Lineage-specific change in craniofacial gene regulatory networks of syngnathid fishes revealed by integrating multiomics across fishes

Cranial neural crest (CNC) cells are essential developmental contributors to the remarkable diversity of vertebrate skull shapes, yet how underlying gene regulatory networks (GRNs) evolve to produce highly derived morphologies remains a challenging question. Syngnathid fishes (seahorses, pipefishes, pipehorses, and seadragons) are an opportune family of species in which to address this problem because of their unusual and extensive cranial diversity and their loss of craniofacial patterning genes, fgf3 and fgf4 . Here we investigated whether syngnathid craniofacial evolution experienced only a few localized network changes or required global rewiring of CNC GRNs. Using comparative single-cell RNA sequencing, ATAC-seq and whole genome alignments across Gulf pipefish, threespine stickleback, and zebrafish, we found that the core pharyngeal arch CNC gene network is notably conserved in syngnathids despite their derived morphology. However, we identified key local changes including expression of fgf22 in percomorph CNC-derived pharyngeal arch cells that is not shared with more basally diverging zebrafish, as well as syngnathid-specific changes in conserved regulatory elements associated with the genes ece1 and spry2 . We propose that, while loss of fgf3 expression causes severe craniofacial defects in zebrafish, pharyngeal CNC expression of fgf22 in the percomorph fish lineage provided functional redundancy and relaxed constraint on fgf3/4 , and that altered regulation of Fgf pathway modulators could contribute to craniofacial elaboration. Our findings support a model in which local GRN modifications, rather than widespread network rewiring, underlie the evolution of derived syngnathid craniofacial structures.