Identif. of oscillatory genes in somitogenesis from functional genomic analysis of C2C12 myoblast line
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE7012 | GEO | 2007/05/01
SECONDARY ACCESSION(S): PRJNA104043
REPOSITORIES: GEO
Similar Datasets
Project description: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. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. 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. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Keywords: time series
2007-05-01 | GSE7015 | GEO
Project description: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. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. 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. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Experiment Overall Design: Synchronization of C2C12 myoblasts 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 5% FBS then incubated in DMEM with only 0.2% FBS for 24 hours, and returned to DMEM supplemented with FBS. Samples were collected every 30 min. for 8 hours.
2008-06-15 | E-GEOD-7012 | biostudies-arrayexpress
Project description: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. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 hour period, consistent with available data on the rate of somitogenesis in humans. 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. Expression patterns of these genes were disrupted in Wnt3atm1Amc mutants but not in Dll3pu mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro. Experiment Overall Design: Synchronization of human umbilical cord blood derived stem/stromal cell (population 1) 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 30 min. intervals from 0 to 8 hours, and then at 1 hour intervals from 9 to 24 hours.
2008-06-15 | E-GEOD-7015 | biostudies-arrayexpress
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 GeneChips®). 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.
2008-10-21 | E-GEOD-11854 | biostudies-arrayexpress
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 GeneChips®). 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. Keywords: time-course
2008-06-23 | GSE11854 | GEO
Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.
2009-03-11 | GSE15153 | GEO
Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.
2009-03-11 | GSE15152 | GEO
Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function. Experiment Overall Design: To enrich for genes in the presomitic mesoderm that are specifically disrupted by Dll3 mutation, we compared microdissected tissues from wild-type and Dll3 mutant embryos. We generated biological replicate pools from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of six pools. Microarray analysis using Affymetrix MOE430A arrays was carried out on the biological pool triplicates for both wild-type and mutant genotypes.
2009-03-14 | E-GEOD-15152 | biostudies-arrayexpress
Project description:Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rphn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a; regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function. Experiment Overall Design: To enrich for genes in the somite level tissues that are specifically disrupted by Dll3 mutation, we compared microdissected tissues from wild-type and Dll3 mutant embryos. We generated biological replicate pools from Dll3+/+ (wild-type) or Dll3neo/neo embryos for a total of six pools. Microarray analysis using Affymetrix MOE430A arrays was carried out on the biological pool triplicates for both wild-type and mutant genotypes.
2009-03-14 | E-GEOD-15153 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series:; GSE7012: Identif. of oscillatory genes in somitogenesis from functional genomic analysis of C2C12 myoblast line; GSE7015: Identif. of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model Experiment Overall Design: Refer to individual Series
2008-06-15 | E-GEOD-7054 | biostudies-arrayexpress