The Cis-Regulatory Code for Kelch-like 21/30 Specific Expression in Ciona robusta Sensory Organs

The tunicateCiona robustais an emerging model system to study the evolution of the nervous system. Due to their small embryos and compact genomes, tunicates, likeCiona robusta, have great potential to comprehend genetic circuitry underlying cell specific gene repertoire, among different neuronal cells. Their simple larvae possess a sensory vesicle comprising two pigmented sensory organs, the ocellus and the otolith. We focused here onKlhl21/30, a gene belonging toKelchfamily, that, inCiona robusta, starts to be expressed in pigmented cell precursors, becoming specifically maintained in the otolith precursor during embryogenesis. Evolutionary analyses demonstrated the conservation ofKlhl21/30in all the chordates.Cis-regulatory analyses and CRISPR/Cas9 mutagenesis of potential upstream factors, revealed thatKlhl21/30expression is controlled by the combined action of three transcription factors, Mitf, Dmrt, and Msx, which are downstream of FGF signaling. The central role of Mitf is consistent with its function as a fundamental regulator of vertebrate pigment cell development. Moreover, our results unraveled a new function for Dmrt and Msx as transcriptional co-activators in the context of theCionaotolith.