Axolotls are uniquely able to mobilize neural stem cells to regenerate all missing regions of the spinal cord. How a neural stem cell under homeostasis converts after injury to a highly regenerative cell remains unknown. Here, we show that during regeneration, axolotl neural stem cells repress neurogenic genes and reactivate a transcriptional program similar to embryonic neuroepithelial cells. This dedifferentiation includes the acquisition of rapid cell cycles, the switch from neurogenic to proliferative divisions, and the re-expression of planar cell polarity (PCP) pathway components. We show that PCP induction is essential to reorient mitotic spindles along the anterior-posterior axis of elongation, and orthogonal to the cell apical-basal axis. Disruption of this property results in premature neurogenesis and halts regeneration. Our findings reveal a key role for PCP in coordinating the morphogenesis of spinal cord outgrowth with the switch from a homeostatic to a regenerative stem cell that restores missing tissue.

Plasmid construction is central to life science research, and sequence verification is arguably its costliest step. Long-read sequencing has emerged as a competitor to Sanger sequencing, with the principal benefit that whole plasmids can be sequenced in a single run. Nevertheless, the current cost of nanopore sequencing is still prohibitive for routine sequencing during plasmid construction. We develop a computational approach termed Simple Algorithm for Very Efficient Multiplexing of Oxford Nanopore Experiments for You (SAVEMONEY) that guides researchers to mix multiple plasmids and subsequently computationally de-mixes the resultant sequences. SAVEMONEY defines optimal mixtures in a pre-survey step, and following sequencing, executes a post-analysis workflow involving sequence classification, alignment, and consensus determination. By using Bayesian analysis with prior probability of expected plasmid construction error rate, high-confidence sequences can be obtained for each plasmid in the mixture. Plasmids differing by as little as two bases can be mixed as a single sample for nanopore sequencing, and routine multiplexing of even six plasmids per 180 reads can still maintain high accuracy of consensus sequencing. SAVEMONEY should further democratize whole-plasmid sequencing by nanopore and related technologies, driving down the effective cost of whole-plasmid sequencing to lower than that of a single Sanger sequencing run.