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Gene expression profiling in Ciona TVCs with FGF-MAPK perturbation [bulk RNA-seq]

ABSTRACT: 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.

ORGANISM(S): Ciona robusta

PROVIDER: GSE99845 | GEO | 2017/06/15

SECONDARY ACCESSION(S): PRJNA389948

REPOSITORIES: GEO

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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. 1823 FACS purified Ciona cardiopharyngeal progenitor cells at successive developmental stages (12, 14, 16, 18, 20 hpfs) have been sequenced in this research, encompassing the developmental spectrum from single multipotent progenitors to diverse fate-restricted progenitor cells. 1796 out of 1823 cells have reads successfully mapped to Ciona genome (i.e only 1796 samples have FPKM data in the *txt processed data files). We adopted multiple quality control criteria to filter out low quality single cell transcriptomes, the contaminating subpopulations and the doublets. Eventually, 848 high-quality cells were retained for further analysis. Based on previously identified cell type specific markers and the well established lineage tree, we identified all five cardiopharyngeal progenitor subtypes (TVC, STVC, ASM, FHP, SHP) and in silico reconstructed three unidirectional trajectories corresponding to the specification of pharyngeal and cardiac fate. Our study enabled us to characterize the global gene expression patterns of heterogeneous cardiopharyngeal progenitors, and interrogate the spatial-temporal dynamics of cardiopharyngeal specification.

Project description:In vertebrates, pluripotent pharyngeal mesoderm progenitors produce the cardiac precursors of the second heart field as well as the branchiomeric head muscles and associated stem cells. However, the cellular and molecular mechanisms underlying the transition from multipotent progenitors to distinct heart and muscle precursors remain obscured by the complexity of vertebrate embryos. Here, using the ascidian Ciona intestinalis as a simple chordate model for cardiopharyngeal development, we show that bipotent progenitors are transcriptionally primed to activate both heart and pharyngeal muscle regulatory programs, which become restricted to the corresponding precursors following a conserved pattern of asymmetric divisions. Localized expression of COE (Collier/OLF1/EBF) then orchestrates the transition to a pharyngeal muscle fate both by promoting an MRF (Myogenic Regulatory Factor)-associated core myogenic program in myoblasts and by maintaining an undifferentiated state in their sister precursors through Notch-mediated lateral inhibition. Using single cell lineage tracing, we show that the latter are stem-like muscle precursors, which form most of the juvenile body wall muscles following proliferation, self-renewal, re-activation of MRF, and migration. We discuss the implications of our findings for the development and evolution of the cardiopharyngeal mesoderm in chordates. We combined fluorescence-activated cell sorting (FACS) and whole genome transcription profiling following perturbations of COE function to characterize the transcriptional dynamics underlying the specification of heart and ASM precursors in the ascidian cardiopharyngeal lineage. We used whole genome transcription profiling of FACS-purified cell populations isolated from 21 hpf larvae expressing FoxF>COE, FoxF>COE::WRPW or the FoxF>NLS::lacZ control. To gain insights into the transcriptional dynamics underlying fate specification in the cardiopharyngeal lineage, we also purified B7.5-lineage cells from control embryos and larvae collected every two hours from 8 to 28 hpf. This time window encompasses all developmental transitions from early TVC specification till ASM ring formation and initial differentiation.

Project description:During development, stem and progenitor cells divide and transition through germ layer- and lineage-specific multipotent states to generate the diverse cell types that compose an animal. Defined changes in biomolecular composition underlie the progressive loss of potency and acquisition of lineage-specific characteristics. For example, multipotent cardiopharyngeal progenitors display multilineage transcriptional priming, whereby both the cardiac and pharyngeal muscle programs are partially active and coexist in the same progenitor cells, while their daughter cells engage in a cardiac or pharyngeal muscle differentiation path only after cell division. Here, using the tunicate Ciona, we studied the acquisition of multilineage competence and the coupling between fate decisions and cell cycle progression. We showed that multipotent cardiopharyngeal progenitors acquire the competence to produce distinct Tbx1/10 (+) and (-) daughter cells shortly before mitosis, which is necessary for Tbx1/10 activation. By combining transgene-based sample barcoding with single cell RNA-seq (scRNA-seq), we uncovered transcriptome-wide dynamics in migrating cardiopharyngeal progenitors as cells progress through G1, S and G2 phases. We termed this process “transcriptome maturation”, and identified candidate “mature genes”, including the Rho GAP-coding gene Depdc1, which peak in late G2. Functional assays indicated that transcriptome maturation fosters cardiopharyngeal competence, in part through multilineage priming and proper oriented and asymmetric division that influences subsequent fate decisions, illustrating the concept of “behavioral competence”. Both classic feedforward circuits and coupling with cell cycle progression drive transcriptome maturation, uncovering distinct levels of coupling between cell cycle progression and fateful molecular transitions. We propose that coupling competence and fate decision with the G2 and G1 phases, respectively, ensures the timely deployment of lineage-specific programs.

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Gene expression profiling in Ciona TVCs with FGF-MAPK perturbation [bulk RNA-seq]

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