Cell type transcriptome atlas for the planarian Schmidtea mediterranea.

INTRODUCTION The complete sequence of animal genomes has had a transformative impact on biological research. Whereas the genome sequence of an organism contains the information for its development and physiology, the transcriptomes (the sets of actively transcribed genes) of the cell types in an organism define how the genome is used for the unique functions of its cells. Cell number and complexity have historically made the identification of all cell types, much less their transcriptomes, an extreme challenge for most multicellular organisms. Recent advances in single-cell RNA sequencing (SCS) have greatly enhanced the ability to determine cell type transcriptomes, with SCS of thousands of cells readily achievable. RATIONALE We reasoned that it might be possible, given these advances, to determine the transcriptomes of essentially every cell type of a complete organism possessing an unknown number of cell types. The planarian Schmidtea mediterranea , famous for its regeneration ability, is an attractive case study for such an undertaking. Planarians possess a complex anatomy with diverse differentiated cell types, including many found across animals. Furthermore, planarians contain a proliferating cell population called neoblasts that includes pluripotent stem cells. Neoblasts mediate regeneration and constitutive tissue turnover. Consequently, lineage precursors for essentially all differentiated cells types are also present in adults. Finally, planarians constitutively express positional information guiding tissue turnover. Therefore, comprehensive SCS at a single time point (the adult) could allow transcriptome determination for all differentiated cell types and for lineage precursors, and could identify patterning information that guides new cell production and organization. Capturing this information in most organisms would require sampling adults and many transient embryonic stages. RESULTS We used the SCS method Drop-seq to determine the transcriptomes for 66,783 individual cells from adult planarians. We locally saturated cell type coverage by iteratively sequencing distinct body regions and assessing the frequency of known rare cell types in the data. Clustering the cells by shared gene expression grouped cells into broad tissue classes. Subclustering of each broad tissue type in isolation enabled separation of cells into the cell populations constituting each tissue. These analyses enabled the identification of a previously unidentified tissue group and the classification of poorly characterized tissues into their constituent cell types, including numerous previously unknown cell types. Transcriptomes were identified for many rare cell types, including those that exist as rarely as ~10 cells in an animal that has 10 5 to 10 6 cells, which suggests that near-to-complete cellular saturation was reached. In addition, transcriptomes for known and novel lineage precursors, from pluripotent stem cell to differentiated cell types, were generated. Precursor transcriptomes identified transcription factors required for maintenance of associated differentiated cells during homeostatic cell turnover. Finally, the data were used to identify genes regionally expressed in muscle, which is the site of planarian patterning gene expression. CONCLUSION We successfully used SCS to generate transcriptomes for most to all cells of a complete organism. This resource provides a wealth of data regarding the cellular site of expression of thousands of conserved genes and the transcriptomes for cell types widely used in animals. These data will inform studies of these genes and cell types broadly, and will provide a resource for the fields of planarian biology and comparative evolutionary biology. This work also provides a template for the generation of cell type transcriptome atlases, which can be applied to a large array of organisms.