DEVELOPMENT 3231 RESEARCH ARTICLE INTRODUCTIONThe mammalian skeleton forms from three distinct cell lineages (Nakashima and de Crombrugghe, 2003). Facial bones and the cranium are derived from the neural crest. The base of the skull, the parietal bones and the axial elements of the ribs and vertebrae are derived from paraxial mesoderm. The sternum and long bones are formed from lateral plate mesoderm. Although several cell lineages contribute to skeletal structures, each gives rise to a common bone matrix-secreting cell type, the osteoblast. The requirement of this cell type for the production of bone is clearly demonstrated by the targeted mutation of Runx2 (Komori et al., 1997;Ducy et al., 1997;Otto et al., 1997) and osterix1 (Osx1; Sp7 -Mouse Genome Informatics) (Nakashima et al., 2002), transcriptional regulators expressed in osteoblast progenitors where both mutants lack all bone.Bone can be produced by two distinct mechanisms, direct differentiation of osteoblasts from mesenchymal progenitors (intramembranous ossification, e.g. skull and face) or by formation of bone on a cartilage scaffold (endochondral ossification, e.g. remainder of the skeleton). Endochondral ossification, the principle focus of this study, is initiated by the condensation of multipotent mesenchymal progenitor cells into structures that anticipate skeletal elements of the adult (reviewed by Kronenberg, 2003). Chondrocytes are the first cell type to form, starting out as immature proliferative cells that express type 2 collagen (Col2a1) that subsequently mature into postmitotic type 10 collagen (Col10a1)-expressing hypertrophic chondrocytes. Osteoblast progenitors can first be identified in the inner layer of perichondrial cells that lie immediately adjacent to the zone of hypertrophic chondrocytes, the periosteum, where the first bone matrix is deposited. Death of hypertrophic chondrocytes and vascular invasion result in the formation of a new area of mineralization, the primary spongiosa, within the shaft of the long bones.Several lines of evidence implicate Hedgehog (Hh) and canonical Wnt signaling in the regulation of endochondral ossification (reviewed by Kronenberg, 2003;Karsenty, 2003). Indian hedgehog (Ihh) is expressed by prehypertrophic chondrocytes and plays an essential role in coordinating the growth and differentiation of chondrocytes, both directly and through the control of other factors, notably parathyroid hormone-related peptide (Pthrp; Pthlh -Mouse Genome Informatics). In addition, Ihh appears to act directly on perichondrial mesenchyme to initiate an osteogenic program in osteoblast progenitors; in the absence of an Ihh input, these cells adopt an alternate chondrogenic fate (Long et al., 2004). The failure of activation of Runx2, a crucial early determinant of the osteoblast lineage, indicates that Hh signaling acts to initiate an osteogenic program. Whether Hh signaling is required at later stages of the osteogenic program has not been addressed.Initial in vivo evidence for canonical Wnt activity in osteogenesis c...
Osteosarcoma is the most common primary malignant tumor of bone. Analysis of familial cancer syndromes and sporadic cases has strongly implicated both p53 and pRb in its pathogenesis; however, the relative contribution of these mutations to the initiation of osteosarcoma is unclear. We describe here the generation and characterization of a genetically engineered mouse model in which all animals develop short latency malignant osteosarcoma. The genetically engineered mouse model is based on osteoblast-restricted deletion of p53 and pRb. Osteosarcoma development is dependent on loss of p53 and potentiated by loss of pRb, revealing a dominance of p53 mutation in the development of osteosarcoma. The model reproduces many of the defining features of human osteosarcoma including cytogenetic complexity and comparable gene expression signatures, histology, and metastatic behavior. Using a novel in silico methodology termed cytogenetic region enrichment analysis, we demonstrate high conservation of gene expression changes between murine osteosarcoma and known cytogentically rearranged loci from human osteosarcoma. Due to the strong similarity between murine osteosarcoma and human osteosarcoma in this model, this should provide a valuable platform for addressing the molecular genetics of osteosarcoma and for developing novel therapeutic strategies.[Keywords: Cancer; mouse model; osteocarcinoma] Supplemental material is available at http://www.genesdev.org.
Small noncoding RNAs, microRNAs (miRNAs), bind to messenger RNAs through base pairing to suppress gene expression. Despite accumulating evidence that miRNAs play critical roles in various biological processes across diverse organisms, their roles in mammalian skeletal development have not been demonstrated. Here, we show that Dicer, an essential component for biogenesis of miRNAs, is essential for normal skeletal development. Dicer-null growth plates show a progressive reduction in the proliferating pool of chondrocytes, leading to severe skeletal growth defects and premature death of mice. The reduction of proliferating chondrocytes in Dicer-null growth plates is caused by two distinct mechanisms: decreased chondrocyte proliferation and accelerated differentiation into postmitotic hypertrophic chondrocytes. These defects appear to be caused by mechanisms downstream or independent of the Ihh-PTHrP signaling pathway, a pivotal signaling system that regulates chondrocyte proliferation and differentiation. Microarray analysis of Dicer-null chondrocytes showed limited expression changes in miRNA-target genes, suggesting that, in the majority of cases, chondrocytic miRNAs do not directly regulate target RNA abundance. Our results demonstrate the critical role of the Dicer-dependent pathway in the regulation of chondrocyte proliferation and differentiation during skeletal development.microRNA ͉ skeletal development E ndochondral bone development is composed of the initial formation of a cartilage template and its subsequent replacement by mineralized bone. Longitudinal bone growth is driven by regulated proliferation and differentiation of chondrocytes in the growth plate cartilage. In developing growth plates, periarticular chondrocytes proliferate and differentiate into flat columnar chondrocytes that proliferate further to form orderly columns. Columnar chondrocytes stop proliferating and then differentiate into postmitotic hypertrophic chondrocytes. This process is tightly controlled by multiple layers of regulatory mechanisms, thus allowing persistent longitudinal bone growth.Regulation of gene expression is the major mechanism to control a variety of cellular functions, including proliferation and differentiation. Small noncoding microRNAs (miRNAs) encoded in the genome regulate gene expression at the posttranscriptional level. Genetic ablation of miRNA genes has demonstrated that loss of single miRNAs can results in significant physiological consequences in mice (1-4). In addition, germ-line ablation of genes encoding components for miRNA biogenesis results in embryonic or perinatal lethality in mice (5-7). These examples suggest that posttranscriptional gene regulation by miRNAs plays a critical role in regulating fundamental cellular functions in mice. miRNAs are generated from long primary transcripts (primiRNAs) through multiple processing steps (8). primiRNAs are cleaved into small-hairpin premiRNAs by the microprocessor complex containing Drosha and DGCR8. premiRNAs are exported into the cytoplasm, where ...
Although endochondral ossification of the limb and axial skeleton is relatively well-understood, the development of dermal (intramembranous) bone featured by many craniofacial skeletal elements is not nearly as well-characterized. We analyzed the expression domains of a number of markers that have previously been associated with endochondral skeleton development to define the cellular transitions involved in the dermal ossification process in both chick and mouse. This led to the recognition of a series of distinct steps in the dermal differentiation pathways, including a unique cell type characterized by the expression of both osteogenic and chondrogenic markers. Several signaling molecules previously implicated in endochondrial development were found to be expressed during specific stages of dermal bone formation. Three of these were studied functionally using retroviral misexpression. We found that activity of bone morphogenic proteins (BMPs) is required for neural crest-derived mesenchyme to commit to the osteogenic pathway and that both Indian hedgehog (IHH) and parathyroid hormone-related protein (PTHrP, PTHLH) negatively regulate the transition from preosteoblastic progenitors to osteoblasts. These results provide a framework for understanding dermal bone development with an aim of bringing it closer to the molecular and cellular resolution available for the endochondral bone development.
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