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. Overall design: 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.