Retinoic acid, an essential component of the RP organizer, promotes the spatio-temporal segregation of dorsal neural fates

Dorsal neural tube-derived retinoic acid promotes the end of neural crest production and transition into a definitive roof plate. Here we analyze how this impacts the segregation of central and peripheral lineages, a process essential for tissue patterning and function. Localized in-ovo inhibition of retinoic acid activity followed by single cell transcriptomics unraveled a comprehensive list of differentially expressed genes relevant to these processes. Importantly, progenitors co-expressed neural crest, roof plate and di1 interneuron markers indicating a failure in proper lineage segregation. Furthermore, we found that separation between roof plate and dI1 interneurons is mediated by Notch activity downstream of retinoic acid, highlighting their critical role in establishing the roof plate-dI1 boundary. Within the peripheral branch, where absence of retinoic acid resulted in neural crest production and emigration extending into the roof plate stage, sensory progenitors failed to separate from melanocytes leading to formation of a common glia-melanocyte cell with aberrant migratory patterns. Together, we uncover and characterize a molecular mechanism responsible for segregation of dorsal neural fates during development. Overall design: To enrich for dorsal NT cell types, quail E2.5 neural tubes were electroporated at the flank region of the axis with Msx1-Citrine combined with either a control vector, or RARa403, a dominant negative retinoic acid receptor. For each group, ten neural tubes with associated mesoderm were mechanically microdissected at E4 and single cell suspensions prepared. Fluorescent cells were then sorted and subjected to droplet-based scRNA-seq (10x Genomics Chromium) to generate a gene expression profile of control and treated dorsal neural tube cells