Modeling Genetic Networks from Clonal Analysis

In this report a systematic approach is used to determine the approximate genetic network and robust dependencies underlying differentiation. The data considered is in the form of a binary matrix and represent the expression of the nine genes across the ninety-nine colonies. The report is divided into two parts: the first part identifies significant pair-wise dependencies from the given binary matrix using linear correlation and mutual information. A new method is proposed to determine statistically significant dependencies estimated using the mutual information measure. In the second, a Bayesian approach is used to obtain an approximate description (equivalence class) of network structures. The robustness of linear correlation, mutual information and the equivalence class of networks is investigated with perturbation and decreasing colony number. Perturbation of the data was achieved by generating bootstrap realizations. The results are refined with biological knowledge. It was found that certain dependencies in the network are immune to perturbation and decreasing colony number and may represent robust features, inherent in the differentiation program of osteoblast progenitor cells. The methods to be discussed are generic in nature and not restricted to the experimental paradigm addressed in this study.Comment: 59 pahes, 11 figures, 3 table

The PPARgamma-selective ligand BRL-49653 differentially regulates the fate choices of rat calvaria versus rat bone marrow stromal cell populations

Abstract Background Osteoblasts and adipocytes are derived from a common mesenchymal progenitor and an inverse relationship between expression of the two lineages is seen with certain experimental manipulations and in certain diseases, i.e., osteoporosis, but the cellular pathway(s) and developmental stages underlying the inverse relationship is still under active investigation. To determine which precursor mesenchymal cell types can differentiate into adipocytes, we compared the effects of BRL-49653 (BRL), a selective ligand for peroxisome proliferators-activated receptor (PPAR)γ, a master transcription factor of adipogenesis, on osteo/adipogeneis in two different osteoblast culture models: the rat bone marrow (RBM) versus the fetal rat calvaria (RC) cell system. Results BRL increased the number of adipocytes and corresponding marker expression, such as lipoprotein lipase, fatty acid-binding protein (aP2), and adipsin, in both culture models, but affected osteoblastogenesis only in RBM cultures, where a reciprocal decrease in bone nodule formation and osteoblast markers, e.g., osteopontin, alkaline phosphatase (ALP), bone sialoprotein, and osteocalcin was seen, and not in RC cell cultures. Even though adipocytes were histologically undetectable in RC cultures not treated with BRL, RC cells expressed PPAR and CCAAT/enhancer binding protein (C/EBP) mRNAs throughout osteoblast development and their expression was increased by BRL. Some single cell-derived BRL-treated osteogenic RC colonies were stained not only with ALP/von Kossa but also with oil red O and co-expressed the mature adipocyte marker adipsin and the mature osteoblast marker OCN, as well as PPAR and C/EBP mRNAs. Conclusion The data show that there are clear differences in the capacity of BRL to alter the fate choices of precursor cells in stromal (RBM) versus calvarial (RC) cell populations and that recruitment of adipocytes can occur from multiple precursor cell pools (committed preadipocyte pool, multi-/bipotential osteo-adipoprogenitor pool and conversion of osteoprogenitor cells or osteoblasts into adipocytes (transdifferentiation or plasticity)). They also show that mechanisms beyond activation of PPARγ by its ligand are required for changing the fate of committed osteoprogenitor cells and/or osteoblasts into adipocytes

The EP4-ERK-dependent pathway stimulates osteo-adipogenic progenitor proliferation resulting in increased adipogenesis in fetal rat calvaria cell cultures

We previously reported that fetal rat calvaria (RC) cells are osteo-adipogenic bipotential and that PGE2 receptors EP2 and EP4 are involved in bone nodule formation via both common and distinct MAPK pathways in RC cell cultures. Because PGE2 participates in multiple biological processes including adipogenesis, it is of interest to determine the additional role(s) of PGE2 in RC cells. PGE2 increased the number of adipocyte colonies when RC cells were treated during proliferation but not other development stages. Of four EP agonists tested, the EP4 agonist ONO-AE1-437 (EP4A) was the most effective in promoting adipogenesis. Concomitantly, EP4A increased the number of cells with BrdU labeling and gene expression of CCAAT/enhancer binding protein (C/EBP)δ and c-fos but not peroxisome proliferator-activated receptor γ2 and C/EBPα. Amongst MAPK inhibitors, U0126, an inhibitor of MEK1/2, abrogated the EP4A-dependent effects. Our results suggest that the PGE2–EP4-ERK pathway increases the number of osteo-adipogenic bipotential progenitor cells, with a resultant increase in adipogenesis in RC cell cultures.This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan (13771074 to YY)and Ono Pharmaceutical Co. (to YY), and the Canadian Institutes of Health Research (CIHR; FRN 83704 to JEA)

1α,25-dihydroxyvitamin D3 acts predominately in mature osteoblasts under conditions of high extracellular phosphate to increase fibroblast growth factor 23 production in vitro

Osteoblasts/osteocytes are the principle sources of fibroblast growth factor 23 (FGF23), a phosphaturic hormone, but the regulation of FGF23 expression during osteoblast development remains uncertain. Because 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and inorganic phosphate (Pi) may act as potent activators of FGF23 expression, we estimated how these molecules regulate FGF23 expression during rat osteoblast development in vitro. 1,25(OH)2D3-dependent FGF23 production was restricted largely to mature cells in correlation with increased vitamin D receptor (VDR) mRNA levels, in particular, when Pi was present. Pi alone and more so in combination with 1,25(OH)2D3 increased FGF23 production and VDR mRNA expression. Parathyroid hormone, stanniocalcin 1, prostaglandin E2, FGF2, and foscarnet did not increase FGF23 mRNA expression. Thus, these results suggest that 1,25(OH)2D3 may exert its largest effect on FGF23 expression/production when exposed to high levels of extracellular Pi in osteoblasts/osteocytes

Soluble Klotho causes hypomineralization in Klotho-deficient mice

The type I transmembrane protein αKlotho (Klotho) serves as a coreceptor for the phosphaturic hormone fibroblast growth factor 23 (FGF23) in kidney, while a truncated form of Klotho (soluble Klotho, sKL) is thought to exhibit multiple activities, including acting as a hormone, but whose mode(s) of action in different organ systems remains to be fully elucidated. FGF23 is expressed primarily in osteoblasts/osteocytes and aberrantly high levels in the circulation acting via signaling through an FGF receptor (FGFR)-Klotho coreceptor complex cause renal phosphate wasting and osteomalacia. We assessed the effects of exogenously added sKL on osteoblasts and bone using Klotho-deficient (kl/kl) mice and cell and organ cultures. sKL induced FGF23 signaling in bone and exacerbated the hypomineralization without exacerbating the hyperphosphatemia, hypercalcemia and hypervitaminosis D in kl/kl mice. The same effects were seen in rodent bone models in vitro, in which we also detected formation of a sKL complex with FGF23-FGFR and decreased Phex (gene responsible for X-linked hypophosphatemic rickets (XLH)/osteomalacia) expression. Further, sKL-FGF23-dependent hypomineralization in vitro was rescued by soluble PHEX. These data suggest that exogenously added sKL directly participates in FGF23 signaling in bone and that PHEX is a downstream effector of the sKL-FGF23-FGFR axis in bone.This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture, Japan (18592001 and 20592139 to Y Y and 21791788 to T M), and the Canadian Institutes of Health Research (MOP 83704 to J E A)

Conservation of core gene expression in vertebrate tissues

Abstract Background Vertebrates share the same general body plan and organs, possess related sets of genes, and rely on similar physiological mechanisms, yet show great diversity in morphology, habitat and behavior. Alteration of gene regulation is thought to be a major mechanism in phenotypic variation and evolution, but relatively little is known about the broad patterns of conservation in gene expression in non-mammalian vertebrates. Results We measured expression of all known and predicted genes across twenty tissues in chicken, frog and pufferfish. By combining the results with human and mouse data and considering only ten common tissues, we have found evidence of conserved expression for more than a third of unique orthologous genes. We find that, on average, transcription factor gene expression is neither more nor less conserved than that of other genes. Strikingly, conservation of expression correlates poorly with the amount of conserved nonexonic sequence, even using a sequence alignment technique that accounts for non-collinearity in conserved elements. Many genes show conserved human/fish expression despite having almost no nonexonic conserved primary sequence. Conclusions There are clearly strong evolutionary constraints on tissue-specific gene expression. A major challenge will be to understand the precise mechanisms by which many gene expression patterns remain similar despite extensive cis-regulatory restructuring

Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis.

International audienceBone sialoprotein (BSP) and osteopontin (OPN) are both highly expressed in bone, but their functional specificities are unknown. OPN knockout ((-/-)) mice do not lose bone in a model of hindlimb disuse (tail suspension), showing the importance of OPN in bone remodeling. We report that BSP(-/-) mice are viable and breed normally, but their weight and size are lower than wild-type (WT) mice. Bone is undermineralized in fetuses and young adults, but not in older (>/=12 mo) BSP(-/-) mice. At 4 mo, BSP(-/-) mice display thinner cortical bones than WT, but greater trabecular bone volume with very low bone formation rate, which indicates reduced resorption, as confirmed by lower osteoclast surfaces. Although the frequency of total colonies and committed osteoblast colonies is the same, fewer mineralized colonies expressing decreased levels of osteoblast markers form in BSP(-/-) versus WT bone marrow stromal cultures. BSP(-/-) hematopoietic progenitors form fewer osteoclasts, but their resorptive activity on dentin is normal. Tail-suspended BSP(-/-) mice lose bone in hindlimbs, as expected. In conclusion, BSP deficiency impairs bone growth and mineralization, concomitant with dramatically reduced bone formation. It does not, however, prevent the bone loss resulting from loss of mechanical stimulation, a phenotype that is clearly different from OPN(-/-) mice

The functional landscape of mouse gene expression

BACKGROUND: Large-scale quantitative analysis of transcriptional co-expression has been used to dissect regulatory networks and to predict the functions of new genes discovered by genome sequencing in model organisms such as yeast. Although the idea that tissue-specific expression is indicative of gene function in mammals is widely accepted, it has not been objectively tested nor compared with the related but distinct strategy of correlating gene co-expression as a means to predict gene function. RESULTS: We generated microarray expression data for nearly 40,000 known and predicted mRNAs in 55 mouse tissues, using custom-built oligonucleotide arrays. We show that quantitative transcriptional co-expression is a powerful predictor of gene function. Hundreds of functional categories, as defined by Gene Ontology 'Biological Processes', are associated with characteristic expression patterns across all tissues, including categories that bear no overt relationship to the tissue of origin. In contrast, simple tissue-specific restriction of expression is a poor predictor of which genes are in which functional categories. As an example, the highly conserved mouse gene PWP1 is widely expressed across different tissues but is co-expressed with many RNA-processing genes; we show that the uncharacterized yeast homolog of PWP1 is required for rRNA biogenesis. CONCLUSIONS: We conclude that 'functional genomics' strategies based on quantitative transcriptional co-expression will be as fruitful in mammals as they have been in simpler organisms, and that transcriptional control of mammalian physiology is more modular than is generally appreciated. Our data and analyses provide a public resource for mammalian functional genomics