A Spatio-Temporal-Dependent Requirement of Sonic Hedgehog in the Early Development of Sclerotome-Derived Vertebrae and Ribs.

Derived from axial structures, Sonic Hedgehog (Shh) is secreted into the paraxial mesoderm, where it plays crucial roles in sclerotome induction and myotome differentiation. Through conditional loss-of-function in quail embryos, we investigate the timing and impact of Shh activity during early formation of sclerotome-derived vertebrae and ribs, and of lateral mesoderm-derived sternum. To this end, Hedgehog interacting protein (Hhip) was electroporated at various times between days 2 and 5. While the vertebral body and rib primordium showed consistent size reduction, rib expansion into the somatopleura remained unaffected, and the sternal bud developed normally. Additionally, we compared these effects with those of locally inhibiting BMP activity. Transfection of Noggin in the lateral mesoderm hindered sternal bud formation. Unlike Hhip, BMP inhibition via Noggin or Smad6 induced myogenic differentiation of the lateral dermomyotome lip, while impeding the growth of the myotome/rib complex into the somatic mesoderm, thus affirming the role of the lateral dermomyotome epithelium in rib guidance. Overall, these findings underscore the continuous requirement for opposing gradients of Shh and BMP activity in the morphogenesis of proximal and distal flank skeletal structures, respectively. Future research should address the implications of these early interactions to the later morphogenesis and function of the musculo-skeletal system and of possible associated malformations.

The intraflagellar transport protein IFT52 associated with short-rib thoracic dysplasia is essential for ciliary function in osteogenic differentiation in vitro and for sensory perception in Drosophila.

Primary cilia are non-motile sensory cell-organelle that are essential for organismal development, differentiation, and postnatal homeostasis. Their biogenesis and function are mediated by the intraflagellar transport (IFT) system. Pathogenic variants in IFT52, a central component of the IFT-B complex is associated with short-rib thoracic dysplasia with or without polydactyly 16 (SRTD16), with major skeletal manifestations, in addition to other features. Here we sought to examine the role of IFT52 in osteoblast differentiation. Using lentiviral shRNA interference Ift52 was depleted in C3H10T1/2 mouse mesenchymal stem cells. This led to the disruption of the IFT-B anterograde trafficking machinery that impaired primary ciliogenesis and blocked osteogenic differentiation. In Ift52 silenced cells, Hedgehog (Hh) pathway upregulation during osteogenesis was attenuated and despite Smoothened Agonist (SAG) based Hh activation, osteogenic differentiation was incompletely restored. Further we investigated IFT52 activity in Drosophila, wherein the only ciliated somatic cells are the bipolar sensory neurons of the peripheral nervous system. Knockdown of IFT52 in Drosophila neuronal tissues reduced lifespan with the loss of embryonic chordotonal cilia, and produced severe locomotion, auditory and proprioceptive defects in larva and adults. Together these findings improve our knowledge of the role of IFT52 in various physiological contexts and its associated human disorder.

Different Requirements of CBFB and RUNX2 in Skeletal Development among Calvaria, Limbs, Vertebrae and Ribs.

RUNX proteins, such as RUNX2, regulate the proliferation and differentiation of chondrocytes and osteoblasts. Haploinsufficiency of RUNX2 causes cleidocranial dysplasia, but a detailed analysis of Runx2+/- mice has not been reported. Furthermore, CBFB is required for the stability and DNA binding of RUNX family proteins. CBFB has two isoforms, and CBFB2 plays a major role in skeletal development. The calvaria, femurs, vertebrae and ribs in Cbfb2-/- mice were analyzed after birth, and compared with those in Runx2+/- mice. Calvarial development was impaired in Runx2+/- mice but mildly delayed in Cbfb2-/- mice. In femurs, the cortical bone but not trabecular bone was reduced in Cbfb2-/- mice, whereas both the trabecular and cortical bone were reduced in Runx2+/- mice. The trabecular bone in vertebrae increased in Cbfb2-/- mice but not in Runx2+/- mice. Rib development was impaired in Cbfb2-/- mice but not in Runx2+/- mice. These differences were likely caused by differences in the indispensability of CBFB and RUNX2, the balance of bone formation and resorption, or the number and maturation stage of osteoblasts. Thus, different amounts of CBFB and RUNX2 were required among the bone tissues for proper bone development and maintenance.

Linking gene expression and phenotypic changes in the developmental and evolutionary origins of osteosclerosis in the ribs of bowhead whales (Balaena mysticetus).

Bowhead whales are among the longest-lived mammals with an extreme lifespan of about 211 years. During the first 25 years of their lives, rib bones increase in mineral density and the medulla transitions from compact to trabecular bone. Molecular drivers associated with these phenotypic changes in bone remain unknown. This study assessed expression levels of osteogenic genes from samples of rib bones of bowheads. Samples were harvested from prenatal to 86-year-old whales, representing the first third of the bowhead lifespan. Fetal to 2-year-old bowheads showed expression levels consistent with the rapid deposition of the bone extracellular matrix. Sexually mature animals showed expression levels associated with low rates of osteogenesis and increased osteoclastogenesis. After the first 25 years of life, declines in osteogenesis corresponded with increased expression of EZH2, an epigenetic regulator of osteogenesis. These findings suggest EZH2 may be at least one epigenetic modifier that contributes to the age-related changes in the rib bone phenotype along with the transition from compact to trabecular bone. Ancient cetaceans and their fossil relatives also display these phenotypes, suggesting EZH2 may have shaped the skeleton of whales in evolutionary history.

Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Tendons are an essential part of the musculoskeletal system, connecting muscle and skeletal elements to enable force generation. The transcription factor scleraxis marks vertebrate tendons from early specification. Scleraxis-null mice are viable and have a range of tendon and bone defects in the trunk and limbs but no described cranial phenotype. We report the expression of zebrafish scleraxis orthologs: scleraxis homolog (scx)-a and scxb in cranial and intramuscular tendons and in other skeletal elements. Single mutants for either scxa or scxb, generated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), are viable and fertile as adult fish. Although scxb mutants show no obvious phenotype, scxa mutant embryos have defects in cranial tendon maturation and muscle misalignment. Mutation of both scleraxis genes results in more severe defects in cranial tendon differentiation, muscle and cartilage dysmorphogenesis and paralysis, and lethality by 2-5 wk, which indicates an essential function of scleraxis for craniofacial development. At juvenile and adult stages, ribs in scxa mutants fail to mineralize and/or are small and heavily fractured. Scxa mutants also have smaller muscle volume, abnormal swim movement, and defects in bone growth and composition. Scleraxis function is therefore essential for normal craniofacial form and function and vital for fish development.-Kague, E., Hughes, S. M., Lawrence, E. A., Cross, S., Martin-Silverstone, E., Hammond, C. L., Hinits, Y. Scleraxis genes are required for normal musculoskeletal development and for rib growth and mineralization in zebrafish.

Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity.

Formation of the vertebrate axial skeleton requires coordinated Hox gene activity. Hox group 6 genes are involved in the formation of the thoracic area owing to their unique rib-promoting properties. Here we show that the linker region (LR) connecting the homeodomain and the hexapeptide is essential for Hoxb6 rib-promoting activity in mice. The LR-defective Hoxb6 protein was still able to bind a target enhancer together with Pax3, producing a dominant-negative effect, indicating that the LR brings additional regulatory factors to target DNA elements. We also found an unexpected association between Hoxb6 and segmentation in the paraxial mesoderm. In particular, Hoxb6 can disturb somitogenesis and anterior-posterior somite patterning by dysregulation of Lfng expression. Interestingly, this interaction occurred differently in thoracic versus more caudal embryonic areas, indicating functional differences in somitogenesis before and after the trunk-to-tail transition. Our results suggest the requirement of precisely regulated Hoxb6 expression for proper segmentation at tailbud stages.

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development.

BACKGROUND: Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. RESULTS: The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P < 0.05), which was in accordance with the results from histological structures. In comparison groups of 1-IB-vs-2-IB and 1-Rib-vs-2-Rib, 33 and 51 differently expressed proteins were identified and the function annotation results showed that these proteins were involved in regulating bone development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. CONCLUSIONS: Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.

A review of craniofacial disorders caused by spliceosomal defects.

The spliceosome is a large ribonucleoprotein complex that removes introns from pre-mRNA transcripts. Mutations in EFTUD2, encoding a component of the major spliceosome, have recently been identified as the cause of mandibulofacial dysostosis, Guion-Almeida type (MFDGA), characterized by mandibulofacial dysostosis, microcephaly, external ear malformations and intellectual disability. Mutations in several other genes involved in spliceosomal function or linked aspects of mRNA processing have also recently been identified in human disorders with specific craniofacial malformations: SF3B4 in Nager syndrome, an acrofacial dysostosis (AFD); SNRPB in cerebrocostomandibular syndrome, characterized by Robin sequence and rib defects; EIF4A3 in the AFD Richieri-Costa-Pereira syndrome, characterized by Robin sequence, median mandibular cleft and limb defects; and TXNL4A in Burn-McKeown syndrome, involving specific craniofacial dysmorphisms. Here, we review phenotypic and molecular aspects of these syndromes. Given the apparent sensitivity of craniofacial development to defects in mRNA processing, it is possible that mutations in other proteins involved in spliceosomal function will emerge in the future as causative for related human disorders.

PDCD10 (CCM3) regulates brain endothelial barrier integrity in cerebral cavernous malformation type 3: role of CCM3-ERK1/2-cortactin cross-talk.

Impairment of brain endothelial barrier integrity is critical for cerebral cavernous malformation (CCM) lesion development. The current study investigates changes in tight junction (TJ) complex organization when PDCD10 (CCM3) is mutated/depleted in human brain endothelial cells. Analysis of lesions with CCM3 mutation and brain endothelial cells transfected with CCM3 siRNA (CCM3-knockdown) showed little or no increase in TJ transmembrane and scaffolding proteins mRNA expression, but proteins levels were generally decreased. CCM3-knockdown cells had a redistribution of claudin-5 and occludin from the membrane to the cytosol with no alterations in protein turnover but with diminished protein-protein interactions with ZO-1 and ZO-1 interaction with the actin cytoskeleton. The most profound effect of CCM3 mutation/depletion was on an actin-binding protein, cortactin. CCM3 depletion caused cortactin Ser-phosphorylation, dissociation from ZO-1 and actin, redistribution to the cytosol and degradation. This affected cortical actin ring organization, TJ complex stability and consequently barrier integrity, with constant hyperpermeability to inulin. A potential link between CCM3 depletion and altered cortactin was tonic activation of MAP kinase ERK1/2. ERK1/2 inhibition increased cortactin expression and incorporation into the TJ complex and improved barrier integrity. This study highlights the potential role of CCM3 in regulating TJ complex organization and brain endothelial barrier permeability.

A single change in the aptamer of the Lactiplantibacillus plantarum rib operon riboswitch severely impairs its regulatory activity and leads to a vitamin B2 - overproducing phenotype.

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods.

Detection of age-related duplications in mtDNA from human muscles and bones.

Several studies have demonstrated the age-related accumulation of duplications in the D-loop of mitochondrial DNA (mtDNA) extracted from skeletal muscle. This kind of mutation had not yet been studied in bone. The detection of age-related mutations in bone tissue could help to estimate age at death within the context of legal medicine or/and anthropological identification procedures, when traditional osteological markers studied are absent or inefficient. As we detected an accumulation of a point mutation in mtDNA from an older individual's bones in a previous study, we tried here to identify if three reported duplications (150, 190, 260 bp) accumulate in this type of tissue. We developed a sensitive method which consists in the use of back-to-back primers during amplification followed by an electrophoresis capillary analysis. The aim of this study was to confirm that at least one duplication appears systematically in muscle tissue after the age of 20 and to evaluate the duplication age appearance in bones extracted from the same individuals. We found that the number of duplications increase from 38 years and that at least one duplicated fragment is present in 50% of cases after 70 years in this tissue. These results confirm that several age-related mutations can be detected in the D-loop of mtDNA and open the way for the use of molecular markers for age estimation in forensic and/or anthropological identification.

Lead levels in Eurasian otters decline with time and reveal interactions between sources, prevailing weather, and stream chemistry.

The uptake of contaminants by biota varies spatially and temporally due to a complex range of interacting environmental variables, but such complexities are typically disregarded in studies of temporal change. Here, we use linear modeling to explore spatial and temporal variation in bone Pb levels measured in samples taken from 329 Eurasian otters (Lutra lutra) found dead in southwest England. Between 1992 and 2004 Pb levels in otters fell by 73%, following UK legislative control of Pb emissions implemented since the mid 1980s. Spatial variation in bone Pb was positively correlated with modeled Pb emissions and stream sediment Pb, which interacted negatively with wind-speed and sediment Ca, respectively. Opportunistic collection of samples from wildlife mortalities provided a valuable opportunity for monitoring environmental contamination, interpretation of which was aided by spatially explicit analysis of environmental variables.

Shaping skeletal growth by modular regulatory elements in the Bmp5 gene.

Cartilage and bone are formed into a remarkable range of shapes and sizes that underlie many anatomical adaptations to different lifestyles in vertebrates. Although the morphological blueprints for individual cartilage and bony structures must somehow be encoded in the genome, we currently know little about the detailed genomic mechanisms that direct precise growth patterns for particular bones. We have carried out large-scale enhancer surveys to identify the regulatory architecture controlling developmental expression of the mouse Bmp5 gene, which encodes a secreted signaling molecule required for normal morphology of specific skeletal features. Although Bmp5 is expressed in many skeletal precursors, different enhancers control expression in individual bones. Remarkably, we show here that different enhancers also exist for highly restricted spatial subdomains along the surface of individual skeletal structures, including ribs and nasal cartilages. Transgenic, null, and regulatory mutations confirm that these anatomy-specific sequences are sufficient to trigger local changes in skeletal morphology and are required for establishing normal growth rates on separate bone surfaces. Our findings suggest that individual bones are composite structures whose detailed growth patterns are built from many smaller lineage and gene expression domains. Individual enhancers in BMP genes provide a genomic mechanism for controlling precise growth domains in particular cartilages and bones, making it possible to separately regulate skeletal anatomy at highly specific locations in the body.

Study on tensile, bending, fatigue, and in vivo behavior of porous SHS-TiNi alloy used as a bone substitute.

Intermetallic porous SHS-TiNi alloys exhibit tangled and specific stress-strain characteristics. This article aims to evaluate the findings emanating from experiments using standard and proprietary instruments. Fatigue testing under repeated complex loading was used to measure the total number of load cycles before failure of the SHS-TiNi samples occurred. Of the tested samples, seventy percent passed through 106 cycles without failure due to the reversible martensite transformation in the TiNi phase, one of the prevailing constituents of a multiphase matrix. The fractured surfaces were analyzed using scanning electron microscopy and confocal laser scanning instruments. Microscopy studies showed that the entire surface of the sample is concealed by miscellaneous strata that result from the SHS processand effectively protect the porous alloy in a corrosive environment. Numerous non-metallic inclusions, which are also attributed to the SHS reaction, do not have a significant impact on the deformation behavior and fatigue performance. In this context, the successful in vivo functioning of porous grafts assessed in a canine rib-plasty model allows the bone substitute to be congruentially deformed in the body without rejection or degradation; it thus has a long operational life, often greater than 17 ×106 (22 × 60 × 24 × 540) cycles. It acknowledges the potential benefits of SHS-TiNi as a superior osteoplastic material and its high resistance to corrosion fatigue.

Intraspecies disparity in growth rate is associated with differences in expression of local growth plate regulators.

Disparities in longitudinal growth within a species can be partly explained by endocrinological differences. We hypothesized that regulatory networks acting locally in the growth plate may also be important. We tested this hypothesis by evaluating the IGF/IGFBP expression, the vitamin D pathway, and the PTHrP-Indian hedgehog (IHH) feedback loop in rib growth plates from 10- and 21-wk-old small- (Miniature Poodles, MP) and large-breed dogs (Great Danes, GD) using immunohistochemistry and quantitative (q)PCR. The rib growth plates of GD were 1.7 times thicker compared with those of MP, with larger proliferative (in absolute terms) and larger hypertrophic (in absolute and relative terms) zones. IGF/IGFBP gene expression profiling of the growth plates revealed decreased gene expression of igfbp2, -4, and -6 and an unaltered expression of igf-I and igf-II and their respective receptors in GD vs. MP. Immunohistochemistry and qPCR findings showed that the vitamin D pathway was more active in GD than in MP. Staining for 1α- and 24-hydroxylase was more abundant and intense in GD and the gene expressions of 1α-hydroxylase and the vitamin D receptor-driven 24-hydroxylase were six- and eightfold higher in GD vs. MP, respectively. Consistent with the immunohistochemistry findings, the expression of mRNA for components of the parathyroid hormone-related peptide (PTHrP)-IHH loop was different in GD compared with MP, with there being a relative threefold downregulation of Pthrp and a tenfold upregulation of Ihh in GD vs MP. These differences suggest that the effects of IHH in the regulation of chondrocyte proliferation and hypertrophy, both independently of PTHrP, can become more dominant during rapid growth rates. In conclusion, our data suggest that, in addition to modest endocrine differences, more pronounced changes in the expression of locally acting regulatory networks, such as the IGF system, vitamin D pathway, and PTHrP-IHH feedback loop are important contributors to within-species disparities in growth rates.

Mll partial tandem duplication induces aberrant Hox expression in vivo via specific epigenetic alterations.

We previously identified a rearrangement of mixed-lineage leukemia (MLL) gene (also known as ALL-1, HRX, and HTRX1), consisting of an in-frame partial tandem duplication (PTD) of exons 5 through 11 in the absence of a partner gene, occurring in approximately 4%-7% of patients with acute myeloid leukemia (AML) and normal cytogenetics, and associated with a poor prognosis. The mechanism by which the MLL PTD contributes to aberrant hematopoiesis and/or leukemia is unknown. To examine this, we generated a mouse knockin model in which exons 5 through 11 of the murine Mll gene were targeted to intron 4 of the endogenous Mll locus. Mll(PTD/WT) mice exhibit an alteration in the boundaries of normal homeobox (Hox) gene expression during embryogenesis, resulting in axial skeletal defects and increased numbers of hematopoietic progenitor cells. Mll(PTD/WT) mice overexpress Hoxa7, Hoxa9, and Hoxa10 in spleen, BM, and blood. An increase in histone H3/H4 acetylation and histone H3 lysine 4 (Lys4) methylation within the Hoxa7 and Hoxa9 promoters provides an epigenetic mechanism by which this overexpression occurs in vivo and an etiologic role for MLL PTD gain of function in the genesis of AML.

Radiotherapy for breast cancer induced long-term diminished accumulation of radiotracer on bone scan of the irradiated ribs.

Radiotherapy may result in long term effects and composition alterations in bones. Bone scintigraphy after radiotherapy may demonstrate decreased skeletal uptake; however, this is a transient effect with bone scan normalized after a few years. We describe a case of a 31-year-old female patient treated for left breast cancer with chemotherapy and radiotherapy, exhibiting reduced and diffuse diphosphonate uptake in the heavily irradiated sections of left ribs, even twelve years post-treatment. Similarly, quantitative computed tomography indicated altered bone composition. To our knowledge this is the first case describing such a long radiation side effect in breast cancer treatment.

Collagen maturity, glycation induced-pentosidine, and mineralization are increased following 3-year treatment with incadronate in dogs.

UNLABELLED: Collagen cross-linking is a determinant of bone quality. A three-year treatment of bisphosphonate-incadronate disodium-in beagles increased degree of mineralization, collagen maturity, and pentosidine, a compound with advanced glycation end products. The treatment had no effect on the total amount of enzymatic cross-link formation. INTRODUCTION: Collagen cross-linking is a determinant of bone quality. Recently, we reported that long-term treatment with bisphosphonate increased microdamage accumulation. The aim of this study was to clarify the effect of a three-year treatment with bisphosphonate on degree of mineralization and immature and mature enzymatic cross-links and non-enzymatic collagen cross-link, pentosidine, in cortical bone in the same dogs. METHODS: Twenty-nine 1-year-old beagles (15 males, 14 females) were divided into three groups that daily were given vehicle or incadronate at doses of 0.3 or 0.6 mg/kg/day orally for three years. A cortex of a rib was fractionated into low- and high-density portions. The contents of calcium, phosphorus, enzymatic immature and mature cross-links, and the non-enzymatic glycation product pentosidine were determined in each fraction. RESULTS: Calcium, phosphorus, and pentosidine contents and the ratio of mature to immature cross-links increased significantly with incadronate in a dose-dependent manner, but the total amount of enzymatic cross-links was unchanged. The pentosidine content correlated inversely with cortical activation frequency (p < 0.01). CONCLUSION: Long-term suppression of bone remodeling by bisphosphonate increases degree of mineralization, collagen maturity, and non-enzymatic cross-linking.

Primary adamantinoma of the rib. Unusual presentation for a bone neoplasm of uncertain origin.

Adamantinomas are rare, low-grade malignant intra-osseous tumors composed of epithelial and mesenchymal elements, which show a marked predilection for the tibia and fibula of young adult male patients. Although cases of adamantinoma located to the axial skeleton have been reported either as recurrent or metastatic disease, only two cases of primary adamantinoma located to the thoracic wall have been previously described. In this study we present the clinical, radiological and histopathological features of a 24-year-old male with a slow growing, solid-cystic, painful mass, located to the right 11th rib, which was morphological and immunohistochemically diagnosed as a primary classic adamantinoma. Radiological studies showed a multiloculated lesion with a solid component. The patient underwent a whole surgical resection of the lesion. Histologically, multiple foci of epithelial cells with basaloid and squamous components were found intermixed within a fibrous stromal tissue. Immunohistochemical analysis demonstrated expression of cytokeratins, EMA, vimentin and other epithelial markers. Primary affection of the rib is an unusual feature of classic adamantinomas.