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SummaryNeural circuit formation and function require that diverse neurons are specified in appropriate numbers. Known strategies for controlling neuronal numbers involve regulating either cell proliferation or survival. We used the Drosophila visual system to probe how neuronal numbers are set. Photoreceptors from the eye-disc induce their target field, the lamina, such that for every unit eye there is a corresponding lamina unit (column). Although each column initially contains ∼6 post-mitotic lamina precursors, only 5 differentiate into neurons, called L1-L5; the ‘extra’ precursor, which is invariantly positioned above the L5 neuron in each column, undergoes apoptosis. Here, we showed that a glial population called the outer chiasm giant glia (xgO), which resides below the lamina, secretes multiple ligands to induce L5 differentiation in response to EGF from photoreceptors. By forcing neuronal differentiation in the lamina, we uncovered that though fated to die, the ‘extra’ precursor is specified as an L5. Therefore, two precursors are specified as L5s but only one differentiates during normal development. We found that the row of precursors nearest to xgO differentiate into L5s and, in turn, antagonise differentiation signalling to prevent the ‘extra’ precursors from differentiating, resulting in their death. Thus, an intricate interplay of glial signals and feedback from differentiating neurons defines an invariant and stereotyped pattern of neuronal differentiation and programmed cell death to ensure that lamina columns each contain exactly one L5 neuron.

Morphology is a defining feature of neuronal identity. Like neurons, glia display diverse morphologies, both across and within glial classes, but are also known to be morphologically plastic. Here, we explored the relationship between glial morphology and transcriptional signature using theDrosophilacentral nervous system, where glia are categorized into five main classes (outer and inner surface glia, cortex glia, ensheathing glia, and astrocytes), which show within-class morphological diversity. We analysed and validated single cell RNA sequencing data ofDrosophilaglia in two well-characterized tissues from distinct developmental stages, containing distinct circuit types: the embryonic ventral nerve cord (motor) and the adult optic lobes (sensory). Our analysis identified a new morphologically and transcriptionally distinct surface glial population in the ventral nerve cord. However, many glial morphological categories could not be distinguished transcriptionally, and indeed, embryonic and adult astrocytes were transcriptionally analogous despite differences in developmental stage and circuit type. While we did detect extensive within-class transcriptomic diversity for optic lobe glia, this could be explained entirely by glial residence in the most superficial neuropil (lamina) and an associated enrichment for immune-related gene expression. In summary, we generated a single-cell transcriptomic atlas of glia inDrosophila, and our extensivein vivovalidation revealed that glia exhibit more diversity at the morphological level than was detectable at the transcriptional level. This atlas will serve as a resource for the community to probe glial diversity and function.

Defining the origin of neuronal diversity is a major challenge in developmental neurobiology. The Drosophila visual system is an excellent paradigm to study how cellular diversity is generated. Photoreceptors from the eye disc grow their axons into the optic lobe and secrete Hedgehog (Hh) to induce the lamina, such that for every unit eye there is a corresponding lamina unit made up of post-mitotic precursors stacked into columns. Each differentiated column contains five lamina neuron types (L1-L5), making it the simplest neuropil in the optic lobe, yet how this diversity is generated was unknown. Here, we found that Hh pathway activity is graded along the distal-proximal axis of lamina columns and further determined that this gradient in pathway activity arises from a gradient of Hh ligand. We manipulated Hh pathway activity cell-autonomously in lamina precursors and non-cell autonomously by inactivating the Hh ligand, and by knocking it down in photoreceptors. These manipulations showed that different thresholds of activity specify unique cell identities, with more proximal cell types specified in response to progressively lower Hh levels. Thus, our data establish that Hh acts as a morphogen to pattern the lamina. Although, this is the first such report during Drosophila nervous system development, our work uncovers a remarkable similarity with the vertebrate neural tube, which is patterned by Sonic Hedgehog. Altogether, we show that differentiating neurons can regulate the neuronal diversity of their distant target fields through morphogen gradients.

Time-lapse imaging is an essential tool to study dynamic biological processes that cannot be discerned from fixed samples alone. However, imaging cell- and tissue-level processes in intact animals poses numerous challenges if the organism is opaque and/or motile. Explant cultures of intact tissues circumvent some of these challenges, but sample drift remains a considerable obstacle. We employed a simple yet effective technique to immobilize tissues in medium-bathed agarose. We applied this technique to study multipleDrosophilatissues from first-instar larvae to adult stages in various orientations and with no evidence of anisotropic pressure or stress damage. Using this method, we were able to image fine features for up to 18 hours and make novel observations. Specifically, we report that fibers characteristic of quiescent neuroblasts are inherited by their basal daughters during reactivation; that the lamina in the developing visual system is assembled roughly 2-3 columns at a time; that lamina glia positions are dynamic during development; and that the nuclear envelopes of adult testis cyst stem cells do not break down completely during mitosis. In all, we demonstrate that our protocol is well-suited for tissue immobilization and long-term live imaging, enabling new insights into tissue and cell dynamics inDrosophila.

Wiring up the eye During development, sensory systems must build topographic maps by connecting neurons at different levels within a circuit. Fernandes et al. now open a window into how the Drosophila eye develops these maps (see the Perspective by Isaacman-Beck and Clandinin). The authors show that glial cells that ensheath axons relay cues from photoreceptors to induce the differentiation of the photoreceptor target field—the so-called lamina neurons—in the Drosophila visual system. Thus, glia can play an instructive role in differentiation, helping to direct the spatiotemporal patterning of neurogenesis. Science , this issue p. 886 ; see also p. 867

SUMMARY How neuronal and glial fates are specified from neural precursor cells is an important question for developmental neurobiologists. We address this question in the Drosophila optic lobe, composed of the lamina, medulla, and lobula complex. We show that two gliogenic regions posterior to the prospective lamina also produce lamina wide-field (Lawf) neurons, which share common progenitors with lamina glia. These progenitors express neither canonical neuroblast nor lamina precursor cell markers. They bifurcate into two sub-lineages in response to Notch signaling, generating lamina glia or Lawf neurons, respectively. The newly born glia and Lawfs then migrate tangentially over substantial distances to reach their target tissue. Thus, Lawf neurogenesis, which includes a common origin with glia, as well as neuronal migration, resembles several aspects of vertebrate neurogenesis., Graphical abstract

Various regions of the developing brain coordinate their construction so that the correct types and numbers of cells are generated to build a functional network. We previously discovered that wrapping glia in the Drosophila visual system are essential for coordinating retinal and lamina development. We showed that wrapping glia, which ensheath photoreceptor axons, respond to an epidermal growth factor cue from photoreceptors by secreting insulins. Wrapping glial insulins activate the mitogen-activated protein kinase (MAPK) pathway downstream of insulin receptor in lamina precursors to induce neuronal differentiation. The signaling relay via wrapping glia introduces a delay that allows the lamina to assemble the correct stoichiometry and physical alignment of precursors before differentiating and imposes a stereotyped spatiotemporal pattern that is relevant for specifying the individual lamina neuron fates. Here, we further describe how wrapping glia morphogenesis correlates with the timing of lamina neuron differentiation by 2-photon live imaging. We also show that although MAPK activity in lamina precursors drives neuronal differentiation, the upstream receptor driving MAPK activation in lamina precursors and the ligand secreted by wrapping glia to trigger it differentially affect lamina neuron differentiation. These results highlight differences in MAPK signaling properties and confirm that communication between photoreceptors, wrapping glia, and lamina precursors must be precisely controlled to build a complex neural network.

Neuronal birth and specification must be coordinated across the developing brain to generate the neurons that constitute neural circuits. We used the

Ants are some of the most abundant and familiar animals on Earth, and they play vital roles in most terrestrial ecosystems. Although all ants are eusocial, and display a variety of complex and fascinating behaviors, few genomic resources exist for them. Here, we report the draft genome sequence of a particularly widespread and well-studied species, the invasive Argentine ant ( Linepithema humile ), which was accomplished using a combination of 454 (Roche) and Illumina sequencing and community-based funding rather than federal grant support. Manual annotation of >1,000 genes from a variety of different gene families and functional classes reveals unique features of the Argentine ant's biology, as well as similarities to Apis mellifera and Nasonia vitripennis . Distinctive features of the Argentine ant genome include remarkable expansions of gustatory (116 genes) and odorant receptors (367 genes), an abundance of cytochrome P450 genes (>110), lineage-specific expansions of yellow/major royal jelly proteins and desaturases, and complete CpG DNA methylation and RNAi toolkits. The Argentine ant genome contains fewer immune genes than Drosophila and Tribolium , which may reflect the prominent role played by behavioral and chemical suppression of pathogens. Analysis of the ratio of observed to expected CpG nucleotides for genes in the reproductive development and apoptosis pathways suggests higher levels of methylation than in the genome overall. The resources provided by this genome sequence will offer an abundance of tools for researchers seeking to illuminate the fascinating biology of this emerging model organism.

During development a limited number of progenitors generate diverse cell types that comprise the nervous system. Neuronal diversity, which arises largely at the level of neural stem cells, is critical for brain function. Often these cells exhibit temporal patterning: they sequentially produce neurons of distinct cell fates as a consequence of intrinsic and/or extrinsic cues. Here, we review recent advances in temporal patterning during neuronal specification, focusing on conserved players and mechanisms in invertebrate and vertebrate models. These studies underscore temporal patterning as an evolutionarily conserved strategy to generate neuronal diversity. Understanding the general principles governing temporal patterning and the molecular players involved will improve our ability to direct neural progenitors towards specific neuronal fates for brain repair.

Generating cellular heterogeneity is crucial to the development of complex organs. Organ-fate selector genes and signalling pathways generate cellular diversity by subdividing and patterning naïve tissues to assign them regional identities. The Drosophila eye–antennal imaginal disc is a well-characterised system in which to study regional specification; it is first divided into antennal and eye fates and subsequently retinal differentiation occurs within only the eye field. During development, signalling pathways and selector genes compete with and mutually antagonise each other to subdivide the tissue. Wingless (Wg) signalling is the main inhibitor of retinal differentiation; it does so by promoting antennal/head-fate via selector factors and by antagonising Hedgehog (Hh), the principal differentiation-initiating signal. Wg signalling must be suppressed by JAK/STAT at the disc posterior in order to initiate retinal differentiation. Ras/MEK/MAPK signalling has also been implicated in initiating retinal differentiation but its mode of action is not known. We find that compromising Ras/MEK/MAPK signalling in the early larval disc results in expanded antennal/head cuticle at the expense of the compound eye. These phenotypes correspond both to perturbations in selector factor expression, and to de-repressed wg. Indeed, STAT activity is reduced due to decreased mobility of the ligand Unpaired (Upd) along with a corresponding loss in Dally-like protein (Dlp), a heparan sulphate proteoglycan (HSPG) that aids Upd diffusion. Strikingly, blocking HSPG biogenesis phenocopies compromised Ras/MEK/MAPK, while restoring HSPG expression rescues the adult phenotype significantly. This study identifies a novel mode by which the Ras/MEK/MAPK pathway regulates regional-fate specification via HSPGs during development.

Combinations of spatially and temporally restricted transcription factors are shown to coordinate movement in nematode worms by controlling the formation of synaptic connections to and from motor neurons. See Letter p.83 It is important for neuronal synapses to be formed at the right time and in the right place during embryonic development. This is exemplified by the by the DD and VD motor neurons of the round worm Caenorhabditis elegans, where DD neurons undergo dorso-ventral rewiring at the first larval stage and VD motor neurons maintain an unchanged innervation pattern differing from that of the DD motor neurons. Here Oliver Hobert and colleagues identify three gene-regulatory factors (UNC-30, LIN-14 and UNC-55 DNA-binding proteins) and their target (OIG-1, a previously unknown extracellular 'synaptic organizer' protein), as controlling the proper wiring and rewiring of DD and VD neurons. The work reveals an 'intersectional' strategy, whereby UNC-30 works with temporally — but not spatially — controlled LIN-14 in DD neurons, and with spatially — but not temporally — controlled UNC-55 in VD neurons.

We report the draft genome sequence of the red harvester ant, Pogonomyrmex barbatus . The genome was sequenced using 454 pyrosequencing, and the current assembly and annotation were completed in less than 1 y. Analyses of conserved gene groups (more than 1,200 manually annotated genes to date) suggest a high-quality assembly and annotation comparable to recently sequenced insect genomes using Sanger sequencing. The red harvester ant is a model for studying reproductive division of labor, phenotypic plasticity, and sociogenomics. Although the genome of P. barbatus is similar to other sequenced hymenopterans ( Apis mellifera and Nasonia vitripennis ) in GC content and compositional organization, and possesses a complete CpG methylation toolkit, its predicted genomic CpG content differs markedly from the other hymenopterans. Gene networks involved in generating key differences between the queen and worker castes (e.g., wings and ovaries) show signatures of increased methylation and suggest that ants and bees may have independently co-opted the same gene regulatory mechanisms for reproductive division of labor. Gene family expansions (e.g., 344 functional odorant receptors) and pseudogene accumulation in chemoreception and P450 genes compared with A. mellifera and N. vitripennis are consistent with major life-history changes during the adaptive radiation of Pogonomyrmex spp., perhaps in parallel with the development of the North American deserts.

Many genetic networks are astonishingly robust to quantitative variation, allowing these networks to continue functioning in the face of mutation and environmental perturbation. However, the evolution of such robustness remains poorly understood for real genetic networks. Here we explore whether and how ploidy and recombination affect the evolution of robustness in a detailed computational model of the segment polarity network. We introduce a novel computational method that predicts the quantitative values of biochemical parameters from bit sequences representing genotype, allowing our model to bridge genotype to phenotype. Using this, we simulate 2,000 generations of evolution in a population of individuals under stabilizing and truncation selection, selecting for individuals that could sharpen the initial pattern of engrailed and wingless expression. Robustness was measured by simulating a mutation in the network and measuring the effect on the engrailed and wingless patterns; higher robustness corresponded to insensitivity of this pattern to perturbation. We compared robustness in diploid and haploid populations, with either asexual or sexual reproduction. In all cases, robustness increased, and the greatest increase was in diploid sexual populations; diploidy and sex synergized to evolve greater robustness than either acting alone. Diploidy conferred increased robustness by allowing most deleterious mutations to be rescued by a working allele. Sex (recombination) conferred a robustness advantage through “survival of the compatible”: those alleles that can work with a wide variety of genetically diverse partners persist, and this selects for robust alleles., Author Summary Most so-called “higher organisms” are diploid (have two copies of each gene) and reproduce sexually. Diploidy may be advantageous if one functional copy can mask the effects of a mutation in the other copy; however, it is a liability if most mutations are dominant. Sex can increase genetic diversity and the rate of evolution by creating new combinations of alleles that might function better together but can also disrupt working combinations. Given these trade-offs, why are sex and diploidy so common, and why do they occur so often together? We hypothesize that sex and diploidy allow gene networks to evolve to function more robustly in the face of genetic and environmental variation. This robustness would be advantageous because organisms are exposed to constantly changing environments and all genes undergo mutation. To test this hypothesis, we simulated evolution in a model of the segment polarity network, a well-studied group of genes essential for proper development in many organisms. We compared the robustness of haploid and diploid populations that reproduced either sexually or asexually. Sexually reproducing diploid populations evolved the greatest robustness, suggesting an explanation for the selective advantage of diploid sexual reproduction.

During neurogenesis, conserved tissue-specific proneural factors establish a cell׳s competence to take on neural fate from within a field of unspecified cells. Proneural genes encode basic helix-loop-helix transcription factors that promote the expression of ‘core’ and subtype-specific target genes. Target genes include both pan-neuronal genes and genes that aid in the process of refinement, known as lateral inhibition. In this process, proneural gene expression is increased in the neural progenitor while simultaneously down-regulated in the surrounding cells, in a Notch signalling-dependent manner. Here, we identify nemo (nmo) as a target of members of both Drosophila Atonal and Achaete-Scute proneural factor families and find that mammalian proneural homologs induce Nemo-like-kinase (Nlk) expression in cell culture. We find that nmo loss of function leads to reduced expression of Notch targets and to perturbations in Notch-mediated lateral inhibition. Furthermore, Notch hyperactivity can compensate for nmo loss in the Drosophila eye. Thus nmo promotes Notch-mediated lateral inhibition downstream of proneural factors during neurogenesis.