Decoding transcriptional identity during Neuron-Astroglia Cell Fate driven by RAR-specific agonists [scRNA-seq]

How cells respond to different signals leading to defined lineages is an open question to understand physiological differentiation leading to the formation of organs and tissues. Among the various morphogens, retinoic acid signaling, via the RXR/RAR nuclear receptors activation, is a key morphogen of nervous system development and brain homeostasis. Here we analyze gene expression in ~80,000 cells covering 16 days of monolayer mouse stem cell differentiation driven by the pan-RAR agonist all-trans retinoic acid, the RAR-alpha agonist BMS753 or the activation of both RAR-beta and RAR-gamma receptors (BMS641+BMS961). Furthermore, we have elucidated the role of these retinoids for driving nervous tissue formation within 90 days of brain organoid cultures, by analyzing > 8,000 distinct spatial regions over 28 brain organoids. Despite a delayed progression in BMS641+BMS961, RAR-specific agonists led to a variety of neuronal subtypes, astrocytes and oligodendrocyte precursors. Spatially-resolved transcriptomics performed in organoids revealed spatially distinct RAR isotype expression leading to specialization signatures associated to matured tissues, including a variety of neuronal subtypes, retina-like tissue structure signatures and even the presence of microglia. Overall design: mouse embryonic stem cells were cultured in monoloayer in presence of either the pan-RAR agonist ATRA, the RAR-alpha agonist BMS753, or the combination of the RAR-beta and RAR-gamma agonists (BMS641+BMS961). Monolayer cultures driven into differentiation by the action of these RAR ligands were analyzed by single-cell transcriptomics after 4, 8 and 16 days of treatment. Samples collected after 16 days of differentiation were also used for Cut&tag assays for revealing histone modification signatures associated to promoter regions as a way to reveal active, repressed or bivalent promoter chromatin states. To further reveal the role of retinoids action in nervous tissue development, we have cultured mouse stem cells in 3-dimensional conditions. After 3 months of culture, the obtained brain organoid tissues were anlaysed by spatial transcriptomics.