Identification of enhancers that drive the spatially restricted expression of Vsx1 and Rx in the outer proliferation center of the developing Drosophila optic lobe.

Combinatorial spatial and temporal patterning of stem cells is a powerful mechanism for the generation of neural diversity in insect and vertebrate nervous systems. In the developing Drosophila medulla, the neural stem cells of the outer proliferation center (OPC) are spatially patterned by the mutually exclusive expression of three homeobox transcription factors: Vsx1 in the center of the OPC crescent (cOPC), Optix in the main arms (mOPC), and Rx in the posterior tips (pOPC). These spatial factors act together with a temporal cascade of transcription factors in OPC neuroblasts to specify the greater than 80 medulla cell types. Here, we identify the enhancers that are sufficient to drive the spatially restricted expression of the Vsx1 and Rx genes in the OPC. We show that removal of the cOPC enhancer in the Muddled inversion mutant leads to the loss of Vsx1 expression in the cOPC. Analysis of the evolutionarily conserved sequences within these enhancers suggests that direct repression by Optix may restrict the expression of Vsx1 and Rx to the cOPC and pOPC, respectively.

Imp and Syp mediated temporal patterning of neural stem cells in the developing Drosophila CNS.

The assembly of complex neural circuits requires that stem cells generate diverse types of neurons in the correct temporal order. Pioneering work in the Drosophila embryonic ventral nerve cord has shown that neural stem cells are temporally patterned by the sequential expression of rapidly changing transcription factors to generate diversity in their progeny. In recent years, a second temporal patterning mechanism, driven by the opposing gradients of the Imp and Syp RNA-binding proteins, has emerged as a powerful way to generate neural diversity. This long-range temporal patterning mechanism is utilized in the extended neural stem cell lineages of the postembryonic fly brain. Here, we review the role played by Imp and Syp gradients in several neural stem cell lineages, focusing on how they specify sequential neural fates through the post-transcriptional regulation of target genes, including the Chinmo and Mamo transcription factors. We further discuss how upstream inputs, including hormonal signals, modify the output of these gradients to couple neurogenesis with the development of the organism. Finally, we review the roles that the Imp and Syp gradients play beyond the generation of diversity, including the regulation of stem cell proliferation, the timing of neural stem cell lineage termination, and the coupling of neuronal birth order to circuit assembly.

Dissection, Immunohistochemistry and Mounting of Larval and Adult Drosophila Brains for Optic Lobe Visualization.

The Drosophila optic lobe, comprised of four neuropils: the lamina, medulla, lobula and lobula plate, is an excellent model system for exploring the developmental mechanisms that generate neural diversity and drive circuit assembly. Given its complex three-dimensional organization, analysis of the optic lobe requires that one understand how its adult neuropils and larval progenitors are positioned relative to each other and the central brain. Here, we describe a protocol for the dissection, immunostaining and mounting of larval and adult brains for optic lobe imaging. Special emphasis is placed on the relationship between mounting orientation and the spatial organization of the optic lobe. We describe three mounting strategies in the larva (anterior, posterior and lateral) and three in the adult (anterior, posterior and horizontal), each of which provide an ideal imaging angle for a distinct optic lobe structure.