Unknown,Transcriptomics,Genomics,Proteomics

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Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation [frog data]

ABSTRACT: Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to specific Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (Axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. Xenopus laevis embryos were fertilized in vitro according to standard protocols (Sive 2000) in 0.1X Marcâs Modified Ringerâs (MMR; 10mM Na+, 0.2mM K+,10.5mM Cl, 0.2 mM Ca2+, pH 7.8). Intracellular ion concentrations in Xenopus embryo are: 21mM Na+, 90mM K+, 60mM Cl-, 0.5mM Ca2+ (Gillespie 1983). Xenopus embryos were housed at 14-18oC and staged according to Nieuwkoop and Faber (Nieuwkoop 1967). All experiments were approved by the Tufts University Animal Research Committee (M2014-79) in accordance with the guide for care and use of laboratory animals. Capped synthetic mRNAs generated using mMessage mMachine kit (Ambion) were dissolved in nuclease free water and injected into the embryos in 3% Ficoll using standard methods (Sive 2000). Each injection delivered between 1-2 nL or 1â 2 ng of mRNA (per blastomere) into the embryos, usually at 4-cell stage into the middle of the cell in the animal pole. Constructs used were: GlyR (Davies et al. 2003) and 666 chimera (Hough et al. 2000).

ORGANISM(S): Xenopus laevis

SUBMITTER: Christopher Martyniuk

PROVIDER: E-GEOD-72097 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Depolarization of resting membrane potential in select cells in Xenopus larvae induces striking hyperpigmentation due to dysregulation of melanocytes. Here, we show that this non-cell-autonomous process is mediated by cAMP, CREB, and the transcription factors Sox10 and Slug. Our microarray analysis reveals specific transcripts responsive to Vmem levels within a few hours of depolarization, and a set of 517 transcripts whose expression remains altered during the full hyperpigmented phenotype over a week later, linking instructor cell-depolarization to a range of developmental processes and disease states. We also show that voltage-dependent conversion of melanocytes involves the MSH-secreting melanotrope cells of the pituitary, and formulate a model for the molecular pathway linking the bioelectric properties of melanocyte cellsâ microenvironment in vivo to the genetic and cellular changes induced in this melanoma-like phenotype. Remarkably, the phenotype is all-or-none: each individual animal either undergoes melanocyte conversion or not, as a whole. This group decision is stochastic, resulting in varying percentages of hyperpigmented individuals for a given experimental treatment. To understand the stochasticity and dynamic properties of this complex signaling system, we developed a novel computational method that automates the reverse-engineering of stochastic dynamic signaling models. We used this method to discover a network model that quantitatively explained our complex dataset, and even made correct predictions for new experiments that we validated in vivo. Taken together, these data (1) reveal new molecular details about a novel trigger of metastatic-like developmental cell behavior in vivo, (2) suggest new targets for biomedical intervention, and (3) demonstrate proof-of-principle of a computational method for understanding stochastic decision-making by cells during embryonic development and metastasis. Gene expression analysis was performed using samples treated with ivermectin from NF stage 10 onwards, collected at two developmental stages; stage 15 (early neurula) and stage 45 (free-swimming tadpole). Embryos were collected in eppendorf tubes (N=50 for stage 15, N=5 for stage 45) and frozen at -80Â°C. RNA extraction and microarray analysis were performed by the Beth Israel Deaconess Medical Center Genomics and Proteomics Center (Harvard) according to standard Affymetrix protocol, using a high throughput hybridization and scanning system. Microarray hybridization was performed using the Affy 3â IVT Express Kit. Fragmented and biotin labeled and amplified RNA was hybridized to the GeneChipÂ® Xenopus laevis Genome 2.0 array as per manufacturerâs protocol. The Affymetrix GeneChipÂ® X. laevis Genome 2.0 Array, has 32,400 probe sets representing more than 29,900 X. laevis transcripts.

Project description:Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to specific Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (Axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. All axolotls used in these experiments were bred in the axolotl facility at the University of Minnesota under the IACUC protocol #1201A08381. Axolotls of 2â3 cm were used for all in vivo experiments, and animals were kept in separate containers and fed daily with artemia; water was changed daily. Animals were anesthetized in 0.01% p-amino benzocaine (Sigma) before microinjection was performed. There were 9 microarrays conducted, and these were ivermectin-treated (n=3). Experimental Design: Ivermectin injection Ivermectin or vehicle only (water) was pressure injected into the central canal of the spinal cord, and this was visualized by the addition of Fast Green into the solution. Directly after injection, a portion of the spinal cord was surgically removed and the animals were placed back into water in individual containers. One day post injury (1dpi) animals were anesthetized again and the area of the injury was removed. Tissue from 10 animals were pooled for each microarray replicate. Mature - Uninjured spinal cord tissue Sc Crush- spinal cord tissue 1 day after injury Ivermectin - Ivermectin injected spinal cord tissue 1 day after injury.

Project description:Endogenous bioelectric signaling via changes in cellular resting potential (Vmem) is a key regulator of patterning during regeneration and embryogenesis in numerous model systems. Depolarization of Vmem has been functionally implicated in de-differentiation, tumorigenesis, anatomical re-specification, and appendage regeneration. However, no unbiased analyses have been performed to understand genome-wide transcriptional responses to Vmem change in vivo. Moreover, it is unknown which genes or gene networks represent conserved targets of bioelectrical signaling across different patterning contexts and species. Here, we use microarray analysis to comparatively analyze transcriptional responses to specific Vmem depolarization. We compare the response of the transcriptome during embryogenesis (Xenopus development), regeneration (Axolotl regeneration), and stem cell differentiation (human mesenchymal stem cells in culture) to identify common networks across model species that are associated with depolarization. Both sub-network enrichment and PANTHER analyses identified a number of key genetic modules as targets of Vmem change, and also revealed important (well-conserved) commonalities in bioelectric signal transduction, despite highly diverse experimental contexts and species. Depolarization regulates specific transcriptional networks across all three germ layers (ectoderm, mesoderm and endoderm) such as cell differentiation and apoptosis, and this information will be used for developing mechanistic models of bioelectric regulation of patterning. Moreover, our analysis reveals that Vmem change regulates transcripts related to important disease pathways such as cancer and neurodegeneration, which may represent novel targets for emerging electroceutical therapies. Whole bone marrow aspirate from a 25 year-old healthy man was purchased from Lonza through their Research Bone Marrow Donor Program, following approved guidelines of informed consent as previously documented (Sundelacruz et al. 2008, 2013b). Aspirate was plated at a density of 10 mL of aspirate per cm2 in control medium (DMEM with 10% fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 mg/mL), and 0.1 mM nonessential amino acids) supplemented with basic fibroblast growth factor (1 ng/mL) (Invitrogen). Cells were maintained in a humidified incubator at 37Â°C with 5% CO2. The hMSCs were isolated on the basis of their adherence to tissue culture plastic and were used for experiments between passages two and four. Undifferentiated hMSCs were cultured in control medium. Osteogenic (OS) differentiation medium consisted of Î±MEM medium supplemented with 10% FBS, penicillin (100 U/mL), streptomycin (100 mg/mL), 10mM Î²-glycerophosphate, 0.05mM L-ascorbic acid-2-phosphate, and 100nM dexamethasone (Sigma-Aldrich). Vmem was depolarized by (1) addition of Na+/K+ - ATPase-inhibitor ouabain (OB, 10 nM; Sigma-Aldrich) to differentiation medium or (2) elevation of extracellular K+ by adding potassium gluconate (High K+, HK, 40 mM; Sigma-Aldrich) to differentiation medium. Depolarization induced by these concentrations of OB and K+ has been confirmed using voltage-sensitive dyes and/or sharp intracellular recordings [(Sundelacruz et al. 2008) and unpublished data]. OS cells were pre-differentiated for three weeks before the addition of OB or HK for an additional three weeks of culture. The depolarizers were added to the differentiation medium and replenished in subsequent media changes.

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Genome-wide analysis reveals conserved transcriptional responses downstream of resting potential change in Xenopus embryos, axolotl regeneration, and human mesenchymal cell differentiation [frog data]

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