Augmin complex activity finetunes dendrite morphology through non-centrosomal microtubule nucleation in vivo.

During development, neurons achieve a stereotyped neuron type-specific morphology, which relies on dynamic support by microtubules (MTs). An important player is the augmin complex (hereafter augmin), which binds to existing MT filaments and recruits the γ-tubulin ring complex (γ-TuRC), to form branched MTs. In cultured neurons, augmin is important for neurite formation. However, little is known about the role of augmin during neurite formation in vivo. Here, we have revisited the role of mammalian augmin in culture and then turned towards the class four Drosophila dendritic arborization (c4da) neurons. We show that MT density is maintained through augmin in cooperation with the γ-TuRC in vivo. Mutant c4da neurons show a reduction of newly emerging higher-order dendritic branches and in turn also a reduced number of their characteristic space-filling higher-order branchlets. Taken together, our data reveal a cooperative function for augmin with the γ-TuRC in forming enough MTs needed for the appropriate differentiation of morphologically complex dendrites in vivo.

Direct evaluation of neuroaxonal degeneration with the causative genes of neurodegenerative diseases in Drosophila using the automated axon quantification system, MeDUsA.

Drosophila is an excellent model organism for studying human neurodegenerative diseases (NDs). However, there is still almost no experimental system that could directly observe the degeneration of neurons and automatically quantify axonal degeneration. In this study, we created MeDUsA (a 'method for the quantification of degeneration using fly axons'), a standalone executable computer program based on Python that combines a pre-trained deep-learning masking tool with an axon terminal counting tool. This software automatically quantifies the number of retinal R7 axons in Drosophila from a confocal z-stack image series. Using this software, we were able to directly demonstrate that axons were degenerated by the representative causative genes of NDs for the first time in Drosophila. The fly retinal axon is an excellent experimental system that is capable of mimicking the pathology of axonal degeneration in human NDs. MeDUsA rapidly and accurately quantifies axons in Drosophila photoreceptor neurons. It enables large-scale research into axonal degeneration, including screening to identify genes or drugs that mediate axonal toxicity caused by ND proteins and diagnose the pathological significance of novel variants of human genes in axons.

A Quantitative Model of Sporadic Axonal Degeneration in the Drosophila Visual System.

In human neurodegenerative diseases, neurons undergo axonal degeneration months to years before they die. Here, we developed a system modeling early degenerative events in Drosophila adult photoreceptor cells. Thanks to the stereotypy of their axonal projections, this system delivers quantitative data on sporadic and progressive axonal degeneration of photoreceptor cells. Using this method, we show that exposure of adult female flies to a constant light stimulation for several days overcomes the intrinsic resilience of R7 photoreceptors and leads to progressive axonal degeneration. This was not associated with apoptosis. We furthermore provide evidence that loss of synaptic integrity between R7 and a postsynaptic partner preceded axonal degeneration, thus recapitulating features of human neurodegenerative diseases. Finally, our experiments uncovered a role of postsynaptic partners of R7 to initiate degeneration, suggesting that postsynaptic cells signal back to the photoreceptor to maintain axonal structure. This model can be used to dissect cellular and circuit mechanisms involved in the early events of axonal degeneration, allowing for a better understanding of how neurons cope with stress and lose their resilience capacities.SIGNIFICANCE STATEMENT Neurons can be active and functional for several years. In the course of aging and in disease conditions leading to neurodegeneration, subsets of neurons lose their resilience and start dying. What initiates this turning point at the cellular level is not clear. Here, we developed a model allowing to systematically describe this phase. The loss of synapses and axons represents an early and functionally relevant event toward degeneration. Using the ordered distribution of Drosophila photoreceptor axon terminals, we assembled a system to study sporadic initiation of axon loss and delineated a role for non-cell-autonomous activity regulation in the initiation of axon degeneration. This work will help shed light on key steps in the etiology of nonfamilial cases of neurodegenerative diseases.

Transient localization of the Arp2/3 complex initiates neuronal dendrite branching in vivo.

The formation of neuronal dendrite branches is fundamental for the wiring and function of the nervous system. Indeed, dendrite branching enhances the coverage of the neuron's receptive field and modulates the initial processing of incoming stimuli. Complex dendrite patterns are achieved in vivo through a dynamic process of de novo branch formation, branch extension and retraction. The first step towards branch formation is the generation of a dynamic filopodium-like branchlet. The mechanisms underlying the initiation of dendrite branchlets are therefore crucial to the shaping of dendrites. Through in vivo time-lapse imaging of the subcellular localization of actin during the process of branching of Drosophila larva sensory neurons, combined with genetic analysis and electron tomography, we have identified the Actin-related protein (Arp) 2/3 complex as the major actin nucleator involved in the initiation of dendrite branchlet formation, under the control of the activator WAVE and of the small GTPase Rac1. Transient recruitment of an Arp2/3 component marks the site of branchlet initiation in vivo These data position the activation of Arp2/3 as an early hub for the initiation of branchlet formation.

Modulators of hormonal response regulate temporal fate specification in the Drosophila brain.

Neuronal diversity is at the core of the complex processing operated by the nervous system supporting fundamental functions such as sensory perception, motor control or memory formation. A small number of progenitors guarantee the production of this neuronal diversity, with each progenitor giving origin to different neuronal types over time. How a progenitor sequentially produces neurons of different fates and the impact of extrinsic signals conveying information about developmental progress or environmental conditions on this process represent key, but elusive questions. Each of the four progenitors of the Drosophila mushroom body (MB) sequentially gives rise to the MB neuron subtypes. The temporal fate determination pattern of MB neurons can be influenced by extrinsic cues, conveyed by the steroid hormone ecdysone. Here, we show that the activation of Transforming Growth Factor-ß (TGF-ß) signalling via glial-derived Myoglianin regulates the fate transition between the early-born α'ß' and the pioneer αß MB neurons by promoting the expression of the ecdysone receptor B1 isoform (EcR-B1). While TGF-ß signalling is required in MB neuronal progenitors to promote the expression of EcR-B1, ecdysone signalling acts postmitotically to consolidate theα'ß' MB fate. Indeed, we propose that if these signalling cascades are impaired α'ß' neurons lose their fate and convert to pioneer αß. Conversely, an intrinsic signal conducted by the zinc finger transcription factor Krüppel-homolog 1 (Kr-h1) antagonises TGF-ß signalling and acts as negative regulator of the response mediated by ecdysone in promoting α'ß' MB neuron fate consolidation. Taken together, the consolidation of α'ß' MB neuron fate requires the response of progenitors to local signalling to enable postmitotic neurons to sense a systemic signal.

Glycerophospholipids - Emerging players in neuronal dendrite branching and outgrowth.

Dendrites are the input compartment of the neuron, receiving and integrating incoming information. Dendritic trees are often highly complex and branched. Their branch extension and distribution are tightly correlated with their role and interactions within neuronal networks. Thus, intense research has focused on understanding the mechanisms that govern dendrite elaboration. Recent reports highlight the importance of specific lipids for these processes. In particular, glycerophospholipids and several of their interacting proteins are involved in various steps of dendrite growth, including the initiation and elongation of dendritic branches and dendritic spines. The aim of this review is to provide a general overview about which particular lipids are involved in shaping dendrite morphology during neuronal differentiation. Additionally, it summarizes recent studies, which helped to gain insights into the mechanisms by which glycerophospholipids and their associated proteins contribute to establishing correct dendritic morphologies.

The gap junction protein Innexin3 is required for eye disc growth in Drosophila.

The Drosophila compound eye develops from a bilayered epithelial sac composed of an upper peripodial epithelium layer and a lower disc proper, the latter giving rise to the eye itself. During larval stages, complex signalling events between the layers contribute to the control of cell proliferation and differentiation in the disc. Previous work in our lab established the gap junction protein Innexin2 (Inx2) as crucial for early larval eye disc growth. By analysing the contribution of other Innexins to eye size control, we have identified Innexin3 (Inx3) as an important growth regulator. Depleting inx3 during larval eye development reduces eye size, while elevating inx3 levels increases eye size, thus phenocopying the inx2 loss- and gain-of-function situation. As demonstrated previously for inx2, inx3 regulates disc cell proliferation and interacts genetically with the Dpp pathway, being required for the proper activation of the Dpp pathway transducer Mad at the furrow and the expression of Dpp receptor Punt in the eye disc. At the developmental timepoint corresponding to eye disc growth, Inx3 colocalises with Inx2 in disc proper and peripodial epithelium cell membranes. In addition, we show that Inx3 protein levels critically depend on inx2 throughout eye development and that inx3 modulates Inx2 protein levels in the larval eye disc. Rescue experiments demonstrate that Inx3 and Inx2 cooperate functionally to enable eye disc growth in Drosophila. Finally, we demonstrate that expression of Inx3 and Inx2 is not only needed in the disc proper but also in the peripodial epithelium to regulate growth of the eye disc. Our data provide a functional demonstration that putative Inx2/Inx3 heteromeric channels regulate organ size.

Fascin controls neuronal class-specific dendrite arbor morphology.

The branched morphology of dendrites represents a functional hallmark of distinct neuronal types. Nonetheless, how diverse neuronal class-specific dendrite branches are generated is not understood. We investigated specific classes of sensory neurons of Drosophila larvae to address the fundamental mechanisms underlying the formation of distinct branch types. We addressed the function of fascin, a conserved actin-bundling protein involved in filopodium formation, in class III and class IV sensory neurons. We found that the terminal branchlets of different classes of neurons have distinctive dynamics and are formed on the basis of molecularly separable mechanisms; in particular, class III neurons require fascin for terminal branching whereas class IV neurons do not. In class III neurons, fascin controls the formation and dynamics of terminal branchlets. Previous studies have shown that transcription factor combinations define dendrite patterns; we find that fascin represents a downstream component of such programs, as it is a major effector of the transcription factor Cut in defining class III-specific dendrite morphology. Furthermore, fascin defines the morphological distinction between class III and class IV neurons. In fact, loss of fascin function leads to a partial conversion of class III neurons to class IV characteristics, while the reverse effect is obtained by fascin overexpression in class IV neurons. We propose that dedicated molecular mechanisms underlie the formation and dynamics of distinct dendrite branch types to elicit the accurate establishment of neuronal circuits.

Presynapses in Kenyon cell dendrites in the mushroom body calyx of Drosophila.

Plastic changes at the presynaptic sites of the mushroom body (MB) principal neurons called Kenyon cells (KCs) are considered to represent a neuronal substrate underlying olfactory learning and memory. It is generally believed that presynaptic and postsynaptic sites of KCs are spatially segregated. In the MB calyx, KCs receive olfactory input from projection neurons (PNs) on their dendrites. Their presynaptic sites, however, are thought to be restricted to the axonal projections within the MB lobes. Here, we show that KCs also form presynapses along their calycal dendrites, by using novel transgenic tools for visualizing presynaptic active zones and postsynaptic densities. At these presynapses, vesicle release following stimulation could be observed. They reside at a distance from the PN input into the KC dendrites, suggesting that regions of presynaptic and postsynaptic differentiation are segregated along individual KC dendrites. KC presynapses are present in γ-type KCs that support short- and long-term memory in adult flies and larvae. They can also be observed in α/ß-type KCs, which are involved in memory retrieval, but not in α'/ß'-type KCs, which are implicated in memory acquisition and consolidation. We hypothesize that, as in mammals, recurrent activity loops might operate for memory retrieval in the fly olfactory system. The newly identified KC-derived presynapses in the calyx are, inter alia, candidate sites for the formation of memory traces during olfactory learning.

The branching code: A model of actin-driven dendrite arborization.

The cytoskeleton is crucial for defining neuronal-type-specific dendrite morphologies. To explore how the complex interplay of actin-modulatory proteins (AMPs) can define neuronal types in vivo, we focused on the class III dendritic arborization (c3da) neuron of Drosophila larvae. Using computational modeling, we reveal that the main branches (MBs) of c3da neurons follow general models based on optimal wiring principles, while the actin-enriched short terminal branches (STBs) require an additional growth program. To clarify the cellular mechanisms that define this second step, we thus concentrated on STBs for an in-depth quantitative description of dendrite morphology and dynamics. Applying these methods systematically to mutants of six known and novel AMPs, we revealed the complementary roles of these individual AMPs in defining STB properties. Our data suggest that diverse dendrite arbors result from a combination of optimal-wiring-related growth and individualized growth programs that are neuron-type specific.

Dendrite enlightenment.

Neuronal dendrites acquire complex morphologies during development. These are not just the product of cell-intrinsic developmental programs; rather they are defined in close interaction with the cellular environment. Thus, to understand the molecular cascades that yield appropriate morphologies, it is essential to investigate them in vivo, in the actual complex tissue environment encountered by the differentiating neuron in the developing animal. Particularly, genetic approaches have pointed to factors controlling dendrite differentiation in vivo. These suggest that localized and transient molecular cascades might underlie the formation and stabilization of dendrite branches with neuron type-specific characteristics. Here, I highlight the need for studies of neuronal dendrite differentiation in the animal, the challenges provided by such an approach, and the promising pathways that have recently opened.

Drosophila Dendritic Arborisation Neurons: Fantastic Actin Dynamics and Where to Find Them.

Neuronal dendrites receive, integrate, and process numerous inputs and therefore serve as the neuron's "antennae". Dendrites display extreme morphological diversity across different neuronal classes to match the neuron's specific functional requirements. Understanding how this structural diversity is specified is therefore important for shedding light on information processing in the healthy and diseased nervous system. Popular models for in vivo studies of dendrite differentiation are the four classes of dendritic arborization (c1da-c4da) neurons of Drosophila larvae with their class-specific dendritic morphologies. Using da neurons, a combination of live-cell imaging and computational approaches have delivered information on the distinct phases and the time course of dendrite development from embryonic stages to the fully developed dendritic tree. With these data, we can start approaching the basic logic behind differential dendrite development. A major role in the definition of neuron-type specific morphologies is played by dynamic actin-rich processes and the regulation of their properties. This review presents the differences in the growth programs leading to morphologically different dendritic trees, with a focus on the key role of actin modulatory proteins. In addition, we summarize requirements and technological progress towards the visualization and manipulation of such actin regulators in vivo.

The anterior paired lateral neuron normalizes odour-evoked activity in the Drosophila mushroom body calyx.

To identify and memorize discrete but similar environmental inputs, the brain needs to distinguish between subtle differences of activity patterns in defined neuronal populations. The Kenyon cells (KCs) of the Drosophila adult mushroom body (MB) respond sparsely to complex olfactory input, a property that is thought to support stimuli discrimination in the MB. To understand how this property emerges, we investigated the role of the inhibitory anterior paired lateral (APL) neuron in the input circuit of the MB, the calyx. Within the calyx, presynaptic boutons of projection neurons (PNs) form large synaptic microglomeruli (MGs) with dendrites of postsynaptic KCs. Combining electron microscopy (EM) data analysis and in vivo calcium imaging, we show that APL, via inhibitory and reciprocal synapses targeting both PN boutons and KC dendrites, normalizes odour-evoked representations in MGs of the calyx. APL response scales with the PN input strength and is regionalized around PN input distribution. Our data indicate that the formation of a sparse code by the KCs requires APL-driven normalization of their MG postsynaptic responses. This work provides experimental insights on how inhibition shapes sensory information representation in a higher brain centre, thereby supporting stimuli discrimination and allowing for efficient associative memory formation.

Circuit reorganization in the Drosophila mushroom body calyx accompanies memory consolidation.

The formation and consolidation of memories are complex phenomena involving synaptic plasticity, microcircuit reorganization, and the formation of multiple representations within distinct circuits. To gain insight into the structural aspects of memory consolidation, we focus on the calyx of the Drosophila mushroom body. In this essential center, essential for olfactory learning, second- and third-order neurons connect through large synaptic microglomeruli, which we dissect at the electron microscopy level. Focusing on microglomeruli that respond to a specific odor, we reveal that appetitive long-term memory results in increased numbers of precisely those functional microglomeruli responding to the conditioned odor. Hindering memory consolidation by non-coincident presentation of odor and reward, by blocking protein synthesis, or by including memory mutants suppress these structural changes, revealing their tight correlation with the process of memory consolidation. Thus, olfactory long-term memory is associated with input-specific structural modifications in a high-order center of the fly brain.

Achieving functional neuronal dendrite structure through sequential stochastic growth and retraction.

Class I ventral posterior dendritic arborisation (c1vpda) proprioceptive sensory neurons respond to contractions in the Drosophila larval body wall during crawling. Their dendritic branches run along the direction of contraction, possibly a functional requirement to maximise membrane curvature during crawling contractions. Although the molecular machinery of dendritic patterning in c1vpda has been extensively studied, the process leading to the precise elaboration of their comb-like shapes remains elusive. Here, to link dendrite shape with its proprioceptive role, we performed long-term, non-invasive, in vivo time-lapse imaging of c1vpda embryonic and larval morphogenesis to reveal a sequence of differentiation stages. We combined computer models and dendritic branch dynamics tracking to propose that distinct sequential phases of stochastic growth and retraction achieve efficient dendritic trees both in terms of wire and function. Our study shows how dendrite growth balances structure-function requirements, shedding new light on general principles of self-organisation in functionally specialised dendrites.

Slit and Robo regulate dendrite branching and elongation of space-filling neurons in Drosophila.

Space-filling neurons extensively sample their receptive fields with fine dendritic branches. In this study we show that a member of the conserved Robo receptor family, Robo, and its ligand Slit regulate the dendritic differentiation of space-filling neurons. Loss of Robo or Slit function leads to faster elongating and less branched dendrites of the complex and space-filling class IV multi-dendritic dendrite-arborization (md-da) neurons in the Drosophila embryonic peripheral nervous system, but not of the simpler class I neurons. The total dendrite length of Class IV neurons is not modified in robo or slit mutant embryos. Robo mediates this process cell-autonomously. Upon Robo over-expression in md-da neurons the dendritic tree is simplified and time-lapse analysis during larval stages indicates that this is due to reduction in the number of newly formed branches. We propose that Slit, through Robo, provides an extrinsic signal to coordinate the growth rate and the branching level of space-filling neurons, thus allowing them to appropriately cover their target field.

The Drosophila myosin VI Jaguar is required for basal protein targeting and correct spindle orientation in mitotic neuroblasts.

Asymmetric cell divisions generate cellular diversity. In Drosophila, embryonic neuroblasts target cell fate determinants basally, rotate their spindles by 90 degrees to align with the apical-basal axis, and divide asymmetrically in a stem cell-like fashion. In this process, apically localized Bazooka recruits Inscuteable and other proteins to form an apical complex, which then specifies spindle orientation and basal localization of the cell fate determinants and their adapter proteins such as Miranda. Here we report that Miranda localization requires the unconventional myosin VI Jaguar (Jar). In jar null mutant embryos, Miranda is delocalized and the spindle is misoriented, but the Inscuteable crescent remains apical. Miranda directly binds to Jar, raising the possibility that Miranda and its associated proteins are translocated basally by this actin-based motor. Our studies demonstrate that a class VI myosin is necessary for basal protein targeting and spindle orientation in neuroblasts.

Structural aspects of plasticity in the nervous system of Drosophila.

Neurons extend and retract dynamically their neurites during development to form complex morphologies and to reach out to their appropriate synaptic partners. Their capacity to undergo structural rearrangements is in part maintained during adult life when it supports the animal's ability to adapt to a changing environment or to form lasting memories. Nonetheless, the signals triggering structural plasticity and the mechanisms that support it are not yet fully understood at the molecular level. Here, we focus on the nervous system of the fruit fly to ask to which extent activity modulates neuronal morphology and connectivity during development. Further, we summarize the evidence indicating that the adult nervous system of flies retains some capacity for structural plasticity at the synaptic or circuit level. For simplicity, we selected examples mostly derived from studies on the visual system and on the mushroom body, two regions of the fly brain with extensively studied neuroanatomy.

Steroid Hormone Ecdysone Signaling Specifies Mushroom Body Neuron Sequential Fate via Chinmo.

The functional variety in neuronal composition of an adult brain is established during development. Recent studies proposed that interactions between genetic intrinsic programs and external cues are necessary to generate proper neural diversity [1]. However, the molecular mechanisms underlying this developmental process are still poorly understood. Three main subtypes of Drosophila mushroom body (MB) neurons are sequentially generated during development and provide a good example of developmental neural plasticity [2]. Our present data propose that the environmentally controlled steroid hormone ecdysone functions as a regulator of early-born MB neuron fate during larval-pupal transition. We found that the BTB-zinc finger factor Chinmo acts upstream of ecdysone signaling to promote a neuronal fate switch. Indeed, Chinmo regulates the expression of the ecdysone receptor B1 isoform to mediate the production of γ and α'ß' MB neurons. In addition, we provide genetic evidence for a regulatory negative feedback loop driving the α'ß' to αß MB neuron transition in which ecdysone signaling in turn controls microRNA let-7 depression of Chinmo expression. Thus, our results uncover a novel interaction in the MB neural specification pathway for temporal control of neuronal identity by interplay between an extrinsic hormonal signal and an intrinsic transcription factor cascade.