Project description:Morphogenesis of the mammalian neural tube (NT) is reliant on precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube defects (NTDs). MicroRNAs (miRNAs) as well as their downstream messenger RNA (mRNA) targets are key modulators of cell and tissue differentiation. To define potential roles of miRNAs and mRNAs in development of the murine neural tube (NT), total RNA preparations from gestation day (GD)-8.5, -9.0 and -9.5 (crtitcal period of NT developemnt) mouse embryos were used to hybridize both microRNA arrays and messenger RNA arrays, to identify miRNAs and mRNAs co-expressed in the same developing NT tissue samples.
Project description:Morphogenesis of the mammalian neural tube (NT) is reliant on precise, spatio-temporal expression of numerous genes and coordinated interaction of signal transduction and gene regulatory networks, disruption of which may contribute to the etiology of neural tube defects (NTDs). MicroRNAs (miRNAs) as well as their downstream messenger RNA (mRNA) targets are key modulators of cell and tissue differentiation. To define potential roles of miRNAs and mRNAs in development of the murine neural tube (NT), total RNA preparations from gestation day (GD)-8.5, -9.0 and -9.5 (crtitcal period of NT developemnt) mouse embryos were used to hybridize both microRNA arrays and messenger RNA arrays, to identify miRNAs and mRNAs co-expressed in the same developing NT tissue samples.
Project description:At an incidence of approximately 1/1000 births, neural tube defects (NTDs) comprise one of the most common and devastating congenital disorders. In an attempt to enhance and expand our understanding of neural tube closure, we undertook a high-throughput gene expression analysis of the neural tube as it was forming in the mouse embryo. Open and closed sections of the developing neural tube were micro-dissected from mouse embryos, and hybridized to Affymetrix mouse expression arrays. Clustering of genes differentially regulated in open and closed sections of the developing neural tube highlighted molecular processes previously recognized to be involved in neural tube closure and neurogenesis. Analysis of the genes in these categories identified potential candidates underlying neural tube closure. In addition, we identified approximately 25 novel genes, of unknown function, that were significantly up-regulated in the closed neural tube. Based on their expression patterns in the developing neural tube, five novel genes are proposed as interesting candidates for involvement in neurogenesis. The high-throughput expression analysis of the neural tube as it forms allows for better characterization of pathways involved in neural tube closure and neurogenesis, and hopefully will strengthen the foundation for further research along the pathways dictating neural tube development. Embryos were dissected at days E8.5 and E9.5, and the neuroepithelium/ neural tube were mechanically detached from underlying tissues, and then separated into two regions: 1) M-bM-^@M-^\open neuroepitheliumM-bM-^@M-^]: neuroepithelial tissue caudal to the open/closed junction, and 2) M-bM-^@M-^\closed neural tubeM-bM-^@M-^], extending from a somiteM-bM-^@M-^Ys breadth rostral to the open/closed junction, up to the level of the fifth- or sixth-to-last somite. Samples consisted of biological triplicates of RNA extract from the above tissues (pooled by litter, and representing a total of 111 embryos): E8.5 open neuroepithelium, E8.5 closed neural tube, E9.5 open neuroepithelium, and E9.5 closed neural tube. Thus, a total of 12 samples (representing 111 embryos) were hybridized to the GeneChip Mouse Genome 430 2.0 Array (Affymetrix Inc., Santa Clara, CA, USA). One of the samples (06, closed E8.5) deviated significantly from the others in quality assessment and was therefore removed from subsequent analysis and not submitted to GEO.
Project description:RNA-seq differential gene expression analysis was accomplished in E9.5 pooled(n = approximately 30) microdissected heart tubes from Sox7-null embryos and a wild-type littermates.
Project description:At an incidence of approximately 1/1000 births, neural tube defects (NTDs) comprise one of the most common and devastating congenital disorders. In an attempt to enhance and expand our understanding of neural tube closure, we undertook a high-throughput gene expression analysis of the neural tube as it was forming in the mouse embryo. Open and closed sections of the developing neural tube were micro-dissected from mouse embryos, and hybridized to Affymetrix mouse expression arrays. Clustering of genes differentially regulated in open and closed sections of the developing neural tube highlighted molecular processes previously recognized to be involved in neural tube closure and neurogenesis. Analysis of the genes in these categories identified potential candidates underlying neural tube closure. In addition, we identified approximately 25 novel genes, of unknown function, that were significantly up-regulated in the closed neural tube. Based on their expression patterns in the developing neural tube, five novel genes are proposed as interesting candidates for involvement in neurogenesis. The high-throughput expression analysis of the neural tube as it forms allows for better characterization of pathways involved in neural tube closure and neurogenesis, and hopefully will strengthen the foundation for further research along the pathways dictating neural tube development.
Project description:Purpose: Study the developing neural cell fate of the D1 blastomere in the 8-cell embryo. Determine what transcripts are dependant on cell/cell signaling and which are inherit to the single cell and do not depend on the rest of the embryo. Methods: D1 blastomere was either injected with fluorescent dye or explanted away from the developing embryo. Transcript profile was taken at 7 time points: 8-cell, 16-cell, 32-cell, blastula, gastrula, early neurula, and mid/late neurula. 3 biological triplicates were taken at each time point for both the dissect and explant condition. Results: Up until blastula stage, dissect and explant transcript profiles remain fairly similar. But following this the profiles completely diverge, with the dissect tissue heading towards the neural fate while the explant samples head towards an ectoderm/epithelial fate.
Project description:In this work, we developed a microprobe capillary electrophoresis high-resolution mass spectrometry approach to identify proteins in single identified cells in live embryos of the South African clawed frog (Xenopus laevis) and also zebrafish. We used a pulled borosilicate capillary to aspirate a calibrated portion of the cell of interest. Proteins in the collected aspirate were detected and quantified via a bottom-up proteomic workflow that was downscaled to the single cells. The microprobe mass spectrometry platform is scalable to smaller cells in live, morphologically complex, developing embryos. As an example, we used this approach to analyze proteomic changes in cells that form a clone in the early developing Xenopus laevis embryo.