Unknown,Transcriptomics,Genomics,Proteomics

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Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

ABSTRACT: WNT signaling is critical in most aspects of skeletal development and homeostasis, and antagonists of WNT signaling are emergning as key regulatory proteins with great promise as therapeutic agents for bone disorders. Until recently Sost and its paralog Sostdc1 have been described as growth factors with highly restricted expression in the adult where Sost was assumed 'osteocyte-' and Sostdc1 'kidney-' specific. Here we show that these two proteins emerged throgh ancestral genome duplication and their expression patterns have diverged to span complimentary domains in most organ systems including musculoskeletal, cardiovascular, nervous, digestive, reproductive and respiratory. In the developing limb, Sost and Sostdc1 display dynamic expression patterns with Sost being restricted to the distal ectoderm and Sostdc1 to the proximal ectoderm and the mesenchyme. While Sostdc1-/- mice lack any obvious limb and skeletal defects, Sost-/- mice recapitulate the hand defects described for sclerosteosis patients. However, elevated WNT signaling in Sost-/-; Sostdc1-/- mice causes misregulation of SHH signaling, ectopic activation of Sox9 in the digit 1 field and ultimately preaxial polydactyly. In addition, we show that the syndactyly documented in Sclerosteosis is present in both Sost-/- and Sost-/-; Sostdc1-/- mice, and is driven by misregulation of Fgf8 in the AER, a region lacking Sost and Sostdc1 expression. This study highlights the complexity of WNT signaling in skeletal biology and disease and emphasizes how redundant mechanisms and non-cell autonomous effects can synergize to unveil new intricate phenotypes caused by elevated WNT signaling. Five arrays were analyzed, consisting of two total embryonic fore-limb RNA (experimental) and three total embryonic forelimb RNA (reference) samples at E11.5 DPC mouse (C57Bl6 strain). Embryos were not pooled to generate samples. Each time point has 3 to 5 biological replicates for limb bud samples, duplicates for whole embryos. Comparisons were made between limb bud samples and whole embryo at the same stage, fore-limb samples of different stages, hind-limb samples of different stages, and fore-limb samples compared to hind-limb samples at the same or the next stage.

ORGANISM(S): Mus musculus

SUBMITTER: Gabriela Loots

PROVIDER: E-GEOD-44325 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Inhibitors of Wnt signaling have been previously shown to be involved in prostate cancer (PC) metastasis; however the role of Sclerostin (Sost) has not yet been explored. Here we show that elevated Wnt signaling derived from Sost deficient osteoblasts (OBSOSTKO) promotes PC invasion while rhSOST has an inhibitory effect. In contrast, rhDKK1 promotes PC elongation and filopodia formation, morphological changes characteristic of an invasive phenotype. Furthermore, rhDKK1 was found to activate canonical Wnt signaling in PC3 cells as quantified by TOPFLASH reporter and b-catenin activity, suggesting that SOST and DKK1 have opposing roles on Wnt signaling in this context. Gene expression analysis of PC3 cells co-cultured with OBs exhibiting varying amounts of Wnt signaling identified CRIM1 as one of the transcripts up-regulated under highly invasive conditions. Following further analysis we found that CRIM1 increases PC3 invasion, complexes with b-catenin, and promotes cell-adhesion, suggesting that elevated Wnt signaling secreted from the bone may promote PC tropism by promoting CRIM1 expression and facilitating cancer cell invasion and adhesion to bone. We concluded that SOST and DKK1 have opposing effects on PC3 cell invasion and that bone-derived Wnt signaling positively contributes to the invasive phenotypes of metastatic cancer cells by activating CRIM1 expression and facilitating PC-OB physical interaction. As such, we investigated the effects of high concentrations of SOST in vivo. We found that PC3-cells overexpressing SOST injected via tail vein did not readily metastasize in NSG xenografts, suggesting that targeting the molecular bone environment may influence bone metastatic outcome in clinical settings. PC3 cells were cultured for 48 hours in specified conditions prior to RNA extraction and hybridization on Affymetrix Human Genome U133 Plus 2.0 Array. All experiments were performed in at least duplicates. Total RNA was collected from PC3 cells cultured in normal growth media, cocultured with WT mouse osteoblasts supplemented with recombinant human DKK1, cells cocultured with WT mouse osteoblasts supplemented with recombinant human SOST, or cocultured with SostKO mouse osteoblasts.

Project description:Detailed information about stage-specific changes in gene expression is crucial for understanding the gene regulatory networks underlying development and the various signal transduction pathways contributing to morphogenesis. Here, we describe the global gene expression dynamics during early murine limb development, when cartilage, tendons, muscle, joints, vasculature, and nerves are specified and the musculoskeletal system of the limbs is established. We used whole-genome microarrays to identify genes with differential expression at 5 stages of limb development (E9.5 to 13.5), during fore-limb and hind-limb patterning. We found that the onset of limb formation is characterized by an up-regulation of transcription factors, which is followed by a massive activation of genes during E10.5 and E11.5 which tampers off at later time points. Among 3520 genes identified as significantly up-regulated in the limb, we find ~30% to be novel, dramatically expanding the repertoire of candidate genes likely to function in the limb. Hierarchical and stage-specific clustering identified expression profiles that correlate with functional programs during limb development and are likely to provide new insights into specific tissue patterning processes. Here we provide for the first time, a comprehensve analysis of developmentally regulated genes during murine limb development, and provide some novel insights into the expression dynamics governing limb morphogenesis. Fifty- one arrays were analyzed, consisting of whole fore-limb and hind-limb bud RNA (experimental) and whole embryo RNA (reference) samples from E9.5 to E13.5 DPC mouse (FVB strain). Embryos were not pooled to generate samples. Each time point has 3 to 5 biological replicates for limb bud samples, duplicates for whole embryos. Comparisons were made between limb bud samples and whole embryo at the same stage, fore-limb samples of different stages, hind-limb samples of different stages, and fore-limb samples compared to hind-limb samples at the same or the next stage.

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Sost and its paralog Sostdc1 coordinate digit number in a Gli3-dependent manner

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