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Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling

ABSTRACT: 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.

ORGANISM(S): Ciona robusta

PROVIDER: GSE250325 | GEO | 2023/12/21

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. 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.

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.

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Lhx3/4 initiates a cardiopharyngeal-specific transcriptional program in response to widespread FGF signaling

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