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

Abstract

Despite its conserved basic structure, the morphology of the insect brain and the timing of its development underwent evolutionary adaptations. However, little is known about the developmental processes that create this diversity. The central complex is a brain centre required for multimodal information processing and 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. These neural stem cells are believed to be conserved among insects, but little is known on their molecular characteristics in insects other than flies. Tribolium castaneum has emerged as a valuable new insect model for brain development and evolution. However, type-II neuroblast lineages and their role in central complex development have so far not been studied in this beetle. Using CRISPR-Cas9 we created a fluorescent enhancer trap marking expression of Tribolium fez/earmuff, a key marker for type-II neuroblast derived intermediate progenitors. Using combinatorial labelling of further markers including Tc-pointed, Tc-deadpan, Tc-asense and Tc-prospero we characterized the type-II neuroblast lineages present in the Tribolium embryo and their sub-cell-types. Intriguingly, we found 9 type-II neuroblast lineages per hemisphere in the Tribolium embryo while Drosophila produces only 8 per brain hemisphere. In addition, these lineages are significantly larger at the embryonic stage of Tribolium than they are in Drosophila and contain more intermediate progenitors. Finally, we mapped these lineages to the domains of early expressed head patterning genes. Notably, Tc-otd is absent from all type-II neuroblasts and intermediate progenitors, whereas Tc-six3 marks an anterior subset of the type-II-lineages. The placodal marker Tc-six4 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 which is not found in the fly. These findings stipulate further research on the causal link between timing of stem cell activity and temporal and structural differences in central complex development.