No abstract

In this study, we utilize fluorescent activated cell sorting (FACS) of cells from transgenic zebrafish coupled with microarray analysis to globally analyze expression of cell type specific genes. We find that it is possible to isolate cell populations from Tg(fli1:egfp)(y1) zebrafish embryos that are enriched in vascular, hematopoietic and pharyngeal arch cell types. Microarray analysis of GFP+ versus GFP- cells isolated from Tg(fli1:egfp)(y1) embryos identifies genes expressed in hematopoietic, vascular and pharyngeal arch tissue, consistent with the expression of the fli1:egfp transgene in these cell types. Comparison of expression profiles from GFP+ cells isolated from embryos at two different time points reveals that genes expressed in different fli1+ cell types display distinct temporal expression profiles. We also demonstrate the utility of this approach for gene discovery by identifying numerous previously uncharacterized genes that we find are expressed in fli1:egfp-positive cells, including new markers of blood, endothelial and pharyngeal arch cell types. In parallel, we have developed a database to allow easy access to both our microarray and in situ results. Our results demonstrate that this is a robust approach for identification of cell type specific genes as well as for global analysis of cell type specific gene expression in zebrafish embryos.

Steroid hormones including (1,25)-dihydroxyvitamin D3, estrogens, and glucocorticoids control bone development and homeostasis. We show here that the osteogenic transcription factor Runx2 controls genes involved in sterol/steroid metabolism, including Cyp11a1, Cyp39a1, Cyp51, Lss, and Dhcr7 in murine osteoprogenitor cells. Cyp11a1 (P450scc) encodes an approximately 55-kDa mitochondrial enzyme that catalyzes side-chain cleavage of cholesterol and is rate limiting for steroid hormone biosynthesis. Runx2 is coexpressed with Cyp11a1 in osteoblasts as well as nonosseous cell types (e.g. testis and breast cancer cells), suggesting a broad biological role for Runx2 in sterol/steroid metabolism. Notably, osteoblasts and breast cancer cells express an approximately 32-kDa truncated isoform of Cyp11a1 that is nonmitochondrial and localized in both the cytoplasm and the nucleus. Chromatin immunoprecipitation analyses and gel shift assays show that Runx2 binds to the Cyp11a1 gene promoter in osteoblasts, indicating that Cyp11a1 is a direct target of Runx2. Specific Cyp11a1 knockdown with short hairpin RNA increases cell proliferation, indicating that Cyp11a1 normally suppresses osteoblast proliferation. We conclude that Runx2 regulates enzymes involved in sterol/steroid-related metabolic pathways and that activation of Cyp11a1 by Runx2 may contribute to attenuation of osteoblast growth.

Aim. Runx2 (AML3) transcription factor is the key regulator of osteoblastic lineage progression and isindispensable for the formation of mineral bones. Runx2 expression increases during differentiation of osteoblasts to induce osteoblast-specific genes necessary for the production and deposition of bone mineral matrix. However, Runx2 is also expressed at a lower level in early osteoprogenitors, where its function is less understood. Here we study how Runx2 determines the early stages of osteoblastic commitment using the model system of Runx2 re-introduction in mouse calvaria cells with Runx2 null background. Method. Affymetrix analysis, Western blot analysis and quantitative real-time reverse transcriptase PCR (qRT-PCR) analysis were employed. Results. Gene expression profiling by Affymetrix microarrays revealed that along with the induction of extracellular matrix and bone mineral deposition related phenotypic markers, Runx2 regulates several cell programs related to signaling and metabolism in the early osteoprogenitors. Particularly, Runx2 regulates transcription of genes involved in G-protein coupled signaling network, FGF and BMP/TGF beta signaling pathways and in biogenesis and metabolism pathways of steroid hormones. Conclusion. The data indicate that the lineage specific program, regulated by the master regulatory transcription factor, includes the regulation of cellular signaling and metabolism which may allow the committed cell to react and behave differently in the same microenvironment

The osteogenic and cell cycle regulatory functions of Runt‐related transcription factor 2 (Runx2) are perturbed in osteosarcoma cells. We investigated which target genes of Runx2 control proliferation of normal osteoblasts and osteosarcoma cells by Affymetrix expression profiling and ChIP‐on‐chip analysis using NimbleGen arrays. Target gene analysis shows that Runx2 modulates normal osteoblast proliferation by regulating distinct programs of genes involved in G protein‐coupled receptor signaling, sterol/steroid metabolism, the fibroblast growth factor/proteoglycan signaling axis, RNA helicases and heat shock proteins. Strikingly, our data reveal that the gene expression programs controlled by Runx2 in osteoprogenitors are fundamentally different from those in osteosarcomas. Thus cancer‐related perturbations in normal osteoblast growth and differentiation switch the biological function of Runx2 from a growth suppressor to a putative oncoprotein that supports the tumorigenic and/or metastatic potential of osteosarcoma cells.