Transcription profiling of B7.5 lineage cells in the tunicate Ciona intestinalis to study cardiac cell migration
ABSTRACT: Eight different types of sample (three samples each) were used in this study of cardiac cell migration in the tunicate Ciona intestinalis. The B7.5 lineage cells were isolated by Fluorescent Activated Cell Sorting (FACS) at the tailbud stage, when the cardiac precursors (anterior B7.5 lineage cells) are migrating into the trunk, while their sister cells (posterior 7.5 lineage cells) remain in the tail, where they form muscle cells. Cells were sorted at the late gastrula stage, prior to migration induction and after manipulation of the FGF-MAPK-Ets signaling pathway (targeted expression of dominant negative FGF receptor and Ets:VP16 fusion protein, targeted expression was achieved using the Mesp cis-regulatory DNA), FoxF function (a direct target of FGF-MAPK-Ets signaling, required primarily for cell migration) by targeted expression of FoxF:VP16 (constitutive activator) or FoxF:WRPW (constitutive repressor, dominant negative), Mesp function (over-expression of Mesp:VP16 inhibits primarily cell migration). Finally, the "whole embryo" samples were collected from dissociated whole tailbud embryos, without FACS. in summary the eight conditions are: "wt (files LC-wt-1,-2,-3)": wild_type B7.5 lineage cells, expressing GFP, sorted at the tailbud stage "Ets:VP16 (files LC-EV-1,-2,-3)": B7.5 lineage cells, expressing GFP and Ets:VP16, sorted at the tailbud stage "DnFGFR (files LC-dnFGFR-1,-2,-3)": B7.5 lineage cells, expressing GFP and dominant-negative FGF receptor, sorted at the tailbud stage. "FoxF:VP16 (files LC-FV-1,-2,-3)": B7.5 lineage cells, expressing GFP and FoxF:VP16, sorted at the tailbud stage. "FoxF:WRPW (files LC-FW-1,-2,-3)": B7.5 lineage cells, expressing GFP and FoxF:WRPW, sorted at the tailbud stage". "Mesp:VP16 (files LC-MV-1,-2,-3)": B7.5 lineage cells, expressing GFP and Mesp:VP16, sorted at the tailbud stage. "Late Gastrula (files LC-LG-1,-2,-3)": wild_type B7.5 lineage cells, expressing GFP, sorted at the late gastrula stage "whole embryo (files LC-whole-1,-2,-3": whole embryos, dissociated at the tailbud stage, not sorted.
Gene regulatory networks direct the progressive determination of cell fate during embryogenesis, but how they control cell behavior during morphogenesis remains largely elusive. Cell sorting, microarrays, and targeted molecular manipulations were used to analyze cardiac cell migration in the ascidian Ciona intestinalis. The heart network regulates genes involved in most cellular activities required for migration, including adhesion, cell polarity, and membrane protrusions. We demonstrated that f ...[more]
Project description:Its known that FGF signaling induces pigment cell precursors formation. We perturbed the endogenous FGF signaling cascade by using a dominant-negative form of the unique Ci-FGF receptor (FGFRDN) and a constitutively active form of Ci-Ets1/2 (Ets:Vp16), a transcriptional effector of FGF/MAP Kinase cascade. Targeted expression of dominant negative FGF receptor and Ets:VP16 fusion protein was achieved in PCPs using the Tyrosinase-related protein 1/2a (Tyrp1/2a) cis-regulatory region. The PCPs were isolated by Fluorescent Activated Cell Sorting (FACS) as GFP+ cells at two developmental stages: 8 hpf at ~16C (neurula stage) corresponding to early FGF-mediated induction and 12 hpf at ~18C (tailbud stage) where PCPs are already fate restricted as pigment cells. To avoid a contamination by mesenchyme cells where Ciona enhancers are often ectopically activated, we co-electroporated the MyoD905>YFP construct and counter-selected YFP+ cells. In summary the conditions used are: FGFRDN, Ets:VP16 and Control samples at 8 and 12 hpf (four samples for each condition).
Project description:Despite being found in the notochord of several chordates, the roles of the Tbx2 subfamily of T-box transcription factors in the development of this tissue remain largely unknown. We explored the targets of the only Tbx2 subfamily member in Ciona intestinalis, Ci-Tbx2/3, by expressing mutant forms of the transcriptional regulator using the Ci-Bra promoter region. We produced a dominant interfering version of Ci-Tbx2/3 (Tbx2/3DBD) through expression of a truncated version consisting only of its DNA-binding domain (DBD) and a constitutive activator form by attaching the T-box of Ci-Tbx2/3 to the VP16 transactivation domain (Tbx2/3VP16). These constructs were introduced into 1-cell stage embryos and grown to the neurula (N) or mid-tailbud (mTb) stage to capture targets regulated throughout notochord morphogenesis. Ci-Tbx2/3 targets were ascertained using whole-genome custom Affymetrix microarrays to compare the transcription levels of Tbx2/3DBD and Tbx2/3VP16 expressing embryos to wild-type controls (Bra>GFP) at the neurula or mTb stages.
Project description:The Ciona heart progenitor lineage (TVC, trunk ventral cells) is first specified by Fibroblast Growth Factor/Map Kinase (FGF/MapK) activation of the transcription factor Ets1/2 (Ets). For this analysis, B7.5 lineage cells were labeled with the Mesp-GFP reporter. In the first experiment, we targeted a dominant-negative form of the sole Ciona FGF receptor (FGFRdn) to the B7.5 lineage using the Mesp enhancer (Mesp-FGFRdn). In the second experiment, we targeted a dominant repressor form of Ets1/2 to the B7.5 lineage using the Mesp enhancer (Mesp-EtsWRPW). This construct is designed to repress Ets1/2 target gene transcription and has previously been shown to abolish TVC induction. We employed whole-genome microarray analysis of sorted B7.5 lineage cells to identify primary FGF:MapK:Ets1/2 target genes.
Project description:This study examined transcripts that are enriched in neonatal mouse cochlear hair cells. Hair cells were purified by FACS sorting for GFP fluorescence from the cochleas of transgenic mice in which the endogenous Atoh1 gene was fused with GFP Two replicates of GFP+ hair cells were compared with all other cochlear cell types that were GFP-
Project description:Epidermis of Xenopus embryos forms a mucociliary epithelium constituted of basal, scattered, secreting and ciliated cells and is histologically similar to human airway mucociliary epithelium. We compared microRNAs signatures of epidermis of Xenopus embryos at stage 11.5 (gastrula, non ciliated epidermis) and at stage 26 (tailbud, ciliated epidermis). 2 technical replicates of a pool of 50 explants for each stage 11.5 (non ciliated) and 26 (ciliated) of Xenopus laevis development
Project description:Title: Regulation of gene expression through mitogen-activated protein kinase cascades in cardiac myocytes.<br/> Description: We aim to identify the changes in gene expression induced in rat <br/> neonatal ventricular myocytes, a well established cell culture model, <br/> by phenylepherine, a known hypertrophic agonist, and to determine which <br/> of the changes are mediated through the ERK cascade. In addition, we <br/> intend to extend the previous studies (EXP_TKAC_0102_01 and <br/> EXP_TKAC_1103_02) on endothelin-1 induced hypertrophy and the oxidative <br/> stress induced by H2O2 by using additional concentrations and timepoints.
Project description:Title: Changes in gene expression affected by H2O2 in cardiac myocytes.<br/> Description: We aim to identify the changes in gene expression in response to <br/> oxidative stress in rat neonatal ventricular myocytes.<br/> Oxidative stress will be induced by dosing neonatal ventricular myocyte<br/> cultures with 0.2, 0.1 and 0.04mM hydrogen peroxide at 2, 4 and 8 hr time<br/> points using unstimulated myocytes as control.
Project description:We previously showed that eRF3a depletion in the human colon carcinoma cell line HCT116 p53+/+ induces mTOR signaling pathway inhibition (Chauvin et al., 2007) suggesting that eRF3a belongs to a regulatory mechanism that modulate mTOR activity. However, this mechanism and the precise role of eRF3a remain to be determined. Here, to elucidate the mechanism dictating mTOR signaling pathway inhibition, we aimed to define the transcript expression and polysome profiles of HCT116 cells after short interfering RNA (siRNA)-mediated depletion of eRF3a. Three days after HCT116 cells electroporation with either sh-3a1 which was previously shown for effectively silence eRF3a (Chauvin et al., 2005) or control shRNA (sh-Ctrl), total and polysome-associated RNAs were extracted and we conducted a microarray analysis of differentially expressed genes using the Illumina HumanHT-12 Expression BeadChips.
Project description:Title: Regulation of gene expression through mitogen-activated protein kinase cascades in cardiac myocytes.<br/> Description: The aim of this study is to identify the changes in gene expression induced in rat neonatal <br/> ventricular myocytes, a well-established cell culture model, by endothelin-1, <br/> a known hypertrophic agonist, and to determine which of the changes are <br/> mediated through the ERK cascade. This continues TKACs previous study of the <br/> effects of oxidative stress, which induces cardiac myocyte apoptosis.<br/>