Nunez, S. A. | Search bioRxiv

bioRxiv2026

The developing midbrain hindbrain boundary contains molecularly distinct cell populations

BackgroundDuring vertebrate embryogenesis, the isthmic region spans the midbrain-hindbrain boundary of the neural tube and includes an organizer (IsO) that is essential for proper formation of adjacent brain regions, yet the molecular and cellular composition of the isthmic region remains unresolved. ResultsWe employed combined single-nucleus ATAC-seq and RNA-seq (scMultiome) in 13 and 16 hours-post-fertilization zebrafish embryos to molecularly resolve cell populations in the isthmic region and validated our findings in vivo by RNA fluorescence in situ hybridization. We identified two distinct isthmic cell populations - isthmic midbrain (IsMB) and isthmic hindbrain (IsHB) - that share expression of canonical isthmic genes, but that differ in their expression of midbrain vs hindbrain genes. We also uncovered a previously unrecognized heterogeneity within the IsHB, reflecting a canonical fgf8-expressing population anteriorly (IsO/r0a), and a novel fgf8-negative population posteriorly (r0p). We find that inhibition of Fgf signaling disrupts formation of the isthmic region, leading to loss of isthmic cell populations except a residual population characterized by a mixed neural identity. ConclusionsUsing transcriptional and epigenetic characterization, we expand on prior anatomical and genetic analyses of the isthmic region to refine our understanding of its cellular organization and demonstrate that it consists of several subdomains.

developmental biology↗

bioRxiv2025

Epigenetic priming of neural progenitors by Notch enhances Sonic hedgehog signaling and establishes gliogenic competence

The remarkable cell diversity of multicellular organisms relies on the ability of multipotent progenitor cells to generate distinct cell types at the right times and locations during embryogenesis. A key question is how progenitors establish competence to respond to the different environmental signals required to produce specific cell types at critical developmental timepoints. We addressed this in the mouse developing forebrain, where neural progenitor cells must switch from producing neurons to making oligodendrocytes in response to increased Sonic Hedgehog (SHH) signaling during late embryogenesis. We show that progenitor responses to SHH are regulated by Notch signaling, thus permitting proper timing of the neuron-oligodendrocyte switch. Notch activity epigenetically primes genes associated with the oligodendrocyte lineage and SHH pathway, enabling amplified transcriptional responses to endogenous SHH and robust oligodendrogenesis. These results reveal a critical role for Notch in facilitating progenitor competence states and influencing cell fate transitions at the epigenetic level.

developmental biology↗