A protocadherin mediates oral placode morphogenesis in the tunicate <i>Ciona</i>

In chordate embryos, placodes are ectodermal thickenings around the borders of the neural plate that give rise to various sensory organs and cell types. While generally thought to be a vertebrate-specific innovation, homologous placodes are proposed to exist in non-vertebrate chordates as well. In Ciona robusta, a solitary tunicate, the adult mouth (the oral siphon) is derived from one such 'cranial-like' placode in the larva, which we term the oral siphon placode (OSP). At embryonic and larval stages, the OSP consists of a small rosette of cells that form from the neuropore at the anteriormost extent of neural tube closure. While the morphogenesis of the OSP and its physical separation from other surface ectoderm structures have been described in detail, how this is regulated at the molecular level is currently unknown. Here, we show the involvement of protocadherin-mediated cell-cell interactions in the proper morphogenesis of the OSP. Protocadherin.e (Pcdh.e) is expressed specifically in the OSP, but not in other surface ectoderm cells. CRISPR/Cas9-mediated disruption of Pcdh.e in these cells results in the failure of the OSP to physically separate from other structures derived from the same cell lineage. Overexpression of Pcdh.e throughout the anterior surface ectoderm results in a similar loss of a physically separate and distinct OSP territory. This effect is likely mediated by homophilic adhesion in trans, as Pcdh.e with scrambled extracellular domains failed to recapitulate the phenotype. Finally, we show that Pcdh.e expression in the OSP depends on oral placode-specific transcription factors such as Six1/2 and Pitx. Our results suggest that OSP morphogenesis requires precise regulation of a homotypic cell-cell adhesion molecule, which might reflect a conserved mechanism for placode formation in chordates.