Regeneration requires coordination of stem cells, their progeny, and distant differentiated tissues. Here, we present a comprehensive atlas of whole-body regeneration in Schmidtea mediterranea and identify wound-induced cell states. Analysis of 299,998 single-cell transcriptomes captured from regeneration-competent and regeneration-incompetent fragments identified transient regeneration-activated cell states (TRACS) in the muscle, epidermis, and intestine. TRACS were stem-cell-division-independent with distinct spatiotemporal distributions and RNAi depletion of TRACS-enriched genes produced regeneration defects. Muscle expression of notum , follistatin , evi/wls , glypican-1 , and junctophilin-1 was required for tissue polarity. Epidermal expression of agat-1/2/3 , cyp3142a1 , zfhx3 , and atp1a1 was important for stem cell proliferation. Finally, expression of spectrinβ and atp12a in intestinal basal cells and lrrk2 , cathepsinB , myosin1e , polybromo-1 , and talin-1 in intestinal enterocytes regulated stem cell proliferation and tissue remodeling. respectively. Our results identify cell types and molecules important for regeneration, indicating that regenerative capacity can emerge from coordinated transcriptional plasticity across all three germ layers.

Summary The expression of chloroplast genes relies on a host of nucleus‐encoded proteins. Identification of such proteins and elucidation of their functions are ongoing challenges. We used ribosome profiling to revisit the function of the pentatricopeptide repeat protein LPE1, reported to stimulate translation of the chloroplast psbA mRNA in Arabidopsis. Mutation of the maize LPE1 ortholog causes a photosystem II (PSII) deficiency and a defect in translation of the chloroplast psbJ open reading frame (ORF) but has no effect on psbA expression. To reflect this function, we named the maize LPE1 ortholog Translation of psbJ 1 (TPJ1). Arabidopsis lpe1 mutants likewise exhibit a loss of psbJ translation, and have, in addition, a decrease in psbN translation. We detected a small decrease in ribosome occupancy on the psbA mRNA in Arabidopsis lpe1 mutants, but ribosome profiling analyses of other PSII mutants (hcf107 and hcf173) in conjunction with in vitro RNA binding data strongly suggest that this is a secondary effect of their PSII deficiency. We conclude that maize TPJ1 promotes PSII synthesis by activating translation of the psbJ ORF, that this function is conserved in Arabidopsis LPE1, and that an additional role for LPE1 in psbN translation contributes to the PSII deficiency in lpe1 mutants.

Regeneration requires functional coordination of stem cells, their progeny, and differentiated cells. Past studies have focused on regulation of stem cell identity and proliferation near to the wound-site, but less is known about contributions made by differentiated cells distant to the injury. Here, we present a comprehensive atlas of whole-body regeneration over time and identify rare, transient, somatic cell states induced by injury and required for regeneration. To characterize amputation-specific signaling across a whole animal, 299,998 single-cell transcriptomes were captured from planarian tissue fragments competent and incompetent to regenerate. Amputation-specific cell states were rare, non-uniformly distributed across tissues, and particularly enriched in muscle (mesoderm), epidermis (ectoderm), and intestine (endoderm). Moreover, RNAi-mediated knockdown of genes up-regulated in amputation-specific cell states drastically reduced regenerative capacity. These results identify novel cell states and molecules required for whole-body regeneration and indicate that regenerative capacity requires transcriptional plasticity in a rare subset of differentiated cells.

SUMMARYRegeneration of missing or damaged tissue requires the concerted activities of both pre-existing and newly generated cells. While previous studies identified regeneration-induced gene expression programs in tissues and single cells1–6, emerging spatial transcriptomic methods can place these programs into their organismal context at near cellular resolution7–11. Here we report a spatial transcriptomic atlas of whole-body regeneration and identify changes in gene expression patterning and tissue architecture during regeneration. We produced spatially resolved RNA-seq data from regenerating tail fragments of the free-living planarian Schmidtea mediterranea. Comparison of fragments 6 and 48 hours after amputation revealed time-dependent changes in gene expression patterns and cellular associations. Notable examples include re-localization of agat-1+ epidermal cells from lateral edges to both lateral and anterior edges and the discovery of wound-induced association of stem cells with cells expressing the matrix remodeling gene matrix metalloproteinase-1. Our results thoroughly detail changes in gene expression patterns and uncover interactions between stem cells and their microenvironment, demonstrating the unique ability of unbiased spatial profiling methods to reveal the biology of regeneration.

Spatial transcriptomic techniques such as Slide-seqV2 uncover novel relationships and interactions between cell types by coupling gene expression and spatial data. Here we discuss two unexpected sources of error in Slide-seqV2 data, one physical and one computational. To address this we present an analysis pipeline augmentation, Syrah, which corrects for these errors and show that it improves both data quantity and quality over the standard pipeline alone or in combination with additional sequencing.