Ascidians with very similar embryos but highly divergent genomes are thought to have undergone extensive developmental system drift. We compared, in four species ( and for Phlebobranchia, and for Stolidobranchia), gene expression and gene regulation for a network of six transcription factors regulating peripheral nervous system (PNS) formation in . All genes, but one in , were expressed in the PNS with some differences correlating with phylogenetic distance. Cross-species transgenesis indicated strong levels of conservation, except in , in gene regulation despite lack of sequence conservation of the enhancers. Developmental system drift in ascidians is thus higher for gene regulation than for gene expression and is impacted not only by phylogenetic distance, but also in a clade-specific manner and unevenly within a network. Finally, considering that is divergent in our analyses, this suggests deep conservation of developmental mechanisms in ascidians after 390 My of separate evolution.