Indeed, Hox11 expression (primarily Hoxa11) was intimately coupled to such morphogenetic processes and, in particular, to the topographical rearrangement of chondrocytes into columns within the intermediate and deep zones of tibial plateau that normally endures maximal mechanical loads. Revealingly, these expression patterns were maintained even at 6 months of age. In sum, our data indicate that Hox11 genes remain engaged well beyond embryonic synovial joint patterning and are specifically tied to postnatal articular cartilage morphogenesis into a zonal and resilient tissue. The data demonstrate that Hox11 genes characterize adult, terminally differentiated, articular chondrocytes and maintain region-specificity established in the embryo.

Premature Growth Plate Closure Caused by a Hedgehog Cancer Drug Is Preventable by Co-Administration of a Retinoid Antagonist in Mice.

The growth plates are key engines of skeletal development and growth and contain a top reserve zone followed by maturation zones of proliferating, prehypertrophic, and hypertrophic/mineralizing chondrocytes. Trauma or drug treatment of certain disorders can derange the growth plates and cause accelerated maturation and premature closure, one example being anti-hedgehog drugs such as LDE225 (Sonidegib) used against pediatric brain malignancies. Here we tested whether such acceleration and closure in LDE225-treated mice could be prevented by co-administration of a selective retinoid antagonist, based on previous studies showing that retinoid antagonists can slow down chondrocyte maturation rates. Treatment of juvenile mice with an experimental dose of LDE225 for 2 days (100 mg/kg by gavage) initially caused a significant shortening of long bone growth plates, with concomitant decreases in chondrocyte proliferation; expression of Indian hedgehog, Sox9, and other key genes; and surprisingly, the number of reserve progenitors. Growth plate involution followed with time, leading to impaired long bone lengthening. Mechanistically, LDE225 treatment markedly decreased the expression of retinoid catabolic enzyme Cyp26b1 within growth plate, whereas it increased and broadened the expression of retinoid synthesizing enzyme Raldh3, thus subverting normal homeostatic retinoid circuitries and in turn accelerating maturation and closure. All such severe skeletal and molecular changes were prevented when LDE-treated mice were co-administered the selective retinoid antagonist CD2665 (1.5 mg/kg/d), a drug targeting retinoid acid receptor γ, which is most abundantly expressed in growth plate. When given alone, CD2665 elicited the expected maturation delay and growth plate expansion. In vitro data showed that LDE225 acted directly to dampen chondrogenic phenotypic expression, a response fully reversed by CD2665 co-treatment. In sum, our proof-of-principle data indicate that drug-induced premature growth plate closures can be prevented or delayed by targeting a separate phenotypic regulatory mechanism in chondrocytes. The translation applicability of the findings remains to be studied. © 2021 American Society for Bone and Mineral Research (ASBMR).

Activin A promotes the development of acquired heterotopic ossification and is an effective target for disease attenuation in mice.

Heterotopic ossification (HO) is a common, potentially debilitating pathology that is instigated by inflammation caused by tissue damage or other insults, which is followed by chondrogenesis, osteogenesis, and extraskeletal bone accumulation. Current remedies are not very effective and have side effects, including the risk of triggering additional HO. The TGF-ß family member activin A is produced by activated macrophages and other inflammatory cells and stimulates the intracellular effectors SMAD2 and SMAD3 (SMAD2/3). Because HO starts with inflammation and because SMAD2/3 activation is chondrogenic, we tested whether activin A stimulated HO development. Using mouse models of acquired intramuscular and subdermal HO, we found that blockage of endogenous activin A by a systemically administered neutralizing antibody reduced HO development and bone accumulation. Single-cell RNA-seq analysis and developmental trajectories showed that the antibody treatment reduced the recruitment of Sox9+ skeletal progenitors, many of which also expressed the gene encoding activin A (Inhba), to HO sites. Gain-of-function assays showed that activin A enhanced the chondrogenic differentiation of progenitor cells through SMAD2/3 signaling, and inclusion of activin A in HO-inducing implants enhanced HO development in vivo. Together, our data reveal that activin A is a critical upstream signaling stimulator of acquired HO in mice and could represent an effective therapeutic target against forms of this pathology in patients.

SOX9 keeps growth plates and articular cartilage healthy by inhibiting chondrocyte dedifferentiation/osteoblastic redifferentiation.

Cartilage is essential throughout vertebrate life. It starts developing in embryos when osteochondroprogenitor cells commit to chondrogenesis, activate a pancartilaginous program to form cartilaginous skeletal primordia, and also embrace a growth-plate program to drive skeletal growth or an articular program to build permanent joint cartilage. Various forms of cartilage malformation and degeneration diseases afflict humans, but underlying mechanisms are still incompletely understood and treatment options suboptimal. The transcription factor SOX9 is required for embryonic chondrogenesis, but its postnatal roles remain unclear, despite evidence that it is down-regulated in osteoarthritis and heterozygously inactivated in campomelic dysplasia, a severe skeletal dysplasia characterized postnatally by small stature and kyphoscoliosis. Using conditional knockout mice and high-throughput sequencing assays, we show here that SOX9 is required postnatally to prevent growth-plate closure and preosteoarthritic deterioration of articular cartilage. Its deficiency prompts growth-plate chondrocytes at all stages to swiftly reach a terminal/dedifferentiated stage marked by expression of chondrocyte-specific (Mgp) and progenitor-specific (Nt5e and Sox4) genes. Up-regulation of osteogenic genes (Runx2, Sp7, and Postn) and overt osteoblastogenesis quickly ensue. SOX9 deficiency does not perturb the articular program, except in load-bearing regions, where it also provokes chondrocyte-to-osteoblast conversion via a progenitor stage. Pathway analyses support roles for SOX9 in controlling TGFß and BMP signaling activities during this cell lineage transition. Altogether, these findings deepen our current understanding of the cellular and molecular mechanisms that specifically ensure lifelong growth-plate and articular cartilage vigor by identifying osteogenic plasticity of growth-plate and articular chondrocytes and a SOX9-countered chondrocyte dedifferentiation/osteoblast redifferentiation process.

Joints in the appendicular skeleton: Developmental mechanisms and evolutionary influences.

The joints are a diverse group of skeletal structures, and their genesis, morphogenesis, and acquisition of specialized tissues have intrigued biologists for decades. Here we review past and recent studies on important aspects of joint development, including the roles of the interzone and morphogenesis of articular cartilage. Studies have documented the requirement of interzone cells in limb joint initiation and formation of most, if not all, joint tissues. We highlight these studies and also report more detailed interzone dissection experiments in chick embryos. Articular cartilage has always received special attention owing to its complex architecture and phenotype and its importance in long-term joint function. We pay particular attention to mechanisms by which neonatal articular cartilage grows and thickens over time and eventually acquires its multi-zone structure and becomes mechanically fit in adults. These and other studies are placed in the context of evolutionary biology, specifically regarding the dramatic changes in limb joint organization during transition from aquatic to land life. We describe previous studies, and include new data, on the knee joints of aquatic axolotls that unlike those in higher vertebrates, are not cavitated, are filled with rigid fibrous tissues and resemble amphiarthroses. We show that when axolotls metamorph to life on land, their intra-knee fibrous tissue becomes sparse and seemingly more flexible and the articular cartilage becomes distinct and acquires a tidemark. In sum, there have been considerable advances toward a better understanding of limb joint development, biological responsiveness, and evolutionary influences, though much remains unclear. Future progress in these fields should also lead to creation of new developmental biology-based tools to repair and regenerate joint tissues in acute and chronic conditions.

DNMT3A and TET1 cooperate to regulate promoter epigenetic landscapes in mouse embryonic stem cells.

BACKGROUND: DNA methylation is a heritable epigenetic mark, enabling stable but reversible gene repression. In mammalian cells, DNA methyltransferases (DNMTs) are responsible for modifying cytosine to 5-methylcytosine (5mC), which can be further oxidized by the TET dioxygenases to ultimately cause DNA demethylation. However, the genome-wide cooperation and functions of these two families of proteins, especially at large under-methylated regions, called canyons, remain largely unknown. RESULTS: Here we demonstrate that DNMT3A and TET1 function in a complementary and competitive manner in mouse embryonic stem cells to mediate proper epigenetic landscapes and gene expression. The longer isoform of DNMT3A, DNMT3A1, exhibits significant enrichment at distal promoters and canyon edges, but is excluded from proximal promoters and canyons where TET1 shows prominent binding. Deletion of Tet1 increases DNMT3A1 binding capacity at and around genes with wild-type TET1 binding. However, deletion of Dnmt3a has a minor effect on TET1 binding on chromatin, indicating that TET1 may limit DNA methylation partially by protecting its targets from DNMT3A and establishing boundaries for DNA methylation. Local CpG density may determine their complementary binding patterns and therefore that the methylation landscape is encoded in the DNA sequence. Furthermore, DNMT3A and TET1 impact histone modifications which in turn regulate gene expression. In particular, they regulate Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 enrichment to constrain gene expression from bivalent promoters. CONCLUSIONS: We conclude that DNMT3A and TET1 regulate the epigenome and gene expression at specific targets via their functional interplay.

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

Hox11 Function Is Required for Region-Specific Fracture Repair.

The processes that govern fracture repair rely on many mechanisms that recapitulate embryonic skeletal development. Hox genes are transcription factors that perform critical patterning functions in regional domains along the axial and limb skeleton during development. Much less is known about roles for these genes in the adult skeleton. We recently reported that Hox11 genes, which function in zeugopod development (radius/ulna and tibia/fibula), are also expressed in the adult zeugopod skeleton exclusively in PDGFRα+/CD51+/LepR+ mesenchymal stem/stromal cells (MSCs). In this study, we use a Hoxa11eGFP reporter allele and loss-of-function Hox11 alleles, and we show that Hox11 expression expands after zeugopod fracture injury, and that loss of Hox11 function results in defects in endochondral ossification and in the bone remodeling phase of repair. In Hox11 compound mutant fractures, early chondrocytes are specified but show defects in differentiation, leading to an overall deficit in the cartilage production. In the later stages of the repair process, the hard callus remains incompletely remodeled in mutants due, at least in part, to abnormal bone matrix organization. Overall, our data supports multiple roles for Hox11 genes following fracture injury in the adult skeleton. © 2017 American Society for Bone and Mineral Research.

Hox genes in the adult skeleton: Novel functions beyond embryonic development.

Hox genes encode evolutionarily conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in regionally restricted domains and function to regulate the morphology of specific vertebral and long bone elements. Recent work has provided evidence that Hox genes continue to be regionally expressed in adult tissues. Fibroblasts cultured from adult tissues show broadly maintained Hox gene expression patterns. In the adult skeleton, Hox genes are expressed in progenitor-enriched populations of mesenchymal stem/stromal cells (MSCs), and genetic loss-of-function analyses have provided evidence that Hox genes function during the fracture healing process. This review will highlight our current understanding of Hox expression in the adult animal and its function in skeletal regeneration. Developmental Dynamics 246:310-317, 2017. © 2016 Wiley Periodicals, Inc.

Regionally Restricted Hox Function in Adult Bone Marrow Multipotent Mesenchymal Stem/Stromal Cells.

Posterior Hox genes (Hox9-13) are critical for patterning the limb skeleton along the proximodistal axis during embryonic development. Here we show that Hox11 paralogous genes, which developmentally pattern the zeugopod (radius/ulna and tibia/fibula), remain regionally expressed in the adult skeleton. Using Hoxa11EGFP reporter mice, we demonstrate expression exclusively in multipotent mesenchymal stromal cells (MSCs) in the bone marrow of the adult zeugopod. Hox-positive cells express PDGFRα and CD51, are marked by LepR-Cre, and exhibit colony-forming unit fibroblast activity and tri-lineage differentiation in vitro. Loss of Hox11 function leads to fracture repair defects, including reduced cartilage formation and delayed ossification. Hox mutant cells are defective in osteoblastic and chondrogenic differentiation in tri-lineage differentiation experiments, and these defects are zeugopod specific. In the stylopod (humerus and femur) and sternum, bone marrow MSCs express other regionally restricted Hox genes, and femur fractures heal normally in Hox11 mutants. Together, our data support regional Hox expression and function in skeletal MSCs.

Inducible cassette exchange: a rapid and efficient system enabling conditional gene expression in embryonic stem and primary cells.

Genetic modification is critically enabling for studies addressing specification and maintenance of cell fate; however, methods for engineering modifications are inefficient. We demonstrate a rapid and efficient recombination system in which an inducible, floxed cre allele replaces itself with an incoming transgene. We target this inducible cassette exchange (ICE) allele to the (HPRT) locus and demonstrate recombination in murine embryonic stem cells (ESCs) and primary cells from derivative ICE mice. Using lentivectors, we demonstrate recombination at a randomly integrated ICE locus in human ESCs. To illustrate the utility of this system, we insert the myogenic regulator, Myf5, into the ICE locus in each platform. This enables efficient directed differentiation of mouse and human ESCs into skeletal muscle and conditional myogenic transdetermination of primary cells cultured in vitro. This versatile tool is thus well suited to gain-of-function studies probing gene function in the specification and reprogramming of cell fate.

HoxA3 is an apical regulator of haemogenic endothelium.

During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.