Osteoporosis is a mineral bone disease arising from the predominance of osteoclastic bone resorption. Bisphosphonates which inhibit osteoclasts are commonly used in osteoporosis treatment, but are not without severe adverse effects like osteonecrosis of the jaw. The mechanisms behind the development of such phenomena is not well understood. Bone homeostasis is achieved through an intimate cross-talk between osteoclasts and osteoblasts. Thus, it is important to visualise activities of these cells simultaneously in situ. Currently, there are means to visualise osteoclast shape and numbers with tartrate-resistant alkaline phosphatase (TRAP) staining but no practical and accurate methods to quantify osteoclast activity in situ. This investigation aims to establish the use of ELF97, a substrate of TRAP, to visualise and quantify osteoclast activity. This provides vital clues to mechanisms of various bone disorders. TRAP dephosphorylation of ELF97 results in a detectable fluorescent product at areas of osteoclast activity. Osteoclastic activity was initiated in zebrafish by inducing crush injuries in tail fin rays. Colocalisation of ELF97 fluorescence with osteoclast-specific DsRed in transgenic zebrafish, visualised under confocal microscopy, is used to further establish the specificity of ELF97 to sites of osteoclastic activity. Quantification is established by comparing fluorescence between wild type, osteoclast-deficient mutants and bisphosphonate-treated zebrafish. The utility of ELF97 will also be investigated in terms of the stability of the florescent product. The investigation revealed that ELF97 and DsRed fluorescence were found commonly at crush sites with osteoclastic activity. Wild type zebrafish had greater fluorescence compared to osteoclast-deficient (p<0.0001) and bisphosphonate-treated zebrafish (p<0.0001) after 7 and 14 days post-crush, revealing that fluorescence from ELF97 corresponds to expected osteoclastic activity. Fluorescence of tail fins treated with ELF97 did not diminish over a period of 21 days of storage, demonstrating its stability. ELF97 is thus a useful means to visualise osteoclast activity, potentially crucial in more advanced investigations to understand bone disorders. It could be used in combination with other cellular markers in whole biological samples to study and experimentally manipulate bone remodelling

The etiology of adolescent idiopathic scoliosis (AIS) is largely unknown, but clinical observations revealed the role of hereditary and rapid growth in the development of this condition. More recently, several genes were suspected to cause or contribute to AIS. Our group identified gene variants of POC5 centriolar protein in a French and French-Canadian families with multiple members affected with AIS. We sought to expand on this study and to investigate for the role of POC5 gene and mutated protein. In this work, the potential pathogenic effect of mutated POC5 was investigated in vitro (human osteoblats cell culture) and in vivo in a zebrafish animal model. To investigate the role of POC5 in AIS, we investigated subcellular localization of POC5 with respect to cilia in cells overexpressing wt or POC5 variants (C1286T, A429V) and in human osteoblasts from scoliotic patients carrying these POC5 variants and normal control cells (in vitro study). We also created a loss-of-function model in zebrafish (in vivo study). The role of POC5 was investigated by: 1) mass spectroscopy analysis and co-immunoprecipitation to identify differences in binding partners between the wild-type (wt POC5 and mut POC5 protein; 2) immunolocalization of POC5 wt and mut proteins at the cellular level; 3) histology and immunohistochemistry performed on tissues from wt (control) and scoliotic (poc5 mut) zebrafish. Our work identified several interacting partners with POC5, and documented functional connections with respect to cilia and centrosome dysfunction. A number of ciliary proteins were identified to be interacting with wt POC5 but not mut POC5. At the cellular level, localization and co-localisation of wt POC5 and mut POC5 protein with alpha acetylated tubulin (cilia marker), confirmed the consequence of the mutation on subcellular location with respect to cilium structure, length and staining intensity of cilia. In vivo, several defects in the retina were identified in mut poc5 zebrafish compared wt zebrafish. Finally, using different markers for retinal layers and acetylated tubulin, the defects were localized in ganglion cell layer and cones of the retina. Our findings confirm the involvement of POC5 in scoliosis. A role of POC5 with respect to the primary cilia was attributed. These findings open new avenues for the understanding the primary causes of AIS at the molecular and physiological levels

The aim of this study is to clarify the implication of ciliary pathway on the onset of the spinal curvature that occurs in Adolescent Idiopathic Scoliosis (AIS) patients through functional studies of two genes: POC5 and TTLL11. Since the genetic implication for AIS is accepted, many association and candidate gene analysis revealed the implication of ciliary genes. The characterisation of these two proteins was assessed by qPCR, WB and immunofluorescence in vitro using control cells and cells derived from AIS patients. The impact of genetic modification of these genes on the functionality of the proteins in vitro and in vivo was analysed in zebrafish model created by CRISPR/Cas9 using microCT and histologic analysis. Our study revealed that mutant cells, for both gene, were less ciliated and the primary cilia was significantly shorter compared to control cells. We also observed a default in cilia glutamylation by immunofluorescence and Western Blot. Moreover, we observed in both zebrafish model, a 3D spine curvature similar to the spinal deformation in AIS. Interestingly, our preliminary results of immunohistology showed a retinal defect, especially at the cone cell layer level. This study strongly supports the implication of the ciliary pathway in the onset of AIS and this is the first time that a mechanism is described for AIS. Indeed, we show that shorter cilia could be less sensitive to environmental factors due to lower glutamylation and result in altered signalling pathway. Identifying the biological mechanism involved is crucial for elucidating AIS pathogenesis

Introduction. Adolescent idiopathic scoliosis (AIS) is the most common spinal deformity in children, and its cause is unknown. Recently, researchers have traced a defect in the gene CHD7 to AIS. CHD7 encodes for a chromodomain helicase of the DNA-binding domain protein family and is thought to have a crucial role in many basic cellular functions. However, the functional role of CHD7 in AIS is still elusive. In this study, we investigated the potential pathogenic effect of gene defects in CHD7 in vivo by evaluating their effect on spine formation and development in zebrafish. Methods. To investigate the function of the CHD7 encoded protein, we generated an antisense morpholino oligonucleotide against the CHD7 gene to disrupt the translation of the gene transcripts and knockdown the levels of its protein. The morpholino was injected into single-cell stage zebrafish embryos. The injected fish were allowed to develop and were then assessed for distinct phenotypes reminiscent of scoliosis by histological stains. Results. Knockdown of CHD7 resulted in a spectrum of ocular and heart anomalies. We noted that 26% of the zebrafish morphants exhibited curvature of the body axis at early stages. Histological stains of the vertebrae at later stages revealed that the spine of the zebrafish morphant had abnormal kinks rather than scoliotic curves. These defects were accompanied by reduced vertebral mineralisation around the kink area. The CHD7 morphant also showed severe cranial nerve abnormalities and had missing or malformed otolith—a part of the vestibular system analogous to the human ear. Conclusions. Our findings indicate a key role of CHD7 in eye, heart, and ear development but not in the onset of skeletal deformities as observed in scoliosis. Our results are similar to the congenital abnormities associated with CHD7 mutations in the CHARGE syndrome. Acknowledgments. This study is supported by the Yves Cotrel Fondation

Osteosarcoma (OS) is the most prevalent bone tumor in children and young adults. Most tumors arise from the metaphysis of the long bones and easily metastasize to the lungs. Current therapeutic strategies of osteosarcoma are routinely surgical resection and chemotherapy, which are limited to the patients suffering from metastatic recurrence. Therefore, to investigate molecular mechanisms that contribute to osteosarcoma progression is very important and may shed light on targeted therapeutic approach to improve the survival of patients with this disease. Several miRNAs have been found expressed differentially in osteosarcoma (OS), In this study, we found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration andinvasion ability in vitro, and inhibited tumor growth and metastasisin vivo. The function and molecular mechanism of miR-144 in Osteosarcoma was further investigated. Tissue samples from fifty-one osteosarcoma patients were obtained from Shanghai Ninth People's Hospital. The in vitro function of miR-144 in Osteosarcoma was investigated by cell viability assay, wound healing assay, invasion assay, the molecular mechanism was identified by Biotin-coupled miRNA capture, Dual-luciferase reporter assays, etc. the in vivo function of miR-144 in osteosarcoma was confirmed by osteosarcoma animal model and miR-144−/− zebrafish model. Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) were both identified as direct targets of miR-144. Moreover, the negative co-relation between downregulated miR-144 and upregulated ROCK1/RhoA was verified both in the osteosarcoma cell lines and clinical patients' specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on the miR-144 inhibited cell growth, migration and invasion abilities, while individual overexpression of ROCK1 had no statistical significance compared with control in miR-144 transfected SAOS2 and U2-OS cells. This study demonstrates that miR-144 inhibited tumor growth and metastasis in osteosarcoma via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment ofosteosarcoma

Purpose. Despite enormous progress regarding the genetic regulation of limb development, little attention has been paid to the cellular and tissue mechanisms that govern outgrowth. How does the limb bud acquire its peculiar shape? Previous models have focused on isotropic growth resulting from distally based proliferation. However, recent models and proliferation data and models suggest that differential proliferation cannot explaing the morphogenesis of the limb bud. We tested the possibility that oriented cell behaviours underlie early outgrowth. Method. We visualised early limb buds in living mouse and zebrafish embryos at cellular resolution by using transgenic subcellular fluorescent labels that mark either the cell nucleus or the cell membrane. We acquired time lapse and static images using a confocal microscope and generated velocity vector fields to track cell movements, and also tracked cell division planes through the entire tissue. To complement the live imaging, we also undertook lineage tracing experiments in chick and zebrafish embryos. The molecular determinants of these cell movements were tested by crossing the reporter transgenes onto mutant backgrounds. Results. Lateral plate contributes mesoderm to the early limb bud through directional cell movement. The direction of cell motion, longitudinal cell axes and bias in cell division planes lie largely parallel to one another along the rostrocaudal (head-tail) axis in lateral plate mesoderm. Transition of these parameters from a rostrocaudal to a mediolateral (outward from the body wall) orientation accompanies early limb bud outgrowth. Furthermore, we provide evidence that Wnt5a established cell polarity that likely underlies the oriented cell behaviours. Conclusion. Anisotroic, oriented cell behaviours likely underlie early limb bud outgrowth. The driving forces of cell movements have yet to be determined. This morphogenetic perspective will also help to place information regarding genetic regulation of limb development and human mutations into a dynamic context. Of great interest to us is how dynamic cell movements might contribute to the establishment of early limb prepattern, prior to overt differentiation of the skeletal template

Introduction. Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity. It occurs mainly in girls and progresses during pre-pubertal and pubertal growth, which is a crucial period for bone mass acquisition. The cause and molecular mechanisms of AIS are not clear; at present the consensus is that AIS has a multifactor cause, with many genetic factors. During the past 5 years, considerable effort has been devoted to identify a gene or genes that cause a predisposition to AIS. Many loci for this disorder have been mapped to different chromosome regions, but no genes have been clearly identified as being responsible for AIS, and, most importantly, the resulting protein defects remain to be shown. We aimed to identify the gene(s) that could be involved in AIS and to validate their involvement by both genetic and functional analyses. Methods. A large multiplex AIS French family was chosen for this study on the basis of clinical and radiological data. Whole genome genotyping of the 20 members of this family led to the mapping of a dominant disease-causing gene to two critical genomic intervals (Edery and colleagues, Eur J Hum Genet, accepted [2011]), but the causative mutation remains to be identified. In parallel, gene expression profiling was investigated by microarray analysis in RNA samples isolated from osteoblasts derived from healthy individuals and those with AIS. RNA samples were extracted from osteoblasts, purified, fluorescently labelled, and then hybridised to gene expression microarrays with the Illumina expression BeadChips technology containing more than 46 000 probes for the human genome (HumanHT-12). Data analysis in R version 2.10.1 (Bioconductor packages oligo and limma) was done, and genes that had at least 1·5-fold change in expression were considered differentially regulated relative to controls. AIS candidate genes within the critical intervals were selected on the basis of their mRNA expression in AIS individuals and by their known functions. The coding regions of these candidate genes were then sequenced to identify potential mutations. The biological activity of mutant proteins is under evaluation by in-vivo functional studies in zebrafish. Results. In the AIS family, a maximum LOD score of 3·01 was reached on two specific chromosomal regions. The interval lengths of these regions were 7cM and 12cM. These two regions contain several genes that might be responsible for AIS. Microarray analysis showed many genes that are differentially regulated in AIS osteoblasts compared with control osteoblasts. We recorded that 2·6% of the 24000 genes examined were upregulated in AIS osteoblasts, whereas 2·16% of them were downregulated. We observed a roughly 3-fold increase or decrease in the transcripts of many genes in AIS osteoblasts. Some of the differentially regulated genes are located within the two chromosomal candidate regions. The sequencing of the candidate genes' coding sequences was done on the family members. Sequence analysis showed two rare SNPs located on the coding regions of a gene that we called CH5G1. These two SNPs are located on the C-terminal region of the CH5G1 protein and affect its structure and probably its cellular activity and biological process leading to the disease. The C-terminal region of this protein interacts with the mRNA of a gene whose defects cause scoliosis as a secondary phenotype. The pathogenic nature of these SNPs is being investigated in the zebrafish model. The results suggest that CH5G1 gene's defects could be associated with AIS in this family. Conclusions. Identification of susceptibility genes for AIS will facilitate the understanding of underlying biochemical pathways (functional studies) and ultimately the development of specific therapies (pharmacological studies). This is likely to have important implications, since the cause of AIS is unknown. Acknowledgments. This study is supported by the Fondation Yves Cotrel, Institut de France

While classically bone tumors were classified according to the differentiation and cellular morphology at the light microscopical level, it has become clear that over the past decade the array of knowledge which became available on the cytogenetic and molecular genetic level should influence tumor classification. This lead to an integrated approach in drafting the 2002 WHO nomenclature for bone tumors in which morphology was combined with the evolving genetic information. With regard to bone tumorigenesis distinct patterns can be recognized with regard to underlying genetics:. balanced translocations such as in Ewing sarcoma, or aneurysmal bone cysts. numerical chromosomal abnormalities like in adamantinoma. complex karyotypes with as yet no identified starting point from a genetic point of view such as in osteosarcomas or central chondrosarcomas. single gene events such as in fibrous dysplasia or osteochondroma. These different pathways in oncogenesis open an array of possibilities for studying the drivers in oncogenesis. Especially those pathways essential in keeping the proliferative state as opposed to the ones governing differentiation might proof to be potential targets for tailored drug treatment. Here high throughput screens focusing on kinase activity appear especially of interest. Within the EuroBoNeT consortium (. www.eurobonet.eu. ) several working groups act transnationally together in unraveling these events. Important key elements of study are the molecular-genetic networks involved in skeletogenesis as well, which amongst others are studied within the consortium using in vitro systems as well as in vivo models using xenografted mouse models, mesenchymal stem cell models and models exploring zebrafish development