Next Generation Sequencing Analysis of gene expression profile in Dgcr8 conditional KO embryonic hearts
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE96646 | GEO | 2018/09/12
REPOSITORIES: GEO
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Project description:Purpose: To compare the single cell transcriptome of wild type and Mesp1Cre+/Dgcr8-/- embryonic heart at embryonic stage E9.5 Methods: For single cell sample preparation, Ventricular part of E9.5 heart tubes were dissected and digested into single cells by 0.04% Trypsin combined with 0.05% Collagenase IV, and then transferred into DMEM containing 10% fetal bovine serum for termination. After washed in PBS without Ca2+, the single cells were manually transferred into cell lysis buffer with a mouth pipette. Total RNA was reverse transcribed and amplified using Smart-seq2 protocol (Picelli et al., 2014), followed by Tn5 tagmentation and PCR enrichment to generate libraries using TruePrep DNA Library Prep Kit V2 for illumina (Vazyme, TD501-503) according to the manufacturer’s suggested protocol. Clean reads were mapped to mouse genome (mm10) using Salmon. Results: Genes significantly differentially expressed in the E9.5 Dgcr8 cKO single cardiomyocyte compared with wild type group were identified. Conclusions: Dgcr8 cKO caused important gene expression changes in E9.5 cardiomyocytes at single cell level
2018-09-12 | GSE110265 | GEO
Project description:Purpose: To compare the E9.5 Dgcr8 conditional knockout embryonic heart cells transfected with NC miRNA and miR-541 mimics Methods: In vitro cultured E9.5 Dgcr8 conditional KO heart cells transfected with miR-541-5p and NC miRNA were extracted with TRIZOL 48hrs after transfection, and 10ng total RNA was reverse transcribed and amplified by Smart-seq2 protocol as described (Picelli et al., 2014). Duplicated biological samples were analyzed using Illumina HiSeqX10, Clean reads were mapped to mouse genome (mm9) using BWA software. Results: Genes differentially expressed in E9.5 Dgcr8 cKO embryonic heart cells transfected with NC miRNA and miR-541 were identified. Conclusions: miRNA-541 significantly changes the gene expression profiles of E9.5 Dgcr8 cKO embryonic heart cells and promote the cardiac function
2018-09-12 | GSE96647 | GEO
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. For microarray analysis of CM-CKO embryos, embryo heads of four genotypes were collected at E8.5 (5-7 somites) and E9.5 (18- 20 somites): CM-CKO (Twist1flox/del; Mesp1Cre/+), CM-Het (Twist1flox/wt; Mesp1Cre/+), Het (Twist1flox/del; Mesp1+/+) and Control (Twist1flox/wt; Mesp1+/+). Sample sizes for E8.5 embryos were as follows: Control, n=4 CM-CKO, n=4; Het, n=3; CM-Hets, n=3).
2016-08-23 | E-GEOD-80334 | biostudies-arrayexpress
Project description:TWIST1, a basic helix-loop-helix transcription factor is essential for the development of cranial mesoderm and cranial neural crest-derived craniofacial structures. Our previous work showed that, in the absence of TWIST1, some cells within the cranial mesoderm adopt an abnormal epithelial configuration. Here, we show by transcriptome analysis that loss of TWIST1 in the cranial mesoderm is accompanied by a reduction in the expression of genes that are associated with cell-extracellular matrix interactions and the acquisition of mesenchymal characteristics. By comparing the transcriptional profiles of cranial mesoderm-specific Twist1 loss-of-function mutant and control mouse embryos, we identified a set of genes that are both TWIST1-dependent and predominantly expressed in the mesoderm. By ChIP-seq in a cell line model of a TWIST1-dependent mesenchymal state, we identified, among the downstream genes, three direct transcriptional targets of TWIST1: Ddr2, Pcolce and Tgfbi. Our findings show that the mesenchymal properties of the cranial mesoderm is likely to be regulated by a network of TWIST1 targets genes that influence the extracellular matrix and cell-matrix interactions, and collectively they are required for the morphogenesis of the craniofacial structures. For microarray analysis of CM-CKO embryos, embryo heads of four genotypes were collected at E8.5 (5-7 somites) and E9.5 (18- 20 somites): CM-CKO (Twist1flox/del; Mesp1Cre/+), CM-Het (Twist1flox/wt; Mesp1Cre/+), Het (Twist1flox/del; Mesp1+/+) and Control (Twist1flox/wt; Mesp1+/+). Sample sizes for E9.5 were as follows: Control, n=3; CM-CKO, n=4; Het, n=4; CM-Het, n=4). RNA was extracted using the RNeasy Micro kit (Qiagen) and samples sent to the Australian Genome Research Foundation for labelling and hybridization.
2016-08-23 | E-GEOD-80509 | biostudies-arrayexpress
Project description:Morphogenesis of the heart is a complex process that relies on the precise gene expression and gene expression regulators during embryonic development. Dgcr8 is a gene involved in cardiac morphogenesis and it is in the chromosomal region deleted in 22q11.2DS patients. In order to study Dgcr8 function on heart development, we inactivated this gene in the cardiac progenitor cells of mouse embryos and did expression profiling of the long RNAs and miRNAs. We used microarrays to detail the global programme of gene expression downstream of DGCR8 underlying cardiac development.
2023-10-01 | GSE186064 | GEO
Project description:DiGeorge syndrome critical region 8 (DGCR8) is a critical component of the canonical microprocessor complex for microRNA biogenesis. However, the non-canonical functions of DGCR8 have not been studied. Here, we demonstrate that DGCR8 plays an important role in maintaining heterochromatin organization and attenuating aging. An N-terminal-truncated version of DGCR8 (DR8dex2) accelerated senescence in human mesenchymal stem cells (hMSCs) independent of its miRNA-processing activity, which is mediated by its C-terminal domains. Further studies revealed that DGCR8 maintained heterochromatin organization by interacting with the nuclear envelope protein Lamin B1, and heterochromatin-associated proteins, KAP1 and HP1 Overexpression of any of these proteins, including DGCR8, reversed premature senescent phenotypes in DR8dex2 hMSCs. Finally, DGCR8 was downregulated in pathologically and naturally aged hMSCs, whereas DGCR8 overexpression alleviated hMSC aging and osteoarthritis in mice. Taken together, these analyses uncovered a novel, miRNA processing-independent role for DGCR8 in maintaining heterochromatin organization and attenuating senescence. DGCR8 may therefore represent a new therapeutic target for alleviating human aging-related disorders.
2019-05-28 | PXD013856 | Pride
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.
2014-01-01 | E-GEOD-39442 | biostudies-arrayexpress
Project description:Next Generation Sequencing Analysis of gene expression profile in E9.5 Mesp1Cre/+/Dgcr8-/- embryonic heart cells transfected with NC miRNA and miR-541 mimics
| PRJNA379286 | ENA
Project description:To determine whether DGCR8 is required for maturation of all miRNAs, we performed miRNA microarray analysis. Using RNA from wild-type ES cells as our reference sample, we observed a global loss of miRNAs in DGCR8 knockout cells, but normal levels of expression in DGCR8 heterozygous cells. The similarity in expression levels between wild-type and heterozygous cells suggests that DGCR8 is not limiting in the maintenance of steady-state levels of miRNAs in ES cells. Of the eighty-nine miRNA array probes that showed significant signals with wild-type RNA, eighty-two were drastically reduced in the DGCR8 knockout cells. The remaining seven were not significantly altered in the DGCR8 knockout cells, but at least four of these seven miRNAs appear to be due to unavoidable contamination with RNA from the mouse embryonic fibroblast (MEF) feeder cells used for ES cell culture prior to the isolation of RNA. These miRNAs were highly expressed in the MEFs and decreased when the ES cells were temporarily passaged on gelatin-coated plates without feeders. The remaining three showed similar levels of expression in the heterozygous, knockout and MEF feeder cells. Therefore, these signals may be small RNAs that are not processed with the help of the microprocessor complex. Alternatively, these signals may result from unavoidable degradation products within the purified small RNA population. In either case, our results show that DGCR8 is broadly required for miRNA processing with little evidence for redundancy or bypass mechanisms. Keywords: cell type comparison, genetic modification MicroRNAs extracted from wild-type, DGCR8 heterozygous knockout and DGCR8 homozygous knockout were analyzed by microarray. In each array, wild-type samples serve as reference. Ratios were normalized based on positive control RNAs on the array. To determine if some of signals appeared in DGCR8 knockout ES cells are due to mice embryonic fibroblast (MEF) feeder cell contamination (this is unavoidable because these ES cells were grown on MEFs and passaged off MEF before RNA extraction). Two independent DGCR8 knockout ES cell lines were analyzed. For each cell line, array hybridization was done in duplicates. For DGCR8 heterozygous knockout ES cells, two independent batches of RNA samples were prepared and analyzed.
2010-06-11 | E-GEOD-6586 | biostudies-arrayexpress
Project description:We investigated the effect of Dgcr8-homozygous mutation on microRNA expression profile in mouse embryonic stem cells. MicroRNA expression was substantially impaired, indicating a pivotal role of DGCR8 in microRNA biogenesis.
2010-09-01 | GSE20268 | GEO