A pair-rule-like transcription network coordinates neural tube closure in a proto-vertebrate

Neural tube closure (NTC) is a conserved morphogenetic process in chordates, during which the neural plate folds and fuses to form a closed neural tube. While the mechanical forces and signaling pathways governing NTC have been characterized in vertebrates, the transcriptional programs coordinating cell behaviors during closure remain less understood. Here, we identify a transcriptional circuit involving Lmx1, Cdkn1b, and Msx that regulates dorsal midline dynamics during NTC in the tunicate Ciona. High-resolution HCR in situ hybridization reveals that Lmx1 is dynamically enriched at the zippering point and advances in a posterior-to-anterior transcription wave, while Msx is downregulated in the same region, marking a transition from early neural patterning to morphogenetic execution. As closure progresses, Lmx1 and Cdkn1b exhibit complementary, alternating expression at the dorsal midline, resembling a pair-rule-like pattern. Misexpression studies show that Lmx1 promotes proliferation and activates its own expression, whereas Cdkn1b limits proliferation and impedes closure. Single-cell RNA-seq reveals transcriptionally distinct dorsal neural populations enriched for Lmx1 or Cdkn1b, supporting spatially organized cell-cycle states. These findings suggest that a transcriptional switch from Msx to Lmx1, followed by spatially alternating Lmx1 and Cdkn1b activity, coordinates proliferation and neural fold fusion during NTC. This mechanism may represent a general strategy for regulating epithelial remodeling in animal embryos.