Both activating and null mutations of proteins required for canonical WNT signaling have revealed the importance of this pathway for normal skeletal development. However, tissue-specific transcriptional mechanisms through which WNT signaling promotes the differentiation of bone-forming cells have yet to be identified. Here, we address the hypothesis that canonical WNT signaling and the bone-related transcription factor RUNX2/CBFA1/ AML3 are functionally linked components of a pathway required for the onset of osteoblast differentiation. Our findings show that, in bone of the SFRP1 (secreted frizzled-related protein-1)-null mouse, which exhibits activated WNT signaling and a high bone mass phenotype, there is a significant increase in expression of T-cell factor (TCF)-1, Runx2, and the RUNX2 target gene osteocalcin. We demonstrate by mutational analysis that a functional TCF regulatory element responsive to canonical WNT signaling resides in the promoter of the Runx2 gene (؊97 to ؊93). By chromatin immunoprecipitation, recruitment of ␤-catenin and TCF1 to the endogenous Runx2 gene is shown. Coexpression of TCF1 with canonical WNT proteins resulted in a 2-5-fold activation of Runx2 promoter activity and a 7-8-fold induction of endogenous mRNA in mouse pluripotent mesenchymal and osteoprogenitor cells. This enhancement was abrogated by SFRP1. Taken together, our results provide evidence for direct regulation of Runx2 by canonical WNT signaling and suggest that Runx2 is a target of ␤-catenin/TCF1 for the stimulation of bone formation. We propose that WNT/TCF1 signaling, like bone morphogenetic protein/transforming growth factor-␤ signaling, activates Runx2 gene expression in mesenchymal cells for the control of osteoblast differentiation and skeletal development.During development of the skeleton and formation of bone tissue, several morphogenic growth factor and hormone signaling pathways impinge upon transcriptional regulators to induce the osteogenic phenotype (1, 2). The challenge is to identify how developmental cues and regulatory factors are integrated to accommodate the requirements for biological control of cell differentiation and tissue formation. Here, we have addressed the interaction of two key signals for osteogenesis: the WNT pathway, which contributes to the development of skeletal structures (3, 4), and the transcription factor RUNX2 (CBFA1/AML3), which is required for embryonic bone formation (5, 6).WNT signaling comprises a family of 19 secreted glycoproteins that have functions related to cell specification, formation of the body plan, cell growth, differentiation and apoptosis (7,8). WNT proteins function through Frizzled receptors, which transduce the signal through either the canonical ␤-catenin pathway or non-canonical pathway (7, 8). Activation of the Frizzled receptor complex results in inhibition of a phosphorylation cascade that stabilizes intracellular ␤-catenin levels. ␤-Catenin is subsequently translocated to the nucleus to form a transcriptionally active heterodimeric ␤-catenin TCF 2...

1. The importance of hydrologic variability for shaping the biophysical attributes and functioning of riverine ecosystems is well recognised by ecologists and water resource managers. In addition to the ecological dependences of flow for aquatic organisms, human societies modify natural flow regimes to provide dependable ecological services, including water supply, hydropower generation, flood control, recreation and navigation. Management of scarce water resources needs to be based on sound science that supports the development of environmental flow standards at the regional scale. 2. Hydrological classification has long played an essential role in the ecological sciences for understanding geographic patterns of riverine flow variability and exploring its influence on biological communities, and more recently, has been identified as a critical process in environmental flow assessments. 3. We present the first continental-scale classification of hydrologic regimes for Australia based on 120 metrics describing ecologically relevant characteristics of the natural hydrologic regime derived from discharge data for 830 stream gauges. Metrics were calculated from continuous time series (15-30 years of record constrained within a 36-year period) of mean daily discharge data, and classification was undertaken using a fuzzy partitional method -Bayesian mixture modelling. 4. The analysis resulted in the most likely classification having 12 classes of distinctive flow-regime types differing in the seasonal pattern of discharge, degree of flow permanence (i.e. perennial versus varying degrees of intermittency), variations in flood magnitude and frequency and other aspects of flow predictability and variability. Geographic, climatic and some catchment topographic factors were generally strong discriminators of flow-regime classes. The geographical distribution of flow-regime classes showed varying degrees of spatial cohesion, with stream gauges from certain flow-regime classes often being non-contiguously distributed across the continent. These results support the view that spatial variation in hydrology is determined by interactions among climate, geology, topography and vegetation at multiple spatial and temporal scales. Decision trees were also developed to provide the ability to determine the natural flowregime class membership of new stream gauges based on their key environmental and ⁄ or hydrological characteristics.

MicroRNAs (miRNAs) repress cellular protein levels to provide a sophisticated parameter of gene regulation that coordinates a broad spectrum of biological processes. Bone organogenesis is a complex process involving the differentiation and crosstalk of multiple cell types for formation and remodeling of the skeleton. Inhibition of mRNA translation by miRNAs has emerged as an important regulator of developmental osteogenic signaling pathways, osteoblast growth and differentiation, osteoclast-mediated bone resorption activity and bone homeostasis in the adult skeleton. miRNAs control multiple layers of gene regulation for bone development and postnatal functions, from the initial response of stem/progenitor cells to the structural and metabolic activity of the mature tissue. This Review brings into focus an emerging concept of bone-regulating miRNAs, the evidence for which has been gathered largely from in vivo mouse models and in vitro studies in human and mouse skeletal cell populations. Characterization of miRNAs that operate through tissue-specific transcription factors in osteoblast and osteoclast lineage cells, as well as intricate feedforward and reverse loops, has provided novel insights into the supervision of signaling pathways and regulatory networks controlling normal bone formation and turnover. The current knowledge of miRNAs characteristic of human pathologic disorders of the skeleton is presented with a future goal towards translational studies.

Bone morphogenetic proteins (BMPs) are potent morphogens that activate transcriptional programs for lineage determination. How BMP induction of a phenotype is coordinated with microRNAs (miRNAs) that inhibit biological pathways to control cell differentiation, remains unknown. Here, we show by profiling miRNAs during BMP2 induced osteogenesis of C2C12 mesenchymal cells, that 22 of 25 miRNAs which significantly changed in response to BMP2 are down-regulated. These miRNAs are each predicted to target components of multiple osteogenic pathways. We characterize two representative miRNAs and show that miR-133 directly targets Runx2, an early BMP response gene essential for bone formation, and miR-135 targets Smad5, a key transducer of the BMP2 osteogenic signal, controlled through their 3UTR sequences. Both miRNAs functionally inhibit differentiation of osteoprogenitors by attenuating Runx2 and Smad5 pathways that synergistically contribute to bone formation. Although miR-133 is known to promote MEF-2-dependent myogenesis, we have identified a second complementary function to inhibit Runx2-mediated osteogenesis. Our key finding is that BMP2 controls bone cell determination by inducing miRNAs that target muscle genes but mainly by down-regulating multiple miRNAs that constitute an osteogenic program, thereby releasing from inhibition pathway components required for cell lineage commitment. Thus, our studies establish a mechanism for BMP morphogens to selectively induce a tissuespecific phenotype and suppress alternative lineages.S keletal development requires stringent control of a program for gene activation and suppression in response to physiological cues. The potent osteogenic bone morphogenetic proteins (BMPs) (BMP2/7) and canonical Wnt signaling activate skeletal-related genes for formation of cartilage and bone (1-3). BMPs regulate the differentiation of multiple cell types (4-6) but will also drive commitment of mesenchymal stem cells into a specific lineage. BMP2, for example, supports osteoblastogenesis (7, 8). There has been a principal focus on identifying the mechanisms by which only one cell phenotype is activated by BMP2. BMP2 intracellular receptor Smads, particularly Smad5, activates osteoblast-essential genes, including the transcription factors required for in vivo bone formation Runx2 (Cbfa1/ AML3) and Osterix (9-11). The contribution of global mechanisms for suppression of alternative lineages is equally important for understanding tissue development and diseases.MicroRNAs (miRNAs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation, and differentiation (reviewed in ref. 12) and in the control of tumorigenesis (13,14). miRNAs are small (22-nt) endogenous noncoding RNAs that anneal to the 3Ј UTR of target mRNAs to mediate inhibition of translation and lower protein levels. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype i...

Bone tissue arises from mesenchymal cells induced into the osteoblast lineage by essential transcription factors and signaling cascades. MicroRNAs regulate biological processes by binding to mRNA 3-untranslated region (UTR) sequences to attenuate protein synthesis. Here we performed microRNA profiling and identified miRs that are up-regulated through stages of osteoblast differentiation. Among these are the miR-29, miR-let-7, and miR-26 families that target many collagens and extracellular matrix proteins. We find that miR-29b supports osteoblast differentiation through several mechanisms. miR-29b decreased and anti-miR-29b increased activity of COL1A1, COL5A3, and COL4A2 3-UTR sequences in reporter assays, as well as endogenous gene expression. These results support a mechanism for regulating collagen protein accumulation during the mineralization stage when miR-29b reaches peak levels. We propose that this mechanism prevents fibrosis and facilitates mineral deposition. Our studies further demonstrate that miR-29b promotes osteogenesis by directly down-regulating known inhibitors of osteoblast differentiation, HDAC4, TGF␤3, ACVR2A, CTNNBIP1, and DUSP2 proteins through binding to target 3-UTR sequences in their mRNAs. Thus, miR-29b is a key regulator of development of the osteoblast phenotype by targeting anti-osteogenic factors and modulating bone extracellular matrix proteins.