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Next generation sequencing of chicken otocyst with pharmacologicaly induced loss-of-Wnt signalling

ABSTRACT: Bulk RNA-sequencing of individual E2.5 chicken otocyst incubated in media enriched with Wnt signalling inhibitor IWR-1 or control condition (DMSO) for 24h. Transcripts abundance was mapped to a chicken reference genome using Kallisto package and differentially expressed genes were identify using Sleuth package (genes with p-value < 0.05 were considered as significant). Biological Function enrichment analysis (Toppgene online tool) showed that many of these genes were associated with neurogenesis.

ORGANISM(S): Gallus gallus

PROVIDER: GSE161638 | GEO | 2021/04/01

REPOSITORIES: GEO

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Next generation sequencing of chicken otocyst with pharmacologically induced loss-of-Wnt signalling

Project description:Bulk RNA-sequencing of individual E2.5 chicken otocyst incubated in media enriched with Wnt signalling inhibitor IWR-1 or control condition (DMSO) for 24h. Transcripts abundance was mapped to a chicken reference genome using Kallisto package and differentially expressed genes were identify using Sleuth package (genes with p-value < 0.05 were considered as significant). The expression of 1340 genes was significantly changed:733 genes were up-regulated and 607 were down-regulated. Biological Function enrichment analysis (Toppgene online tool) showed that many of these genes were associated with neurogenesis.

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Next generation sequencing of chicken otocyst with genetically induced loss-of-Wnt signalling

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Project description:Bulk RNA-sequencing of individual chicken otocysts, in which Notch activity was either stimulated or blocked. To stimulate Notch activity, the E2 otic placodes were in ovo transfected with a construct overexpressing Notch1 Intracellular domain (NICD1) or a control plasmid expressing mRFP1 and collected 6h or 24h later. To pharmacologically block Notch activity, E2.5 chicken otocysts were incubated in media enriched with γ-secretase Inhibitor or control condition (DMSO) for 6h or 24h. Transcripts abundance was mapped to a chicken reference genome using Kallisto package.

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Analysis of Gene Expression in the Chicken Otocyst

Project description:The inner ear arises from multipotent placodal precursors that are gradually committed to the otic fate and further differentiate into all inner ear cell types, with the exception of a few neural crest-derived cells. The otocyst has a pivotal role during inner ear development: otic progenitor cells sub-compartmentalize into non-sensory regions and regions giving rise to prosensory domains where subsequently also hair cells differentiate. The genes and pathways underlying this progressive subdivision and differentiation process are not entirely known. The goal of this study was to identify a comprehensive set of genes expressed in the chicken otocyst using the serial analysis of gene expression (SAGE) method. Our analysis revealed several hundred transcriptional regulators, potential signaling proteins, and receptors. We identified a substantial collection of genes that were previously known in the context of inner ear development, but we also found many new candidate genes, such as Sox4, Sox5, Sox7, Sox8, Sox11, and Sox18, which previously were not known to be expressed in the developing inner ear. Despite its obvious limitation of not being all-inclusive, the generated otocyst SAGE library is a practical bioinformatics tool to study otocyst gene expression and to identify candidate genes for developmental studies. Otocysts were dissected from HH stage 18-19 chicken embryos, total RNA was extracted, and subjected to a commercial long-SAGE protocol, resulting in a library of concatemerized tags. 3,512 individual clones of SAGE concatemers were sequenced resulting in 39,326 17-bp tags with tag counts up to 718 for the most abundant tag; 3,292 tags that were represented between two and five times, whereas the majority of tags (11,717) were only found once. Overall, we identified 16,008 unique sequence tags.

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Analysis of Gene Expression in the Chicken Otocyst

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Expression data from E10.5 mouse otocyst

Project description:We established a novel EGFP reporter mouse line (named Tg(ETAR-EGFP)14Imeg), which enables the placode-derived inner ear sensory cell lineage to be visualized and monitored. At E10.5, EGFP expression was detected in the ventral and dorsomedial region of the otocyst. Using this reporter line and FACS-array technology, we performed gene expression profiling focused on the regional specificity of the otocyst. We sorted E10.5 otocyst epithelial cells of heterozygous mice into EGFP-positive and EGFP-negative cells for RNA extraction and hybridization on Affymetrix microarrays. We profiled the expression of the genes in FACS-enriched cell population and conducted transcriptome analysis.

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Expression data from E10.5 mouse otocyst

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Next generation sequencing of chicken otocyst with pharmacologicaly induced loss-of-Wnt signalling

Project description:Next generation sequencing of chicken otocyst with pharmacologicaly induced loss-of-Wnt signalling

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Next generation sequencing of chicken otocyst with pharmacologically induced loss-of-Wnt signalling

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Transcriptomic differences between mouse tumor-infiltrating and splenic regulatory T cells with or without Treg-specific loss of CD177

Project description:The goal of this study was to identify transcriptomic differences between splenic regulatory T cells and tumor-infiltrating regualtory T cells with or without Treg-specific loss of CD177. Methods: MC38 tumors from CD177flox/flox/FOXP3-CreYFP and CD177flox/flox C57Bl/6 mice were ezymatically dissocaited and a single cell suspension was obtained. Spleens from the same mice were collected and single cell suspension was obtained by mechanical dissociation. Regulatory T cells, identified as CD45+ CD4+ CD25hi GITR+, were isolated by flourenscence activated cell sorting. RNA from the regualtory T cell populations were isolated and libraries were generated using the NextSeq500 system. Samples were aligned using Kallisto pseudoalignment protocol and the mm19 mouse genome. Counts were processed and aggregated by gene symbol using the sleuth R package. Results: The pro-survival gene Bcl2l1 is selectivley upregulated in tumor-infiltratign regulatory T cells compared to splenic T cells

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