Differences in size and number of embryonic type II neuroblast lineages correlate with divergent timing of central complex development between beetle and fly.

The insect brain and the timing of its development underwent evolutionary adaptations. However, little is known about the underlying developmental processes. The central complex of the brain is an excellent model to understand neural development and divergence. It is produced in large parts by type II neuroblasts, which produce intermediate progenitors, another type of cycling precursor, to increase their neural progeny. Type II neuroblasts lineages are believed to be conserved among insects, but little is known on their molecular characteristics in insects other than flies. has emerged as a model for brain development and evolution. However, type II neuroblasts have so far not been studied in this beetle. We created a fluorescent enhancer trap marking expression of , a key marker for intermediate progenitors. Using combinatorial labeling of further markers, including we characterized embryonic type II neuroblast lineages. Intriguingly, we found nine lineages per hemisphere in the embryo while produces only eight per brain hemisphere. These embryonic lineages are significantly larger in than they are in and contain more intermediate progenitors. Finally, we mapped these lineages to the domains of head patterning genes. Notably, is absent from all type II neuroblasts and intermediate progenitors, whereas marks an anterior subset of the type II lineages. specifically marks the territory where anterior-medial type II neuroblasts differentiate. In conclusion, we identified a conserved pattern of gene expression in holometabolan central complex forming type II neuroblast lineages, and conserved head patterning genes emerged as new candidates for conferring spatial identity to individual lineages. The higher number and greater lineage size of the embryonic type II neuroblasts in the beetle correlate with a previously described embryonic phase of central complex formation. These findings stipulate further research on the link between stem cell activity and temporal and structural differences in central complex development.