(A) Protein sequence of Tc-FoxQ2. The C-terminus containing 85 amino acids (underlined) has little homology to other proteins in Tribolium and was used for protein expression. (B) Coomassie-blue stained SDS-PAGE gel analysis of expression and purification of Tc-FoxQ2. (-) Before IPTG induction; (+) after IPTG induction. M, marker; lane 1, cell pellet; lane 2, supernatant; lane 3, flow through after Ni2+ chelate affinity chromatography; lane 4, eluted fractions by imidazole; lane 5, before SUMO protease digestion (red arrow); lane 6, after SUMO protease digestion, two bands are observed (red arrows): 6xHis-SUMO and Tc-FoxQ2; lane 7, flow through after re-Ni2+ chelate affinity chromatography which contains Tc-FoxQ2. (C) Expression of Tc-foxQ2 RNA (green) and Tc-FoxQ2 protein (magenta) in the embryo. Tc-foxQ2 RNA is detected throughout the cytoplasm, while Tc-FoxQ2 protein is detected in the nuclei (blue). Tc-foxQ2 RNA and Tc-FoxQ2 protein show a high overlap

The genetic control of anterior brain development is highly conserved throughout animals. For instance, a conserved anterior gene regulatory network specifies the ancestral neuroendocrine center of animals and the apical organ of marine organisms. However, its contribution to the brain in non-marine animals has remained elusive. Here, we study the function of the Tc-foxQ2 forkhead transcription factor, a key regulator of the anterior gene regulatory network of insects. We characterized four distinct types of Tc-foxQ2 positive neural progenitor cells based on differential co-expression with Tc-six3/optix, Tc-six4, Tc-chx/vsx, Tc-nkx2.1/scro, Tc-ey, Tc-rx and Tc-fez1. An enhancer trap line built by genome editing marked Tc-foxQ2 positive neurons, which projected through the primary brain commissure and later through a subset of commissural fascicles. Eventually, they contributed to the central complex. Strikingly, in Tc-foxQ2 RNAi knock-down embryos the primary brain commissure did not split and subsequent development of midline brain structures stalled. Our work establishes foxQ2 as a key regulator of brain midline structures, which distinguish the protocerebrum from segmental ganglia. Unexpectedly, our data suggest that the central complex evolved by integrating neural cells from an ancestral anterior neuroendocrine center.