The developing midbrain-hindbrain boundary contains molecularly distinct cell populations

During 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.

We 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.

Using 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.