SUMMARY The intestinal epithelium is continuously regenerated by highly proliferative Lgr5+ intestinal stem cells (ISCs). The existence of a population of quiescent ISCs has been suggested yet its identity and features remain controversial. Here we describe that the expression of the RNA-binding protein Mex3a labels a subpopulation of Lgr5+ cells that divide less frequently and contribute to regenerate all intestinal lineages with slow kinetics. Single cell transcriptomic analysis revealed two classes of Lgr5-high cells, one of them defined by the Mex3a-expression program and by low levels of proliferation genes. Lineage tracing experiments show that large fraction of Mex3a+ cell population is continuously recalled into the rapidly dividing self-renewing ISC pool in homeostatic conditions. Chemotherapy and radiation target preferentially rapidly dividing Lgr5+ cells but spare the Mex3a-high/Lgr5+ population, which helps sustain the renewal of the intestinal epithelium during treatment.

A variety of single-cell RNA preparation procedures have been described. So far, protocols require fresh material, which hinders complex study designs. We describe a sample preservation method that maintains transcripts in viable single cells, allowing one to disconnect time and place of sampling from subsequent processing steps. We sequence single-cell transcriptomes from >1000 fresh and cryopreserved cells using 3'-end and full-length RNA preparation methods. Our results confirm that the conservation process did not alter transcriptional profiles. This substantially broadens the scope of applications in single-cell transcriptomics and could lead to a paradigm shift in future study designs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1171-9) contains supplementary material, which is available to authorized users.

Planarians can undergo dramatic changes in body size and regenerate their entire body plan from small pieces after cutting. This remarkable morphological plasticity has made them an excellent model in which to analyze phenomena such as morphogenesis, restoration of pattern and polarity, control of tissue proportions and tissue homeostasis. They have a unique population of pluripotent stem cells in the adult that can give rise to all differentiated cell types, including the germ cells. These cellular characteristics provide an excellent opportunity to study the mechanisms involved in the maintenance and differentiation of cell populations in intact and regenerating animals. Until recently, the planarian model system lacked opportunities for genetic analysis; however, this handicap was overcome in the last decade through the development of new molecular methods which have been successfully applied to planarians. These techniques have allowed analysis of the temporal and spatial expression of genes, as well as interference with gene function, generating the first phenotypes by loss or gain of function. Finally, the sequencing of the planarian genome has provided the essential tools for an in-depth analysis of the genomic regulation of this model system. In this review, we provide an overview of planarians as a model system for research into development and regeneration and describe new lines of investigation in this area.