Differential ageing of growth plate cartilage determines skeletal proportions
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ABSTRACT: Bones at different anatomical locations vary dramatically in size. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed growth plate senescence. This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes. Here we use RNA-Seq to compare changes of gene expression with age in the longer bone (tibia, 1- vs 4-wk) with the difference of gene expression between long and short bones (tibia vs phalanx) at 1wk. We found that the developmental program of growth plate senescence is more advanced in the shorter bone and this differential senescence (or aging) underlies the disparities in bone length.
Project description:Affymetrix Chicken Gene 1.0 ST Array (ChiGene-1_1-st) profiles were generated from growth plate cartilage derived from the distal femur and proximal tibia of embryonic chickens at three developmental stages (day 12, 13 and 15) corresponding to stages prior to and immediately after the acqusition of mechano-responsive cartilage growth in the femur but not tibia (which does not respond to mechanical stimuli until later stages). Additionally, array profiles were generated from growth plate cartilage from the distal femur and proximal tibia of E15 chicks which were pharmacologically immobilized by decamethonium bromide treatment, which reduces longitudinal growth of the femur only at this stage.
Project description:Femur and Tibia of Mice 3 months of age were dissected, growth plate removed, and serial digested with collagenase-trypsin to remove endosteal and periosteal cells. The cortical bone tubes, highly enriched in osteocye in their natural bone matrix environment where then cultured with and and with out PTH at 250nM for 24hr, RNA extracted and biotinylated cDNA prepared, and hybrized to Illumina MouseWG-6_V2 arrays. Data was analyzed using Genome Studio-Gene Expression Module from Illumina.
Project description:Appendicular skeletal growth and bone mass acquisition are controlled by a variety of growth factors, hormones, and mechanical forces in a dynamic process called endochondral ossification. In long bones, chondrocytes in the growth plate proliferate and undergo hypertrophy to drive bone lengthening and mineralization. Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 and 2 (Phlpp1 and Phlpp2) are serine/threonine protein phosphatases that regulate cell proliferation, survival, and maturation via Akt, PKC, Raf1, S6k, and other intracellular signaling cascades. Germline deletion of Phlpp1 suppresses bone lengthening in part through parathyroid hormone receptor-dependent signaling in growth plate chondrocytes. Here, we demonstrate that Phlpp2 does not regulate endochondral ossification, and we define the molecular differences between Phlpp1 and Phlpp2 in chondrocytes. Phlpp2-/- mice are phenotypically indistinguishable from their wildtype (WT) littermates, with similar bone length, bone mass, and growth plate dynamics. Deletion of Phlpp2 had moderate effects on the chondrocyte transcriptome and proteome compared to WT cells. By contrast, Phlpp1/2-/- (double knockout) mice resembled Phlpp1-/- mice phenotypically and chondrocyte phospho-proteomes of Phlpp1-/- and Phlpp1/2-/- chondrocytes were different than WT and Phlpp2-/- chondrocyte phospho-proteomes. Data integration via multiparametric analysis identified alterations in Pdpk1 and Pak1/2 signaling pathways in chondrocytes lacking Phlpp1. In conclusion, these data demonstrate that Phlpp1, but not Phlpp2, regulates endochondral ossification through multiple and complex signaling cascades.
Project description:Different bones of the skeleton originate from three distinct embryonic lineages. Osteoblasts derived from bones of different embryonic origin displayed cell intrinsic difference. Osteocytes are terminally differentiated osteoblasts which displays a unique genetic makeup and a distinct morphology. It is not know if osteocytes display a cell intrinsic differences depeding on their origin. To understand the difference in the gene expression profiles of osteocytes of three different embryonic origins we examined osteocytes from three different bones neural crest origin-frontal bone, paraxial mesoderm origin-parietal bone, and lateral plate mesoderm- femur bone.
Project description:C-type natriuretic peptide (CNP) has been recently identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanism mediating these effects are not completely understood. Here we demonstrate that CNP activates the p38 MAP kinase pathway in chondrocytes and that pharmacological inhibition of p38 blocks the anabolic effects of CNP in a tibia organ culture system. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We performed Affymetrix microarray analyses to identify CNP target genes in the organ culture system. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, potentially because cGMP-dependent kinases I and II, important transducers of CNP signaling and are expressed at much higher levels in these cells than in other areas of the tibia. One of the genes most strongly induced by CNP was the Ptgs2 gene, encoding Cox2. Real-time PCR confirmed that Cox2 expression was induced by CNP in hypertrophic chondrocytes, but surprisingly in a p38-independent manner. Moreover, Cox2 inhibition – in contrast to p38 inhibition - did not block the anabolic effects of CNP. In summary, our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral ossification, with potential implications for numerous skeletal diseases. Keywords: Growth plate zone comparison and treatment response analysis
Project description:C-type natriuretic peptide (CNP) has been recently identified as an important anabolic regulator of endochondral bone growth, but the molecular mechanism mediating these effects are not completely understood. Here we demonstrate that CNP activates the p38 MAP kinase pathway in chondrocytes and that pharmacological inhibition of p38 blocks the anabolic effects of CNP in a tibia organ culture system. We further show that CNP stimulates endochondral bone growth largely through expansion of the hypertrophic zone of the growth plate, while delaying mineralization. Both effects are reversed by p38 inhibition. We performed Affymetrix microarray analyses to identify CNP target genes in the organ culture system. These studies confirmed that hypertrophic chondrocytes are the main targets of CNP signaling in the growth plate, potentially because cGMP-dependent kinases I and II, important transducers of CNP signaling and are expressed at much higher levels in these cells than in other areas of the tibia. One of the genes most strongly induced by CNP was the Ptgs2 gene, encoding Cox2. Real-time PCR confirmed that Cox2 expression was induced by CNP in hypertrophic chondrocytes, but surprisingly in a p38-independent manner. Moreover, Cox2 inhibition â in contrast to p38 inhibition - did not block the anabolic effects of CNP. In summary, our data identify novel target genes of CNP and demonstrate that the p38 pathway is a novel, essential mediator of CNP effects on endochondral ossification, with potential implications for numerous skeletal diseases. Experiment Overall Design: Tibiae from E15.5 day old embryonic mice were isolated and cultured in minimal media in the presence of vehicle, BSA/HCl (1mM), or C-type natriuretic peptide, CNP (10-6M). On the sixth day of treatment cultured tibias were micro-dissected into the resting/proliferating, hypertrophic, and mineralized areas. Distinct zones from approximately 24 bones were pooled together, from which RNA was isolated using the Qiagen RNeasy Lipid Extraction Kit. Once the quality of total RNA from three independent trials was determined using the Agilent 2100 bioanalyzer, microarray analyses were performed at the London Regional Genomics Centre using MOE430_2.0 Affymetrix arrays. Results were analyzed using GeneSpring 7.2 software.
Project description:A major question in developmental and regenerative biology is how organ size is controlled by progenitor cells. For example, while limb bones exhibit catch-up growth (recovery of a normal growth trajectory after transient developmental perturbation), it is unclear how this emerges from the behaviour of chondroprogenitors, the cells sustaining the cartilage anlagen that are progressively replaced by bone. Here we show that transient sparse cell death in the mouse foetal cartilage was repaired postnatally, via a two-step process. During injury, progression of chondroprogenitors towards more differentiated states was delayed, leading to altered cartilage cytoarchitecture and impaired bone growth. Then, once cell death was over, chondroprogenitor differentiation was accelerated and cartilage structure recovered, including partial rescue of bone growth. At the molecular level, ectopic activation of mTORC1 correlated with, and was necessary for, part of the recovery, revealing a specific candidate to be explored during normal growth and in future therapies.
Project description:Gli1+ progenitors are considered as metaphyseal mesenchymal progenitors in the distal femur and proximal tibia under the growth plate. We used single cell RNA sequencing (scRNA-seq) to analyze the diversity of Gli1+ progenitors in response to methylprednisolone.
Project description:Local site-specific differences between bones in different regions of the skeleton account for their different properties and functions. To identify mechanisms behind these differences, we have performed a cross-species study comparing RNA transcriptomes of cranial and tibial osteocytes, from bones with very different primary functions and physiological responses, collected from the same individual mouse, rat and rhesus macaque. Bioinformatic analysis was performed to identify 32 genes changed in the same direction between sites and shared across all three species. Several well-established key genes in bone growth and remodelling were upregulated in the tibias of all three species (BMP7, DKK1, FGF1, FRZB, SOST). Many of them associate or crosstalk with the canonical Wnt signalling pathway. These results suggest Wnt signalling-related candidates for different control of regulatory mechanisms in bone homeostasis in the skull and tibia and indicate a different balance between genetically determined structure and feedback mechanisms to strains induced by mechanical loading at the different sites.
Project description:Objective: Craniofacial bone defects caused by injuries and congenital diseases are a formidable challenge to clinicians. Research has shown promise in using bone marrow mesenchymal stem cells (BM-MSCs) from limb bones for craniofacial bone regeneration; yet little is known about the potential of BM-MSCs from craniofacial bones. This study compared BM-MSCs isolated from limb and craniofacial bones in pigs, a preclinical model closely resembling humans. Design: Bone marrow was aspirated from the tibia and mandible of four-month-old pigs (n=4), followed by BM-MSC isolation, culture-expansion and confirmation by flow cytometry. Proliferation rates were compared using population doubling times. Osteogenic differentiation was evaluated by quantifying alkaline phosphatase (ALP) activity. Total mRNA was extracted from freshly isolated BM-MSCs and analyzed to compare gene expressions of tibial and mandibular BM-MSCs using an Affymetrix GeneChip porcine genome array, followed by real-time RT-PCR evaluation of two neural crest markers. Results: BM-MSCs from both locations expressed MSC markers without expression of hematopoietic markers. Mandibular BM-MSCs proliferated significantly faster than tibial BM-MSCs. Without osteogenic inducers, mandibular BM-MSC alkaline phosphatase activities were 3.3-fold greater than those of tibial origin. Microarray analysis identified 383 differentially expressed genes in mandibular and tibial BM-MSCs, including higher expression of cranial neural crest-related genes nestin and BMP-4 in mandibular BM-MSCs, a trend also confirmed by real-time RT-PCR. Among differently expressed genes, only 47 showed greater than 1.5-fold differences in expression. Conclusions: These data indicate that despite many similarities in gene expression, mandibular BM-MSCs express of number of genes differently than tibial BM-MSCs and have a phenotypic profile that may make them advantageous for craniofacial bone regeneration. Bone marrow was aspirated from the mandibular symphyseal region and the tibia of 3 pigs. Mesenchymal stem cells were isolated from the bone marrow and cultured to 80% confluence. Cells were harvested for total RNA extraction and the RNA was analyzed by Affymetrix GeneChip porcine genome array.