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. Tribolium castaneum 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 Tc-fez/earmuff, a key marker for intermediate progenitors. Using combinatorial labeling of further markers, including Tc-pointed, we characterized embryonic type II neuroblast lineages. Intriguingly, we found nine lineages per hemisphere in the Tribolium embryo while Drosophila produces only eight per brain hemisphere. These embryonic lineages are significantly larger in Tribolium than they are in Drosophila and contain more intermediate progenitors. Finally, we mapped these lineages to the domains of 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. 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. These findings stipulate further research on the link between stem cell activity and temporal and structural differences in central complex development.

Fibroblast growth factor (FGF) signaling elicits multiple downstream pathways, most notably the Ras/MAPK cascade facilitated by the adaptor protein Grb2. However, the mechanism by which Grb2 is recruited to the FGF signaling complex remains unresolved. Here, we showed that genetic ablation of FGF signaling prevented murine lens induction by disrupting transcriptional regulation and actin cytoskeletal arrangements, which could be reproduced by deleting the juxtamembrane region of the FGF receptor and rescued by Kras activation. Conversely, mutations affecting the Frs2-binding site on the FGF receptor or the deletion of Frs2 and Shp2 primarily impact later stages of lens vesicle development involving lens fiber cell differentiation. Our study further revealed that the loss of Grb2 abolished MAPK signaling, resulting in a profound arrest of lens development. However, removing Grb2’s putative Shp2 dephosphorylation site (Y209) neither produced a detectable phenotype nor impaired MAPK signaling during lens development. Furthermore, the catalytically inactive Shp2 mutation (C459S) only modestly impaired FGF signaling, whereas replacing Shp2’s C-terminal phosphorylation sites (Y542/Y580) previously implicated in Grb2 binding only caused placental defects, perinatal lethality, and reduced lacrimal gland branching without impacting lens development, suggesting that Shp2 only partially mediates Grb2 recruitment. In contrast, we observed that FGF signaling is required for the phosphorylation of the Grb2-binding sites on Shc1 and the deletion of Shc1 exacerbates the lens vesicle defect caused by Frs2 and Shp2 deletion. These findings establish Shc1 as a critical collaborator with Frs2 and Shp2 in targeting Grb2 during FGF signaling.