Unknown,Transcriptomics,Genomics,Proteomics

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Microarray data of differentiating embryonic stem cells overexpressing the transcription factor Msgn1

ABSTRACT: During mammalian gastrulation, pluripotent epiblast stem cells migrate through the primitive streak to form the multipotent progenitors of the mesoderm and endoderm germ layers. Msgn1 is a bHLH transcription factor and is a direct target gene of the Wnt/bcatenin signaling pathway. Msgn1 is expressed in the mesodermal compartment of the primitive streak and is necessary for the proper development of the mesoderm. Msgn1 mutants show defects in somitogenesis leading to a lack of trunk skeletal muscles, vertebra and ribs. To study the molecular and cellular function of Msgn1 in Embryonic Stem Cells (ESC), we have generated doxycycline inducible gain-of-function ESC to overexpress Msgn1 in ESC. In order to identify Msgn1 targets, we performed transcriptional profiling of Msgn1 expressing ES cells and found that upon induction of Msgn1, multiple genes in the Notch pathway were differentially expressed compared to the uninduced cells. Moreover, Whole Mount Insitu Hybridization analysis in Msgn1 null mutants revealed that these Notch pathway genes required Msgn1 for their proper expression in vivo. Our studies demonstrate that Msgn1 is a critical effector of the Wnt pathway during mammalian somitogenesis, mediating crosstalk between the Wnt and Notch pathways. Inducible A2lox-Flag Msgn1 ES cells were differentiated to form Embryoid bodies (EBs) for 2 days. Flag-Msgn1 was induced on day 2 with doxycycline and samples were collected at three time points, 12h, 24h and 48h after addition of doxycycline. Uninduced cells were used as controls. Experiments were performed in triplicate

ORGANISM(S): Mus musculus

SUBMITTER: Ravindra B Chalamalasetty

PROVIDER: E-GEOD-29848 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Expression of the proendocrine gene neurogenin 3 (Ngn3) is required for the development of pancreatic islets. In order to better characterize the molecular events regulated by Ngn3 during development, we have determined the expression profile of differentiating murine embryonic stem cells (mESCs) uniformly induced to overexpress Ngn3. An ESC line was created that allows for the induction of Ngn3 by adding doxycycline (Dox) to the culture medium. Genome-wide microarray analysis was performed to identify genes regulated by Ngn3 in a variety of both undifferentiated and differentiated conditions. Characterization of pancreatic developmental markers during embryoid body (EB) formation revealed an optimum context for Ngn3 induction. Neuroendocrine genes including neurogenic differentiation 1 (NeuroD1) and single minded 1 (Sim1) were found to be significantly upregulated. Genes regulated by Ngn3 independent of the context were analyzed using systematic gene ontology tools and revealed Notch signaling as the most significantly regulated signaling pathway (p=0.009). This result is consistent with the hypothesis that Ngn3 expression makes the cell competent for Notch signaling to be activated and conversely, more sensitive to Notch signaling inhibition. Indeed, EBs induced to express Ngn3 were significantly more sensitive to gamma-secretase inhibitor-mediated Notch signaling inhibition (p<0.0001). Moreover, we find that Ngn3 induction in differentiating ESCs results in significant increases in insulin, glucagon, and somatostatin transcription. Experiment Overall Design: 16 embryonic stem cell (ESC) samples (8 groups in duplicate) were processed for microarray analysis using the Affymetrix Mouse Genome 430 2.0 GeneChip. The samples included the parental Ainv15 ESC line (1) undifferentiated (Ainv15 ESC), (2) differentiated for 3 days as embryoid bodies (Ainv15 EB3) or (3) for 10 days as embryoid bodies (Ainv15 EB10). The remaining samples included tetracycline inducible Ngn3 ESC line derived from the parental Ainv15 ESC line (4) after differentiation and addition of doxycycline for 3 days without embryoid body formation (Ngn3 ES3 ON), (5 and 6) after differentiation as embryoid bodies for 3 days with (Ngn3 EB3 ON) or without (Ngn3 EB3 OFF) doxycycline, and (7 and 8) after differentiation as embryoid bodies for 10 days with (Ngn3 EB10 ON) or without (Ngn3 EB10 OFF) doxycycline.

Project description:Scoliosis is a common disorder, affecting one out of every twenty females. Some forms of scoliosis are congenital, resulting from disruptions in early spinal development. The spinal column is patterned during a cycling process called somitogenesis, and is the first musculoskeletal structure formed during development. Genes in the notch signaling pathway regulate somitogenesis, and display oscillatory expression in synchrony with somite formation. During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. The periodicity and components of the human clock have recently been described by our group (William et al., Dev Biol, 2007). In that publication, we showed that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression, including known cycling genes such as HES1 that displayed a period of 5 hours. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 hours, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Studies in mouse cell lines, including fibroblasts and PC12 neuronal cells, by R. Kageyema (Kyoto University, Hirata et al., Science, 2002) have shown that a number of mouse cell types can be synchronized to display oscillatory expression. In this study, we extend our analysis of human cells to human primary fibroblasts. Developing an in vitro model of somitogenesis using human mesenchymal stem cells and fibroblasts; a. Induce cycling gene expression in human mesenchymal stem cells (MSC) and fibroblasts and identify oscillatory genes (using Affymetrix U133 GeneChipsÂ®). Compare lists of oscillatory genes identified in these two human cell types. b. Characterize and validate mouse homologues of oscillatory genes for expression during somitogenesis. Using notch and wnt pathway mutants and cultured half embryos, determine if these oscillatory genes also display cycling expression during somitogenesis. We hypothesized that human fibroblasts could be synchronized to display oscillatory expression of notch and wnt pathway genes, including the marker gene HES1. Preliminary quantitative analysis by Taqman-based Real-Time PCR for these samples confirms that HES1 expression displays oscillatory expression. Homo sapiens fibroblasts were maintained in culture medium (DMEM high glucose with 10% fetal bovine serum, FBS), which was replaced twice weekly. The cells were then seeded at a density of 6,000-10,000 cells/cm2 and expanded in DMEM high glucose with 10% FBS. After 5-7 days of incubation at 37C in a humidified atmosphere containing 5% carbon dioxide, cells were detached with 0.05% trypsin for 2 minutes at confluency. Cells were synchronized using low serum treatment, which has been described previously for Hes1 (Hirata et al., 2002). Cell culture synchronization is required to assay oscillatory expression levels, otherwise the oscillations of individual cells would be out of phase and cancel one another. Synchronized cells will gradually desynchronize, leading to diminished oscillatory amplitude. Briefly, synchronization was carried out as follows: T-25 cm flasks were set up in parallel. These cells were grown to 90% confluence in DMEM supplemented with 10% FBS (UCB-MSC), then incubated in DMEM with only 0.2% FBS for 24 hours, and returned to DMEM supplemented with FBS. Samples were collected at 1 hr. intervals from 0 to 12 hours. In addition, an unsynchronized cell culture was sampled. Total RNA was isolated from cells in culture using the RNeasy Mini Kit (Qiagen) and quantitated using a Nanodrop 2000. Experiment Overall Design: A biological replicate of the submitted project (hCycle11) time-series was carried out in parallel has been collected (hCycle12). We have evaluated both biological replicates by Q-PCR. We have selected hCycle11 for HG-U133A Plus 2.0 analysis, and we reserve hCycle12 for possible future microarray analysis or comparison by Q-PCR.

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Microarray data of differentiating embryonic stem cells overexpressing the transcription factor Msgn1

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