Meningeal-derived retinoic acid regulates neurogenesis via suppression of Notch and Sox2

The meninges act as a regulator of brain development by secreting ligands that act on neural cells to regulate neurogenesis and neuronal migration. Meningeal-derived retinoic acid (RA) promotes neocortical neural progenitor cell cycle exit; however, the underlying molecular mechanism is unknown. Here, we used spatial transcriptomics and profiling of retinoic-acid receptor-α (RARα) DNA binding in Foxc1-mutant embryos that lack meninges-derived ligands to identify the neurogenic transcriptional mechanisms of RA signaling in telencephalic neural progenitors. We determined that meningeal-derived RA controls neurogenesis by suppressing progenitor self-renewal pathways Notch signaling and the transcription factor Sox2. We show that RARα binds in the Sox2ot promoter, a long non-coding RNA that regulates Sox2expression, and RA promotes Sox2ot expression in neocortical progenitors. Our findings elucidate a previously unknown mechanism of how meningeal RA coordinates neocortical development and insight into how defects in meningeal development may cause neurodevelopmental disorders.

Methods
Pregnant mice were anesthetized using isoflurane and embryos were collected at E12, E13, or E14 by removing the heads and placing them in 4% paraformaldehyde (PFA) overnight at 4 degrees C. Whole heads were placed into 20% sucrose overnight and embedded in OCT. Mouse heads were sectioned at 12 um increments using a cryostat and sections mounted on glass slides. Sections were blocked in 3% bovine serum albumin (BSA) + 0.05% Triton-X for 1 h at room temperature and incubated with primary antibodies at a dilution of 1:100. Following incubation with primary antibody(s), sections were incubated with appropriate Alexa Fluor-conjugated secondary antibodies and DAPI (Invitrogen, Carlsbad, CA, United States). In situ hybridization was completed using the ACD Bio Multiplex Fluorescent Detection assay kit (Neward, CA) and their probes against Sox2 and Sox2ot.