Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.

Components of the genetic network specifying eye development are conserved from flies to humans, but homologies between individual neuronal cell types have been difficult to identify. In the vertebrate retina, the homeodomain-containing transcription factor Chx10 is required for both progenitor cell proliferation and the development of the bipolar interneurons, which transmit visual signals from photoreceptors to ganglion cells.

Cell and tissue specific gene expression is a defining feature of embryonic development in multi-cellular organisms. However, the range of gene expression patterns, the extent of the correlation of expression with function, and the classes of genes whose spatial expression are tightly regulated have been unclear due to the lack of an unbiased, genome-wide survey of gene expression patterns.

We combined Gal4-UAS and the FLP recombinase-FRT and fluorescent reporters to generate cell clones that provide spatial, temporal and genetic information about the origins of individual cells in Drosophila melanogaster. We named this combination the Gal4 technique for real-time and clonal expression (G-TRACE). The approach should allow for screening and the identification of real-time and lineage-traced expression patterns on a genomic scale.

Mislocalization of axonal proteins can result in misassembly and/or miswiring of neural circuits, causing disease. To date, only a handful of genes that control polarized localization of axonal membrane proteins have been identified. Here we report that Drosophila X11/Mint proteins are required for targeting several proteins, including human amyloid precursor protein (APP) and Drosophila APP-like protein (APPL), to axonal membranes and for their exclusion from dendrites of the mushroom body in Drosophila, a brain structure involved in learning and memory. Axonal localization of APP is mediated by an endocytic motif, and loss of X11/Mint results in a dramatic increase in cell-surface levels of APPL, especially on dendrites. Mutations in genes required for endocytosis show similar mislocalization of these proteins to dendrites, and strongly enhance defects seen in X11/Mint mutants. These results suggest that X11/Mint-dependent endocytosis in dendrites may serve to promote the axonal localization of membrane proteins. Since X11/Mint binds to APP, and abnormal trafficking of APP contributes to Alzheimer's disease, deregulation of X11/Mint may be important for Alzheimer's disease pathogenesis.

The Drosophila central brain is largely composed of lineages, units of sibling neurons derived from a single progenitor cell or neuroblast. During the early embryonic period, neuroblasts generate the primary neurons that constitute the larval brain. Neuroblasts reactivate in the larva, adding to their lineages a large number of secondary neurons which, according to previous studies in which selected lineages were labeled by stably expressed markers, differentiate during metamorphosis, sending terminal axonal and dendritic branches into defined volumes of the brain neuropil. We call the overall projection pattern of neurons forming a given lineage the "projection envelope" of that lineage. By inducing MARCM clones at the early larval stage, we labeled the secondary progeny of each neuroblast. For the supraesophageal ganglion excluding mushroom body (the part of the brain investigated in the present work) we obtained 81 different types of clones. Based on the trajectory of their secondary axon tracts (described in the accompanying paper, Lovick et al., 2013), we assigned these clones to specific lineages defined in the larva. Since a labeled clone reveals all aspects (cell bodies, axon tracts, terminal arborization) of a lineage, we were able to describe projection envelopes for all secondary lineages of the supraesophageal ganglion. This work provides a framework by which the secondary neurons (forming the vast majority of adult brain neurons) can be assigned to genetically and developmentally defined groups. It also represents a step towards the goal to establish, for each lineage, the link between its mature anatomical and functional phenotype, and the genetic make-up of the neuroblast it descends from.

Visual information processing in animals with large image forming eyes is carried out in highly structured retinotopically ordered neuropils. Visual neuropils in Drosophila form the optic lobe, which consists of four serially arranged major subdivisions; the lamina, medulla, lobula and lobula plate; the latter three of these are further subdivided into multiple layers. The visual neuropils are formed by more than 100 different cell types, distributed and interconnected in an invariant highly regular pattern. This pattern relies on a protracted sequence of developmental steps, whereby different cell types are born at specific time points and nerve connections are formed in a tightly controlled sequence that has to be coordinated among the different visual neuropils. The developing fly visual system has become a highly regarded and widely studied paradigm to investigate the genetic mechanisms that control the formation of neural circuits. However, these studies are often made difficult by the complex and shifting patterns in which different types of neurons and their connections are distributed throughout development. In the present paper we have reconstructed the three-dimensional architecture of the Drosophila optic lobe from the early larva to the adult. Based on specific markers, we were able to distinguish the populations of progenitors of the four optic neuropils and map the neurons and their connections. Our paper presents sets of annotated confocal z-projections and animated 3D digital models of these structures for representative stages. The data reveal the temporally coordinated growth of the optic neuropils, and clarify how the position and orientation of the neuropils and interconnecting tracts (inner and outer optic chiasm) changes over time. Finally, we have analyzed the emergence of the discrete layers of the medulla and lobula complex using the same markers (DN-cadherin, Brp) employed to systematically explore the structure and development of the central brain neuropil. Our work will facilitate experimental studies of the molecular mechanisms regulating neuronal fate and connectivity in the fly visual system, which bears many fundamental similarities with the retina of vertebrates.

Similarly to vertebrates, arthropod brains are compartmentalized into centers with specific neurological functions such as cognition, behavior, and memory. The centers can be further subdivided into smaller functional units. This raises the question of how these compartments are formed during development and how they are integrated into brain centers. We show here for the first time how the precheliceral neuroectoderm of the spider Cupiennius salei is compartmentalized to form the distinct brain centers of the visual system: the optic ganglia, the mushroom bodies, and the arcuate body. The areas of the visual brain centers are defined by the formation of grooves and vesicles and express the proneural gene CsASH1, followed by expression of the neural differentiation marker Prospero. Furthermore, the transcription factor dachshund, which is strongly enriched in the mushroom bodies and the outer optic ganglion of Drosophila, is expressed in the optic anlagen and the mushroom bodies of the spider. The developing brain centers are further subdivided into single neural precursor groups, which become incorporated into the grooves and vesicles but remain distinguishable throughout development, suggesting that they encode spatial information for neural subtype identity. Several molecular and morphological aspects of the development of the optic ganglia and the mushroom bodies are similar in the spider and in insects. Furthermore, we show that the primary engrailed head spot contributes neurons to the optic ganglia of the median eyes, whereas the secondary head spot, which has been associated with the optic ganglia in insects and crustaceans, is absent.

The neuropile of the Drosophila brain is subdivided into anatomically discrete compartments. Compartments are rich in terminal neurite branching and synapses; they are the neuropile domains in which signal processing takes place. Compartment boundaries are defined by more or less dense layers of glial cells as well as long neurite fascicles. These fascicles are formed during the larval period, when the approximately 100 neuronal lineages that constitute the Drosophila central brain differentiate. Each lineage forms an axon tract with a characteristic trajectory in the neuropile; groups of spatially related tracts congregate into the brain fascicles that can be followed from the larva throughout metamorphosis into the adult stage. Here we provide a map of the adult brain compartments and the relevant fascicles defining compartmental boundaries. We have identified the neuronal lineages contributing to each fascicle, which allowed us to compare compartments of the larval and adult brain directly. Most adult compartments can be recognized already in the early larval brain, where they form a "protomap" of the later adult compartments. Our analysis highlights the morphogenetic changes shaping the Drosophila brain; the data will be important for studies that link early-acting genetic mechanisms to the adult neuronal structures and circuits controlled by these mechanisms.

How global organ asymmetry and individual cell polarity are connected to each other is a central question in studying planar cell polarity (PCP). In the Drosophila wing, which develops PCP along its proximal-distal (P-D) axis, we previously proposed that the core PCP mediator Frizzled redistributes distally in a microtubule (MT)-dependent manner. Here, we performed organ-wide analysis of MT dynamics by introducing quantitative in vivo imaging. We observed MTs aligning along the P-D axis at the onset of redistribution and a small but significant excess of + ends-distal MTs in the proximal region of the wing. This characteristic alignment and asymmetry of MT growth was controlled by atypical cadherins Dachsous (Ds) and Fat (Ft). Furthermore, the action of Ft was mediated in part by PAR-1. All these data support the idea that the active reorientation of MT growth adjusts cell polarity along the organ axis.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.

The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. Here we characterize the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. We then use clonal MARCM labeling to identify all adult-specific neuroblast lineages in the late larval SEG and find a surprisingly small number of neuroblast lineages, 13 paired and one unpaired. The Hox genes Dfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, our findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain.

Site-specific transcription factors (TFs) bind DNA regulatory elements to control expression of target genes, forming the core of gene regulatory networks. Despite decades of research, most studies focus on only a small number of TFs and the roles of many remain unknown.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

Insect brains are formed by conserved sets of neural lineages whose fibers form cohesive bundles with characteristic projection patterns. Within the brain neuropil, these bundles establish a system of fascicles constituting the macrocircuitry of the brain. The overall architecture of the neuropils and the macrocircuitry appear to be conserved. However, variation is observed, for example, in size, shape, and timing of development. Unfortunately, the developmental and genetic basis of this variation is poorly understood, although the rise of new genetically tractable model organisms such as the red flour beetle Tribolium castaneum allows the possibility to gain mechanistic insights. To facilitate such work, we present an atlas of the developing brain of T. castaneum, covering the first larval instar, the prepupal stage, and the adult, by combining wholemount immunohistochemical labeling of fiber bundles (acetylated tubulin) and neuropils (synapsin) with digital 3D reconstruction using the TrakEM2 software package. Upon comparing this anatomical dataset with the published work in Drosophila melanogaster, we confirm an overall high degree of conservation. Fiber tracts and neuropil fascicles, which can be visualized by global neuronal antibodies like antiacetylated tubulin in all invertebrate brains, create a rich anatomical framework to which individual neurons or other regions of interest can be referred to. The framework of a largely conserved pattern allowed us to describe differences between the two species with respect to parameters such as timing of neuron proliferation and maturation. These features likely reflect adaptive changes in developmental timing that govern the change from larval to adult brain.

Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

Autoimmune gastritis (AIG) is a prevalent chronic inflammatory disease with oncogenic potential that causes destruction of parietal cells and severe mucosal atrophy. We aimed to explore the distinctive gene expression profiles, activated signaling pathways, and their underlying mechanisms.

A wide spectrum of occupied ecological niches and spectacular morphological adaptations make social insects a prime object for comparative neuroanatomical studies. Eusocial insects have evolved complex societies based on caste polyphenism. A diverse behavioral repertoire of morphologically distinct castes of the same species requires a high degree of plasticity in the central nervous system. We have analyzed the central brain neuropils and fiber tract systems of the worker of the ant Cardiocondyla obscurior, a model for the study of social traits. Our analysis is based on whole mount preparations of adult brains labeled with an antibody against Drosophila-Synapsin, which cross-reacts strongly with synapses in Cardiocondyla. Neuropil compartments stand out as domains with a certain texture and intensity of the anti-Synapsin signal. By contrast, fiber tracts, which are composed of bundles of axons accompanied by glia and are devoid of synapses, appear as channels or sheaths with low anti-Synapsin signal. We have generated a digital 3D atlas of the Cardiocondyla brain neuropil. The atlas provides a reference for future studies of brain polymorphisms in distinct castes, brain development or localization of neurotransmitter systems.

The application of the Gal4/UAS system to enhancer and gene trapping screens in zebrafish has greatly increased the ability to label and manipulate cell populations in multiple tissues, including the central nervous system (CNS). However the ability to select existing lines for specific applications has been limited by the lack of detailed expression analysis.