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

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. To test hypotheses regarding properties of the C. robusta cardiopharyngeal lineage determinant Lhx3/4, we ectopically expressed the transcription factor in the C. robusta anterior neural plate lineage. Our control samples (in triplicate: C1, C2, C3) consist of RNA sequencing data from FACS-collected GFP+ cells from embryos electroporated with only Dmrt>GFP. The experimental samples (in triplicate: X1, X2, X3) consist of RNA sequencing data from FACS-collected GFP+ cells from embryos electroporated with Dmrt>GFP and Dmrt>Lhx3/4. We used DESeq2 to analyze Lhx3/4-driven transcriptomic changes.