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Genome-wide analysis of HoxBlinc lncRNA downstream targets in mouse ESC-derived embryonic bodies

ABSTRACT: We find a new non-coding transcript associated with active HoxB locus, which we named HoxBlinc lncRNA. We report that HoxBlinc RNA specifies Flk1+ mesoderm and then promotes the development of HS/PCs and cardiomyocytes. To understand the molecular pathways reguated by HoxBlinc, we generate two doxycycline inducible KD ES R1E cell lines, then differentiate these cell lines to day 6 via embryonic bodies stage. RNA-seq analysis were performed for day 6 EBs with or without doxycycline treatment starting at Flk1+ mesodermal stage. RNA from day 6 EBs targeting with 2 invidiual shRNAs with or without doxycycline treatment were combined together for RNA-seq analysis.

ORGANISM(S): Mus musculus

SUBMITTER: Lei Zhou

PROVIDER: E-GEOD-74894 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Genome-wide analysis of HoxBlinc lncRNA downstream targets in mouse ESC-derived embryonic bodies

Project description:We find a new non-coding transcript associated with active HoxB locus, which we named HoxBlinc lncRNA. We report that HoxBlinc RNA specifies Flk1+ mesoderm and then promotes the development of HS/PCs and cardiomyocytes. To understand the molecular pathways reguated by HoxBlinc, we generate two doxycycline inducible KD ES R1E cell lines, then differentiate these cell lines to day 6 via embryonic bodies stage. RNA-seq analysis were performed for day 6 EBs with or without doxycycline treatment starting at Flk1+ mesodermal stage.

2015-11-11 | GSE74894 | GEO

Expression data from differentiating Flk1- and Flk1+ ES cells expressing Snail during Wnt inhibition

Project description:ES cells differentiated in the presence of the Wnt inhibitor DKK1 fail to express the transcription factor Snail and undergo EMT or mesoderm differentiation. We generated an ES cell line, A2.snail, that induced Snail expression upon addition of doxycycline addition. Microarrays were used to gain a global picture of Flk1- and Flk1+ cells generated one day after Snail was expressed during Wnt inhibition. A2.snail ES cells, which express Snail upon addition of doxycycline, were differentiated as embryoid bodies in differentiation media and DKK1. Snail-induced cultures uniquely develop a select population of Flk1+ cells. Total RNA was harvested from sorted control (no doxycycline) Flk1- cells and sorted Snail-induced (doxycycline at day 2) Flk1- and Flk1+ cells at day 3 of differentiation.

2013-10-23 | E-GEOD-26524 | biostudies-arrayexpress

Transcription profiling of mouse embryoid bodies with HOXB4 induced or not induced to identify its target genes

Project description:To unravel the molecular mechanism by which HOXB4 promotes the expansion of early hematopoietic progenitors within differentiating ES cells, we analzed the gene expression profiles of embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced. A substantial number of the identified HOXB4 target genes are involved in signaling pathways important for controlling self-renewal, maintenance and differentiation of stem cells. Furthermore, we demonstrate that HOXB4 activity and FGF-signaling are intertwined. HOXB4-mediated expansion of ES cell-derived early progenitors was enhanced by specific and complete inhibition of FGF-receptors. In contrast, the expanding activity of HOXB4 on hematopoietic progenitors in day4-6 embryoid bodies was blunted in the presence of basic FGF (FGF2) indicating a dominant negative effect of FGF-signaling on the earliest hematopoietic cells. Taken together, we show that modulation of FGF signaling is an essential feature of HOXB4 activity in the context of embryonic hematopoiesis. Experiment Overall Design: The Hoxb4i ES cell line (Kyba et al. 2002, Cell 109:29-37) contains an integrated âtet-onâ cassette that allows induction of HOXB4 expression upon treatment with doxycycline. These ES cells can be used to produce hematopoietic cells through the formation of embryoid bodies (EBs). Hematopoiesis starts in these EBs at day 4 and the differentiation into hematopoietic fates can be quantified by colony assays on methyl-cellulose using cells dissociated from EBs at day 6 of incubation. The induction of HOXB4 by incubation with doxycycline increases the production of hematopoietic progenitors within EBs by day 6. Using this specific ES cell line, we compared the transcriptome between embryoid bodies (EBs) in which transcription of HOXB4 had been induced or not induced from day 4 to day 6 (48hours). Experiment Overall Design: Biological replicates: 3

2008-06-16 | E-GEOD-9044 | biostudies-arrayexpress

Expression data of control (F/F) and Snai1 knockout (KO) Flk1+/- cells

Project description:Snai1 is a master factor of epithelial to mesenchymal transitioin (EMT), however, its role in embryonic stem cell (ESC) differentiation and lineage commitment remains undefined. We used microarrays to compare the global programme of gene expression between control and Snai1 knockout Flk1+ and Flk1- cells sorted from 4 day EBs. Control and Snai1 knockout ESCs were cultured as embryoid bodies in spotaneous differentiation media, 4 days EBs were dissociated and sorted by anti-Flk1 antibody to separated Flk1+ and Flk1- cells, total RNA were collected for Affymetrix microarrays

2013-12-10 | E-GEOD-53108 | biostudies-arrayexpress

Expression data of control (F/F) and Snai1 knockout (KO) Flk1+/- cells

2013-12-10 | GSE53108 | GEO

Transcription profiling of mouse ES cell derived brachyury+ (T) cell specific transcriptome

Project description:This experiment is part of the FunGenES project (FunGenES - Functional Genomics in Embryonic Stem Cells partially funded by the 6th Framework Programme of the European Union, http://www.fungenes.org). The experiment was conducted at University of Cologne, Cologne, Germany. Goal of the experiment was an analysis the transcriptome of the ES-cell derived pure T-brachyury expressing CGR8 cells. Materials and methods: On day 0, EBs were made from T-brachyury ES (CGR8) cell line by hanging drop protocol. On second day, the hanging drop EBs were transferred into suspension. On third day, the EBs were treated or untreated with puromycin at a concentration of 5ug/ml for 2 days. On 5th day, the medium was changed and added medium with or without puromycin at a concentration of 2 ug/ml for 1 day. On day 6th , RNA was isolated from EBs treated or untreated with puromycin for affymetrix analysis.

2010-05-12 | E-TABM-716 | biostudies-arrayexpress

Induced overexpression of Pdx1 and Ngn3 in a mouse ES cell-derived endoderm population induces pancreatic differentiation

Project description:Induced overexpression of Pdx1 in activin-induced endoderm population resulted in the upregulation of pancreas-related genes such as insulin 1 and 2 at day 20. To enhance the developmental progression from the pancreatic bud to the formation of the endocrine lineages, we next expressed neurogenin3 (Ngn3) together with Pdx-1. Induced overexpression of Pdx1 together with Ngn3 dramatically increased Insulin 1 mRNA by day 9 differentiation. The levels of insulin 1 mRNA present in the induced EBs represented approximately 100 % of that found in insulinoma cell line, betaTC6. We also confirmed insulin and C-peptide staining by immunohistochemistry. These cells process and secrete insulin and respond to various insulin secretagogues. These inductive effects were restricted to c-kit+ endoderm enriched EB-derived populations suggesting that Pdx1/Ngn3 functions at the level of pancreatic specification of endoderm in this model. Microarray analysis showed that Pdx1/Ngn3 regulated the expression of a broad spectrum of pancreatic endocrine cell-related genes. On day 4 of a multiday differentiation protocol, EBs were dissociated and cultured in serum-free growth medium supplemented with differentiation facots, with or without Dox (1 ug/ml). On day 6, EBs were replated on gelatin in 12-well low-cluster dishes (Nunc) to obtain non-adherent floating EBs with or without Dox (1 ug/ml) and cultured out to day 13, at which time RNA was harvested for microarray analysis.

2010-08-24 | E-GEOD-23752 | biostudies-arrayexpress

Scl specifies hemogenic endothelium and inhibits cardiogenesis via primed enhancers [ChIP-seq]

Project description:Scl/Tal1 confers hemogenic competence and prevents cardiomyogenesis in embryonic endothelium. Here we show that Scl both directly activates a broad gene regulatory network required for hematopoietic stem/progenitor cell (HS/PC) development, and represses transcriptional regulators required for cardiogenesis. Cardiac repression occurs during a short developmental window through Scl binding to distant cardiac enhancers that harbor H3K4me1 at this stage. Scl binding to hematopoietic regulators extends throughout HS/PC and erythroid development and spreads from distant enhancers to promoters. Surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic versus cardiac specification and Scl binding to the majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors to facilitate HS/PC emergence from hemogenic endothelium. These results uncover a dual function for Scl in dictating hematopoietic versus cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates. ChIP-seq with Scl, Hand1, Lsd1, Ezh2, H3K4me1 and H3K27ac in different cell types with mesodermal origin. Scl ChIP-seq in WT, SclKO, SclKO-iScl and Gata12KO mES cell derived day4 EB (embryoid body) Flk1+ mesodermal cells, SclKO-iScl ES cells and MEL cells; Hand1 ChIP-seq in WT mES cell derived day4 EB Flk1+ mesodermal cells; Lsd1 and Ezh2 ChIP-seq in WT and SclKO mES cell derived day4 EB Flk1+ mesodermal cells. ChIP-seq of histone modifications H3K4me1 and H3K27ac in WT, SclKO and Gata12KO mES cell derived day4 EB Flk1+ mesodermal cells, HPC7 hematopoietic progenitor cells and HL1 cardiomyogenic cells

2015-01-01 | E-GEOD-47082 | biostudies-arrayexpress

Expression data from mouse embryonic stem cell differentiation experiment

Project description:We differentiated mouse embryonic stem (mES) cells spontaneously into embryoid bodies (EBs). Gene expression of biological replicates of undifferentiated ES cells (0-day), 4-day, 8-day and 14-day EBs were measured by Affymetrix microarrays. Keywords: time course Mouse embroynic stem cells were spontaneously into EBs. The gene expression was measured on undifferentiated mES cells on gelatin (0-day), undifferentiated mES cells sorted by FACS on Oct4 GFP-day (0-day), 4-day, 8-day and 14-day EBs.

2010-03-09 | E-GEOD-12550 | biostudies-arrayexpress

Differentiating i-Mixl1 mESCs cultured in the presence or absence of Doxycycline

Project description:We profiled gene expression changes in differentiating i-Mixl1 ES cells(Willey, Ayuso-Sacido et al., 2006, Blood 107(8): 3122-3130) cultured in the presence or absence of Doxycycline (DOX, 0.1 ug/ml, 3 replicates per treatment/time point). Total RNA was isolated from EBs harvested at day 2, 3 and 4 (DOX added 1 day after plating of ES cells). RNA (1 ug) was subjected to one round of linear amplification (RiboAmp System) to yield 10 ug of RNA. The RNA was indirectly labeled using amino allyl-dUTP('t Hoen, de Kort et al., 2003, Nucleic Acids Res. 31: e20), then conjugated with Cy3 or Cy5. The labeled RNAs were used to screen a 15K mouse developmental cDNA microarray(Tanaka, Jaradat et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97: 9127-9132). Pairwise analysis of hybridization results for EBs cultured with or without DOX was performed for samples harvested on each day. Spotfire(R) software was used for data management and filtering. Gene expression ratios were normalized after filtering the data to remove low-intensity and poor quality spots. Data obtained for replicate samples were in excellent agreement. Six experiments total: three time points (day 2, day 3, day 4); for each time point mRNA was collected in the presence or absence of Doxycycline. Three biological replicas were collected for each of the six experiments

2015-09-01 | E-GEOD-40703 | biostudies-arrayexpress

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