Transcriptional analysis of fluorescence-activated sorted Mesp-GFP-labeled B7.5 founder trunk ventral cell lineage cells from Ciona intestinalis
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ABSTRACT:
ORGANISM(S): Ciona intestinalis
PROVIDER: GSE38313 | GEO | 2012/06/05
SECONDARY ACCESSION(S): PRJNA168038
REPOSITORIES: GEO
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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.
2012-06-04 | E-GEOD-38313 | biostudies-arrayexpress
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).
2014-07-14 | E-MTAB-2759 | biostudies-arrayexpress
Project description: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.
2008-06-01 | E-MEXP-1478 | biostudies-arrayexpress
Project description:In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, likely mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell elongation. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates. Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling, and conclude that Islet is a key regulatory factor governing morphogenesis of the palps. Three biological replicates were analyzed for both sample types (Fox positive and Negative).
2014-05-23 | E-GEOD-57920 | biostudies-arrayexpress
Project description:In Ciona intestinalis, the palps consist of three conical protrusions within a field of thickened epithelium that form late in embryogenesis as tailbuds mature into larvae. The palp protrusions express the LIM-homeodomain transcription factor Islet. Protrusion occurs through differential cell elongation, likely mediated by Islet, as we find that ectopic expression of Islet is sufficient to promote cell elongation. FGF signaling is required for both Islet expression and palp morphogenesis. Importantly, we show that Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling. We conclude that Islet is a key regulatory factor governing morphogenesis of the palps. It is conceivable that Islet is also essential for the cellular morphogenesis of placode-derived sensory neurons in vertebrates. Islet expression can rescue the palp-deficient phenotype that results from inhibition of FGF signaling, and conclude that Islet is a key regulatory factor governing morphogenesis of the palps.
2014-05-23 | GSE57920 | GEO
Project description:Dynamic gene expression programs determine multipotent cell states and fate choices during development. Multipotent progenitors for cardiomyocytes and branchiomeric head muscles populate the pharyngeal mesoderm of vertebrate embryos, but the mechanisms underlying cardiopharyngeal multipotency and heart vs. head muscle fate choices remain elusive. The tunicate Ciona emerged as a simple chordate model to study cardiopharyngeal development with unprecedented spatio-temporal resolution. We analyzed the transcriptome of single cardiopharyngeal lineage cells isolated at successive time points encompassing the transitions from multipotent progenitors to distinct first and second heart, and pharyngeal muscle precursors. We reconstructed the three cardiopharyngeal developmental trajectories, and characterized gene expression dynamics and regulatory states underlying each fate choice. Experimental perturbations and bulk transcriptome analyses revealed that ongoing FGF/MAPK signaling maintains cardiopharyngeal multipotency and promotes the pharyngeal muscle fate, whereas signal termination permits the deployment of a full pan-cardiac program and heart fate specification. We identified the Dach1/2 homolog as a novel evolutionarily conserved second-heart-field-specific factor and demonstrate, through lineage tracing and CRISPR/Cas9 perturbations, that it operates downstream of Tbx1/10 to actively suppress the first heart lineage program. This data indicates that the regulatory state of multipotent cardiopharyngeal progenitors determines the first vs. second heart lineage choice, and that Tbx1/10 acts as a bona fide regulator of cardiopharyngeal multi potency. We performed bulk RNAseq to profile the FACS purified Ciona Robusta Truck Ventral Cells (TVCs) with FGF-MAPK perturbation conditions to address the question- What is the role of FGF signaling pathway during early cardiopharyngeal specification. we performed bulk RNA sequencing of FACS-purified cardiopharyngeal lineage cells isolated from embryos and larvae expressing either a dominant negative form the fibroblast growth factor receptor (dnFGFR), or a constitutively active form of M-Ras (caM-Ras), the sole Ras homolog in Ciona, under the control of TVC-specific enhancers.
2017-06-15 | GSE99845 | GEO
Project description:Ciona intestinalis is an invertebrate animal model system that is well characterized and has many advantages for the study of cardiovascular biology. The regulatory mechanisms of cardiac myocyte proliferation in Ciona are intriguing since Ciona are capable of regeneration throughout their lifespan. To identify important regeneration factors in Ciona, microarray analysis was conducted on RNA from adult Ciona hearts with normal or damaged myocardium using custom Affymetrix GeneChips. After a 24- or 48-hour recovery period, total RNA was isolated from damaged and control hearts. Initial results indicate significant changes in gene expression in hearts damaged by ligation in comparison to cryoinjured or control hearts. Ligation injury shows differential expression of 223 genes as compared to control (fold change >2, p<0.01, Student’s t-test) with limited false discovery (5.8%). Among these 223 genes, 117 have known human orthologs of which 68 were up-regulated and 49 were down-regulated. Notably, FGF 9/16/20 and Ras were significantly upregulated in injured hearts. Histological analyses of injured myocardium were conducted in parallel to the microarray study. Taken together, these studies will coordinate differences in gene expression to cellular changes in the regenerative myocardium of Ciona, which will help to elucidate the regulatory mechanisms of cardiac myocyte proliferation across chordates.
2024-02-06 | GSE244713 | GEO
Project description:Individual signaling pathways, such as fibroblast growth factors (FGFs), can regulate a plethora of inductive events. According to current paradigms, signal-dependent transcription factors, such as FGF/MapK-activated Ets family factors, partner with lineage-determining factors to achieve regulatory specificity. However, many aspects of this model have not been rigorously investigated. One key question relates to whether lineage-determining factors dictate lineage-specific responses to inductive signals or facilitate these responses in collaboration with other inputs. We utilize the chordate model Ciona robusta to investigate mechanisms generating lineage-specific induction. Previous studies in C. robusta have shown that cardiopharyngeal progenitor cells are specified through the combined activity of FGF-activated Ets1/2.b and an inferred ATTA-binding transcriptional cofactor. Here we show that the homeobox transcription factor Lhx3/4 serves as the lineage-determining transcription factor that dictates cardiopharyngeal-specific transcription in response to pleiotropic FGF signaling. Targeted knockdown of Lhx3/4 leads to loss of cardiopharyngeal gene expression. Strikingly, ectopic expression of Lhx3/4 in a neuroectodermal lineage subject to FGF-dependent specification leads to ectopic cardiopharyngeal gene expression in this lineage. Furthermore, ectopic Lhx3/4 expression disrupts neural plate morphogenesis, generating aberrant cell behaviors associated with execution of incompatible morphogenetic programs. Based on these findings we propose that combinatorial regulation by signal-dependent and lineage-determinant factors represents a generalizable, previously uncategorized regulatory subcircuit we term cofactor-dependent induction. Integration of this subcircuit into theoretical models will facilitate accurate predictions regarding the impact of gene regulatory network rewiring on evolutionary diversification and disease ontogeny.
2023-12-21 | GSE250325 | GEO
Project description:XBP transcription factors are well-known regulators of the the unfolded protein response and are required for plasma cell differentiation. However, recent studies have shown that the XBP1 gene is also expressed in the developing notochord of Ciona and various vertebrates, and to date its role in the formation of this organ is largely uncharacterized. We identified putative targets of Ci-XBP1 through a microarray screen, using RNAs extracted from embryos expressing mutant forms of this transcriptional regulator in the notochord. We expressed in the Ciona notochord a dominant-negative version of Ci-XBP1 (XBP DBD::GFP) by cloning its DNA-binding domain (DBD) downstream of the Ci-Brachyury (Ci-Bra) promoter. We created a constitutive activator form of Ci-XBP by fusing its DBD to the VP16 transactivation domain (Bra>XBP DBD::VP16::GFP). These constructs were introduced into 1-cell stage embryos and grown to the initial tailbud (iTb) stage to identify Ci-XBP notochord target genes.
2022-01-20 | GSE46751 | GEO
Project description:Ciona intestinalis, Ciona roulei, and Ciona robusta raw sequence reads from ddRADseq
| PRJNA759193 | ENA