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Identification of Kctd15 target genes in Xenopus animal caps injected with wnt3a and chordin

ABSTRACT: We aimed to identify genes that are inhibited by Kctd15 overexpression during neural crest (NC) induction. Injection of RNAs encoding Wnt3a and chordin into the embryo followed by animal cap explant culture leads to the induction of NC marker genes as well as additional genes expressed in the neural plate border. Our previous study indicated that co-injection of kctd15 inhibits the induction of these markers. We injected four sets of RNAs, lacZ as control (L), wnt3a+chd (WC), wnt3a+chd+kctd15 (WCK), and kctd15 (K). After evaluation of marker gene expression with RT-PCR, we prepared probes with same RNA extract for Affymetrix DNA microarray. The hybridization, washing and scanning were perfomed following the manual from Affymetrix. Partek Genomics Suite was employed for data analysis.

ORGANISM(S): Xenopus laevis

PROVIDER: GSE72391 | GEO | 2015/08/26

SECONDARY ACCESSION(S): PRJNA293938

REPOSITORIES: GEO

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Identification of Kctd15 target genes in Xenopus animal caps injected with wnt3a and chordin

Project description:We aimed to identify genes that are inhibited by Kctd15 overexpression during neural crest (NC) induction. Injection of RNAs encoding Wnt3a and chordin into the embryo followed by animal cap explant culture leads to the induction of NC marker genes as well as additional genes expressed in the neural plate border. Our previous study indicated that co-injection of kctd15 inhibits the induction of these markers. We injected four sets of RNAs, lacZ as control (L), wnt3a+chd (WC), wnt3a+chd+kctd15 (WCK), and kctd15 (K). After evaluation of marker gene expression with RT-PCR, we prepared probes with same RNA extract for Affymetrix DNA microarray. The hybridization, washing and scanning were perfomed following the manual from Affymetrix. Partek Genomics Suite was employed for data analysis. Xenopus embryos were injected with mRNAs at two-cell stage. When developing to stage 9, the animal caps were dissected for RNA extraction and hybridization on Affymetrix microarrays. According to the mRNAs injected, the animal caps (AC) were divided into four groups, including (1) AC injected with lacZ; (2) AC injected with kctd15; (3) AC injected with chordin and wnt3a; (4) AC injected with chordin, wnt3a and kctd15. The injected animal caps were cultured till stage 15 and then collected for RT-PCR and microarray assay. Each group had three repeats.

2015-08-26 | E-GEOD-72391 | biostudies-arrayexpress

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Fam46a is a regulator for gene expression in secretory tissues and plays an important role in development and homeostasis [Chordin]

Project description:In early embryonic development, the organiser functions in multiple developmental processes, including neural induction, dorsoventral patterning of the mesoderm, and formation of the axial mesoderm. Chordin is a central molecule for the activity of the Spemann’s organiser and abundantly secreted from the Spemann’s organiser in Xenopus laevis. However, so far, a whole picture of the gene expression profile regulated by Chordin has not been examined. Here, we perform microarray analysis of chordin overexpression and identify fam46a as an upregulated gene. We demonstrate that fam46a is essential for Xenopus laevis early embryonic development as well as gene induction during cell differentiation in mammalian cell culture models. fam46a is preferentially expressed in tissues with high secretory ability. In Xenopus laevis, fam46a is required for normal neural tube closure and expression of epidermal markers, neural markers and mesodermal markers. In the human Caco-2 cell model, FAM46A is localised in the nucleus and cytoplasm, and regulates gene induction during cell differentiation. In the mouse intestinal organoid model, Fam46a is required for survival of organoids as well as enterocyte differentiation. These results indicate that fam46a is a regulator for gene expression in secretory cells during cell differentiation in early embryonic development and adult homeostasis.

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Endoderm induction by the organizer-secreted factors chordin and noggin in Xenopus animal caps.

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A Robust Method To Derive Functional Neural Crest Cells From Human Pluripotent Stem Cells

2013-08-16 | GSE44727 | GEO

A Robust Method To Derive Functional Neural Crest Cells From Human Pluripotent Stem Cells

Project description:Neural crest (NC) cells contribute to the development of many complex tissues. The abnormal development of NC cells accounts for a number of congenital birth defects. Generating NC cells, and more specifically NC subpopulations such as cranial, cardiac, and trunk NC cells from human induced pluripotent stem (iPS) cells and human embryonic stem (ES) cells presents a valuable tool to model and study human NC development and disease. Here we provide a robust, efficient, and reproducible protocol for the differentiation of human iPS and ES cells into NC cells. The protocol has been validated in multiple human pluripotent stem cell lines and yields relatively pure NC cell populations in eight days. The resulting cells can be propagated and retain NC marker expression over multiple passages. The NC cells show proper cell specification and can develop into NC-derived cell lineages including smooth muscle cells, peripheral neurons, and Schwann cells. Additionally, the NC cells are functional and migrate to appropriate chemoattractants such as SDF-1, Fgf8b, BMP2, and Wnt3a. Importantly, this method generates all NC subpopulations (cranial, cardiac, and trunk) providing a great advantage to readily available NC differentiation methods. Neural crest cells derived from human induced pluripotent stem cells were profiled using Affymetrix Gene 1.0 arrays to identify differential gene expression changes and alternative exons from the open-source software AltAnalyze. An FDR adjusted emperical Bayes moderated t-test p < 0.05 was used to identify differentially expressed Ensembl genes and GO-Elite used to identify biologically relevant, Ontology, pathway and gene-set categories. Alternative exons were obtained using the FIRMA analysis option and default thresholds. Other array neural crest array and RNA-Seq dataset were compared to this to identify common and distinct regulatory mechanisms.

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Expression data that were specifically regulated by lncRNA-LFAR1 in livers of mice undergoing hepatic fibrosis.

Project description:Long noncoding RNAs (lncRNAs) play important roles in various biological processes; however, few have been identified that regulate hepatic stellate cells (HSCs) activation and the progression of liver fibrosis. Through a detailed analysis of the expression of lncRNAs in various tissues, we discovered the existence of a liver enriched lncRNA-LFAR1 (lncRNA-Liver Fibrosis Associated RNA1). To identify the roles of lncRNA-LFAR1 in liver fiboris, we systematically analyzed the regulation of mRNAs in the livers of mice treated with oil in combination with injection of Lenti-NC (NC, n=3), CCl4 in combination with injection of Lenti-NC (NC+CCl4, n=3), oil in combination with injection of Lenti-shLFAR1 (shLFAR1, n=3) and CCl4 in combination with injection of Lenti-shLFAR1 (shLFAR1+CCl4, n=3) by mRNA microarrays, which revealed a panel of mRNAs that were specifically regulated by lncRNA-LFAR1 in livers of mice undergoing hepatic fibrosis. To identify the roles of lncRNAs-LFAR1 in regulating liver fiboris and the potential targets of lncRNA-LFAR1 in liver fiboris,we determined the mRNA expression profiles in the livers of Balb/c mice treated with oil in combination with injection of Lenti-NC (NC, n=3), CCl4 in combination with injection of Lenti-NC (NC+CCl4, n=3), oil in combination with injection of Lenti-shLFAR1 (shLFAR1, n=3) and CCl4 in combination with injection of Lenti-shLFAR1 (shLFAR1+CCl4, n=3) by mRNA microarrays.

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Xenopus laevis

Project description:Identification of Kctd15 target genes in Xenopus animal caps injected with wnt3a and chordin

| PRJNA293938 | ENA