The tripartite neural plate border is a common trait of vertebrates and tunicates

The neural crest and neurogenic placodes, which arise from the neural plate border, give rise to morphological characteristics that distinguish vertebrates from invertebrates (Gans & Northcutt, 1983). Recent studies have suggested that embryos of ascidians, a group of tunicates that are the closest invertebrate relatives of vertebrates, possess cells that share an evolutionary origin with vertebrate neural crest cells (Abitua et al., 2012; Fatieieva et al., 2025; Ishida & Satou, 2024; Stolfi et al., 2015; Todorov et al., 2024; Waki et al., 2015) and neurogenic placode cells (Abitua et al., 2015; Ikeda et al., 2013; Liu et al., 2023; Liu & Satou, 2019; Manni et al., 2004; Mazet et al., 2005; Papadogiannis et al., 2022; Wagner & Levine, 2012). To dissect the neural plate border of ascidian embryos at the molecular level, and to gain deeper insights into evolutionary origins of the neural crest and neurogenic placodes, we comprehensively analyzed expression patterns of transcription factor genes at single-cell resolution. We demonstrated that the ascidian neural plate border consists of three domains with distinct gene expression profiles: the anterior, inner lateral, and outer lateral domains. A cross-species comparison of transcriptomes from ascidians and zebrafish suggests that the anterior domain is homologous to zebrafish neurogenic placodes, the inner lateral domain to the neural crest and tail bud and the outer lateral domain to the median fin fold ectoderm. We propose that a tripartite neural plate border was present in the last common ancestor of vertebrates and tunicates, providing a blueprint for evolutionary emergence of vertebrate morphological novelties.