Zebrafish model for spondylo-megaepiphyseal-metaphyseal dysplasia reveals post-embryonic roles of Nkx3.2 in the skeleton.

The regulated expansion of chondrocytes within growth plates and joints ensures proper skeletal development through adulthood. Mutations in the transcription factor NKX3.2 underlie spondylo-megaepiphyseal-metaphyseal dysplasia (SMMD), which is characterized by skeletal defects including scoliosis, large epiphyses, wide growth plates and supernumerary distal limb joints. Whereas nkx3.2 knockdown zebrafish and mouse Nkx3.2 mutants display embryonic lethal jaw joint fusions and skeletal reductions, respectively, they lack the skeletal overgrowth seen in SMMD patients. Here, we report adult viable nkx3.2 mutant zebrafish displaying cartilage overgrowth in place of a missing jaw joint, as well as severe dysmorphologies of the facial skeleton, skullcap and spine. In contrast, cartilage overgrowth and scoliosis are absent in rare viable nkx3.2 knockdown animals that lack jaw joints, supporting post-embryonic roles for Nkx3.2. Single-cell RNA-sequencing and in vivo validation reveal increased proliferation and upregulation of stress-induced pathways, including prostaglandin synthases, in mutant chondrocytes. By generating a zebrafish model for the skeletal overgrowth defects of SMMD, we reveal post-embryonic roles for Nkx3.2 in dampening proliferation and buffering the stress response in joint-associated chondrocytes.

The RNA helicase DDX3 induces neural crest by promoting AKT activity.

Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many individuals carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3ß, leading to reduced levels of ß-catenin and Snai1: two GSK3ß substrates that are crucial for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by individuals harboring mutations in DDX3 and its downstream effectors in this signaling cascade.

Aggrecan, the Primary Weight-Bearing Cartilage Proteoglycan, Has Context-Dependent, Cell-Directive Properties in Embryonic Development and Neurogenesis: Aggrecan Glycan Side Chain Modifications Convey Interactive Biodiversity.

This review examines aggrecan's roles in developmental embryonic tissues, in tissues undergoing morphogenetic transition and in mature weight-bearing tissues. Aggrecan is a remarkably versatile and capable proteoglycan (PG) with diverse tissue context-dependent functional attributes beyond its established role as a weight-bearing PG. The aggrecan core protein provides a template which can be variably decorated with a number of glycosaminoglycan (GAG) side chains including keratan sulphate (KS), human natural killer trisaccharide (HNK-1) and chondroitin sulphate (CS). These convey unique tissue-specific functional properties in water imbibition, space-filling, matrix stabilisation or embryonic cellular regulation. Aggrecan also interacts with morphogens and growth factors directing tissue morphogenesis, remodelling and metaplasia. HNK-1 aggrecan glycoforms direct neural crest cell migration in embryonic development and is neuroprotective in perineuronal nets in the brain. The ability of the aggrecan core protein to assemble CS and KS chains at high density equips cartilage aggrecan with its well-known water-imbibing and weight-bearing properties. The importance of specific arrangements of GAG chains on aggrecan in all its forms is also a primary morphogenetic functional determinant providing aggrecan with unique tissue context dependent regulatory properties. The versatility displayed by aggrecan in biodiverse contexts is a function of its GAG side chains.

EFTUD2 gene deficiency disrupts osteoblast maturation and inhibits chondrocyte differentiation via activation of the p53 signaling pathway.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM) is characteristic of multiple skeletal anomalies comprising craniofacial anomalies/dysplasia, microcephaly, dysplastic ears, choanal atresia, and short stature. Heterozygous loss of function variants of EFTUD2 was previously reported in MFDM; however, the mechanism underlying EFTUD2-associated skeletal dysplasia remains unclear. RESULTS: We identified a novel frameshift variant of EFTUD2 (c.1030_1031delTG, p.Trp344fs*2) in an MFDM Chinese patient with craniofacial dysmorphism including ear canal structures and microcephaly, mild intellectual disability, and developmental delay. We generated a zebrafish model of eftud2 deficiency, and a consistent phenotype consisting of mandibular bone dysplasia and otolith loss was observed. We also showed that EFTUD2 deficiency significantly inhibited proliferation, differentiation, and maturation in human calvarial osteoblast (HCO) and human articular chondrocyte (HC-a) cells. RNA-Seq analysis uncovered activated TP53 signaling with increased phosphorylation of the TP53 protein and upregulation of five TP53 downstream target genes (FAS, STEAP3, CASP3, P21, and SESN1) both in HCO and in eftud2-/- zebrafish. Additionally, inhibition of p53 by morpholino significantly reduced the mortality of eftud2-/- larvae. CONCLUSIONS: Our results confirm a novel de novo variant of the EFTUD2 gene and suggest that EFTUD2 may participate in the maturation and differentiation of osteoblasts and chondrocytes, possibly via activation of the TP53 signaling pathway. Thus, mutations in this gene may lead to skeletal anomalies in vertebrates.

Cellular Invasion and Matrix Degradation, a Different Type of Matrix-Degrading Cells in the Cartilage of Catfish (Clarias gariepinus) and Japanese Quail Embryos (Coturnix coturnix japonica).

We previously studied the phenomena of the mesenchymal cell-dependent mode of cartilage growth in quail and catfish. Thus, we selected the two cartilage models in which mesenchymal cells participate in their growth. In such models, cartilage degradation occurred to facilitate cellular invasion. The studies do not explain the nature of the cartilage degrading cells. The current study aims to explore the nature of the cartilage-degrading cells using transmission electron microscopy (TEM) and immunohistochemistry. Samples of cartilage have been isolated from the air-breathing organ of catfish and the cartilage of the prospective occipital bone of quail embryos. Samples have been processed for TEM and immunohistochemistry. We found that two different cell types are involved in cartilage degradation; the macrophage in the cartilage of catfish and mesenchymal cells in the cartilage of the quail. Areas of cellular invasion in both catfish cartilage and quail embryo cartilage had an immunological affinity for MMP-9. In catfish, cartilage-degrading cells had identical morphological features of macrophages, whereas in quail embryos, cartilage-degrading cells were mesenchymal-like cells which had cell processes rich in vesicles and expressed CD117. Further study should consider the role of macrophage and mesenchymal cells during cartilage degradation. This could be valuable to be applied to remove the defective cartilage matrix formed in osteoarthritic patients to improve cartilage repair strategies.

Structural Features of Heparan Sulfate from Multiple Osteochondromas and Chondrosarcomas.

Multiple osteochondromas (MO) is a hereditary disorder associated with benign cartilaginous tumors, known to be characterized by absence or highly reduced amount of heparan sulfate (HS) in the extracellular matrix of growth plate cartilage, which alters proper signaling networks leading to improper bone growth. Although recent studies demonstrated accumulation of HS in the cytoplasm of MO chondrocytes, nothing is known on the structural alterations which prevent HS from undergoing its physiologic pathway. In this work, osteochondroma (OC), peripheral chondrosarcoma, and healthy cartilaginous human samples were processed following a procedure previously set up to structurally characterize and compare HS from pathologic and physiologic conditions, and to examine the phenotypic differences that arise in the presence of either exostosin 1 or 2 (EXT1 or EXT2) mutations. Our data suggest that HS chains from OCs are prevalently below 10 kDa and slightly more sulfated than healthy ones, whereas HS chains from peripheral chondrosarcomas (PCSs) are mostly higher than 10 kDa and remarkably more sulfated than all the other samples. Although deeper investigation is still necessary, the approach here applied pointed out, for the first time, structural differences among OC, PCS, and healthy HS chains extracted from human cartilaginous excisions, and could help in understanding how the structural features of HS are modulated in the presence of pathological situations also involving different tissues.

The ribosome biogenesis protein Esf1 is essential for pharyngeal cartilage formation in zebrafish.

Craniofacial malformations are common congenital birth defects and usually caused by abnormal development of the cranial neural crest cells. Some nucleolar ribosome biogenesis factors are implicated in neural crest disorders also known as neurocristopathies. However, the underlying mechanisms linking ribosome biogenesis and neural crest cell (NCC) development remain to be elucidated. Here we report a novel zebrafish model with a CRISPR/Cas9-generated esf1 mutation, which exhibits severe NCC-derived pharyngeal cartilage loss and defects in the eyes, brain, and heart. The expression of several typical NCC markers, including sox10, dlx2a, nrp2b, crestin, vgll2a, and sox9a, was reduced in the head of the esf1 mutants, which indicates that esf1 plays a role in the development of zebrafish NCCs. We demonstrate that, similar to the yeast, loss of esf1 in zebrafish leads to defects in 18S rRNA biogenesis and ribosome biogenesis. We also show strong upregulation of p53 signaling as well as apoptosis, and poor proliferation in mutants. Inactivation of p53 rescues the early tissue defects and pharyngeal cartilage loss observed in esf1 mutants, indicating that increased cell death and pharyngeal cartilage defects observed in esf1 mutants are mediated via upregulated p53 signaling pathways. Based on transplantation analysis, we found esf1 functions in NCC in a cell autonomous fashion. Together, our results suggest that esf1 is required for NCC development and pharyngeal cartilage formation. These studies provide a potential model for investigating the relationship between ribosome biogenesis defects and craniofacial neurocristopathies.

Effects of calcium and estrogen on the development of the ceratohyal cartilage in zebrafish (Danio rerio) larvae upon embryo and maternal cadmium exposure.

The present study is to investigate the reason why the ceratohyal cartilage (CH) angle of zebrafish larvae were larger compared to the control group after their female parents were treated with cadmium (F-Cd). However, the CH angle was smaller compared to the control group when embryos were directly exposed to Cd2+ for 72 h (D-Cd). Results showed that calcium contents of larvae were lower than the control, but the transporter isoforms trpv4 and trpv6 mRNA expressions were significantly increased upon D-Cd treatment. Furthermore, external Ca2+ added during D-Cd treatment reveals that the CH angles of larvae did not appear significantly different compared to the control. On the other hand, E2 (17ß-estradiol) contents were higher around 1.9 folds in the ovaries of females; CH angle were over 25°, and Cd2+ contents were higher around 6 folds than the control group on larvae treated through F-Cd treatment; CH angles and E2 levels on larvae were higher than the control after the larvae were treated with 1.84 µM E2 (D-E2); Estradiol receptor (ER) isoforms ERß1 and ERα mRNA expressions significantly increased when 0 hpf embryos were either treated with D-E2 or D-Cd. According to the results, we suggested that the CH angle of larvae become larger upon F-Cd treatment due to maternal Cd2+ inducing E2 levels. However, the CH angle of larvae appeared to be smaller compared to the control upon D-Cd treatment. We suggested that the CH angle decreased due to the decrease of Ca2+ contents upon Cd2+ exposure.

Msx2 Marks Spatially Restricted Populations of Mesenchymal Precursors.

Craniofacial development requires a set of patterning codes that define the identities of postmigratory mesenchymal cells in a region-specific manner, in which locally expressed morphogens, including fibroblast growth factors (FGFs) and bone morphogenetic proteins (BMPs), provide instructive cues. Msx2, a bona fide target of BMP signaling, is a transcription factor regulating Runx2 and osterix (Osx), whose mutations are associated with cranial deformities in humans. Here we show that Msx2 defines osteo-chondro precursor cells in specific regions of the craniofacial mesenchyme at the postmigratory stage, particularly in the mandibular process and the posterior cranial vault. Analysis of Msx2-creER mice revealed that early mesenchymal cells in proximity to the BMP4-expressing mesenchyme were marked upon tamoxifen injection, and their descendants contributed to diverse types of mesenchymal cells in the later stage, such as chondrocytes and perichondrial cells of the transient cartilage, as well as osteoblasts and suture mesenchymal cells. By contrast, Osx-creER marked osteoblast precursors at the later stage, and their descendants continued to become osteoblasts well into the postnatal stage. Therefore, Msx2 marks spatially restricted populations of mesenchymal precursor cells with diverse differentiation potential, suggesting that extrinsic molecular cues can dictate the nature of postmigratory mesenchymal cells in craniofacial development.

Differentiated embryo chondrocyte plays a crucial role in DNA damage response via transcriptional regulation under hypoxic conditions.

Tumor hypoxia contributes to a biologically aggressive phenotype and therapeutic resistance. Recent studies have revealed that hypoxia reduces expression of several DNA damage recognition and repair (DRR) genes via both hypoxia-inducible factor (HIF)-independent and -dependent pathways, and this induced genomic instability in cancer cells. We show here that one of the HIF-target genes-differentiated embryo chondrocyte (DEC)-plays a role in DNA damage response via transcriptional repression. Comprehensive gene expression and database analyses have revealed systemic repression of DNA-DRR genes in cancer and non-cancer cells under hypoxic conditions. Hypoxic repression in typical cases was confirmed by quantitative RT-PCR and promoter reporter experiments, and knockdown experiments indicated the critical role of DEC2 in such repression. Assessment of histone H2AX phosphorylation revealed that recognition and repair of DNA double-strand breaks (DSBs) induced by bleomycin or γ-ray irradiation were attenuated; moreover, Cleaved Caspase-3 levels were decreased with pre-conditioning under hypoxia: opposing phenomena were ascertained by knockdown of DEC2. Finally, pre-conditioning under hypoxia decreased the sensitivity of cancer cells to DSBs, and knockdown of DEC2 increased γ-ray sensitivity. These data imply that a critical reduction of DNA-DRR occurs via DEC-dependent transcriptional repression and suggest that DEC is a potential molecular target for anti-cancer strategies.

Genes uniquely expressed in human growth plate chondrocytes uncover a distinct regulatory network.

BACKGROUND: Chondrogenesis is the earliest stage of skeletal development and is a highly dynamic process, integrating the activities and functions of transcription factors, cell signaling molecules and extracellular matrix proteins. The molecular mechanisms underlying chondrogenesis have been extensively studied and multiple key regulators of this process have been identified. However, a genome-wide overview of the gene regulatory network in chondrogenesis has not been achieved. RESULTS: In this study, employing RNA sequencing, we identified 332 protein coding genes and 34 long non-coding RNA (lncRNA) genes that are highly selectively expressed in human fetal growth plate chondrocytes. Among the protein coding genes, 32 genes were associated with 62 distinct human skeletal disorders and 153 genes were associated with skeletal defects in knockout mice, confirming their essential roles in skeletal formation. These gene products formed a comprehensive physical interaction network and participated in multiple cellular processes regulating skeletal development. The data also revealed 34 transcription factors and 11,334 distal enhancers that were uniquely active in chondrocytes, functioning as transcriptional regulators for the cartilage-selective genes. CONCLUSIONS: Our findings revealed a complex gene regulatory network controlling skeletal development whereby transcription factors, enhancers and lncRNAs participate in chondrogenesis by transcriptional regulation of key genes. Additionally, the cartilage-selective genes represent candidate genes for unsolved human skeletal disorders.

Topography of the inferior alveolar nerve in human embryos and fetuses: an histomorphological study

The aim of this study is to establish the position of the inferior alveolar nerve in relation to the Meckel's cartilage, the anlage of the mandibular body and primordia of the teeth, and also to trace the change in nerve trunk structure in the human prenatal ontogenesis. serial sections (20µm) from thirty-two 6-12 weeks-old entire human embryos and serial sections (10µm) of six mandibles of 13-20 weeks-old human fetuses without developmental abnormalities were studied. histological sections were impregnated with silver nitrate according to Bilshovsky-Buke and stained with hematoxylin and eosin. during embryonic development, the number of branches of the inferior alveolar nerve increases and its fascicular structure changes. in conclusion, the architecture of intraosseous canals in the body of the mandible, as well as the location of the foramina, is predetermined by the course and pattern of the vessel/nerve branching in the mandibular arch, even before the formation of bony trabeculae. particularly, the formation of the incisive canal of the mandible can be explained by the presence of the incisive nerve as the extension of the inferior alveolar nerve. It has also been established that Meckel's cartilage does not participate in mandibular canal morphogenesis.

Endochondral Ossification Is Accelerated in Cholinesterase-Deficient Mice and in Avian Mesenchymal Micromass Cultures.

Most components of the cholinergic system are detected in skeletogenic cell types in vitro, yet the function of this system in skeletogenesis remains unclear. Here, we analyzed endochondral ossification in mutant murine fetuses, in which genes of the rate-limiting cholinergic enzymes acetyl- (AChE), or butyrylcholinesterase (BChE), or both were deleted (called here A-B+, A+B-, A-B-, respectively). In all mutant embryos bone growth and cartilage remodeling into mineralizing bone were accelerated, as revealed by Alcian blue (A-blu) and Alizarin red (A-red) staining. In A+B- and A-B- onset of mineralization was observed before E13.5, about 2 days earlier than in wild type and A-B+ mice. In all mutants between E18.5 to birth A-blu staining disappeared from epiphyses prematurely. Instead, A-blu+ cells were dislocated into diaphyses, most pronounced so in A-B- mutants, indicating additive effects of both missing ChEs in A-B- mutant mice. The remodeling effects were supported by in situ hybridization (ISH) experiments performed on cryosections from A-B- mice, in which Ihh, Runx2, MMP-13, ALP, Col-II and Col-X were considerably decreased, or had disappeared between E18.5 and P0. With a second approach, we applied an improved in vitro micromass model from chicken limb buds that allowed histological distinction between areas of cartilage, apoptosis and mineralization. When treated with the AChE inhibitor BW284c51, or with nicotine, there was decrease in cartilage and accelerated mineralization, suggesting that these effects were mediated through nicotinic receptors (α7-nAChR). We conclude that due to absence of either one or both cholinesterases in KO mice, or inhibition of AChE in chicken micromass cultures, there is increase in cholinergic signalling, which leads to increased chondroblast production and premature mineralization, at the expense of incomplete chondrogenic differentiation. This emphasizes the importance of cholinergic signalling in cartilage and bone formation.

Cartilage rings contribute to the proper embryonic tracheal epithelial differentiation, metabolism, and expression of inflammatory genes.

The signaling cross talk between the tracheal mesenchyme and epithelium has not been researched extensively, leaving a substantial gap of knowledge in the mechanisms dictating embryonic development of the proximal airways by the adjacent mesenchyme. Recently, we reported that embryos lacking mesenchymal expression of Sox9 did not develop tracheal cartilage rings and showed aberrant differentiation of the tracheal epithelium. Here, we propose that tracheal cartilage provides local inductive signals responsible for the proper differentiation, metabolism, and inflammatory status regulation of the tracheal epithelium. The tracheal epithelium of mesenchyme-specific Sox9Δ/Δ mutant embryos showed altered mRNA expression of various epithelial markers such as Pb1fa1, surfactant protein B (Sftpb), secretoglobulin, family 1A, member 1 (Scgb1a1), and trefoil factor 1 (Tff1). In vitro tracheal epithelial cell cultures confirmed that tracheal chondrocytes secrete factors that inhibit club cell differentiation. Whole gene expression profiling and ingenuity pathway analysis showed that the tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-ß (TGF-ß) signaling pathways were significantly altered in the Sox9 mutant trachea. TNF-α and IFN-γ interfered with the differentiation of tracheal epithelial progenitor cells into mature epithelial cell types in vitro. Mesenchymal knockout of Tgf-ß1 in vivo resulted in altered differentiation of the tracheal epithelium. Finally, mitochondrial enzymes involved in fat and glycogen metabolism, cytochrome c oxidase subunit VIIIb (Cox8b) and cytochrome c oxidase subunit VIIa polypeptide 1 (Cox7a1), were strongly upregulated in the Sox9 mutant trachea, resulting in increases in the number and size of glycogen storage vacuoles. Our results support a role for tracheal cartilage in modulation of the differentiation and metabolism and the expression of inflammatory-related genes in the tracheal epithelium by feeding into the TNF-α, IFN-γ, and TGF-ß signaling pathways.

Characterising the effects of in vitro mechanical stimulation on morphogenesis of developing limb explants.

Mechanical forces due to fetal movements play an important role in joint shape morphogenesis, and abnormalities of the joints relating to abnormal fetal movements can have long-term health implications. While mechanical stimulation during development has been shown to be important for joint shape, the relationship between the quantity of mechanical stimulation and the growth and shape change of developing cartilage has not been quantified. In this study, we culture embryonic chick limb explants in vitro in order to reveal how the magnitude of applied movement affects key aspects of the developing joint shape. We hypothesise that joint shape is affected by movement magnitude in a dose-dependent manner, and that a movement regime most representative of physiological fetal movements will promote characteristics of normal shape development. Chick hindlimbs harvested at seven days of incubation were cultured for six days, under either static conditions or one of three different dynamic movement regimes, then assessed for joint shape, cell survival and proliferation. We demonstrate that a physiological magnitude of movement in vitro promotes the most normal progression of joint morphogenesis, and that either under-stimulation or over-stimulation has detrimental effects. Providing insight into the optimal level of mechanical stimulation for cartilage growth and morphogenesis is pertinent to gaining a greater understanding of the etiology of conditions such as developmental dysplasia of the hip, and is also valuable for cartilage tissue engineering.

HES factors regulate specific aspects of chondrogenesis and chondrocyte hypertrophy during cartilage development.

RBPjκ-dependent Notch signaling regulates multiple processes during cartilage development, including chondrogenesis, chondrocyte hypertrophy and cartilage matrix catabolism. Select members of the HES- and HEY-families of transcription factors are recognized Notch signaling targets that mediate specific aspects of Notch function during development. However, whether particular HES and HEY factors play any role(s) in the processes during cartilage development is unknown. Here, for the first time, we have developed unique in vivo genetic models and in vitro approaches demonstrating that the RBPjκ-dependent Notch targets HES1 and HES5 suppress chondrogenesis and promote the onset of chondrocyte hypertrophy. HES1 and HES5 might have some overlapping function in these processes, although only HES5 directly regulates Sox9 transcription to coordinate cartilage development. HEY1 and HEYL play no discernable role in regulating chondrogenesis or chondrocyte hypertrophy, whereas none of the HES or HEY factors appear to mediate Notch regulation of cartilage matrix catabolism. This work identifies important candidates that might function as downstream mediators of Notch signaling both during normal skeletal development and in Notch-related skeletal disorders.

Comparison of fetal cartilage-derived progenitor cells isolated at different developmental stages in a rat model.

Fetal cartilage-derived progenitor cells (FCPCs) could be a useful cell source in cell-based therapies for cartilage disorders. However, their characteristics can vary depending on the developmental stages. The aim of this study was to compare the characteristics of rat FCPCs from the hind limb on embryonic day 14 (E14), E16 and E20 regarding proliferation, pluripotency, and differentiation. Morphologically, rat fetal cartilage tissue showed an increase in cartilaginous differentiation features (Safranin-O, type II collagen) and decrease in pluripotency marker (Sox2) in the order of E14, E16 and E20. E14 FCPCs showed significantly higher doubling time compared to E16 and E20 FCPCs. While the E14 FCPCs expressed pluripotent genes (Sox2, Oct4, Nanog), the E16 and E20 FCPCs expressed chondrogenic markers (Sox9, Col2a1, Acan). E20 FCPCs showed the highest ability to both chondrogenic and adipogenic differentiation and E14 FCPCs showed relatively better activity in osteogenic differentiation. Further analysis showed that E20 FCPCs expressed both adipogenic (C/ebpß) and osteogenic (Runx2, Sp7, Taz) transcription factors as well as chondrogenic transcription factors. Our results show an inverse relationship overall between the expression of pluripotency genes and that of chondrogenic and lineage-specific genes in FCPCs under development. Due to its exceptional proliferation and chondrogenic differentiation ability, fetal cells from epiphyseal cartilage (E20 in rats) may be a suitable cell source for cartilage regeneration.

Stable 5-Hydroxymethylcytosine (5hmC) Acquisition Marks Gene Activation During Chondrogenic Differentiation.

Regulation of gene expression changes during chondrogenic differentiation by DNA methylation and demethylation is little understood. Methylated cytosines (5mC) are oxidized by the ten-eleven-translocation (TET) proteins to 5-hydroxymethylcytosines (5hmC), 5-formylcytosines (5fC), and 5-carboxylcytosines (5caC), eventually leading to a replacement by unmethylated cytosines (C), ie, DNA demethylation. Additionally, 5hmC is stable and acts as an epigenetic mark by itself. Here, we report that global changes in 5hmC mark chondrogenic differentiation in vivo and in vitro. Tibia anlagen and growth plate analyses during limb development at mouse embryonic days E 11.5, 13.5, and 17.5 showed dynamic changes in 5hmC levels in the differentiating chondrocytes. A similar increase in 5hmC levels was observed in the ATDC5 chondroprogenitor cell line accompanied by increased expression of the TET proteins during in vitro differentiation. Loss of TET1 in ATDC5 decreased 5hmC levels and impaired differentiation, demonstrating a functional role for TET1-mediated 5hmC dynamics in chondrogenic differentiation. Global analyses of the 5hmC-enriched sequences during early and late chondrogenic differentiation identified 5hmC distribution to be enriched in the regulatory regions of genes preceding the transcription start site (TSS), as well as in the gene bodies. Stable gains in 5hmC were observed in specific subsets of genes, including genes associated with cartilage development and in chondrogenic lineage-specific genes. 5hmC gains in regulatory promoter and enhancer regions as well as in gene bodies were strongly associated with activated but not repressed genes, indicating a potential regulatory role for DNA hydroxymethylation in chondrogenic gene expression.

Citalopram and sertraline exposure compromises embryonic bone development.

Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed treatments for depression and, as a class of drugs, are among the most used medications in the world. Concern regarding possible effects of SSRI treatment on fetal development has arisen recently as studies have suggested a link between maternal SSRI use and an increase in birth defects such as persistent pulmonary hypertension, seizures and craniosynostosis. Furthermore, SSRI exposure in adults is associated with decreased bone mineral density and increased fracture risk, and serotonin receptors are expressed in human osteoblasts and osteoclasts. To determine possible effects of SSRI exposure on developing bone, we treated both zebrafish, during embryonic development, and human mesenchymal stem cells (MSCs), during differentiation into osteoblasts, with the two most prescribed SSRIs, citalopram and sertraline. SSRI treatment in zebrafish decreased bone mineralization, visualized by alizarin red staining and decreased the expression of mature osteoblast-specific markers during embryogenesis. Furthermore, we showed that this inhibition was not associated with increased apoptosis. In differentiating human MSCs, we observed a decrease in osteoblast activity that was associated with a decrease in expression of the osteoblast-specific genes Runx2, Sparc and Spp1, measured with quantitative real-time PCR (qRT-PCR). Similar to the developing zebrafish, no increase in expression of the apoptotic marker Caspase 3 was observed. Therefore, we propose that SSRIs inhibit bone development by affecting osteoblast maturation during embryonic development and MSC differentiation. These results highlight the need to further investigate the risks of SSRI use during pregnancy in exposing unborn babies to potential skeletal abnormalities.

Cartilage development requires the function of Estrogen-related receptor alpha that directly regulates sox9 expression in zebrafish.

Estrogen-related receptor alpha (ESRRa) regulates a number of cellular processes including development of bone and muscles. However, direct evidence regarding its involvement in cartilage development remains elusive. In this report, we establish an in vivo role of Esrra in cartilage development during embryogenesis in zebrafish. Gene expression analysis indicates that esrra is expressed in developing pharyngeal arches where genes necessary for cartilage development are also expressed. Loss of function analysis shows that knockdown of esrra impairs expression of genes including sox9, col2a1, sox5, sox6, runx2 and col10a1 thus induces abnormally formed cartilage in pharyngeal arches. Importantly, we identify putative ESRRa binding elements in upstream regions of sox9 to which ESRRa can directly bind, indicating that Esrra may directly regulate sox9 expression. Accordingly, ectopic expression of sox9 rescues defective formation of cartilage induced by the knockdown of esrra. Taken together, our results indicate for the first time that ESRRa is essential for cartilage development by regulating sox9 expression during vertebrate development.