A Systematic Nomenclature for the Drosophila Ventral Nerve Cord.

Drosophila melanogaster is an established model for neuroscience research with relevance in biology and medicine. Until recently, research on the Drosophila brain was hindered by the lack of a complete and uniform nomenclature. Recognizing this, Ito et al. (2014) produced an authoritative nomenclature for the adult insect brain, using Drosophila as the reference. Here, we extend this nomenclature to the adult thoracic and abdominal neuromeres, the ventral nerve cord (VNC), to provide an anatomical description of this major component of the Drosophila nervous system. The VNC is the locus for the reception and integration of sensory information and involved in generating most of the locomotor actions that underlie fly behaviors. The aim is to create a nomenclature, definitions, and spatial boundaries for the Drosophila VNC that are consistent with other insects. The work establishes an anatomical framework that provides a powerful tool for analyzing the functional organization of the VNC.

Extensive and diverse patterns of cell death sculpt neural networks in insects.

Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death (PCD) appears to play a key role. We show that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Mimicking an evolutionary role for increasing cell numbers, we artificially block in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of 'undead' neurons with complex arborisations and distinct neurotransmitter identities. Activation of these 'undead' neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Focusing on two dipterans, which have lost flight during evolution, we reveal that reductions in populations of flight interneurons are likely caused by increased cell death during development. Our findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations.

Just like a sculptor chips away at a block of granite to make a statue, the nervous system reaches its mature state by eliminating neurons during development through a process known as programmed cell death. In vertebrates, this mechanism often involves newly born neurons shrivelling away and dying if they fail to connect with others during development. Most studies in insects have focused on the death of neurons that occurs at metamorphosis, during the transition between larva to adult, when cells which are no longer needed in the new life stage are eliminated. Pop et al. harnessed a newly designed genetic probe to point out that, in fruit flies, programmed cell death of neurons at metamorphosis is not the main mechanism through which cells die. Rather, the majority of cell death takes place as soon as neurons are born throughout all larval stages, when most of the adult nervous system is built. To gain further insight into the role of this 'early' cell death, the neurons were stopped from dying, showing that these cells were able to reach maturity and function. Together, these results suggest that early cell death may be a mechanism fine-tuned by evolution to shape the many and varied nervous systems of insects. To explore this, Pop et al. looked for hints of early cell death in relatives of fruit flies that are unable to fly: the swift lousefly and the bee lousefly. This analysis showed that early cell death is likely to occur in these two insects, but it follows different patterns than in the fruit fly, potentially targeting the neurons that would have controlled flight in these flies' ancestors. Brains are the product of evolution: learning how neurons change their connections and adapt could help us understand how the brain works in health and disease. This knowledge may also be relevant to work on artificial intelligence, a discipline that often bases the building blocks and connections in artificial 'brains' on how neurons communicate with one another.

Developmental organization of central neurons in the adult Drosophila ventral nervous system.

We have used MARCM to reveal the adult morphology of the post embryonically produced neurons in the thoracic neuromeres of the Drosophila VNS. The work builds on previous studies of the origins of the adult VNS neurons to describe the clonal organization of the adult VNS. We present data for 58 of 66 postembryonic thoracic lineages, excluding the motor neuron producing lineages (15 and 24) which have been described elsewhere. MARCM labels entire lineages but where both A and B hemilineages survive (e.g., lineages 19, 12, 13, 6, 1, 3, 8, and 11), the two hemilineages can be discriminated and we have described each hemilineage separately. Hemilineage morphology is described in relation to the known functional domains of the VNS neuropil and based on the anatomy we are able to assign broad functional roles for each hemilineage. The data show that in a thoracic hemineuromere, 16 hemilineages are primarily involved in controlling leg movements and walking, 9 are involved in the control of wing movements, and 10 interface between both leg and wing control. The data provide a baseline of understanding of the functional organization of the adult Drosophila VNS. By understanding the morphological organization of these neurons, we can begin to define and test the rules by which neuronal circuits are assembled during development and understand the functional logic and evolution of neuronal networks.

Neurexin and Neuroligin-based adhesion complexes drive axonal arborisation growth independent of synaptic activity.

Building arborisations of the right size and shape is fundamental for neural network function. Live imaging in vertebrate brains strongly suggests that nascent synapses are critical for branch growth during development. The molecular mechanisms underlying this are largely unknown. Here we present a novel system in Drosophila for studying the development of complex arborisations live, in vivo during metamorphosis. In growing arborisations we see branch dynamics and localisations of presynaptic proteins very similar to the 'synaptotropic growth' described in fish/frogs. These accumulations of presynaptic proteins do not appear to be presynaptic release sites and are not paired with neurotransmitter receptors. Knockdowns of either evoked or spontaneous neurotransmission do not impact arbor growth. Instead, we find that axonal branch growth is regulated by dynamic, focal localisations of Neurexin and Neuroligin. These adhesion complexes provide stability for filopodia by a 'stick-and-grow' based mechanism wholly independent of synaptic activity.

More alive than dead: non-apoptotic roles for caspases in neuronal development, plasticity and disease.

Nervous systems are arguably the most fascinating and complex structures in the known universe. How they are built, changed by experience and then degenerate are some of the biggest questions in biology. Regressive phenomena, such as neuron pruning and programmed cell death, have a key role in the building and maintenance of the nervous systems. Both of these cellular mechanisms deploy the caspase family of protease enzymes. In this review, we highlight the non-apoptotic function of caspases during nervous system development, plasticity and disease, particularly focussing on their role in structural remodelling. We have classified pruning as either macropruning, where complete branches are removed, or micropruning, where individual synapses or dendritic spines are eliminated. Finally we discuss open questions and possible future directions within the field.

Postembryonic lineages of the Drosophila ventral nervous system: Neuroglian expression reveals the adult hemilineage associated fiber tracts in the adult thoracic neuromeres.

During larval life most of the thoracic neuroblasts (NBs) in Drosophila undergo a second phase of neurogenesis to generate adult-specific neurons that remain in an immature, developmentally stalled state until pupation. Using a combination of MARCM and immunostaining with a neurotactin antibody, Truman et al. (2004; Development 131:5167-5184) identified 24 adult-specific NB lineages within each thoracic hemineuromere of the larval ventral nervous system (VNS), but because of the neurotactin labeling of lineage tracts disappearing early in metamorphosis, they were unable extend the identification of these lineages into the adult. Here we show that immunostaining with an antibody against the cell adhesion molecule neuroglian reveals the same larval secondary lineage projections through metamorphosis and bfy identifying each neuroglian-positive tract at selected stages we have traced the larval hemilineage tracts for all three thoracic neuromeres through metamorphosis into the adult. To validate tract identifications we used the genetic toolkit developed by Harris et al. (2015; Elife 4) to preserve hemilineage-specific GAL4 expression patterns from larval into the adult stage. The immortalized expression proved a powerful confirmation of the analysis of the neuroglian scaffold. This work has enabled us to directly link the secondary, larval NB lineages to their adult counterparts. The data provide an anatomical framework that 1) makes it possible to assign most neurons to their parent lineage and 2) allows more precise definitions of the neuronal organization of the adult VNS based in developmental units/rules. J. Comp. Neurol. 524:2677-2695, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

An ESCRT module is required for neuron pruning.

Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. Here, we reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. Our data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore we show that this ESCRT module requires a direct, in vivo, interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.

An interaction screen identifies headcase as a regulator of large-scale pruning.

Large-scale pruning, the removal of long neuronal processes, is deployed widely within the developing nervous system and is essential for proper circuit formation. In Drosophila the dendrites of the class IV dendritic arborization sensory neuron ddaC undergo large-scale pruning by local degeneration controlled by the steroid hormone ecdysone. The molecular mechanisms that control such events are largely unknown. To identify new molecules that orchestrate this developmental degeneration, we performed a genetic interaction screen. Our approach combines the strength of Drosophila forward genetics with detailed in vivo imaging of ddaC neurons. This screen allowed us to identify headcase (hdc) as a new gene involved in dendrite pruning. hdc is evolutionarily conserved, but the protein's function is unknown. Here we show that hdc is expressed just before metamorphosis in sensory neurons that undergo remodeling. hdc is required in a cell-autonomous manner to control dendrite severing, the first phase of pruning. Our epistasis experiments with known regulators of dendrite pruning reveal hdc as a founding member of a new pathway downstream of ecdysone signaling.

The BTB/POZ zinc finger protein Broad-Z3 promotes dendritic outgrowth during metamorphic remodeling of the peripheral stretch receptor dbd.

BACKGROUND: Various members of the family of BTB/POZ zinc-finger transcription factors influence patterns of dendritic branching. One such member, Broad, is notable because its BrZ3 isoform is widely expressed in Drosophila in immature neurons around the time of arbor outgrowth. We used the metamorphic remodeling of an identified sensory neuron, the dorsal bipolar dendrite sensory neuron (dbd), to examine the effects of BrZ3 expression on the extent and pattern of dendrite growth during metamorphosis. RESULTS: Using live imaging of dbd in Drosophila pupae, we followed its normal development during metamorphosis and the effect of ectopic expression of BrZ3 on this development. After migration of its cell body, dbd extends a growth-cone that grows between two muscle bands followed by branching and turning back on itself to form a compact dendritic bundle. The ectopic expression of the BrZ3 isoform, using the GAL4/UAS system, caused dbd's dendritic tree to transform from its normal, compact, fasciculated form into a comb-like arbor that spread over on the body wall. Time-lapse analysis revealed that the expression of BrZ3 caused the premature extension of the primary dendrite onto immature myoblasts, ectopic growth past the muscle target region, and subsequent elaboration onto the epidermis. To control the timing of expression of BrZ3, we used a temperature-sensitive GAL80 mutant. When BrZ3 expression was delayed until after the extension of the primary dendrite, then a normal arbor was formed. By contrast, when BrZ3 expression was confined to only the early outgrowth phase, then ectopic arbors were subsequently formed and maintained on the epidermis despite the subsequent absence of BrZ3. CONCLUSIONS: The adult arbor of dbd is a highly branched arbor whose branches self-fasciculate to form a compact dendritic bundle. The ectopic expression of BrZ3 in this cell causes a premature extension of its growth-cone, resulting in dendrites that extend beyond their normal muscle substrate and onto the epidermis, where they form a comb-shaped, ectopic arbor. Our quantitative data suggest that new ectopic arbor represents an 'unpacking' of the normally fasciculated arbor onto the epidermis. These data suggest that the nature of their local environment can change dendrite behavior from self-adhesion to self-avoidance.

Role of Notch signaling in establishing the hemilineages of secondary neurons in Drosophila melanogaster.

The secondary neurons generated in the thoracic central nervous system of Drosophila arise from a hemisegmental set of 25 neuronal stem cells, the neuroblasts (NBs). Each NB undergoes repeated asymmetric divisions to produce a series of smaller ganglion mother cells (GMCs), which typically divide once to form two daughter neurons. We find that the two daughters of the GMC consistently have distinct fates. Using both loss-of-function and gain-of-function approaches, we examined the role of Notch signaling in establishing neuronal fates within all of the thoracic secondary lineages. In all cases, the 'A' (Notch(ON)) sibling assumes one fate and the 'B' (Notch(OFF)) sibling assumes another, and this relationship holds throughout the neurogenic period, resulting in two major neuronal classes: the A and B hemilineages. Apparent monotypic lineages typically result from the death of one sibling throughout the lineage, resulting in a single, surviving hemilineage. Projection neurons are predominantly from the B hemilineages, whereas local interneurons are typically from A hemilineages. Although sibling fate is dependent on Notch signaling, it is not necessarily dependent on numb, a gene classically involved in biasing Notch activation. When Numb was removed at the start of larval neurogenesis, both A and B hemilineages were still generated, but by the start of the third larval instar, the removal of Numb resulted in all neurons assuming the A fate. The need for Numb to direct Notch signaling correlated with a decrease in NB cell cycle time and may be a means for coping with multiple sibling pairs simultaneously undergoing fate decisions.

Temporal patterns of broad isoform expression during the development of neuronal lineages in Drosophila.

BACKGROUND: During the development of the central nervous system (CNS) of Drosophila, neuronal stem cells, the neuroblasts (NBs), first generate a set of highly diverse neurons, the primary neurons that mature to control larval behavior, and then more homogeneous sets of neurons that show delayed maturation and are primarily used in the adult. These latter, 'secondary' neurons show a complex pattern of expression of broad, which encodes a transcription factor usually associated with metamorphosis, where it acts as a key regulator in the transitions from larva and pupa. RESULTS: The Broad-Z3 (Br-Z3) isoform appears transiently in most central neurons during embryogenesis, but persists in a subset of these cells through most of larval growth. Some of the latter are embryonic-born secondary neurons, whose development is arrested until the start of metamorphosis. However, the vast bulk of the secondary neurons are generated during larval growth and bromodeoxyuridine incorporation shows that they begin expressing Br-Z3 about 7 hours after their birth, approximately the time that they have finished outgrowth to their initial targets. By the start of metamorphosis, the oldest secondary neurons have turned off Br-Z3 expression, while the remainder, with the exception of the very youngest, maintain Br-Z3 while they are interacting with potential partners in preparation for neurite elaboration. That Br-Z3 may be involved in early sprouting is suggested by ectopically expressing this isoform in remodeling primary neurons, which do not normally express Br-Z3. These cells now sprout into ectopic locations. The expression of Br-Z3 is transient and seen in all interneurons, but two other isoforms, Br-Z4 and Br-Z1, show a more selective expression. Analysis of MARCM clones shows that the Br-Z4 isoform is expressed by neurons in virtually all lineages, but only in those cells born during a window during the transition from the second to the third larval instar. Br-Z4 expression is then maintained in this temporal cohort of cells into the adult. CONCLUSION: These data show the potential for diverse functions of Broad within the developing CNS. The Br-Z3 isoform appears in all interneurons, but not motoneurons, when they first begin to interact with potential targets. Its function during this early sorting phase needs to be defined. Two other Broad isoforms, by contrast, are stably expressed in cohorts of neurons in all lineages and are the first examples of persisting molecular 'time-stamps' for Drosophila postembryonic neurons.

Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map.

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. Here we reveal that this fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Our developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. We demonstrate that the medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections.

Delta expression in post-mitotic neurons identifies distinct subsets of adult-specific lineages in Drosophila.

The Drosophila ventral nerve cord is comprised of numerous neuronal lineages, each derived from a stereotypically positioned neuroblast (NB). At the embryonic stage the unique identities of each NB, and several of their neuronal progeny, are well characterized by spatial and temporal expression patterns of molecular markers. These patterns of expression are not preserved at the larval stage and thus the identity of adult-specific lineages remains obscure. Recent clonal analysis using MARCM has identified 24 adult-specific lineages arising from thoracic NBs at the larval stage. In this study, we have explored a role for the Delta protein in development of the post-embryonic Drosophila ventral nerve cord. We find that Delta expression identifies 7 of the 24 adult-specific lineages of the thoracic ganglia by being highly enriched in clusters of newly born post-mitotic neurons and their neurite bundles. The Delta lineages constitute the majority of bundles projecting to the ventral neuropil, consistent with a role in processing leg sensory information. Targeted knockdown of Delta in neurons using RNAi results in significantly decreased leg chemosensory response and a relatively unaffected leg mechanosensory response. Delta RNAi knockdown in Delta lineages also gives a more diffuse bundle terminal morphology while the overall path-finding of neurite bundles is unaffected. We also identify a male-specific Delta lineage in the terminal abdominal ganglia, implicating a role for Delta in development of sexually dimorphic neural networks. Examples of Delta-expressing neurites contacting Notch-expressing glia are also seen, but are not common to all Delta lineages. Altogether, these data reveal a fundamental pattern of Delta expression that is indicative of an underlying developmental program that confers identity to adult lineage neurons.

Local caspase activity directs engulfment of dendrites during pruning.

Pruning is important for sculpting neural circuits, as it removes excessive or inaccurate projections. Here we show that the removal of sensory neuron dendrites during pruning in Drosophila melanogaster is directed by local caspase activity. Suppressing caspase activity prevented dendrite removal, whereas a global activation of caspases within a neuron caused cell death. A new genetically encoded caspase probe revealed that caspase activity is confined to the degenerating dendrites of pruning neurons.

Cellular mechanisms of dendrite pruning in Drosophila: insights from in vivo time-lapse of remodeling dendritic arborizing sensory neurons.

Regressive events that refine exuberant or inaccurate connections are critical in neuronal development. We used multi-photon, time-lapse imaging to examine how dendrites of Drosophila dendritic arborizing (da) sensory neurons are eliminated during early metamorphosis, and how intrinsic and extrinsic cellular mechanisms control this deconstruction. Removal of the larval dendritic arbor involves two mechanisms: local degeneration and branch retraction. In local degeneration, major branch severing events entail focal disruption of the microtubule cytoskeleton, followed by thinning of the disrupted region, severing and fragmentation. Retraction was observed at distal tips of branches and in proximal stumps after severing events. The pruning program of da neuron dendrites is steroid induced; cell-autonomous dominant-negative inhibition of steroid action blocks local degeneration, although retraction events still occur. Our data suggest that steroid-induced changes in the epidermis may contribute to dendritic retraction. Finally, we find that phagocytic blood cells not only engulf neuronal debris but also attack and sever intact branches that show signs of destabilization.

Developmental architecture of adult-specific lineages in the ventral CNS of Drosophila.

In Drosophila most thoracic neuroblasts have two neurogenic periods: an initial brief period during embryogenesis and a second prolonged phase during larval growth. This study focuses on the adult-specific neurons that are born primarily during the second phase of neurogenesis. The fasciculated neurites arising from each cluster of adult-specific neurons express the cell-adhesion protein Neurotactin and they make a complex scaffold of neurite bundles within the thoracic neuropils. Using MARCM clones, we identified the 24 lineages that make up the scaffold of a thoracic hemineuromere. Unlike the early-born neurons that are strikingly diverse in both form and function, the adult specific cells in a given lineage are remarkably similar and typically project to only one or two initial targets, which appear to be the bundled neurites from other lineages. Correlated changes in the contacts between the lineages in different segments suggest that these initial contacts have functional significance in terms of future synaptic partners. This paper provides an overall view of the initial connections that eventually lead to the complex connectivity of the bulk of the thoracic neurons.

Fish as bioreactors: transgene expression of human coagulation factor VII in fish embryos.

A plasmid containing human coagulation factor VII (hFVII) complementary DNA regulated by a cytomegalovirus promoter was microinjected into fertilized eggs of zebrafish, African catfish, and tilapia. The active form of hFVll was detected in the fish embryos by various assays. This positive expression of human therapeutic protein in fish embryos demonstrates the possibility of exploitation of transgenic fish as bioreactors.

Mechanisms of dendritic elaboration of sensory neurons in Drosophila: insights from in vivo time lapse.

In vivo time-lapse multiphoton microscopy was used to analyze the remodeling of the dendritic arborizing (da) sensory neuron known as dorsal dendritic arborizing neuron E (ddaE) during metamorphosis. After its larval processes have been removed, the cell body of ddaE repositions itself on the body wall between 25 and 40 hr after puparium formation (APF) and begins its adult outgrowth at 40 hr APF. The scaffold of the arbor is laid down between 40 and 54 hr APF, when growth is characterized by high filopodial activity at both terminal and interstitial positions and by branch retraction along with branch establishment. Later in development, filopodial activity remains high but is confined to terminal branches, and branch retraction is no longer seen. Treatment with the insect hormone juvenile hormone (JH), a key regulator of metamorphosis, alters the shape and complexity of the adult dendritic tree in a time-dependent manner. Early treatments with juvenile hormone mimic (JHm) appear to repress extension programs and maintain retraction programs. With later JHm treatments, extension programs appear normal, but retraction programs are maintained beyond their normal time. The JH treatments show the importance of retraction programs in establishing the overall arbor shape.

Persistent larval sensory neurones are required for the normal development of the adult sensory afferent projections in Drosophila.

We have tested the hypothesis that larval neurones guide growth of adult sensory axons in Drosophila. We show that ablation of larval sensory neurones causes defects in the central projections of adult sensory neurones. Spiralling axons and ectopic projections indicate failure in axon growth guidance. We show that larval sensory neurones are required for peripheral pathfinding, entry into the CNS and growth guidance within the CNS. Ablation of subsets of neurones shows that larval sensory neurones serve specific guidance roles. Dorsal neurones are required for axon guidance across the midline, whereas lateral neurones are required for posterior growth. We conclude that larval sensory neurones pioneer the assembly of sensory arrays in adults.