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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.

Summary 1.Ionizing radiation produces oxidative stress, but organisms can adapt to their exposure with physiological adaptive responses. However, the role of radioadaptive responses in wild populations remains poorly known. 2. At Chernobyl, studies of birds and other taxa including humans show that chronic exposure to radiation depletes antioxidants and increases oxidative damage. Here, we present analyses of levels of the most important intracellular antioxidant (i.e. glutathione, GSH), its redox status, DNA damage and body condition in 16 species of birds exposed to radiation at Chernobyl. We use an approach that allows considering the individual bird as the sampling unit while controlling for phylogenetic effects, thus increasing the statistical power by avoiding the use of species means as done for most previous comparative studies. 3. As a consequence, we found a pattern radically different from previous studies in wild populations, showing that GSH levels and body condition increased, and oxidative stress and DNA damage decreased, with increasing background radiation. Thus, when several species are considered, the overall pattern indicates that birds are not negatively affected by chronic exposure to radiation and may even obtain beneficial hormetic effects following an adaptive response. Analysis of the phylogenetic signal supports the existence of adaptation in the studied traits, particularly in GSH levels and DNA damage. 4. We also show that, under equal levels of radiation, the birds that produce larger amounts of the pigment pheomelanin and lower amounts of eumelanin pay a cost in terms of decreased GSH levels, increased oxidative stress and DNA damage, and poorer body condition. Radiation, however, diminished another potential cost of pheomelanin, namely its tendency to produce free radicals when exposed to radiation, because it induced a change towards the production of less pro-oxidant forms of pheomelanin with higher benzothiazole-to-benzothiazine ratios, which may have facilitated the acclimation of birds to radiation exposure. 5. Our findings represent the first evidence of adaptation to ionizing radiation in wild animals, and confirm that pheomelanin synthesis represents an evolutionary constraint under stressful environmental conditions because it requires GSH consumption.