Abstract
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.
Highlights
E ndochondral bone development is composed of the initial formation of a cartilage template and its subsequent replacement by mineralized bone
We show that Dicer plays a critical role in maintaining the proliferating pool of chondrocytes through regulation of chondrocyte proliferation and inhibition of premature differentiation to postmitotic hypertrophic chondrocytes
To assess the efficiency of Dicer removal, we quantified the mRNA level of Dicer mRNA by quantitative RT-PCR using RNA isolated form microdissected hindlimb cartilage of 3-day-old mice
Summary
E ndochondral bone development is composed of the initial formation of a cartilage template and its subsequent replacement by mineralized bone. Columnar chondrocytes stop proliferating and differentiate into postmitotic hypertrophic chondrocytes This process is tightly controlled by multiple layers of regulatory mechanisms, allowing persistent longitudinal bone growth. Germ-line ablation of genes encoding components for miRNA biogenesis results in embryonic or perinatal lethality in mice [5,6,7] These examples suggest that posttranscriptional gene regulation by miRNAs plays a critical role in regulating fundamental cellular functions in mice. PremiRNAs are exported into the cytoplasm, where the RNase III, Dicer, removes the loop region of the hairpin This step is essential for generation of mature miRNAs. To investigate the role of Dicer-dependent small RNAs in skeletal development, we conditionally disrupted the Dicer gene in this study. We show that Dicer plays a critical role in maintaining the proliferating pool of chondrocytes through regulation of chondrocyte proliferation and inhibition of premature differentiation to postmitotic hypertrophic chondrocytes
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