Neurotransmitter classification from electron microscopy images at synaptic sites in Drosophila melanogaster.

High-resolution electron microscopy of nervous systems has enabled the reconstruction of synaptic connectomes. However, we do not know the synaptic sign for each connection (i.e., whether a connection is excitatory or inhibitory), which is implied by the released transmitter. We demonstrate that artificial neural networks can predict transmitter types for presynapses from electron micrographs: a network trained to predict six transmitters (acetylcholine, glutamate, GABA, serotonin, dopamine, octopamine) achieves an accuracy of 87% for individual synapses, 94% for neurons, and 91% for known cell types across a D. melanogaster whole brain. We visualize the ultrastructural features used for prediction, discovering subtle but significant differences between transmitter phenotypes. We also analyze transmitter distributions across the brain and find that neurons that develop together largely express only one fast-acting transmitter (acetylcholine, glutamate, or GABA). We hope that our publicly available predictions act as an accelerant for neuroscientific hypothesis generation for the fly.

Developmental plasticity, straight from the worm's mouth.

Developmental plasticity in response to environmental conditions (polyphenism) plays an important role in evolutionary theory. Analyzing the nematode taxon Pristionchus, Ragsdale et al. demonstrate that a single gene underlies the nematode's ability to develop distinct mouth forms in response to environmental changes.

Rewarding Capacity of Optogenetically Activating a Giant GABAergic Central-Brain Interneuron in Larval Drosophila.

Larvae of the fruit fly Drosophila melanogaster are a powerful study case for understanding the neural circuits underlying behavior. Indeed, the numerical simplicity of the larval brain has permitted the reconstruction of its synaptic connectome, and genetic tools for manipulating single, identified neurons allow neural circuit function to be investigated with relative ease and precision. We focus on one of the most complex neurons in the brain of the larva (of either sex), the GABAergic anterior paired lateral neuron (APL). Using behavioral and connectomic analyses, optogenetics, Ca2+ imaging, and pharmacology, we study how APL affects associative olfactory memory. We first provide a detailed account of the structure, regional polarity, connectivity, and metamorphic development of APL, and further confirm that optogenetic activation of APL has an inhibiting effect on its main targets, the mushroom body Kenyon cells. All these findings are consistent with the previously identified function of APL in the sparsening of sensory representations. To our surprise, however, we found that optogenetically activating APL can also have a strong rewarding effect. Specifically, APL activation together with odor presentation establishes an odor-specific, appetitive, associative short-term memory, whereas naive olfactory behavior remains unaffected. An acute, systemic inhibition of dopamine synthesis as well as an ablation of the dopaminergic pPAM neurons impair reward learning through APL activation. Our findings provide a study case of complex circuit function in a numerically simple brain, and suggest a previously unrecognized capacity of central-brain GABAergic neurons to engage in dopaminergic reinforcement.SIGNIFICANCE STATEMENT The single, identified giant anterior paired lateral (APL) neuron is one of the most complex neurons in the insect brain. It is GABAergic and contributes to the sparsening of neuronal activity in the mushroom body, the memory center of insects. We provide the most detailed account yet of the structure of APL in larval Drosophila as a neurogenetically accessible study case. We further reveal that, contrary to expectations, the experimental activation of APL can exert a rewarding effect, likely via dopaminergic reward pathways. The present study both provides an example of unexpected circuit complexity in a numerically simple brain, and reports an unexpected effect of activity in central-brain GABAergic circuits.

One too many: the surprising heterogeneity of Drosophila macrophages.

Macrophages are motile cells that roam the extracellular spaces within organs or the body cavity and carry out essential functions in organ development and immunity. New work published in The EMBO Journal adds surprising new insights into the heterogeneity of Drosophila macrophages revealing many similarities to their vertebrate counterparts.

Ultrastructural analysis and 3D reconstruction of the frontal sensory-glandular complex and its neural projections in the platyhelminth Macrostomum lignano.

The marine microturbellarian Macrostomum lignano (Platyhelminthes, Rhabditophora) is an emerging laboratory model used by a growing community of researchers because it is easy to cultivate, has a fully sequenced genome, and offers multiple molecular tools for its study. M. lignano has a compartmentalized brain that receives sensory information from receptors integrated in the epidermis. Receptors of the head, as well as accompanying glands and specialized epidermal cells, form a compound sensory structure called the frontal glandular complex. In this study, we used semi-serial transmission electron microscopy (TEM) to document the types, ultrastructure, and three-dimensional architecture of the cells of the frontal glandular complex. We distinguish a ventral compartment formed by clusters of type 1 (multiciliated) sensory receptors from a central domain where type 2 (collar) sensory receptors predominate. Six different types of glands (rhammite glands, mucoid glands, glands with aster-like and perimaculate granula, vacuolated glands, and buckle glands) are closely associated with type 1 sensory receptors. Endings of a seventh type of gland (rhabdite gland) define a dorsal domain of the frontal glandular complex. A pair of ciliary photoreceptors is closely associated with the base of the frontal glandular complex. Bundles of dendrites, connecting the receptor endings with their cell bodies which are located in the brain, form the (frontal) peripheral nerves. Nerve fibers show a varicose structure, with thick segments alternating with thin segments, and are devoid of a glial layer. This distinguishes platyhelminths from larger and/or more complex invertebrates whose nerves are embedded in prominent glial sheaths.

Orthopedia expression during Drosophila melanogaster nervous system development and its regulation by microRNA-252.

During brain development of Drosophila melanogaster many transcription factors are involved in regulating neural fate and morphogenesis. In our study we show that the transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, plays an important role in this process. Otp is expressed in a stable pattern in defined lineages from mid-embryonic stages into the adult brain and therefore a very stable marker for these lineages. We determined the abundance of the two different otp transcripts in the brain and hindgut during development using qPCR. CRISPR/Cas9 generated otp mutants of the longer protein form significantly affect the expression of Otp in specific areas. We generated an otp enhancer trap strain by gene targeting and reintegration of Gal4, which mimics the complete expression of otp during development except the embryonic hindgut expression. Since in the embryo, the expression of Otp is posttranscriptionally regulated, we looked for putative miRNAs interacting with the otp 3'UTR, and identified microRNA-252 as a candidate. Further analyses with mutated and deleted forms of the microRNA-252 interacting sequence in the otp 3'UTR demonstrate an in vivo interaction of microRNA-252 with the otp 3'UTR. An effect of this interaction is seen in the adult brain, where Otp expression is partially abolished in a knockout strain of microRNA-252. Our results show that Otp is another important factor for brain development in Drosophila melanogaster.

Lineages to circuits: the developmental and evolutionary architecture of information channels into the central complex.

The representation and integration of internal and external cues is crucial for any organism to execute appropriate behaviors. In insects, a highly conserved region of the brain, the central complex (CX), functions in the representation of spatial information and behavioral states, as well as the transformation of this information into desired navigational commands. How does this relatively invariant structure enable the incorporation of information from the diversity of anatomical, behavioral, and ecological niches occupied by insects? Here, we examine the input channels to the CX in the context of their development and evolution. Insect brains develop from ~ 100 neuroblasts per hemisphere that divide systematically to form "lineages" of sister neurons, that project to their target neuropils along anatomically characteristic tracts. Overlaying this developmental tract information onto the recently generated Drosophila "hemibrain" connectome and integrating this information with the anatomical and physiological recording of neurons in other species, we observe neuropil and lineage-specific innervation, connectivity, and activity profiles in CX input channels. We posit that the proliferative potential of neuroblasts and the lineage-based architecture of information channels enable the modification of neural networks across existing, novel, and deprecated modalities in a species-specific manner, thus forming the substrate for the evolution and diversification of insect navigational circuits.

The role of cell lineage in the development of neuronal circuitry and function.

Complex nervous systems have a modular architecture, whereby reiterative groups of neurons ("modules") that share certain structural and functional properties are integrated into large neural circuits. Neurons develop from proliferating progenitor cells that, based on their location and time of appearance, are defined by certain genetic programs. Given that genes expressed by a given progenitor play a fundamental role in determining the properties of its lineage (i.e., the neurons descended from that progenitor), one efficient developmental strategy would be to have lineages give rise to the structural modules of the mature nervous system. It is clear that this strategy plays an important role in neural development of many invertebrate animals, notably insects, where the availability of genetic techniques has made it possible to analyze the precise relationship between neuronal origin and differentiation since several decades. Similar techniques, developed more recently in the vertebrate field, reveal that functional modules of the mammalian cerebral cortex are also likely products of developmentally defined lineages. We will review studies that relate cell lineage to circuitry and function from a comparative developmental perspective, aiming at enhancing our understanding of neural progenitors and their lineages, and translating findings acquired in different model systems into a common conceptual framework.

Identification of Dopaminergic Neurons That Can Both Establish Associative Memory and Acutely Terminate Its Behavioral Expression.

An adaptive transition from exploring the environment in search of vital resources to exploiting these resources once the search was successful is important to all animals. Here we study the neuronal circuitry that allows larval Drosophila melanogaster of either sex to negotiate this exploration-exploitation transition. We do so by combining Pavlovian conditioning with high-resolution behavioral tracking, optogenetic manipulation of individually identified neurons, and EM data-based analyses of synaptic organization. We find that optogenetic activation of the dopaminergic neuron DAN-i1 can both establish memory during training and acutely terminate learned search behavior in a subsequent recall test. Its activation leaves innate behavior unaffected, however. Specifically, DAN-i1 activation can establish associative memories of opposite valence after paired and unpaired training with odor, and its activation during the recall test can terminate the search behavior resulting from either of these memories. Our results further suggest that in its behavioral significance DAN-i1 activation resembles, but does not equal, sugar reward. Dendrogram analyses of all the synaptic connections between DAN-i1 and its two main targets, the Kenyon cells and the mushroom body output neuron MBON-i1, further suggest that the DAN-i1 signals during training and during the recall test could be delivered to the Kenyon cells and to MBON-i1, respectively, within previously unrecognized, locally confined branching structures. This would provide an elegant circuit motif to terminate search on its successful completion.SIGNIFICANCE STATEMENT In the struggle for survival, animals have to explore their environment in search of food. Once food is found, however, it is adaptive to prioritize exploiting it over continuing a search that would now be as pointless as searching for the glasses you are wearing. This exploration-exploitation trade-off is important for animals and humans, as well as for technical search devices. We investigate which of the only 10,000 neurons of a fruit fly larva can tip the balance in this trade-off, and identify a single dopamine neuron called DAN-i1 that can do so. Given the similarities in dopamine neuron function across the animal kingdom, this may reflect a general principle of how search is terminated once it is successful.

Structural aspects of the aging invertebrate brain.

Aging is characterized by a decline in neuronal function in all animal species investigated so far. Functional changes are accompanied by and may be in part caused by, structurally visible degenerative changes in neurons. In the mammalian brain, normal aging shows abnormalities in dendrites and axons, as well as ultrastructural changes in synapses, rather than global neuron loss. The analysis of the structural features of aging neurons, as well as their causal link to molecular mechanisms on the one hand, and the functional decline on the other hand is crucial in order to understand the aging process in the brain. Invertebrate model organisms like Drosophila and C. elegans offer the opportunity to apply a forward genetic approach to the analysis of aging. In the present review, we aim to summarize findings concerning abnormalities in morphology and ultrastructure in invertebrate brains during normal aging and compare them to what is known for the mammalian brain. It becomes clear that despite of their considerably shorter life span, invertebrates display several age-related changes very similar to the mammalian condition, including the retraction of dendritic and axonal branches at specific locations, changes in synaptic density and increased accumulation of presynaptic protein complexes. We anticipate that continued research efforts in invertebrate systems will significantly contribute to reveal (and possibly manipulate) the molecular/cellular pathways leading to neuronal aging in the mammalian brain.

Serial electron microscopic reconstruction of the drosophila larval eye: Photoreceptors with a rudimentary rhabdomere of microvillar-like processes.

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 µm diameter) alternating with thin segments (>0.1 µm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

An Emerging System to Study Photosymbiosis, Brain Regeneration, Chronobiology, and Behavior: The Marine Acoel Symsagittifera roscoffensis.

The acoel worm Symsagittifera roscoffensis, an early offshoot of the Bilateria and the only well-studied marine acoel that lives in a photosymbiotic relationship, exhibits a centralized nervous system, brain regeneration, and a wide repertoire of complex behaviors such as circatidal rhythmicity, photo/geotaxis, and social interactions. While this animal can be collected by the thousands and is studied historically, significant progress is made over the last decade to develop it as an emerging marine model. The authors here present the feasibility of culturing it in the laboratory and describe the progress made on different areas, including genomic and tissue architectures, highlighting the associated challenges. In light of these developments, and on the ability to access abundant synchronized embryos, the authors put forward S. roscoffensis as a marine system to revisit questions in the areas of photosymbiosis, regeneration, chronobiology, and the study of complex behaviors from a molecular and evolutionary perspective.

Mechanisms of cnidocyte development in the moon jellyfish Aurelia.

Stinging cells called cnidocytes are a defining trait of the cnidarians (sea anemones, corals, jellyfish, and their relatives). In hydrozoan cnidarians such as Hydra, cnidocytes develop from interstitial stem cells set aside in the ectoderm. It is less clear how cnidocytes develop outside the Hydrozoa, as other cnidarians appear to lack interstitial stem cells. We addressed this question by studying cnidogenesis in the moon jellyfish (Aurelia) through the visualization of minicollagen-a protein associated with cnidocyte development-as well as transmission electron microscopy. We discovered that developing cnidoblasts are rare or absent in feeding structures rich in mature cnidocytes, such as tentacles and lappets. Using transmission electron microscopy, we determined that the progenitors of cnidocytes have traits consistent with epitheliomuscular cells. Our data suggests a dynamic where cnidocytes develop at high concentrations in the epithelium of more proximal regions, and subsequently migrate to more distal regions where they exhibit high usage and turnover. Similar to some anthozoans, cnidocytes in Aurelia do not appear to be generated by interstitial stem cells; instead, epitheliomuscular cells appear to be the progenitor cell type. This observation polarizes the evolution of cnidogenesis, and raises the question of how interstitial stem cells came to regulate cnidogenesis in hydrozoans.

Spatio-temporal pattern of neuronal differentiation in the Drosophila visual system: A user's guide to the dynamic morphology of the developing optic lobe.

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.

bHLH proneural genes as cell fate determinants of entero-endocrine cells, an evolutionarily conserved lineage sharing a common root with sensory neurons.

Entero-endocrine cells involved in the regulation of digestive function form a large and diverse cell population within the intestinal epithelium of all animals. Together with absorptive enterocytes and secretory gland cells, entero-endocrine cells are generated by the embryonic endoderm and, in the mature animal, from a pool of endoderm derived, self-renewing stem cells. Entero-endocrine cells share many structural/functional and developmental properties with sensory neurons, which hints at the possibility of an ancient evolutionary relationship between these two cell types. We will survey in this article recent findings that emphasize the similarities between entero-endocrine cells and sensory neurons in vertebrates and insects, for which a substantial volume of data pertaining to the entero-endocrine system has been compiled. We will then report new findings that shed light on the specification and morphogenesis of entero-endocrine cells in Drosophila. In this system, presumptive intestinal stem cells (pISCs), generated during early metamorphosis, undergo several rounds of mitosis that produce the endocrine cells and stem cells (ISCs) with which the fly is born. Clonal analysis demonstrated that individual pISCs can give rise to endocrine cells expressing different types of peptides. Immature endocrine cells start out as unpolarized cells located basally of the gut epithelium; they each extend an apical process into the epithelium which establishes a junctional complex and apical membrane specializations contacting the lumen of the gut. Finally, we show that the Drosophila homolog of ngn3, a bHLH gene that defines the entero-endocrine lineage in mammals, is expressed and required for the differentiation of this cell type in the fly gut.

Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells.

Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. In this paper we investigated the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. Our results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation.

Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation.

The visceral musculature of the Drosophila intestine plays important roles in digestion as well as development. Detailed studies investigating the embryonic development of the visceral muscle exist; comparatively little is known about postembryonic development and metamorphosis of this tissue. In this study we have combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. We could not obtain evidence that cells apart from the dedifferentiated larval visceral muscle contributed to the adult muscle, nor does it appear that the number of adult fibers (or nuclei per fiber) is increased over that of the larva by proliferation. In contrast to the musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes.

A conserved plan for wiring up the fan-shaped body in the grasshopper and Drosophila.

The central complex comprises an elaborate system of modular neuropils which mediate spatial orientation and sensory-motor integration in insects such as the grasshopper and Drosophila. The neuroarchitecture of the largest of these modules, the fan-shaped body, is characterized by its stereotypic set of decussating fiber bundles. These are generated during development by axons from four homologous protocerebral lineages which enter the commissural system and subsequently decussate at stereotypic locations across the brain midline. Since the commissural organization prior to fan-shaped body formation has not been previously analyzed in either species, it was not clear how the decussating bundles relate to individual lineages, or if the projection pattern is conserved across species. In this study, we trace the axonal projections from the homologous central complex lineages into the commissural system of the embryonic and larval brains of both the grasshopper and Drosophila. Projections into the primordial commissures of both species are found to be lineage-specific and allow putatively equivalent fascicles to be identified. Comparison of the projection pattern before and after the commencement of axon decussation in both species reveals that equivalent commissural fascicles are involved in generating the columnar neuroarchitecture of the fan-shaped body. Further, the tract-specific columns in both the grasshopper and Drosophila can be shown to contain axons from identical combinations of central complex lineages, suggesting that this columnar neuroarchitecture is also conserved.

Hydroxyurea-mediated neuroblast ablation establishes birth dates of secondary lineages and addresses neuronal interactions in the developing Drosophila brain.

The Drosophila brain is comprised of neurons formed by approximately 100 lineages, each of which is derived from a stereotyped, asymmetrically dividing neuroblast. Lineages serve as structural and developmental units of Drosophila brain anatomy and reconstruction of lineage projection patterns represents a suitable map of Drosophila brain circuitry at the level of neuron populations ("macro-circuitry"). Two phases of neuroblast proliferation, the first in the embryo and the second during the larval phase (following a period of mitotic quiescence), produce primary and secondary lineages, respectively. Using temporally controlled pulses of hydroxyurea (HU) to ablate neuroblasts and their corresponding secondary lineages during the larval phase, we analyzed the effect on development of primary and secondary lineages in the late larval and adult brain. Our findings indicate that timing of neuroblast re-activation is highly stereotyped, allowing us to establish "birth dates" for all secondary lineages. Furthermore, our results demonstrate that, whereas the trajectory and projection pattern of primary and secondary lineages is established in a largely independent manner, the final branching pattern of secondary neurons is dependent upon the presence of appropriate neuronal targets. Taken together, our data provide new insights into the degree of neuronal plasticity during Drosophila brain development.

Origin and development of neuropil glia of the Drosophila larval and adult brain: Two distinct glial populations derived from separate progenitors.

Glia comprise a conspicuous population of non-neuronal cells in vertebrate and invertebrate nervous systems. Drosophila serves as a favorable model to elucidate basic principles of glial biology in vivo. The Drosophila neuropil glia (NPG), subdivided into astrocyte-like (ALG) and ensheathing glia (EG), extend reticular processes which associate with synapses and sheath-like processes which surround neuropil compartments, respectively. In this paper we characterize the development of NPG throughout fly brain development. We find that differentiated neuropil glia of the larval brain originate as a cluster of precursors derived from embryonic progenitors located in the basal brain. These precursors undergo a characteristic migration to spread over the neuropil surface while specifying/differentiating into primary ALG and EG. Embryonically-derived primary NPG are large cells which are few in number, and occupy relatively stereotyped positions around the larval neuropil surface. During metamorphosis, primary NPG undergo cell death. Neuropil glia of the adult (secondary NPG) are derived from type II lineages during the postembryonic phase of neurogliogenesis. These secondary NPG are much smaller in size but greater in number than primary NPG. Lineage tracing reveals that both NPG subtypes derive from intermediate neural progenitors of multipotent type II lineages. Taken together, this study reveals previously uncharacterized dynamics of NPG development and provides a framework for future studies utilizing Drosophila glia as a model.