The study of how transcriptional control and cell signaling influence neurons and glia to acquire their differentiated properties is fundamental to understanding CNS development and function. The Drosophila CNS midline cells are an excellent system for studying these issues because they consist of a small population of diverse cells with well-defined gene expression profiles. In this paper, the origins and differentiation of midline neurons and glia were analyzed. Midline precursor (MP) cells each divide once giving rise to two neurons; here, we use a combination of single-cell gene expression mapping and time-lapse imaging to identify individual MPs, their locations, movements and stereotyped patterns of division. The role of Notch signaling was investigated by analyzing 37 midline-expressed genes in Notch pathway mutant and misexpression embryos. Notch signaling had opposing functions: it inhibited neurogenesis in MP1,3,4 and promoted neurogenesis in MP5,6. Notch signaling also promoted midline glial and median neuroblast cell fate. This latter result suggests that the median neuroblast resembles brain neuroblasts that require Notch signaling, rather than nerve cord neuroblasts, the formation of which is inhibited by Notch signaling. Asymmetric MP daughter cell fates also depend on Notch signaling. One member of each pair of MP3-6 daughter cells was responsive to Notch signaling. By contrast,the other daughter cell asymmetrically acquired Numb, which inhibited Notch signaling, leading to a different fate choice. In summary, this paper describes the formation and division of MPs and multiple roles for Notch signaling in midline cell development, providing a foundation for comprehensive molecular analyses.

Horizontal cells are inhibitory interneurons with laterally oriented dendrites that overlap one another, contacting the pedicles of cone photoreceptors. Because of their regular spacing, the network of horizontal cells provides a uniform coverage of the retinal surface. The developmental processes establishing these network properties are undefined, but cell-intrinsic instructions and interactions with other cells have each been suggested to play a role. Here, we show that the intercellular spacing of horizontal cells is essentially independent of genetic background and is predicted by local density, suggesting that horizontal cell positioning is modulated by proximity to other horizontal cells. Dendritic field area compensates for this variation in intercellular spacing, maintaining constant dendritic coverage between strains. Functional dendritic overlap is achieved anatomically at the level of the pedicles, where horizontal cells interact with one another to establish their connectivity: the number of dendritic terminals contacting a pedicle changes, reciprocally, between neighboring horizontal cells during development based on their relative proximity to each pedicle. Cellular morphology is also shown to be regulated by the afferents themselves: afferent elimination before innervation does not alter dendritic field size nor stratification but compromises dendritic branching and prevents terminal formation. Afferent and homotypic interactions therefore generate the morphology, spacing, and connectivity of horizontal cells underlying their functional coverage of the retina.

SUMMARYThe Drosophila CNS contains a variety of glia, including highly specialized glia that reside at the CNS midline and functionally resemble the midline floor plate glia of the vertebrate spinal cord. Both insect and vertebrate midline glia play important roles in ensheathing axons that cross the midline and secreting signals that control a variety of developmental processes. The Drosophila midline glia consist of two spatially and functionally distinct populations. The anterior midline glia (AMG) are ensheathing glia that migrate, surround and send processes into the axon commissures. By contrast, the posterior midline glia (PMG) are nonensheathing glia. Together, the Notch and hedgehog signaling pathways generate AMG and PMG from midline neural precursors. Notch signaling is required for midline glial formation and for transcription of a core set of midline glial-expressed genes. The Hedgehog morphogen is secreted from ectodermal cells adjacent to the CNS midline and directs a subset of midline glia to become PMG. Two transcription factor genes, runt and engrailed, play important roles in AMG and PMG development. The runt gene is expressed in AMG, represses engrailed and maintains AMG gene expression. The engrailed gene is expressed in PMG, represses runt and maintains PMG gene expression. In addition, engrailed can direct midline glia to a PMG-like nonensheathing fate. Thus, two signaling pathways and runt-engrailed mutual repression initiate and maintain two distinct populations of midline glia that differ functionally in gene expression, glial migration, axon ensheathment, process extension and patterns of apoptosis.

The present investigation has sought to determine whether the population of retinal horizontal cells undergoes an increase in the precision of its mosaic patterning during postnatal development, and if so, whether this increase is compatible with three different mechanistic accounts of retinal mosaic formation. Horizontal cells were labeled with antibodies to neurofilaments or calbindin at different developmental stages, and then visualized in retinal wholemounts. Multiple fields were sampled from each retina to determine horizontal cell density, while the X-Y coordinates of each cell in a field were determined. An estimate of total horizontal cell number was calculated for each retina, while the Voronoi domain regularity index and the packing factor were computed for each field. Two strains of mice showing a two-fold difference in the size of their horizontal cell population in maturity were sampled, C57BL/6J and A/J. Horizontal cell number in C57BL/6J was approximately twice that observed in A/J at all postnatal stages, with neither strain showing an effect of age on horizontal cell number. In both strains, however, the Voronoi domain regularity index and the packing factor were significantly lower at P-1 relative to later developmental stages. These results show that accounts of mosaic formation proposing the selective death of irregularly positioned cells, or the periodic occurrence of fate-determining events, are insufficient to establish the final patterning achieved by horizontal cells. Rather, they support the hypothesis that tangential dispersion enhances mosaic patterning during postnatal development.

Background: The Drosophila CNS midline cells are an excellent model system to study neuronal and glial development because of their diversity of cell types and the relative ease in identifying and studying the function of midline-expressed genes. In situ hybridization experiments generated a large dataset of midline gene expression patterns. To help synthesize these data and make them available to the scientific community, we developed a web-accessible database.