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Understanding the generation of neuronal and glial diversity is one of the major goals of developmental neuroscience. The Drosophila CNS midline cells constitute a simple neurogenomic system to study neurogenesis, cell fate acquisition, and neuronal function. Previously, we identified and determined the developmental expression profiles of 224 midline-expressed genes. Here, the expression of 59 transcription factors, signaling proteins, and neural function genes was analyzed using multi-label confocal imaging, and their expression patterns mapped at the single-cell level at multiple stages of CNS development. These maps uniquely identify individual cells and predict potential regulatory events and combinatorial protein interactions that may occur in each midline cell type during their development. Analysis of neural function genes, including those encoding peptide neurotransmitters, neurotransmitter biosynthetic enzymes, transporters, and neurotransmitter receptors, allows functional characterization of each neuronal cell type. This work is essential for a comprehensive genetic analysis of midline cell development that will likely have widespread significance given the high degree of evolutionary conservation of the genes analyzed.

The Drosophila columnar genes are key regulators of neural precursor formation and patterning along the dorsal–ventral axis of the developing CNS and include ventral nerve cord defective (vnd), intermediate nerve cord defective (ind), muscle segment homeodomain (msh), and Epidermal growth factor receptor (Egfr). To investigate the evolution of neural pattern formation, we identified and determined the expression patterns of Tribolium vnd, ind, and msh, and found that they are expressed in the medial, intermediate, and lateral columns of the developing CNS, respectively, in patterns similar, but not identical, to their Drosophila orthologs. The pattern of Egfr activity suggests that the genetic regulatory mechanisms that initiate Tc-vnd expression are similar in Drosophila and Tribolium, whereas those that initiate Tc-ind have diverged. RNAi analyses of gene function show that Tc-vnd and Tc-ind promote the formation of medial and intermediate column neural precursors and that vnd-mediated repression of ind establishes the boundary between the medial and intermediate columns. These data suggest that columnar gene expression and function underlie neural pattern formation in Drosophila, Tribolium, and potentially all insects, but that subtle spatiotemporal differences in expression of these genes may produce species-specific morphological differences.