High-throughput identification of the spatial origins of Drosophila optic lobe neurons using single-cell mRNA-sequencing.

The medulla is the largest neuropil of the Drosophila optic lobe. It contains about 100 neuronal types that have been comprehensively characterized morphologically and molecularly. These neuronal types are specified from a larval neuroepithelium called the Outer Proliferation Center (OPC) via the integration of temporal, spatial, and Notch-driven mechanisms. Although we recently characterized the temporal windows of origin of all medulla neurons, as well as their Notch status, their spatial origins remained unknown. Here, we isolated cells from different OPC spatial domains and performed single-cell mRNA-sequencing to identify the neuronal types produced in these domains. This allowed us to characterize in a high-throughput manner the spatial origins of all medulla neurons and to identify two new spatial subdivisions of the OPC. Moreover, our work shows that the most abundant neuronal types are produced from epithelial domains of different sizes despite being present in a similar number of copies. Combined with our previously published scRNA-seq developmental atlas of the optic lobe, our work opens the door for further studies on how specification factor expression in progenitors impacts gene expression in developing and adult neurons.

Spalt and disco define the dorsal-ventral neuroepithelial compartments of the developing Drosophila medulla.

Spatial patterning of neural stem cell populations is a powerful mechanism by which to generate neuronal diversity. In the developing Drosophila medulla, the symmetrically dividing neuroepithelial cells of the outer proliferation center crescent are spatially patterned by the nonoverlapping expression of 3 transcription factors: Vsx1 in the center, Optix in the adjacent arms, and Rx in the tips. These spatial genes compartmentalize the outer proliferation center and, together with the temporal patterning of neuroblasts, act to diversify medulla neuronal fates. The observation that the dorsal and ventral halves of the outer proliferation center also grow as distinct compartments, together with the fact that a subset of neuronal types is generated from only one half of the crescent, suggests that additional transcription factors spatially pattern the outer proliferation center along the dorsal-ventral axis. Here, we identify the spalt (salm and salr) and disco (disco and disco-r) genes as the dorsal-ventral patterning transcription factors of the outer proliferation center. Spalt and Disco are differentially expressed in the dorsal and ventral outer proliferation center from the embryo through to the third instar larva, where they cross-repress each other to form a sharp dorsal-ventral boundary. We show that hedgehog is necessary for Disco expression in the embryonic optic placode and that disco is subsequently required for the development of the ventral outer proliferation center and its neuronal progeny. We further demonstrate that this dorsal-ventral patterning axis acts independently of Vsx1-Optix-Rx and thus propose that Spalt and Disco represent a third outer proliferation center patterning axis that may act to further diversify medulla fates.

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.

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.