BackgroundThe planarian Schmidtea mediterranea is a master regenerator with a large adult stem cell compartment. The lack of transgenic labeling techniques in this animal has hindered the study of lineage progression and has made understanding the mechanisms of tissue regeneration a challenge. However, recent advances in single-cell transcriptomics and analysis methods allow for the discovery of novel cell lineages as differentiation progresses from stem cell to terminally differentiated cell.ResultsHere we apply pseudotime analysis and single-cell transcriptomics to identify adult stem cells belonging to specific cellular lineages and identify novel candidate genes for future in vivo lineage studies. We purify 168 single stem and progeny cells from the planarian head, which were subjected to single-cell RNA sequencing (scRNAseq). Pseudotime analysis with Waterfall and gene set enrichment analysis predicts a molecularly distinct neoblast sub-population with neural character (νNeoblasts) as well as a novel alternative lineage. Using the predicted νNeoblast markers, we demonstrate that a novel proliferative stem cell population exists adjacent to the brain.ConclusionsscRNAseq coupled with in silico lineage analysis offers a new approach for studying lineage progression in planarians. The lineages identified here are extracted from a highly heterogeneous dataset with minimal prior knowledge of planarian lineages, demonstrating that lineage purification by transgenic labeling is not a prerequisite for this approach. The identification of the νNeoblast lineage demonstrates the usefulness of the planarian system for computationally predicting cellular lineages in an adult context coupled with in vivo verification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0937-9) contains supplementary material, which is available to authorized users.

The asexual freshwater planarian is a constitutive adult, whose central nervous system (CNS) is in a state of constant homeostatic neurogenesis. However, very little is known about the extrinsic signals that act on planarian stem cells to modulate rates of neurogenesis. We have identified two planarian homeobox transcription factors, Smed-nkx2.1 and Smed-arx, which are required for the maintenance of cholinergic, GABAergic, and octopaminergic neurons in the planarian CNS. These very same neurons also produce the planarian hedgehog ligand (Smed-hh), which appears to communicate with brain-adjacent stem cells to promote normal levels of neurogenesis. Planarian stem cells nearby the brain express core hh signal transduction genes, and consistent hh signaling levels are required to maintain normal production of neural progenitor cells and new mature cholinergic neurons, revealing an important mitogenic role for the planarian hh signaling molecule in the adult CNS.DOI: http://dx.doi.org/10.7554/eLife.19735.001

Graphical AbstractHighlights d soxB1-2-deficient planarians exhibit movement defects and abnormal sensory function d Ectodermal-lineage progenitor cells express soxB1-2 d soxB1-2 activity is required for differentiation and function of sensory neurons d Inhibition of genes downstream of soxB1-2 recapitulates sensory regeneration defects SUMMARY SoxB1 genes play fundamental roles in neurodevelopmental processes and maintaining stem cell multipotency, but little is known about their function in regeneration. We addressed this question by analyzing the activity of the SoxB1 homolog soxB1-2 in the planarian Schmidtea mediterranea.Expression and functional analysis revealed that soxB1-2 marks ectodermal-lineage progenitors, and its activity is required for differentiation of subsets of ciliated epidermal and neuronal cells. Moreover, we show that inhibiting soxB1-2 or its candidate target genes leads to abnormal sensory neuron regeneration that causes planarians to display seizure-like movements or phenotypes associated with the loss of sensory modalities. Our analyses highlight soxB1-2-regulated genes that are expressed in sensory neurons and are homologous to factors implicated in epileptic disorders in humans and animal models of epilepsy, indicating that planarians can serve as a complementary model to investigate genetic causes of epilepsy.