Cross-species single cell transcriptomics in fly and beetle reveals the genetic core of brain neuroblast specification

The brain is essential for animal survival and based on its conserved Bauplan, an impressive adaptive diversity has evolved. However, the genetic mechanisms regulating brain development and diversification remain enigmatic. The insect neural stem cells (neuroblasts, NBs) acquire different identities through the combinatorial expression of transcription factors (TFs), but this code is unknown for the brain. Here, we define the conserved core of TFs expressed in insect brain NBs by a combined analysis of single-cell expression from NBs derived from two holometabolous insects, the fly Drosophila melanogaster and the beetle Tribolium castaneum. In Tribolium, we established a Gal4 enhancer trap system to identify a line that marks NBs. From 37,137 sequenced NBs, we identified 10,425 brain NBs. In Drosophila, we sequenced 32,112 NB nuclei, identifying 12,389 brain NBs. Analysing the combined dataset strongly increased the sensitivity in specifying the core of 188 brain-specific TFs. We found two atypical clusters with some similarity to Type II NBs and identified seven transcription factors not previously associated with or confirmed in NBs (Hmx, CG15696, CG32532, dmrt99B, fD59A, TfAP-2, and Fer1). Our data reveals fundamental differences between brain and ventral nerve cord specification and paves the way to study the development and evolution of brain specific structures.