SummaryThe diversity of cell types and regulatory states in the brain, and how these change during aging, remains largely unknown. We present a single-cell transcriptome atlas of the entire adult Drosophila melanogaster brain sampled across its lifespan. Cell clustering identified 87 initial cell clusters that are further subclustered and validated by targeted cell-sorting. Our data show high granularity and identify a wide range of cell types. Gene network analyses using SCENIC revealed regulatory heterogeneity linked to energy consumption. During aging, RNA content declines exponentially without affecting neuronal identity in old brains. This single-cell brain atlas covers nearly all cells in the normal brain and provides the tools to study cellular diversity alongside other Drosophila and mammalian single-cell datasets in our unique single-cell analysis platform: SCope (http://scope.aertslab.org). These results, together with SCope, allow comprehensive exploration of all transcriptional states of an entire aging brain.

For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae , that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type–related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.

Melanoma cells can switch between a melanocytic and mesenchymal-like state. Scattered evidence indicates that additional, intermediate state(s) may exist. To search for such states and decipher their underlying gene regulatory network (GRN), we studied ten melanoma cultures by single-cell RNA-seq, and 26 additional cultures by bulk RNA-seq. Although each culture exhibited a unique transcriptome, we identified shared GRNs that underlie the extreme melanocytic and mesenchymal states, and the intermediate state. This intermediate state is corroborated by a distinct chromatin landscape and governed by the transcription factors SOX6, NFATC2, EGR3, ELF1 and ETV4. Single-cell migration assays confirmed its intermediate migratory phenotype. By time-series sampling of single cells after knockdown of SOX10, we unravelled the sequential and recurrent arrangement of GRNs during phenotype switching. Jointly, these analyses indicate that an intermediate state exists and is driven by a distinct and stable "mixed" GRN rather than being a symbiotic, heterogeneous mix of cells.