The Interactive Fly

Zygotically transcribed genes

Dendritic Development

  • Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila
  • Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons
  • Structural homeostasis: Compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input
  • Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system
  • Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites
  • The bHLH-PAS protein Spineless is necessary for the diversification of dendrite morphology of Drosophila dendritic arborization neurons
  • Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning
  • The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance
  • The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway
  • The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons
  • Drosophila Valosin-containing protein is required for dendrite pruning through a regulatory role in mRNA metabolism
  • Wnt5 and drl/ryk gradients pattern the Drosophila olfactory dendritic map
  • Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map
  • Dendritic arbor reduction 1, zinc finger transcription factor that promotes dendrite growth in part by suppressing the expression of the microtubule-severing protein Spastin
  • Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites
  • The Me31B DEAD-box helicase localizes to postsynaptic foci and regulates expression of a CaMKII reporter mRNA in dendrites of Drosophila olfactory projection neurons
  • The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting
  • The RhoGEF trio functions in sculpting class specific dendrite morphogenesis in Drosophila sensory neurons
  • Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons
  • Centrosomin represses dendrite branching by orienting microtubule nucleation
  • GM130 is required for compartmental organization of dendritic Golgi outposts
  • Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw
  • Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila
  • Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber
  • Drosophila Hook-Related Protein (Girdin) is essential for sensory dendrite formation
  • The Kruppel-like factor Dar1 determines multipolar neuron morphology
  • Spindle-F is the central mediator of Ik2 kinase-dependent dendrite pruning in Drosophila sensory neurons
  • Functions of the SLC36 transporter Pathetic in growth control
  • The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons
  • Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions
  • A genome-wide screen for dendritically localized RNAs identifies genes required for dendrite morphogenesis
  • The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion
  • Intra-neuronal competition for synaptic partners conserves the amount of dendritic building material
  • Mitochondrial dysfunction induces dendritic loss via eIF2α phosphorylation
  • Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development
  • Dendritic growth gated by a steroid hormone receptor underlies increases in activity in the developing Drosophila locomotor system
  • Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling
  • Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila.
  • Dendrites are dispensable for basic motoneuron function but essential for fine tuning of behavior
  • In vivo dendrite regeneration after injury is different from dendrite development
  • Nutrient-dependent increased dendritic arborization of somatosensory neurons
  • A bidirectional circuit switch reroutes pheromone signals in male and female brains
  • The stum gene is essential for mechanical sensing in proprioceptive neurons
  • The RNA-binding protein Caper is required for sensory neuron development in Drosophila melanogaster
  • Neuronal processing of noxious thermal stimuli mediated by dendritic Ca influx in somatosensory neurons
  • Enclosure of dendrites by epidermal cells restricts branching and permits coordinated development of spatially overlapping sensory neurons

    Secretory Pathway and Dendrites
  • Growing dendrites and axons differ in their reliance on the secretory pathway
  • Nak regulates localization of clathrin sites in higher-order dendrites to promote local dendrite growth
  • Endocytic pathways downregulate the L1-type cell adhesion molecule Neuroglian to promote dendrite pruning in Drosophila
  • Synaptic control of secretory trafficking in dendrites
  • Regulation of dendrite growth and maintenance by exocytosis
  • Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons
  • Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons

    Embryonic origins of a motor system: Motor dendrites form a myotopic map in Drosophila

    The organisational principles of locomotor networks are less well understood than those of many sensory systems, where in-growing axon terminals form a central map of peripheral characteristics. Using the neuromuscular system of the Drosophila embryo as a model and retrograde tracing and genetic methods, principles underlying the organisation of the motor system have been uncovered. Dendritic arbors of motor neurons, rather than their cell bodies, are partitioned into domains to form a myotopic map, which represents centrally the distribution of body wall muscles peripherally. While muscles are segmental, the myotopic map is parasegmental in organisation. It forms by an active process of dendritic growth independent of the presence of target muscles, proper differentiation of glial cells, or (in its initial partitioning) competitive interactions between adjacent dendritic domains. The arrangement of motor neuron dendrites into a myotopic map represents a first layer of organisation in the motor system. This is likely to be mirrored, at least in part, by endings of higher-order neurons from central pattern-generating circuits, which converge onto the motor neuron dendrites. These findings will greatly simplify the task of understanding how a locomotor system is assembled. These results suggest that the cues that organise the myotopic map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

    The analysis began by correlating the positions of motor neuron dendrites with the distribution of their muscle targets in the periphery. Motor neurons were retrogradely labelled in a pairwise fashion and the positions of their dendritic arbors were mapped. Because of an interest in the mechanisms that underlie the assembly of the motor system, focus was placed on stages when each motor neuron first establishes a characteristic domain of arborisation within the neuropile (early stage 17, 15h after egg-laying [AEL]) (Landgraf, 2003).

    Motor axons project into the muscle field via two main nerves, the intersegmental (ISN) and the segmental nerve (SN). The transverse nerve (TN) runs along the segment border and has few motor axons. Choice of nerve root is one of several features that divide the motor neurons into two principal sets, the ISN and SN. (1) The cell bodies of SN motor neurons are located in the same segment as the muscles that they innervate, whereas ISN motor neuron somata are located in the segment next anterior (with the exception of the RP2 and two neuromodulatory efferent ventral unpaired median [VUM] neurons. (2) ISN motor neurons innervate internal muscles, which span a segment from anterior to posterior, whereas SN (and the TN) motor neurons innervate external muscles. External muscles are distinct from the internal set in several respects: (1) they are generally transverse; (2) unlike internal muscles, they require wingless (wg) signalling for their specification; (3) external (but not internal) muscles and their innervating motor neurons express the cell adhesion molecule (CAM) Connectin, with the single exception of muscle ventral transverse 1 (VT1) (Landgraf, 2003 and references therein).

    In addition, ISN and SN motor neurons elaborate their dendrites in distinct regions of the neuropile. Dendrites of ISN motor neurons occupy a domain extending posteriorly from the posterior part of one neuromere into the anterior part of the next. SN motor neuron dendrites occupy a domain that lies between the domains of ISN motor neuron arbors (Landgraf, 2003).

    Thus, the organisation of the body wall muscles into internal and external sets is reflected centrally in patterns of motor neuron arborisations. The innervating motor neurons project their axons through different nerves and elaborate their dendritic fields in distinct regions of the neuropile. Although dendritic arbors become progressively more elaborate and extensive over developmental time, their separate domains remain clearly recognisable and appear to be maintained at least until the motor system is fully functional (18 h AEL) (Landgraf, 2003).

    Having established that there is a central representation of the muscle field, the organisation of the motor neuron dendrites was analyzed in greater detail. (1) The set of external muscles and their innervating (SN) motor neurons were examined. Muscles of similar anteroposterior positions, such as the ventral acute muscle (VA3) and the segment border muscle (SBM), are innervated by motor neurons whose dendritic arbors lie in a common region of the neuropile. Conversely, motor neurons supplying the anterior (lateral transverse 1-2 [LT1-LT2]) versus the posterior (SBM) muscles have dendritic arbors that are correspondingly separated in the anteroposterior axis of the CNS (Landgraf, 2003).

    To put the idea of a regular map to the test, focus was placed on an unusual external motor neuron-muscle pair. Muscle VT1 is innervated by a TN rather than an SN motor neuron. However, VT1 lies at the same place in the anteroposterior axis as the SBM, although VT1 is ventral and the SBM more dorsal. The VT1 motor neuron dendritic field is found to overlaps with that of the SBM motor neuron. For the external set, it is concluded that differences in target muscle location in the anteroposterior axis are mapped centrally as regular differences in dendritic position, but dorsoventral distinctions are not (Landgraf, 2003).

    It was next asked whether there is a similarly regular representation of the internal muscles in the developing CNS. While most external muscles are transverse and have unique anteroposterior locations, the internal muscles span the width of a segment so that positional distinctions between them are solely in the dorsoventral axis. It was found that the set of internal muscles is represented centrally by three dendritic domains. Motor neurons innervating ventral internal muscles elaborate their dendritic arbors in the anterior half of the ISN dendritic domain. Motor neurons with dorsolateral internal muscle targets (lateral longitudinal [LL] 1, dorsal acute [DA] 3, dorsal oblique 3-5 [DO3-DO5]) put their arbors into the posterior part of the ISN dendritic domain. Finally, dorsal muscles are represented by a motor neuron dendritic domain that lies between those representing ventral (anterior) and dorsolateral (posterior) internal muscle groups. Thus, the internal muscles are represented in the neuropile by three domains of dendritic arborisation that reflect their different dorsoventral locations in the periphery. Once again, it is concluded that there is a regular mapping of muscle position in the neuropile: in this case, it is positions in the dorsoventral axis peripherally that are represented centrally as differences in the anteroposterior locations of dendrites (Landgraf, 2003).

    To test the idea that dendritic arbor positions relate to the distribution of muscles, an atypical motor neuron-muscle pair was examined. The RP2 motor neuron is reported to innervate dorsal muscle DA2, yet its dendrites span the domains that represent both dorsal and dorsolateral internal muscles. However, on careful analysis it was found that DA2 is, in fact, specifically innervated by a U neuron whose dendrites lie in the dorsal internal domain, whereas the RP2 axon forms endings generally on all dorsolateral and dorsal muscles by 19 h AEL. These seem to correspond to the type 1s boutons found in late larvae. Thus, the RP2 neuron puts its dendrites into a region of the neuropile that does indeed represent its targets, namely the dorsolateral and dorsal internal muscles (Landgraf, 2003).

    Like the muscle field itself, the map of motor neuron dendrites is metamerically repeated. However, the boundaries of these two units are out of register with one another, since the dendrites of the motor neurons innervating internal muscles lie in the next anterior neuromere. The anterior border of the dendritic map, as defined by the extent of these anterior dendrites, coincides with the anterior margin of engrailed (en) expression. Thus, while the muscles are segmental in their organisation, the domains occupied by the dendrites of their innervating motor neurons are parasegmental (Landgraf, 2003).

    To test whether genes that implement the parasegmental pattern in the epidermis are also required for the formation of the parasegmental organisation of the neuromuscular system, the formation of SN and ISN dendritic fields was studied in embryos singly mutant for the following segment polarity genes: en/invected (Df(enE)), wg (wgCX4), naked (nkd2), patched (ptc9), hedgehog (hh21), and gooseberry (Df2R(gsb)). Every one of the six different mutants that were analysed has partially aberrant patterns of neuroblasts (NBs). Nevertheless, SN and ISN motor neurons still form and can be identified by their characteristic axonal projections into the periphery. In addition, it was found that the fundamental separation between SN and ISN dendritic domains is present despite often severe perturbations in CNS structure. For example, in gsb mutant embryos, both nerve roots are frequently fused so that the SN and ISN share a common CNS exit point. Nevertheless, SN and ISN axons as well as their dendritic fields do not intermingle but remain separate. These results suggest that the subdivision of the neuropile into the principal ISN and SN dendritic domains is a robust feature of the system, which appears to be specified early in development, since the embryo subdivides into parasegmental units (Landgraf, 2003).

    It was next asked what mechanisms underlie the formation of the myotopic map. Because ISN and SN motor neurons lie at different positions in the CNS and their axons grow out into the muscle field through different nerves, it is reasonable to suppose that at least the major subdivision of dendritic arborisations into internal and external domains could be a byproduct of the locations at which the motor neurons are generated and the paths taken by their growing axons. This ‘passive mapping' explanation can be excluded by considering a single motor neuron-muscle pair, namely dorsal transverse 1 (DT1) and its innervating motor neuron. DT1 is an external muscle (by position, orientation, wg dependence, and Connectin expression), yet its motor neuron is clustered with the internal muscle innervating set and its axon (uniquely for the external muscles) grows out through the ISN. Despite its packing within the ‘internal motor neuron' set, the DT1 motor neuron makes a long posterior projection through the internal muscle domain of the myotopic map to reach the external domain, where it arborises appropriately, reflecting the orientation and external nature of its target muscle. In contrast, motor neurons derived from the same NB as DT1 innervate neighboring internal muscles DO3-DO5 and put their dendrites in a more anterior region characteristic of the dorsolateral muscles. These findings strongly suggest that the mapping of the muscle field within the CNS is an active process of growth and arborisation that partitions dendrites into subdomains of the neuropile that are appropriate to their function, rather than a passive subdivision of available space by position of origin or axon trajectory (Landgraf, 2003).

    Since dendritic arbors form after motor axons have reached their targets, the muscles could be instrumental in dictating the organisation of the central map. To test this idea, the UAS/GAL4 system was used to misexpress an activated form of Notch (Kidd et al. 1998) in the developing mesoderm, suppressing the formation of muscle founder cells while leaving other tissues intact. In such muscleless embryos, the main nerve trunks, SN and ISN, still form and project into the periphery. Retrograde labellings of these nerves show that SN and ISN motor neurons form relatively normal dendritic arbors that consistently conform to the characteristic separation of SN and ISN dendrites. Thus, the neuropile is partitioned into distinct fields of dendritic arborisation independently of the muscles. It is concluded that the mapping process is likely to be an autonomous property of the motor neurons and their neighboring cells (Landgraf, 2003).

    It was next asked whether motor neuron dendritic fields could be patterned by the substrates on which they grow. In the Drosophila ventral nerve cord (VNC), motor neuron dendrites form in the dorsal-most region of the neuropile, sandwiched between longitudinal glia above and the underlying scaffold of axons. Glial cells can act as substrates for supporting and guiding axonal growth. To test whether they might also be required for the growth and spatial patterning of dendritic fields, dendritic arbors were analysed in glial cells missing (gcm) mutant embryos, which are defective in glial cell differentiation. Although the structure of the nervous system is disrupted in gcm mutant embryos and the dendritic arbors are abnormal, they continue to form in their characteristic locations and the fundamental distinction between the ISN and SN motor neuron dendritic fields is maintained. Remarkably, even the long posterior dendritic projection of the DT1 motor neuron forms and reaches its target region, the SN external muscle dendritic domain. These results suggest that the patterning of the neuropile into distinct motor neuron dendritic domains is a process that appears to be intrinsic to the motor neurons and their neighboring neurons, but does not require proper glial cell differentiation (Landgraf, 2003).

    One likely explanation for the division of dendrites into separate domains is that there is a process of mutual exclusion between the arborisations of neighboring cells. Such a process of dendritic ‘tiling' has so far only been documented between particular classes of sensory neurons, but could also occur in the motor system. The idea of tiling was tested by considering two groups of motor neurons whose axons have a common trajectory, but whose dendritic fields form in adjacent territories. The DO3-DO5 and DT1 motor neurons project their dendrites posteriorly, and at their most-anterior point, these dendrites meet the axons and dendrites of the anterior corner cell (aCC) and U/CQ neurons. To show whether the aCC and U/CQ axons and/or dendrites inhibit the growth of DO3-DO5 and DT1 dendrites anteriorly, these neurons (as well as RP2 and the posterior corner cell [pCC] interneuron) were selectively ablated. Using anti-Even-skipped (Eve) staining as a marker for aCC, RP2, and U/CQs (there are an additional two medially located eve-expressing interneurons, pCC and friend of pCC [fpCC], it was found that these neurons can be selectively ablated before they form dendrites (at approximately 11 h AEL): on average, by 10.5 h AEL all but 0.6 and by 12 h AEL all but 0.06 of the seven medially located eve-expressing neurons have been ablated per half-neuromere. In no instance was a concomitant anterior expansion of the DO3-DO5 and DT1 motor neuron dendrites into the regions vacated by the aCC and U/CQ dendrites observed. It is concluded that, at least in this instance, the initial dendritic territory of one set of motor neurons (DO3-DO5 and DT1) is not defined by a process of tiling, in which they are excluded by neighboring (aCC and U/CQ) dendritic arbors. However, it is possible that the elaboration of motor neuron dendritic arbors during later developmental stages may involve interactions between neighboring dendritic territories, activity-dependent processes, or both (Landgraf, 2003).

    Thus, in summary, these results suggest that the mechanisms that subdivide the neuropile into distinct dendritc domains are very robust and refractory to perturbations. They further suggest that the cues that organise the map may be laid down early in development as the embryo subdivides into parasegmental units (Landgraf, 2003).

    The patterning of the motor neuron dendritic arbors in the Drosophila embryo represents a first layer of organisation in the motor system. This is likely in part to be mirrored by the endings of higher-order neurons of central pattern generating circuits, which converge onto the myotopic map. While motor neuron cell body positions may, as has been proposed for vertebrate systems, relate to the ontogeny of target muscles, the operation of mature muscles is reflected by the allegiance of corresponding motor neuron dendrites to a particular territory in the neuropile. Thus, changes in muscle operation could be accommodated by a change of allegiance of the appropriate motor neuron dendrites from one domain to another (e.g., the DT1 motor neuron-muscle pair) without the need for rewiring the underlying higher-order circuitry. Such a model resolves the apparent discrepancy between the distributions of motor neuron cell bodies centrally and target muscles in the periphery. It also implies a considerable degree of flexibility, particularly at the level of motor output, yet suggests that elements of the underlying motor circuitry may have been highly conserved (Landgraf, 2003).

    Dynein is required for polarized dendritic transport and uniform microtubule orientation in axons

    Axons and dendrites differ in both microtubule organization and in the organelles and proteins they contain. This study shows that the microtubule motor dynein has a crucial role in polarized transport and in controlling the orientation of axonal microtubules in Drosophila melanogaster dendritic arborization (da) neurons. Changes in organelle distribution within the dendritic arbors of dynein mutant neurons correlate with a proximal shift in dendritic branch position. Dynein is also necessary for the dendrite-specific localization of Golgi outposts and the ion channel Pickpocket. Axonal microtubules are normally oriented uniformly plus-end-distal; however, without dynein, axons contain both plus- and minus-end distal microtubules. These data suggest that dynein is required for the distinguishing properties of the axon and dendrites: without dynein, dendritic organelles and proteins enter the axon and the axonal microtubules are no longer uniform in polarity (Zheng, 2008).

    Proper cellular morphology and function depends on the polarized localization of organelles and proteins to specific subcellular compartments. This study shows that dynein plays a crucial role in dendrite arbour patterning and in organizing distinct functional compartments (the axon and dendrites) of a neuron. The position of branches within a dendritic arbour has a key role in determining the inputs a neuron receives from pre-synaptic axons or, in the case of sensory neurons, the local environment. This study shows that dynein is necessary for proper positioning of dendritic branches relative to the soma. As a motor, dynein likely influences branch formation by mediating the distribution of cargos that affect branch growth and dynamics. Notwithstanding an overall decrease in dendrite extension and branching in dynein mutants, time-lapse analysis of a few dendrites revealed that they extend normally but have fewer and less stable terminal branches, suggesting that decreased terminal branching is not simply caused by a decrease in dendrite growth. One likely explanation is that 'branching machinery' (including Golgi outposts, endosomes and potentially other proteins and/or organelles) that are normally transported distally for dendrite extension and maintenance become trapped in the proximal arbour in the dynein mutant neurons, resulting in decreased distal branching and the formation of ectopic branches close to the cell body (Zheng, 2008).

    Without dynein, Golgi outposts and Pickpocket (Ppk) are present ectopically in axons, revealing a previously unappreciated role for dynein in mediating the dendrite-specific localization of organelles and proteins. One possible explanation for axonal mislocalization is that Golgi outposts and Ppk interact with a MT plus end-directed motor (e.g., kinesin) that transports them into axons in the absence of dynein. Dynein might normally transport such cargo directly to dendrites; alternatively, it is also possible that cargo first enters axons but that dynein counteracts kinesin and carries this cargo out of axons. Since Golgi outposts moving from the soma into the axon are never seen in wild type neurons, the data favour the former possibility. In contrast to the mislocalization of dendritic protein and organelles, Kin-βgal and proteins destined for the axonal terminal retain their polarized distribution, perhaps to be expected given that kinesin mediates the majority of anterograde axonal transport (Zheng, 2008).

    In mammalian and fly neurons, axonal MTs are arrayed plus end-distal whereas dendritic MT orientation is mixed. A long-standing question concerns the mechanism(s) that establish and maintain different MT orientations in axons and dendrites. Loss of dynein function causes the axonal localization of Nod-βgal and retrograde movement of EB1-GFP, indicating that minus end-distal MTs are present in these mutant axons. How might dynein regulate the orientation of axonal MTs? The sliding filament model of axonal MT transport proposes that a subset of dynein in the axon is stationary (via an interaction with stable MTs and/or actin) and that dynein’s motor domain interacts with short MT polymers, propelling plus end-distal MTs down the axon as the motor moves to the MT minus end. In vivo data support the idea that in addition to transporting MTs, dynein functions as a 'gatekeeper' to move minus end-distal MTs towards the soma, excluding them from the axon. Neurons lacking functional dynein would still transport MTs, likely via kinesin, but now minus end-distal MTs would infiltrate the axon. Proximal axons likely have unique properties, providing a possible explanation for how minus end distal MTs would be excluded from axons but not dendrites (Zheng, 2008).

    Recent studies indicate that the trans Golgi network (TGN), which comprises part of the Golgi outposts, can also function as a MT organizing center (MTOC) and influence MT organization. Although it is conceivable that Golgi outposts mislocalized to dynein mutant axons could alter MT polarity, expressing lava lamp dominant-negative, which prevents Golgi from associating with dynein without affecting dynein function, causes Golgi outposts to mislocalize to axons without altering MT orientation (Ye, 2007). Moreover, the change in axonal MT orientation is not likely to be simply a consequence of altered axon morphology because the loss of dynein function also alters the MT orientation of class I neuron axons, which appear relatively normal. With the current level of understanding, the model in which dynein acts as a 'gatekeeper' is most consistent with the results and the findings of others (Zheng, 2008).

    Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system

    A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. In the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. This study shows that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. This map is 'hard-wired'; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. The midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. These results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. It is further proposed that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common 'meeting regions' (Mauss, 2009).

    How different dendritic morphologies and territories are generated in a motor system was investigated using the neuromuscular system of the Drosophila embryo as a model. Its principal components are segmentally repeated arrays of body wall muscles (30 per abdominal half segment), each innervated by a specific motorneuron. The motorneuron dendrites are the substrate on which connections with presynaptic cholinergic interneurons form. 180 cells (on average 11.25 for each identified motorneuron and a minimum of five) were labelled, and the dendritic morphologies and territories of the motorneurons that innervate the internal muscles were charted using retrograde labelling with the lipophilic tracer dyes 'DiI'and 'DiD.' This was done in the context of independent landmarks, a set of Fasciclin 2-positive axon bundles, at 18.5 h after egg laying (AEL), when the motor system first becomes robustly functional and the geometry of motorneuron dendritic trees has become sufficiently invariant to permit quantitative comparisons (Mauss, 2009).

    Three classes of motorneurons were found based on dendritic arbor morphology and territory with respect to the ventral midline: (1) motorneurons with dendrites in the lateral neuropile (between the lateral and intermediate Fasciclin 2 tracts); (2) in the lateral and intermediate neuropile (between the intermediate and medial Fasciclin 2 tracts), and (3) in the lateral, intermediate plus medial neuropile (posterior commissure) (Mauss, 2009).

    Moreover, the medio-lateral positions of motorneuron dendrites correlate with the dorsal to ventral locations of their target muscles in the periphery. Motorneurons with dorsal targets (DA1, DA3, DO1-5) have their dendrites in the lateral neuropile, while those innervating ventral and lateral muscles (LL1, VL2-4, VO1-2) also have dendrites in the intermediate neuropile. Coverage of the medial neuropile is particular to motorneurons innervating the most ventral group of muscles (VO3-6). These dendritic domains are arranged in the medio-lateral axis of the neuropile in such a way that they form a neural, myotopic representation of the distribution of body wall muscles in the periphery. Only a single motorneuron deviates from this clear-cut correlation between dendritic medio-lateral position and target muscle location: MN-DA2 has dendrites not only in the lateral neuropile, like other motorneurons with dorsal targets, but also in the intermediate neuropile (Mauss, 2009).

    Previously studies have shown that motorneurons in the Drosophila embryo distribute their dendrites in distinct anterior to posterior domains in the neuropile, forming a central representation of target muscle positions in the periphery. The mechanisms required for the generation of this dendritic myotopic map remain elusive. In this study, dendritic myotopic organisation was characterized in a second dimension, with respect to the ventral midline, and the main molecular mechanism that underlies the formation of this dendritic neural map were identified, namely the combinatorial action of the midline signalling systems Slit/Robo and Netrin/Frazzled (Mauss, 2009).

    Neural maps are manifestations of an organisational strategy commonly used by nervous systems to order synaptic connections. The view of these maps has been largely axonocentric and focused on sensory systems, though recent studies have challenged the notion of dendrites as a 'passive' party in arranging the distribution of connections. This study has demonstrated that motorneuron dendrites generate a neural, myotopic map in a motor system and that this manifest regularity can form independently of its presynaptic partner terminals (Mauss, 2009).

    An essential feature of neural maps is the spatial segregation of synaptic connections. In the Drosophila embryonic nerve cord, there is some overlap between dendritic domains in the antero-posterior neuropile axis. Overlap of dendritic territories is also evident in the medio-lateral dimension, since all motorneurons have arborisations in the lateral neuropile, though distinctions arise by virtue of dendrites in additional intermediate and medial neuropile regions. The combination of myotopic mapping in both dimensions may serve to maximise the segregation between dendrites of different motorneuron groups. For example, the dendritic domain of motorneurons with dorsal targets differs from the territory innervated by ventrally projecting motorneurons in the antero-posterior location and the medio-lateral extent. Myotopic mapping in two dimensions could also provide a degree of flexibility that could facilitate wiring up in a combinatorial fashion. For instance, muscle LL1 lies at the interface between the dorsal and ventral muscle field; its motorneuron, MN-LL1, has one part of its dendritic arbor in the lateral domain that is characteristic for dorsally projecting motorneurons, while the other part of the dendritic tree innervates the intermediate neuropile precisely where ventrally projecting motorneurons put their dendrites (Mauss, 2009).

    Myotopic dendritic maps might constitute a general organisational principle in motor systems. In insects, a comparable system of organisation has now been demonstrated also for the adult motor system of Drosophila (Brierley, 2009; Baek, 2009) and a degree of topographic organisation had previously been suggested for the dendrites of motorneurons that innervate the body wall muscles in the moth Manduca sexta. In vertebrates too, there is evidence that different motor pools elaborate their dendrites in distinct regions of the spinal cord in chick, turtle, and mouse. Moreover, elegant work in the mouse has shown that differences in dendritic territories correlate with and may determine the specificity of proprioceptive afferent inputs (Mauss, 2009 and references therein).

    The neural map characterised in this study is composed of three morphological classes of motorneurons with dendrites innervating either (1) the lateral or (2) the lateral and intermediate or (3) the lateral, intermediate, and medial/midline neuropile (Mauss, 2009).

    The motorneuron dendrites are targeted to these medio-lateral territories by the combinatorial, cell-autonomous actions of the midline guidance cue receptors Robo and Frazzled. The formation of dendritic territories by directed, targeted growth appears to be an important mechanism that may be more widespread than previously anticipated, though the underlying mechanisms may vary. Global patterning cues have been implicated in the vertebrate cortex (Sema3A). In the zebrafish retina, live imaging has shown that retinal ganglion cells put their dendrites into specific strata of the inner plexiform layer, but the roles of guidance cues and interactions with partner (amacrine) cells have not yet been studied (Mauss, 2009).

    Slit/Robo and Netrin/Frazzled mediated gating of dendritic midline crossing has been previously documented in Drosophila embryos and zebrafish. This study demonstrated that dendrites are targeted to distinct medio-lateral territories by the combinatorial, opposing actions of Robo and Frazzled and that this is the main mechanism underlying the formation of the myotopic map. Strikingly, the same signalling pathways also regulate dendritic targeting of adult motorneurons in Drosophila, suggesting this to be a conserved mechanism (Brierley, 2009). Robo gates midline crossing of dendrites and in addition, at progressively higher signalling levels, restricts dendritic targeting to intermediate and lateral territories. Frazzled, on the other hand, is required for targeting dendrites towards the midline into intermediate and medial territories. The data argue that Frazzled is expressed by representatives of all three motorneuron types. Recently, Yang (2009) has shown that expression of frazzled leads to a concomitant transcriptional up-regulation of comm, thus linking Frazzled-mediated attraction to the midline with a decrease in Robo-mediated repulsion. While this has been demonstrated for midline crossing of axons in the Drosophila embryo, this study found that, at least until 18.5 h AEL, expression of UAS-frazzled alone was not sufficient to induce midline crossing of dendrites in MN-LL1 and MN-DA3. It is conceivable that differences in expression levels and/or timing between CQ-GAL4 used in this study and egl-GAL4 used by Yang might account for the differences in axonal and dendritic responses to UAS-frazzled expression. Moreover, the widespread expression of Frazzled in motorneurons and other cells in the CNS may point to additional functions, potentially synaptogenesis, as has been shown in C. elegans (Mauss, 2009).

    Strikingly, neither synaptic excitatory activity nor the presynaptic (cholinergic) partner terminals seem to be necessary for the formation of the map. The map is already evident by 15 h AEL, before motorneurons receive synaptic inputs. It also forms in the absence of acetylcholine, the main (and at that stage probably exclusive) neurotransmitter to which motorneurons respond. Moreover, motorneuron dendrites innervate their characteristic dendritic domains when the cholinergic terminals have been displaced to outside the motor neuropile. However, interactions with presynaptic partners seem to contribute to its refinement. First, it was found that dendritic mistargeting phenotypes show a greater degree of penetrance earlier (15 h AEL) than later (18.5 h AEL) in development. Secondly, when interactions with presynaptic partner terminals are reduced or absent, dendritic arbor size increases and the distinction between dendritic territories is less evident than in controls. Fine-tuning of terminal arbors and sets of connections through contact and activity-dependent mechanisms is a well-established feature of neural maps in sensory systems and the current observations suggest that this may also apply to motor systems (Mauss, 2009).

    The formation of the myotopic map is the product of dendritic targeting. It is therefore intimately linked with the question of how cell type-specific dendritic morphologies are specified. For instance, changing the balance between the Robo and Frazzled guidance receptors in motorneurons is sufficient to 'convert' dendritic morphologies from one type to another. The importance of target territories for determining dendritic arbor morphology has recently been explored in a study of lobula plate tangential cells in the blowfly, where the distinguishing parameter between the dendritic trees of four functionally defined neurons were not growth or branching characteristics but the regions where neurons put their dendrites (Mauss, 2009).

    Because Slit/Robo and Netrin/Frazzled signalling have been reported to affect dendritic and axonal branching as well as axonal growth, respectively, it was asked what the effect was on motorneuron dendrites of altered Robo and Frazzled levels. It was found that in the wild-type different motorneurons generate characteristically different amounts of dendritic length and numbers of branch points (MN-DA1/aCC and MN-VO2/RP1, RP2, MN-DA3 and MN-LL1). In the Drosophila embryo and larva, Slit/Robo interactions have been suggested to promote the formation of dendrites and/or branching events, similar to what has been shown for cultured vertebrate neurons. The current data on embryonic motorneurons are not compatible with this interpretation. First, when altering the levels of Robo (or Frazzled) in individual motorneurons and mistargeting their dendrites, no statistically significant changes were detected in total dendritic length or number of branch points. Instead, for MN-DA3 and MN-LL1, it was observed that dendritic arbors respond to changes in the expression levels of midline cue receptors by altering the amount of dendritic length distributed to the medial, intermediate, and lateral neuropile. Secondly, in nerve cords entirely mutant for the Slit receptor Robo an increase is seen in dendrite branching at the midline. These observations suggest that for Drosophila motorneurons Slit/Robo interactions negatively regulate the establishment and branching of dendrites and thus specify dendritic target territories by defining 'exclusion' zones in the neuropile. The quantitative data from this study suggest that dendritic morphology is the product of two intrinsic, genetically separable programmes: one that specifies the total dendritic length to be generated and the frequency of branching; the other implements the distribution of these dendrites in the target territory, presumably by locally modulating rates of extension, stabilisation, and retraction of branches in response to extrinsic signals (Mauss, 2009).

    The question of how neural circuits are generated remains at the heart of developmental neurobiology. At one extreme, one could envisage that every synapse was genetically specified, the product of an exquisitely choreographed sequence of cell-cell interactions. At the other extreme, neural networks might assemble through random cell-cell interactions and feedback processes enabling functional validation. The latter view supposes that neurons inherently generate polarised processes, have a high propensity to form synapses, and arrive at a favourable activity state through homeostatic mechanisms. Current evidence suggests that, at least for most systems, circuits form by a combination of genetic specification and the capacity to self-organise (Mauss, 2009).

    This study has demonstrated that the postsynaptic structures of motorneurons, the dendrites, form a neural map. It was also shown that dendrites are closely apposed to cholinergic presynaptic specialisations in their target territories, suggesting that the segregation of dendrites may be a mechanism that facilitates the formation of specific sets of connections. Strikingly, this map of postsynaptic dendrites appears to be 'hard-wired' in that it can form independently of its presynaptic partners and it is generated in response to a third party, the midline guidance cues Slit and Netrin. A comparable example is the Drosophila antennal lobe, where projection neurons form a neural map independently of their presynaptic olfactory receptor neurons, though in this sensory system the nature and source of the cue(s) remain to be determined. This study complements previous work that demonstrated the positioning of presynaptic axon terminals by midline cues, also independently of their synaptic partners. Together, these results suggest that global patterning cues set up the functional architecture of the nervous system by independently directing pre- and postsynaptic partner terminals towards common 'meeting' areas (Mauss, 2009).

    Clearly, such global guidance systems deliver a relatively coarse level of specificity and there is ample evidence for the existence of codes of cell-adhesion molecules and local receptor-ligand interactions capable of conferring a high degree of synaptic specificity. Therefore, one has to ask what the contribution is of global partitioning systems in establishing patterns of connections that lead to a functional neural network. A recent study in the Xenopus tadpole spinal cord has addressed this issue. Conducting patch clamp recordings from pairs of neurons, it has been found that the actual pattern of connections in the motor circuit reveals a remarkable lack of specificity. Furthermore, the segregation of axons and dendrites into a few broad domains appears to be sufficient to generate the connections that do form and to enable the emergence of a functional network. The implication is that neurons might be intrinsically promiscuous and that targeting nerve terminals to distinct territories by global patterning cues, as has been shown in this study, is important to restrict this synaptogenic potential and thereby confer a degree of specificity that is necessary for the emergence of network function (Mauss, 2009).

    Genome-wide analyses identify transcription factors required for proper morphogenesis of Drosophila sensory neuron dendrites

    Dendrite arborization patterns are critical determinants of neuronal function. To explore the basis of transcriptional regulation in dendrite pattern formation, RNA interference (RNAi) was used to screen 730 transcriptional regulators and 78 genes involved in patterning the stereotyped dendritic arbors of class I da neurons were identified in Drosophila. Most of these transcriptional regulators affect dendrite morphology without altering the number of class I dendrite arborization (da) neurons and fall primarily into three groups. Group A genes control both primary dendrite extension and lateral branching, hence the overall dendritic field. Nineteen genes within group A act to increase arborization, whereas 20 other genes restrict dendritic coverage. Group B genes appear to balance dendritic outgrowth and branching. Nineteen group B genes function to promote branching rather than outgrowth, and two others have the opposite effects. Finally, 10 group C genes are critical for the routing of the dendritic arbors of individual class I da neurons. Thus, multiple genetic programs operate to calibrate dendritic coverage, to coordinate the elaboration of primary versus secondary branches, and to lay out these dendritic branches in the proper orientation (Parrish, 2006; Full text of article).

    To assay for the stereotyped dendrite arborization pattern of class I da neurons (hereafter referred to as class I neurons) in RNAi-based analysis of dendrite development, a Gal4 enhancer trap line (Gal4221) was used that is highly expressed in class I neurons and weakly expressed in class IV neurons during embryogenesis. Because of the simple and stereotyped dendritic arborization patterns of the dorsally located ddaD and ddaE, the studies of dendrite development focused on these two dorsally located class I neurons (Parrish, 2006).

    To establish that RNAi is an efficient method to systematically study dendrite development in the Drosophila embryonic PNS, it was demonstrated that injecting embryos with double-stranded RNA (dsRNA) for green fluorescent protein (gfp) is sufficient to attenuate Gal-4221-driven expression of an mCD8::GFP fusion protein as measured by confocal microscopy. Next whether RNAi could efficiently phenocopy loss-of-function mutants known to affect dendrite development was tested. Similar to the mutant phenotype of short stop (shot), which encodes an actin/microtubule cross-linking protein, shot(RNAi) caused routing defects, dorsal overextension, and a reduction in lateral branching of dorsally extended primary dendrites. Likewise, RNAi of sequoia or flamingo resulted in overextension of ddaD and ddaE, RNAi of hamlet resulted in supernumerary class I neurons, and RNAi of tumbleweed resulted in supernumerary class I neurons and a range of arborization defects, consistent with the reported mutant phenotypes. Thus, RNAi is effective in generating reduction of function phenotypes in embryonic class I dendrites (Parrish, 2006).

    Group A TFs that promote arborization through concerted regulation of outgrowth and branching

    A group of transcriptional regulators, group A, controls the size of the dendritic field of class I neurons. RNAi of 19 TFs resulted in reduction of the field size covered by ddaD and ddaE. A reduction of coverage could be the result of a net reduction in dendrite outgrowth, branching, or both. Group A TFs have effects on both primary dendrite growth and secondary dendrite growth. For example, RNAi of the PAS-domain TF trachealess (trh) caused a minor reduction in both primary branch outgrowth and the number of lateral branches and a more marked reduction in the overall length of lateral branches. Consequently, the most distal regions of the dendritic field, especially the regions covered by lateral branches, are not innervated. By contrast, RNAi of genes such as the zinc-finger TF pygopus or the BTB/POZ-domain TF cg1841 caused more severe reduction of primary branch outgrowth as well as lateral branching and lateral branch length, resulting in a more drastic reduction of receptive field. In an extreme case, RNAi of the high mobility group gene hmgD resulted in an almost complete block of primary dendrite extension and lateral branching. In general, the genes with the most severe effects on primary branch outgrowth also have the most severe effects on branching, suggesting that these genes may function to regulate dendritic arborization overall (Parrish, 2006).

    Although the genes in this class all caused qualitatively similar defects in arborization, some notable phenotypic differences are suggestive of distinct functions for some of these genes in regulating dendrite arborization. RNAi of the nuclear hormone receptors ultraspiracle (usp) and ecdysone receptor (EcR) significantly reduced primary dendrite outgrowth, but caused only modest reduction of lateral branching and lateral branch outgrowth, suggesting that branching is not absolutely dependent on proper outgrowth. Since the Usp/EcR heterodimer is responsible for ecdysone-responsive activation of transcription, as well as ligand-independent transcriptional repression, it is likely that these genes function together to promote dendrite outgrowth (Parrish, 2006).

    RNAi of many group A genes resulted in embryonic lethality at a significantly higher rate than control injections. Thus, many of these genes are likely essential for embryonic development, either due to their involvement in regulating neuronal morphogenesis or due to other aspects of their functions (Parrish, 2006).

    Group A transcriptional regulators that restrict dendrite arborization

    In addition to genes with functions in promoting dendrite arborization, 20 group A genes were identified that regulate dendrite arborization by limiting dendrite growth and/or branching. Consistent with recent reports that loss of function of the BTB/POZ domain TF abrupt (ab) causes an increase in dendritic branching and altered distribution of branches, it was found that ab(RNAi) altered the arborization of class I dendrites. ab(RNAi) caused an increase in the number and length of lateral branches, expanding the coverage field most noticeably along the anteroposterior (AP) axis. In addition to these defects, ab(RNAi) also caused frequent cell death, consistent with the phenotype observed for a hypomorphic allele of ab (Parrish, 2006).

    Increased dendritic branching also resulted from RNAi of several genes known to affect nervous system development, including Adh transcription factor 1 (Adf1), the zinc finger TF nervy (nvy), the basic helix–loop–helix (bHLH) TF deadpan (dpn), as well as genes not previously known to affect neuronal function, such as the putative transcription elongation factor Elongin c. Both Adf1 and dpn mutants have defects in larval locomotion and, in light of recent findings suggesting that da neurons may regulate aspects of larval locomotion, it is possible that dendrite defects underlie these behavioral defects. Consistent with its role in class I dendrite development, dpn is expressed in all PNS neurons. Likewise, nervy has been implicated in regulation of axon branching in motorneurons and is apparently expressed in most neurons. Thus, nervy likely regulates multiple aspects of neuronal differentiation. Finally, Elongin C may regulate transcriptional elongation but also likely functions as a component of a multimeric protein complex that includes the von Hippel-Lindau (VHL) tumor suppressor and targets specific proteins for poly-ubiquitination and degradation. Moreover, BTB/POZ domain proteins (such as cg1841 and ab) function as substrate adaptors for cullin E3 ligases. Interestingly, RNAi of a Drosophila homolog (tango) of a known VHL substrate (HIF-1) also affected dendrite arborization. It thus appears that protein degradation pathways regulate dendrite arborization (Parrish, 2006).

    RNAi of the Polycomb group (PcG) genes Su(z)12, E(z), esc, or Caf1 similarly caused an increase in branch number and an expansion of the receptive field of class I neurons. Consistent with the similar RNAi phenotypes for these genes, Su(z)12, E(z), esc, and Caf1 are components of the multiprotein esc/E(z) polycomb repressor complex. One critical role for PcG-mediated gene silencing is the regulation of hox gene expression. Therefore, Polycomb-mediated regulation of hox gene expression likely contributes to arborization of class I neurons (Parrish, 2006).

    RNAi of several genes affected dendrite arborization primarily by causing an increase in dorsal and lateral dendrite extension without significantly affecting branch number. For example, RNAi of the putative transcriptional repressor cg5684 caused dorsal overextension of the primary dendrite in ddaE and an overall increase in dendritic length in both ddaD and ddaE. In general, RNAi of genes that increased arborization rarely caused dendrites to cross the dorsal midline or segment borders, or increased branching more than twofold as compared with untreated neurons. It thus appears that dendritic outgrowth is further limited by neuronal growth capacity and/or other external constraints (Parrish, 2006).

    Group B TFs with opposing actions on dendrite outgrowth and branching shape dendrite arbors

    In contrast to the genes that coordinately affect dorsal dendrite outgrowth and lateral branching/outgrowth, a group of 21 genes (group B) were identified that have opposing effects on dendrite outgrowth and branching, suggesting that dendrite outgrowth and branching might partially antagonize one another. RNAi of 19 of these genes resulted in dorsal overextension of primary dendrites and a reduction in lateral branching/lateral branch extension. In the most severe cases, such as RNAi of the transcriptional repressor snail, dorsal overextension of almost completely unbranched dendrites was found. Like snail(RNAi), RNAi of the nuclear hormone receptor knirps, the transcriptional repressor l(3)mbt, as well as 15 other genes, all caused dorsal overextension of primary dendrites. As in the case of genes that normally limit arborization, RNAi of these genes rarely caused dendrites to cross the dorsal midline (Parrish, 2006).

    In addition to the effects on primary dendrite extension, RNAi of each of these 18 genes limits the number and length of lateral dendrite branches. RNAi of some genes such as snail or knirps almost completely blocked dendrite branching, whereas RNAi of other genes such l(3)mbt had more modest effects on dendrite branching. In addition, a significant reduction of branching was noticed at the distal tip of the dorsally projected primary dendrite. In control treated stage 17 embryos, branchpoints are distributed along the primary dendrite, with the most distal branchpoint usually located within a few microns of the distal tip of the dendrite. In contrast, branching is rarely observed within 10 microns of the distal dendritic tip following RNAi of these group B genes. In some cases, such as snail(RNAi), knirps(RNAi), or l(3)mbt(RNAi), the most distal branchpoint is located 25 microns or further from the distal tip of the primary dendrite. Therefore, these TFs inhibit primary branch extension but promote lateral branching and lateral branch extension (Parrish, 2006).

    In addition to identifying a large class of TFs that inhibit primary branch extension and promote lateral branching, it was also found that TFs promote dendrite extension and limit dendrite branching. RNAi of two genes, glial cells missing 2 (gcm2) and the histone acetyltransferase pcaf, caused an increase in lateral branching and a marked reduction in dorsal extension of ddaE. Thus, transcriptional pathways exist that have opposing effects on primary branch outgrowth and secondary branching, suggesting that these processes may normally antagonize one another (Parrish, 2006).

    Group C TFs regulate dendrite routing

    Proper dendritic routing is important for primary dendrites of ddaD and ddaE to grow in parallel toward the dorsal midline without crossing each other and for secondary branches of ddaD and ddaE to avoid the space between ddaD and ddaE. Therefore, there must be mechanisms that promote this stereotyped arborization pattern, including signals that promote anterior arborization of ddaD and posterior arborization of ddaE, as well as signals that antagonize posterior arborization of ddaD and anterior arborization of ddaE. Indeed, RNAi of 10 TFs disrupted the dendritic routing patterns of ddaD and ddaE, resulting in aberrantly oriented primary dendrites. RNAi of cg1244, bap55 (brahma associated protein of 55kD), cg9104, cg4328, and cg7417 resulted in inappropriate anterior arborization of ddaE as well as inappropriate posterior arborization of ddaD. Anterior or even ventral displacement of ddaD concomitant with anterior arborization of ddaE was also observed as well as displacement of ddaE arbors concomitant with misrouting of ddaD. Finally, reducing sens function by RNAi or genetic mutation caused extensive mixing of dendritic arbors from ddaD and ddaE, in addition to dorsal overextension of primary dendrites and an overall reduction in the number of class I neurons (Parrish, 2006).

    It is also worth noting that RNAi treatment that caused reduced dendritic outgrowth often caused minor routing defects. For example, pyg(RNAi) or cg1841(RNAi) caused inappropriate routing of ddaE. The routing defects seen with these candidates may reflect a disruption of attractive/repulsive signaling that normally regulates dendrite arborization. The source of such signals is currently unknown, although of great interest (Parrish, 2006).

    TFs likely exert distinct mitotic and post-mitotic functions to regulate neuron morphogenesis

    TFs play critical roles in neurogenesis, and some genes that regulate neurogenesis also affect post-mitotic neuronal differentiation. Because clones of duplicated class I neurons have wild-type dendrite arborization patterns, class I dendritic arbors appear to be insensitive to cell number defects. Indeed, dendrite arborization of class I neurons in embryos carrying the temperature-sensitive neurogenic mutation Notchts (Nts) is unaffected by as much as a fivefold increase in class I neuron number, and is likely insensitive to multiplication of other da neurons as well since Nts experiments caused increased numbers of other da neurons. Furthermore, in cases where only one of the class I neurons is multiplied, the dendrites of neighboring class I neuron are unaffected. In contrast laser ablation of ddaD or ddaE or the occasional cell loss caused by RNAi of various genes did not generally cause defects in arborization of neighboring class I neurons. Therefore, analysis of class I neurons should allow study of post-mitotic functions of genes that affect neuron number (Parrish, 2006).

    RNAi of several genes affected the number of class I neurons as well as morphogenesis of class I dendrites; RNAi of seven genes caused supernumerary cells and RNAi of four genes caused high penetrance cell loss in addition to dendrite defects. For example, RNAi of the zinc finger TF nerfin-1 caused an increase in neurons labeled by Gal4221 with as many as eight neurons visible in some segments. Unlike wild-type class I neurons, neurons from nerfin-1(RNAi)-treated embryos extended mostly unbranched dendrites. In many cases, the routing pattern of the dendrites appeared abnormal, but the cell number defects make it difficult to resolve the projection pattern of individual dendrites or conclusively determine whether each neuron projects the same number of primary dendrites. RNAi of six other genes, including jumeau, a winged-helix TF known to regulate neuroblast cell fate and the number of PNS neurons, similarly caused an increase in neuronal number as well as defects in dendrite morphogenesis (Parrish, 2006).

    In addition to the seven genes that function to restrict class I neuron number and control dendrite morphology, three other genes are required to maintain the number of class I neurons. Reduction of their function caused a reduction of class I neurons and defects in dendrite morphogenesis in the remaining neurons. For example, RNAi of the zinc finger TF senseless (sens) reduced the number of class I neurons, consistent with previous findings that sens is required for development of most cells in the PNS. In addition, sens(RNAi) or a sens loss-of-function mutation caused an increase in dendrite outgrowth and mixing of dendrites in segments with both ddaD and ddaE present. Similarly, RNAi of the proneural bHLH TF atonal (ato) reduced the number of class I neurons, consistent with previous findings that chordotonal organs and some md neurons are absent in embryos lacking ato. Consistent with reports that ato functions in neurite arborization in the larval brain, it was also found that ato(RNAi) caused altered arborization patterns of class I dendrites. Thus, it is likely that multiple TFs that regulate neuron number also regulate aspects of post-mitotic neuronal differentiation (Parrish, 2006).

    Finally, gene was found that appears to be essential for a subset of class I da neurons. RNAi of cubitus interruptus (ci), a component of the hedgehog signaling pathway, caused a loss of dorsal class I da neurons without affecting the ventral class I neuron vpda. Because ci(RNAi) causes embryonic lethality at high concentrations of dsRNA, the phenotypes in surviving embryos likely represent hypomorphic phenotypes. Loss of ddaD and ddaE is not compensated for by a concomitant increase in other md neurons since ci(RNAi) leads to an overall reduction of dorsal md neurons expressing the pan-da marker Gal4109(2)80. Therefore, ci likely promotes differentiation or survival of a subset of da neurons, including ddaD and ddaE, but not of the ventral class I neuron vpda, demonstrating that morphologically similar neurons can be molecularly distinct (Parrish, 2006).

    Some TFs are continuously required to regulate class I dendrite morphology

    As another indication of the hypomorphic nature of many of the alleles and maternal rescue of gene function in mutant embryos, focus was place on dendrite defects that were first apparent during larval stages. For example, a mutant allele of Drosophila Mi-2, which encodes a Hunchback-interacting ATP-dependant chromatin remodeling factor, shows only minor defects in late embryonic stages, but shows an obvious reduction in arborization by 72 h after egg laying. Since Mi-2(RNAi) demonstrates that Mi-2 is required for embryonic dendrite arborization, these findings suggest that Mi-2 is continuously required for class I neurons to maintain proper dendrite arborization patterns. Similarly, the dendritic overbranching associated with a P-element insertion allele of Adf1 was first apparent after embryonic stages, although Adf1(RNAi) caused overbranching in embryos. Class I dendritic arbors of Adf1 mutants are indistinguishable from wild-type neurons until 96 h AEL. By 144 h AEL, ddaE arbors of Adf1 mutants showed a greater than twofold increase in branch number when compared with time-matched wild-type controls. Interestingly, ddaD showed only very minor branching defects in Adf1 mutants, suggesting that ddaD and ddaE might have distinct requirements for Adf1. Similarly, mutant alleles of either E(bx) or Elongin C showed dendrite branching defects only at late larval stages. These findings indicate that Adf1, E(bx), and Elongin C are continuously required to inhibit branching in class I neurons, demonstrating that although class I neurons have very little new branching after embryogenesis, they still retain the capacity to branch (Parrish, 2006).

    RNAi of some group A genes that cause reduced dendrite outgrowth and branching is epistatic to mutation of ab or sens

    Since group A and B TFs regulate aspects of dendritic growth and branching, potential epistatic relationships among TFs was explored in these phenotypic classes. To do this, RNAi was used to knockdown expression of select TFs in Drosophila embryos carrying a loss-of-function mutation in either the group B/C gene senseless (sens) or the group A gene abrupt (ab). sens mutant class I dendrites overextend dorsally and have reduced lateral branching in addition to routing defects. In sens mutants, RNAi of the group A genes Su(z)12 and ab, which cause increased lateral branching following RNAi in wild-type embryos, led to an increase in lateral branching compared with injected controls. Therefore, Su(z)12 and ab function are still required to limit arborization in sens mutants, and the increased dendritic branching as a result of Su(z)12(RNAi) or ab(RNAi) is epistatic to the increased dorsal extension and reduced lateral branching of sens mutants. In contrast, RNAi of the group A genes cg1244 and cg1841, which caused reduced arborization following RNAi in wild-type embryos, led to a reduction in primary dendrite outgrowth and lateral dendrite branching compared with injected controls. Therefore, at least in the instances described above, loss of group A genes is epistatic to loss of group B genes (Parrish, 2006).

    RNAi of group A genes either promoted or antagonized dendrite arborization; therefore, the effect was examined of simultaneously disrupting one group A gene that promoted and one group A gene that antagonized dendrite outgrowth and lateral branching. RNAi or a loss-of-function mutant of the group A gene ab caused increased dendritic branching and extension of class I dendrites. In addition, mutation of ab caused a significant reduction in the number of class I neurons labeled by Gal4221 that was most pronounced in the dorsal cluster of PNS neurons, consistent with the results from RNAi experiments. To facilitate epistasis analysis in ab mutants, dendrite arborization effects in vpda, the ventrally located class I neuron, were assayed. RNAi of the group A gene hmgD, which caused reduced primary dendrite outgrowth and reduced lateral branching when injected into wild-type embryos, caused a striking reduction in the number of dendritic branches and size of the receptive field of vpda in ab mutants. RNAi of the group A gene bap55 had similar effects in ab mutants, demonstrating that, at least in some cases, loss of group A genes that results in reduced arborization is epistatic to loss of group A genes that results in increased arborization. Therefore it is possible that the different classes of group A genes antagonistically regulate a common set of target genes required for dendrite arborization (Parrish, 2006).

    The bHLH-PAS protein Spineless is necessary for the diversification of dendrite morphology of Drosophila dendritic arborization neurons

    Dendrites exhibit a wide range of morphological diversity, and their arborization patterns are critical determinants of proper neural connectivity. How different neurons acquire their distinct dendritic branching patterns during development is not well understood. This study reports that Spineless (Ss), the Drosophila homolog of the mammalian aryl hydrocarbon (dioxin) receptor (Ahr), regulates dendrite diversity in the dendritic arborization (da) sensory neurons. In loss-of-function ss mutants, class I and II da neurons, which are normally characterized by their simple dendrite morphologies, elaborate more complex arbors, whereas the normally complex class III and IV da neurons develop simpler dendritic arbors. Consequently, different classes of da neurons elaborate dendrites with similar morphologies. In its control of dendritic diversity among da neurons, ss likely acts independently of its known cofactor tango and through a regulatory program distinct from those involving cut and abrupt. These findings suggest that one evolutionarily conserved role for Ahr in neuronal development concerns the diversification of dendrite morphology (Kim, 2006).

    The ss protein is present at nearly the same level in all da neurons and acts cell-autonomously to dictate their dendritic complexity, while different da neurons exhibit different sensitivity to the level of Ss, and even the bipolar td neuron can respond to elevated ss activity by increasing dendritic complexity (Kim, 2006).

    Previous studies in C. elegans have demonstrated essential roles for invertebrate homologs of Ahr in neuronal cell fate determination. For example, ahr-1 regulates the differentiation program of a subclass of neurons that contact the pseudocoelomic fluid, and both ahr-1 and aha-1 specify GABAergic neuron cell fate in C. elegans. The dramatic changes in the dendrite morphologies of the da neurons, however, are not due to an all-or-nothing change in cell fate because the da neurons in ss mutants displayed normal class-specific expression patterns of the molecular markers Ab and Cut and normal axon projection patterns characteristic of individual da neurons. However, this also does not assume that a partial cell fate change has not occurred. One reflection of the ss function as a transcription factor is the altered expression levels of GFP in the class I Gal4221 reporter, with increased levels of expression in all class IV neurons and essentially no expression in the dorsal class I neuron ddaD and the ventral class I neuron vpda in ss mutants. It will be of interest to further characterize the genetic basis for this Gal4 reporter, to determine whether this regulation constitutes a partial cell fate alteration or transcriptional regulation of genes downstream from ss in the execution of adjustment of dendritic complexity (Kim, 2006).

    There is an emerging theme that ss functions to diversify neuronal differentiation by expanding the photopigment repertoire of R7 photoreceptors in the Drosophila eye and by diversifying da neuron dendritic morphologies. Recent studies have demonstrated that the entire retinal mosaic pattern required for color vision in Drosophila is regulated by ss. In the Drosophila retina, two types of ommatidia form the wild-type retinal mosaic: 'pale' and 'yellow.' In ssD115.7 mutants, the yellow ommatidial subtype is lost and normally yellow R7 cells are misspecified into the pale subtype. As a result, nearly all R7 cells adopt the pale subtype, leading to loss of the retinal mosaic pattern. Thus, the pale R7 subtype represents the R7 'default state' (Kim, 2006 and references therein).

    The overall lack of dendritic diversity in the da neurons in ss mutants is suggestive of the hypothesis that ss, an ancient, evolutionarily conserved gene, may act to convert a primordial dendrite pattern (perhaps a default state) to different complexities for different neurons in the peripheral nervous system. The loss-of-function ss phenotype in the da neuron dendrites might reflect such a primordial pattern as the dendrites in the mutant are devoid of specific morphological features that define distinct neuronal subclasses. In support of this notion, dendrites of the different classes of da neurons share similar morphological characteristics and elaborate similar numbers of total branches in ss mutants. The ability of ss to regulate the complexity and diversity of this dendrite pattern, by limiting dendritic branching to shape the simpler arbors of the class I and class II neurons and by promoting class-specific terminal branching to shape the more complex arbors of the class III and class IV neurons, is quite unique. Of the many mutants that affect multiple classes of da neurons, the great majority affect da neurons with simple or complex dendritic arbors the same way; that is, causing them to all become simpler or more complex. The ss phenotype of making simple dendritic arbors more complex and complex arbors simpler is very unusual among the many mutants affecting dendrite complexity. It thus seems likely that the distinct dendritic patterns rely not only on a cohort of gene activities specifying the mechanics of dendrite outgrowth and branching, but also a genetic program that diverts the generic primordial mode of dendritic formation to a diverse range of dendritic patterns (Kim, 2006).

    How might spineless exert its functions? Unlike the homeodomain protein Cut, which promotes dendritic complexity in a specific direction, ss functions in an opposing manner in different cell types to regulate dendritic diversity. How might ss function differently in different neuronal cell types? One possibility is that ss is activated by different ligands in different neurons. ss is incapable of binding dioxin and other related compounds, suggesting that other, as yet unidentified ligands are required for its activation. Previous reports have suggested that ss and other invertebrate homologs of Ahr are activated by an endogenous ligand or that no ligand is required at all. Recent studies have shown that Ahr can accumulate in the nucleus upon activation by the second messenger cyclic AMP (cAMP), although it is not yet known whether cAMP signaling can activate ss in Drosophila. Thus, it is conceivable that ss is activated by different upstream factors in different cell types. It will be of interest to test in future studies whether in different neuronal cell types ss is activated by different ligands or upstream second messengers and whether ss acts in concert with regulatory programs for cell fate specification to dictate dendritic complexity (Kim, 2006).

    In the canonical Ahr signaling pathway, Ahr requires the appropriate cofactor for its proper function. Members of the bHLH-PAS protein family are able to heterodimerize with other bHLH-PAS proteins. Previous studies have shown that, upon ligand binding, Ahr is translocated to the nucleus, where it heterodimerizes with Arnt to form a transcriptionally active complex. However, tango, the Drosophila homolog of Arnt, is likely not required for the regulation of dendritic morphogenesis, indicating that ss is probably not acting through its canonical signaling pathway to specify dendritic complexity. In Sf9 cells, ss can act independently of tgo to enhance expression of a reporter in the absence of a ligand. Furthermore, Ahr is unable to interact with Arnt upon activation by cAMP. Although Ahr, Arnt, and the Arnt homolog Arnt2 are widely distributed throughout the rat brain, Ahr does not preferentially colocalize with either Arnt or Arnt2. Ahr is also expressed in specific regions of the rat brain where neither Arnt nor Arnt2 is expressed. These studies support the notion that ss can act independently of tgo in certain developmental contexts. Tgo can heterodimerize with other bHLH-PAS proteins in addition to ss. It is conceivable that ss may act with different heterodimerization partners to mediate its different functions in different cell types (Kim, 2006).

    Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning

    Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, an E2/E3 ubiquitinating enzyme mutant screen was conducted, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme encoding Effete, resulted in retention of C4da neuron dendrites during metamorphosis. Further, UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling (Kuo, 2006).

    The ubiquitin-proteasome system (UPS), evolutionarily conserved for the regulation of protein turnover, targets proteins for degradation via a complex, temporally regulated process that results in proteasome-mediated destruction of polyubiquitinated proteins. There are two distinct steps involved: first, the covalent conjugation of ubiquitin polypeptide to the protein substrates, and second, the destruction of tagged proteins in the proteasome complex. The transfer of ubiquitin to a target molecule slated for degradation involves at least three enzymatic modifications: ubiquitin is first activated by the ubiquitin-activating enzyme E1; ubiquitin is then transferred to a carrier protein, a ubiquitin-conjugating enzyme E2, and finally, ubiquitin is transferred to a protein substrate bound by a ubiquitin ligase E3. There are minor variations to this enzymatic cascade, but overall, these highly specific protein-protein interactions ensure ubiquitin targeting specificity and regulate many aspects of housekeeping protein turnover and cellular maintenance. However, with the multiple regulatory layers, different parts of this complex machinery can break down. Mutations in the UPS pathway causing accumulation of nondegraded proteins have been implicated in a variety of human diseases (Kuo, 2006).

    In the nervous system, aberrations in the UPS pathway have been implicated in disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. One of the common pathological features of neurodegenerative diseases, besides neuronal loss, is local axon degeneration. For example, in the case of Wallerian degeneration in vertebrates, distal parts of a severed axon remain viable and conduct action potentials in vivo for some time before a rapid dismantling of cytoskeletal proteins and axon degeneration, and the initiation of this rapid axon degeneration involves the UPS pathway. It is thought that UPS activation can lead to microtubule depolymerization and subsequent neurofilament degradation, possibly acting in conjunction with the Ca2+-dependent protease calpain. Moreover, inhibiting UPS activity in neurons prior to severing their axons can dramatically retard degradation of the severed axons. These results suggest that a cell-intrinsic UPS pathway regulates axon stability and that pharmaceutical inactivation of the UPS may prevent axonal degeneration in disease states (Kuo, 2006 and references therein).

    In Drosophila, the remodeling of neuronal processes during normal development closely resembles the pathological phenotypes in Wallerian degeneration. In the mushroom body γ neurons, extensive pruning of larval axons occurs during metamorphosis in a process regulated by glia engulfment and neuron-intrinsic UPS activity. Similarly, in the fly peripheral nervous system, the class IV dendritic arborization (C4da) neurons undergo complete pruning of their extensive larval dendrites during metamorphosis, in a process that is also regulated by UPS activity (Kuo, 2005). In both of these examples, severing of neuronal processes is preceded by microtubule depolymerization and followed by cytoplasmic blebbing and degeneration, all phenotypes resembling Wallerian degeneration. Therefore, these fly neurons represent excellent systems in which to understand the roles of the UPS in regulating neuronal axon/dendrite integrity, given the rather limited knowledge of how the UPS participates in the degradation of neuronal processes. It is not known which specific E2 ubiquitin-conjugating enzyme(s) and E3 ubiquitin ligase(s) are involved in UPS-mediated remodeling/degradation of neuronal processes, or their specific downstream target(s) (Kuo, 2006).

    It has been shown that mutations in the fly ubiquitin activation enzyme (uba1) and the proteasome complex (mov34) can prevent efficient pruning of C4da neuron larval dendrites during metamorphosis (Kuo, 2005). To further investigate the role of UPS in C4da neuron dendrite remodeling, a candidate gene screen was conducted to identify the E2 ubiquitin-conjugating enzyme and the E3 ubiquitin ligase required for this process. Analysis of genetic mutants showed that UPS activation in C4da neurons likely results in UbcD1 (an E2 ubiquitin-conjugating enzyme) mediated degradation of Drosophila inhibitor of apoptosis protein 1 (DIAP1), an E3 ligase that antagonizes caspase activity. Degradation of DIAP1 leads to activation of caspase Dronc, which results in local caspase activation and cleavage of proximal dendrites in C4da neurons during metamorphosis. In addition to the identification of a set of E2/E3 ubiquitinating enzymes for C4da neuron dendrite remodeling—with the surprising finding that the UPS mediates degradation of the potent protease inhibitor DIAP1—this study also establishes a mechanistic link between the UPS and caspase pathways in regulating C4da neuron dendrite pruning (Kuo, 2006).

    To identify the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase mediating dendrite pruning of C4da neurons during metamorphosis, a candidate gene approach was taken to systematically test the roles of known E2/E3 ubiquitinating enzymes in Drosophila. A set of putative E2/E3 ubiquitinating enzyme mutations was assembled, and live imaging was used to visualize C4da neurons carrying the E2/E3 mutation via the pickpocket(ppk)-EGFP marker, which specifically labels C4da neurons during Drosophila development. Those mutants with an early lethal phase were characterized by generating mosaic analysis with a repressible cell marker (MARCM) mutant neuronal clones. Since wild-type (wt) C4da neurons during metamorphosis do not retain any larval dendrites following head eversion, as imaged 18–20 hr after puparium formation (APF), mutations that caused larval dendrite retention in C4da neurons at this stage were sought. The candidate genes tested mostly showed no defects in dendrite pruning or neuronal cell death. However, one candidate, ubcD1, showed a modest level of larval dendrite retention at 18 hr APF (Kuo, 2006).

    Live imaging of wt C4da neuron MARCM clones at the start of pupariation (white pupae, WP) showed primary and secondary dendritic branching patterns typical of C4da neurons. Consistent with previous reports (Kuo, 2005; Williams, 2005), wt C4da neurons sever their larval dendrites during early metamorphosis and by 18 hr APF are devoid of any dendrites. The ubcD1 mutant C4da MARCM clones showed similar dendritic morphology to the wt clones at the onset of metamorphosis. However, at 18 hr APF, the mutant clones consistently retained intact, nonsevered larval dendrites. Thus, the UbcD1 E2 ubiquitin-conjugating enzyme is required for proper UPS-mediated dendrite pruning in C4da neurons during metamorphosis (Kuo, 2006).

    UbcD1, encoded by the gene effete, regulates UPS-mediated degradation of the antiapoptotic protein DIAP1 (Treier, 1992; Wang, 1999; Ryoo, 2002). In protecting cells from apoptosis, the DIAP1 E3 ubiquitin ligase antagonizes Dronc caspase activity by regulating ubiquination and degradation of the Dronc protein. Following apoptotic stimuli, UbcD1 mediates self-ubiquination and degradation of DIAP1, allowing for subsequent Dronc caspase activation. The biochemical and genetic interactions between these molecules are well established. The baculovirus p35, which is commonly used to inhibit caspase activity in Drosophila, and does not block C4da neuron dendrite pruning (Kuo, 2005). This may seem to make the involvement of caspases in this process unlikely; however, p35 has only limited activity against the caspase Dronc. To study the effects of dronc mutation on C4da neuron dendrite pruning, two null alleles of Dronc, dronc51 and dronc11, were used. MARCM analysis of dronc mutant clones revealed that the dendrites of mutant C4da neurons appeared normal at larval stages. However, unlike wt clones, without Dronc these neurons failed to properly prune their larval dendrites during metamorphosis, and most showed relatively intact primary and secondary larval dendritic arbors at 18 hr APF. These results show that severing of primary larval dendrites from C4da neurons during early metamorphosis requires the Dronc caspase (Kuo, 2006).

    During apoptosis, Dronc activation requires the degradation of the antiapoptotic/anticaspase protein DIAP1, which is downstream of UbcD1. The requirement of UbcD1 for C4da neuron larval dendrite pruning during metamorphosis, together with the finding that Dronc caspase activity is also essential, raised the question of whether UPS-mediated DIAP1 degradation is a key step that allows for the severing of larval dendrites. Because loss of DIAP1 function causes C4da neuron cell death prior to the onset of metamorphosis, this question was approached using a gain-of-function allele of diap1, diap16-3s, which has a single amino acid mutation that makes DIAP1 an inefficient substrate for UPS-mediated degradation. ppk-EGFP was crossed into the gain-of-function mutant background and live imaging was used to follow C4da neuron dendrite pruning during metamorphosis. The diap16-3s mutation did not significantly affect the ability of C4da neurons to elaborate larval dendrites. However, unlike wt C4da neurons that completely pruned their larval dendrites by 18 hr APF, C4da neurons in the diap16-3s gain-of-function mutants failed to efficiently sever larval dendrites at 18 hr APF. These results suggest that the degradation of DIAP1 during early metamorphosis is required for proper C4da neuron larval dendrite pruning. Quantitatively, mutations in the UPS pathway that modulate Dronc activity (diap16-3s and ubcD1) resulted in less severe dendrite pruning defects than dronc mutants, both in terms of total number of large dendrites attached to soma and in the length of the longest attached dendrite at 18 hr APF (Kuo, 2006).

    The UbcD1-DIAP1-Dronc pathway in apoptosis is well established. Thus, it may be necessary for C4da neurons to restrict the action of this pathway to specific cellular locations in order to prune unwanted dendrites without triggering apoptosis. To address this possibility, the subcellular distribution of DIAP1 and Dronc proteins was examined in ppk-EGFP C4da neurons. During the transition from third instar larvae to white pupae at the onset of metamorphosis, as well as 2 hr APF, there was a consistent induction of nuclear DIAP1 in GFP-labeled C4da neurons. During the same period a concurrent decrease was detected in Dronc staining in the soma of C4da neurons, unlike those from the neighboring cells at 2 hr APF. These results are consistent with previous observations that C4da neurons survive through this stage of metamorphosis. However, the level of antibody staining made it difficult to monitor the distribution of DIAP1 and Dronc within the dendritic structures of the C4da neurons. Because overexpression of Dronc caused C4da neuron to undergo apoptosis prior to metamorphosis, it was not possible to use GFP-tagged Dronc to examine its distribution in these neurons during pupariation. It was therefore necessary to search for alternative means to visualize activated Dronc or its downstream caspases (Kuo, 2006).

    An antibody generated against activated mammalian caspase 3 has been shown to be effective in recognizing activated caspases in Drosophila. Whereas this antibody reportedly recognizes the Drosophila effector caspase Drice, it may also cross react with other activated Drosophila caspases such as Dronc during tissue staining, because of similarities in the sequences of these caspases in the region corresponding to the peptide used to generate this antibody. Therefore this antibody was used to determine whether activated caspase is localized to the dendrites of C4da neurons during the initial severing event. At 4 hr APF, just prior to dendrite severing, antibody staining for activated caspase was consistently observed within the proximal larval dendrites of C4da neurons, especially within dendritic swellings. In the diap16-3s gain-of-function mutant that inhibits Dronc activity, as well as in ubcD1 and dronc mutant MARCM clones, C4da neurons did not show dendritic swellings or activated caspase staining in dendrites during early metamorphosis. Consistent with previous observation that C4da neurons do not remodel their axons during concurrent dendrite pruning, no activated caspase staining was seen within the axons of C4da neurons during dendrite severing. Since overexpression of p35 in these neurons did not block dendrite pruning (Kuo, 2005), it is believed this antibody staining likely recognizes activated Dronc directly or recognizes a p35-resistant caspase that is activated by Dronc. These results show that, concurrent with the nuclear upregulation of DIAP1 in C4da neurons that prevents apoptosis, there is a local activation of caspases in the dendrites, likely as a result of UPS-mediated degradation of DIAP1. The spatially restricted activation of caspases then allows the severing of proximal larval dendrites from the soma (Kuo, 2006).

    This study has shown that the UPS regulates pruning of larval dendrites from C4da neurons in a cell-intrinsic manner. To better understand the molecular pathways regulating UPS-mediated pruning, a candidate E2/E3 ubiquitinating enzyme screen was conducted. In this screen an E2 ubiquitin-conjugating enzyme mutation in was uncovered ubcD1, causing dendrite pruning defects. Taken together with the extensive biochemical characterization of interactions between UbcD1, DIAP1, and Dronc, this study suggests that in C4da neurons, UPS activation leads to UbcD1-mediated degradation of E3 ubiquitin ligase DIAP1, thereby allowing Dronc caspase activation and the subsequent cleavage of larval dendrites. This work not only identifies a set of E2/E3 ubiquitinating enzymes regulating neuronal process remodeling, it also links the UPS to a hitherto unappreciated mechanism for local caspase activation in dendrites during Drosophila metamorphosis (Kuo, 2006).

    The mechanistic link between the UPS and caspase activity in regulating C4da neuron dendrite pruning is unexpected. Although the UPS is known to regulate remodeling and degradation of neuronal processes, it is generally believed that this process is accomplished by degradation of cellular proteins (such as microtubules and neurofilaments) that are required to keep dendrites and axons intact. However, it was found that the UPS in C4da neurons is in fact causing the degradation of an E3 ligase, DIAP1, thereby allowing for subsequent dendrite pruning. In this case, UPS-mediated degradation of a protein does not in and of itself lead to a structural compromise in dendrites, but rather it leads to the activation of another protease that executes dendrite pruning. This two-step activation cascade, which involves both the UPS and the apoptotic machinery, may provide an additional level of control and flexibility that would not be possible if UPS alone regulated the pruning program. After all, these C4da neurons, which are specified during fly embryogenesis, maintain a highly elaborate dendritic field to receive sensory inputs throughout larval development, which lasts for several days. The maintenance of these dendrites over time requires a network of finely tuned cell-intrinsic and -extrinsic pathways. Just as important, the dendritic pruning program enables dramatic neuronal remodeling in response to profound environmental changes during metamorphosis. It is conceivable that C4da neurons evolved this dual control mechanism to prevent any accidental triggering of dendrite pruning prior to metamorphosis. Initiation of C4da neuron dendrite pruning requires cell-intrinsic ecdysone signaling, and ecdysone receptors have been shown to regulate Dronc expression. It will be of interest to determine how this UPS/caspase dendritic pruning pathway is related to the ecdysone signaling cascade (Kuo, 2006).

    During metamorphosis, C4da neurons upregulate DIAP1 expression in the nucleus, which is consistent with this class of neurons surviving early stages of the metamorphosis (only one of the three C4da neurons per hemisegment, the ventral neuron, is lost at a later stage of pupariation). Remarkably, there are activated caspases within the dendrites prior to severing, and a gain-of-function diap1 mutation can block dendrite pruning, strongly implicating a local dendritic program that can activate caspases without causing apoptosis of the neuron. Although mutations in both the Dronc caspase and the UPS pathway that modulate Dronc activity (UbcD1 and DIAP1) result in retention of larval dendrites, their dendrite pruning defects differed somewhat quantitatively. Compared to dronc mutants, diap1 gain-of-function and especially ubcD1 mutants showed less retention of larval dendrites during metamorphosis. This is not surprising for diap1 gain-of-function, as it is an effective Dronc inhibitor but unlikely to be 100% efficient. UbcD1, as an E2 ubiquitin-conjugating enzyme, has wider substrate specificity than E3 ligases. Previous study showed that UbcD1 is involved in mushroom body neuroblast proliferation, so it may be involved in other UPS-mediated pathways during dendrite pruning. It is also conceivable that in the absence of UbcD1 another E2 may trigger a low level of DIAP1 degradation, allowing residual Dronc activation which results in a milder dendrite pruning phenotype in ubcD1 mutants. It is currently unclear whether UbcD1 is also required during DIAP1-mediated degradation of Dronc. However, pruning defects in the ubcD1 mutants suggest that it may not be absolutely required, since undegraded DIAP1 continues to inhibit Dronc, presumably via interaction with another E2 protein (Kuo, 2006).

    How is the specificity of dendrite pruning achieved? Several possible mechanisms are proposed: first, C4da neurons do not change their axonal projections during dendrite pruning, so there could be dendrite-specific trafficking of components of the UPS, such as UbcD1, and/or the caspase Dronc. Of the known proteins that are preferentially trafficked to dendrites, these molecules have not been implicated but warrant further investigation. Second, it is also possible that activated Dronc, or another p35-resistant protease activated by Dronc, could cleave a dendrite-specific substrate. Examples are now emerging from other cellular systems, such as in sperm formation and border cell migration, in which caspases can participate in cleavage of proteins not resulting in apoptosis. Third, the dendritic pruning program takes place during drastic environmental changes that include concurrent degradation and regrowth of the overlying epidermis, activation of extracellular matrix metalloproteases, and blood phagocytes. These environmental cues likely complement the neuronal intrinsic pruning programs, but their exact relationships are not known. Experiments addressing these and other possible mechanisms should provide a greater insight into how the large-scale remodeling of C4da neuron dendrites is achieved (Kuo, 2006).

    In vertebrates, the UPS pathway has been implicated in Wallerian degeneration of severed axons. In the fly, mushroom body γ neurons undergo extensive remodeling of their processes during metamorphosis. The initial stages of axon pruning in these mushroom body neurons closely resemble Wallerian degeneration, and the UPS again plays a critical role. To date, the specific ubiquitin-conjugating enzymes and ligases that mediate target protein degradation have not been identified in these systems. It will be interesting to see whether the UbcD1-DIAP1-Dronc pathway implicated in C4da neuron dendrite pruning also participates in remodeling/degradation of neuronal processes in other systems. It seems likely that more than one pathway would be employed in remodeling different neurons; a previous study excluded UbcD1 as a possible ubiquitin-conjugating enzyme regulating mushroom body γ neuron remodeling, and normal remodeling of mushroom body neuron processes in is seen dronc mutant MARCM clones during metamorphosis (Kuo, 2006).

    A multilayered regulatory machinery for remodeling neurons, as uncovered in this study for C4da neurons, offers versatility and flexibility. It is conceivable that another ubiquitin ligase/caspase pair may function in an analogous UPS pathway during mushroom body neuron remodeling, potentially affording differential regulation of neuronal remodeling. Although pharmacological inhibition of mammalian caspases showed no effect on Wallerian degeneration, it would be important to assess the in vivo effectiveness of the inhibitors against a comprehensive panel of caspases. Moreover, a dual control mechanism, similar to what is proposed for C4da neuron remodeling, may coordinately regulate UPS and another protease that executes axon degradation. Conceivably, instead of having the target of the UPS directly involved in maintaining dendrite/axon stability, the executor of neuronal process degradation may involve a different protease: in the case of C4da neurons it is the caspase Dronc, and in Wallerian degeneration the relevant protease might be the Ca2+-responsive calpain. Future experiments along these lines of thinking may accelerate the identification of specific ubiquitinating enzymes involved in other areas of developmental neuronal remodeling and in diseases where the UPS pathway has been implicated. As target-specific E3 ligases are excellent candidates for pharmaceutical intervention, this approach may also help to find effective treatments for developmental and neurodegenerative diseases that involve degeneration of neuronal processes (Kuo, 2006).

    The tumour suppressor Hippo acts with the NDR kinases in dendritic tiling and maintenance

    Precise patterning of dendritic fields is essential for neuronal circuit formation and function, but how neurons establish and maintain their dendritic fields during development is poorly understood. In Drosophila class IV dendritic arborization neurons, dendritic tiling, which allows for the complete but non-overlapping coverage of the dendritic fields, is established through a 'like-repels-like' behaviour of dendrites mediated by Tricornered (Trc), one of two NDR (nuclear Dbf2-related) family kinases in Drosophila. The other NDR family kinase, the tumour suppressor Warts/Lats (Wts), regulates the maintenance of dendrites; in wts mutants, dendrites initially tile the body wall normally, but progressively lose branches at later larval stages, whereas the axon shows no obvious defects. Biochemical and genetic evidence is provided for the tumour suppressor kinase Hippo (Hpo) as an upstream regulator of Wts and Trc for dendrite maintenance and tiling, respectively, thereby revealing important functions of tumour suppressor genes of the Hpo signalling pathway in dendrite morphogenesis (Emoto, 2006).

    Dendritic arborization patterns are critical to a neuron's ability to receive and process impinging signals. Whereas neurons normally maintain the gross morphology of their dendrites, cortical neurons of Down's syndrome patients gradually lose dendritic branches after initially forming normal dendritic fields. Thus, neurons appear to have separate mechanisms for establishment and maintenance of their dendritic fields (Emoto, 2006).

    Dendritic tiling is an evolutionarily conserved mechanism for neurons of the same type to ensure complete but non-redundant coverage of dendritic fields. In the mammalian visual system, for instance, dendrites of each retinal ganglion cell type cover the entire retina with little overlap, like tiles on a floor. In Drosophila, the dendritic arborization sensory neurons can be divided into four classes (I-IV) based on their dendrite morphology, and the dendritic field of class IV dendritic arborization neurons is shaped, in part, through a like-repels-like tiling behaviour of dendrite terminals. The NDR family kinase Trc and its activator Furry (Fry) has been identified as essential regulators of dendritic tiling and branching of class IV dendritic arborization neurons. These proteins are evolutionarily conserved and probably serve similar functions in neurons of different organisms (Emoto, 2006).

    In addition to Trc, Drosophila has one other NDR family kinase, Wts, which is a tumour suppressor protein that functions in the coordination of cell proliferation and cell death in flies. To uncover the cell-autonomous functions of Wts in neurons, MARCM (mosaic analysis with a repressive cell marker) was ised to generate mCD8-GFP-labelled wts clones in a heterozygous background. Wild-type class IV neurons elaborate highly branched dendrites that cover essentially the entire body wall. Compared to wild-type ddaC (dorsal dendrite arborization neuron C) neurons, wts clones showed a severe and highly penetrant simplification of dendritic trees, with significantly reduced number (wild type, 575.1; wts, 255.6) and length (wild type, 1,457.0; wts, 590.4) of dendritic branches, and hence a greatly reduced dendritic field (Emoto, 2006).

    In contrast to the severe dendritic defects caused by loss of Wts function, wts mutant ddaC axons entered the ventral nerve cord at the appropriate position and showed arborization patterns very similar to wild-type controls, with their axons terminating on the innermost fascicle and sending ipsilateral branches anteriorly and posteriorly and sometimes also a collateral branch towards the midline. Thus, Wts seems to have a crucial role in dendrite-specific morphogenesis in post-mitotic neurons (Emoto, 2006).

    In proliferating cells, Wts is part of a signalling complex for tumour suppression that includes the adaptor protein Salvador (Sav) and the serine/threonine kinase Hpo. sav mutant ddaC MARCM clones were examined and dendritic defects were observed similar to wts MARCM clones. In severely affected clones (3 of 15 clones), most of the high-order branches were missing, whereas moderately affected clones (12 of 15 clones) exhibited a partial loss of their fine branches and major branches (Emoto, 2006).

    To confirm that Wts and Sav function in the same pathway, genetic interaction between wts and sav in regulating dendrite morphogenesis was tested. Whereas heterozygous wts or sav mutants had no obvious dendritic phenotype, trans-heterozygous combinations of wts and sav alleles resulted in simplified dendrites similar to moderately affected sav clones. Furthermore, sav wts double mutant clones showed a severe dendrite defect comparable to wts mutant clones. Thus, Wts and Sav most probably function together in class IV neurons to regulate dendrite morphogenesis (Emoto, 2006).

    The dendritic phenotypes of wts mutants and sav mutants might result from defects in branch formation and/or elongation, or loss of normally formed dendrites. Therefore ddaC dendrites were examined at different time points of larval development using the pickpocket-EGFP reporter, which is specifically expressed in class IV dendritic arborization neurons. Wild-type ddaC neurons elaborated primary and secondary dendritic branches by 24-28 h after egg laying, but large regions of the body wall were not yet covered by dendrites. By 48-52 h after egg laying, the major branches reached the dorsal midline, and the open spaces between major branches were filled with fine branches, resulting in complete dendritic coverage of the body wall. This tiling of dendrites persisted throughout the rest of larval development. In wts and sav mutants, ddaC dendrites were indistinguishable from those of wild-type controls at 24-28 h after egg laying. By 48-52 h after egg laying, wts and sav dendrites tiled the body wall as in wild type. During the next 24 h, however, dendrites of wts and sav mutants no longer tiled the body wall. Therefore, wts and sav seem to be required for maintenance of the already established tiling of dendrites (Emoto, 2006).

    The loss of dendrites was further documented in live mutant larvae imaged for 30 h starting in early second instar larvae (48-50 h after egg laying). In wild-type larvae, ddaC dendrites grew steadily; the number of terminal branches increased by 23.0 over this time period. By contrast, dendrites of wts and sav mutants gradually lost their fine branches (decrease of 27.5 and 31.5, respectively) as well as some of the major branches by 78-80 h after egg laying (Emoto, 2006).

    Class-IV-neuron-specific expression of wts and sav largely rescued the dendritic phenotype of wts and sav mutants, respectively, confirming that Wts and Sav act cell autonomously in class IV neurons. Furthermore, no detectable defect in patterning of the epidermis (anti-Armadillo antibody) or muscle (Tropomyosin::GFP reporter) was observed in wts or sav mutant third instar larvae. Taken together, these results indicate that the Wts/Sav signalling pathway functions in class IV neurons to maintain dendritic arborizations (Emoto, 2006).

    Wts kinase activity is regulated, at least in part, by the Ste20-like serine/threonine kinase Hpo. Indeed, ddaC clones mutant for hpo exhibited simplified dendritic trees in third instar larvae, similar to wts or sav mutant clones, but showed more extensive dendritic arborizations in earlier larval stages (second to early third instar), consistent with the involvement of Hpo in the maintenance of dendrites. Notably, in hpo mutant clones at earlier developmental stages, dendritic branches were often found to overlap. Both the dendritic tiling and maintenance phenotypes were rescued by hpo expression in MARCM clones, consistent with the cell-autonomous function of Hpo in class IV neurons. Because this tiling defect in hpo mutant clones was similar to the tiling defects of trc mutant clones, whether hpo could genetically interact with trc to regulate dendritic tiling was tested. Compared with wild-type controls, trans-heterozygous combinations of trc and hpo exhibited obvious iso-neuronal as well as hetero-neuronal tiling defects, whereas wts and hpo trans-heterozygotes displayed simplified dendrites similar to wts mutants. These dendritic defects were consistently observed in multiple allelic combinations between hpo and trc or wts. In contrast, trans-heterozygous combinations of trc and wts showed no significant dendritic phenotypes. Furthermore, overexpression of wild-type Trc, but not Wts, in hpo MARCM clones partially rescued the dendritic tiling defects in class IV neurons. Thus, Hpo acts through Trc and Wts to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

    Not only did Hpo interact genetically with Trc and Wts, its physical association with these NDR kinases could be detected in vivo. When Flag-tagged Trc was expressed using a nervous-system-specific Gal4 driver, anti-Flag antibodies immunoprecipitated Trc together with Hpo. Similarly, Myc-tagged Wts co-immunoprecipitated with Hpo expressed in embryonic nervous systems. Hpo co-immunoprecipitation appeared to be specific, because Misshapen, another Ste20-like kinase protein present in neurons, was not co-immunoprecipitated by anti-Flag or anti-Myc antibodies in similar experiments. These results suggest that Hpo associates with Trc and Wts in the Drosophila nervous system (Emoto, 2006).

    To examine further the physical interaction between Trc and Hpo, analogous experiments were carried out in Drosophila S2 cells co-transfected with a haemagglutinin (HA)-tagged Trc construct and a Flag-tagged Hpo construct containing the full open reading frame, an amino-terminal fragment containing the kinase domain, or a carboxy-terminal fragment containing the regulatory domain. Full-length Hpo and the C-terminal portion of Hpo, but not the N-terminal fragment, were co-immunoprecipitated with Trc, suggesting that the C-terminal domain of Hpo is sufficient for Trc-Hpo complex formation (Emoto, 2006).

    Hpo physically interacts with Wts and promotes Wts phosphorylation at multiple serine/threonine sites, including two sites, S920 and T1083 of Drosophila Wts, that appear to be necessary for Wts kinase activation. Indeed, Wts protein with mutations in the S920 and T1083 residues was unable to rescue the wts mutant dendritic phenotypes. Given that the corresponding phosphorylation sites in Trc are critical for Trc activation as well as control of dendritic tiling and branching, it was of interest to know whether Hpo may promote Trc phosphorylation at the critical serine and/or threonine residue. Wild-type Hpo, but not catalytically inactive Hpo or the Misshapen kinase, led to substantial incorporation of 32P-labelled phosphate into recombinant Trc or Trc with a mutation at the S292 site (S292A), but not the T449A mutant form of Trc. Analogous results were obtained with Wts. These results support a model in which Hpo associates with and phosphorylates Trc and Wts at a critical threonine residue to regulate dendritic tiling and maintenance, respectively (Emoto, 2006).

    Both genetic and biochemical evidence reveals that Hpo regulates complementary aspects of dendrite development through two distinct downstream signalling pathways: the Trc kinase pathway for tiling and the Wts kinase pathway for maintenance. These studies of class IV dendritic arborization neurons, together with the recent report that Wts signalling is required for cell fate specification of photoreceptor cells in Drosophila retina, demonstrate that the Wts signalling pathway is important for post-mitotic neurons. In proliferating cells, Wts phosphorylates Yorkie (Yki), a transcriptional co-activator, to regulate cell cycle and apoptosis in growing cells. However, Yki is dispensable for Hpo/Wts-mediated dendrite maintenance. Hpo probably functions as an upstream kinase for Trc, as well as Wts, in neurons by phosphorylating a functionally essential threonine, which may also be regulated by MST3, a Ste20-like kinase closely related to Hpo. Given the evolutionary conservation of known components in the Trc and Wts signalling pathways, it will be important to identify their relevant downstream targets and explore mechanisms that coordinate the establishment and maintenance of dendritic fields, and to determine the role of Trc and Wts signalling in the mammalian nervous system (Emoto, 2006).

    The target of rapamycin complex 2 controls dendritic tiling of Drosophila sensory neurons through the Tricornered kinase signalling pathway

    To cover the receptive field completely and non-redundantly, neurons of certain functional groups arrange tiling of their dendrites. In Drosophila class IV dendrite arborization (da) neurons, the NDR family kinase Tricornered (Trc) is required for homotypic repulsion of dendrites that facilitates dendritic tiling. This study reports that Sin1, Rictor, and target of rapamycin (TOR), components of the TOR complex 2 (TORC2), are required for dendritic tiling of class IV da neurons. Similar to trc mutants, dendrites of sin1 and rictor mutants show inappropriate overlap of the dendritic fields. TORC2 components physically and genetically interact with Trc, consistent with a shared role in regulating dendritic tiling. Moreover, TORC2 is essential for Trc phosphorylation on a residue that is critical for Trc activity in vivo and in vitro. Remarkably, neuronal expression of a dominant active form of Trc rescues the tiling defects in sin1 and rictor mutants. These findings suggest that TORC2 likely acts together with the Trc signalling pathway to regulate the dendritic tiling of class IV da neurons, and thus uncover the first neuronal function of TORC2 in vivo (Koike-Kumagai, 2009).

    The microRNA bantam functions in epithelial cells to regulate scaling growth of dendrite arbors in Drosophila sensory neurons

    In addition to establishing dendritic coverage of the receptive field, neurons need to adjust their dendritic arbors to match changes of the receptive field. This study shows that dendrite arborization (da) sensory neurons establish dendritic coverage of the body wall early in Drosophila larval development and then grow in precise proportion to their substrate, the underlying body wall epithelium, as the larva more than triples in length. This phenomenon, referred to as scaling growth of dendrites, requires the function of the microRNA (miRNA) bantam (ban) in the epithelial cells rather than the da neurons themselves. ban in epithelial cells dampens Akt kinase activity in adjacent neurons to influence dendrite growth. This signaling between epithelial cells and neurons receiving sensory input from the body wall synchronizes their growth to ensure proper dendritic coverage of the receptive field (Parrish, 2009).

    Dendrites of class IV da neurons completely and nonredundantly cover the larval body wall early in larval development, a phenomenon referred to as dendritic tiling. Once field coverage is established, dendrites continue to branch and lengthen to maintain tiling as larvae grow, providing a sensitive system for analysis of how neurons first establish and later maintain coverage of the receptive field. This study addressed the question of how late-stage dendrite growth is precisely coordinated with larval growth to maintain proper dendrite coverage of the body wall (Parrish, 2009).

    To examine this process, the pickpocket-EGFP (ppk-EGFP) marker was used to monitor class IV dendrite growth before and after establishment of tiling. To quantitatively assess dendrite coverage, a metric was used that is referred to as the coverage index, the ratio of the territory covered by dendrites of a given da neuron, such as the class IV neuron ddaC, to the area of a hemisegment that harbors the da neurons. Dendrite outgrowth of class IV neurons begins at ~16 hr After Egg Laying (AEL), with class IV dendrites growing rapidly during late embryonic/early larval stages to tile the body wall between 40 and 48 hr AEL and subsequently maintaining this coverage until dendrites are pruned during metamorphosis. Between 48 hr AEL and 120 hr AEL (just prior to metamorphosis), larvae grow nearly 3-fold in length and the dorsal area of class IV receptive fields expands by more than 6-fold. Therefore, class IV dendrites grow extensively and this dendrite growth must be precisely coordinated with larval growth in order to maintain proper coverage of the receptive field (Parrish, 2009).

    Class IV dendrites are located between muscle and epithelial cells. Cell divisions that give rise to larval cells are complete by mid-embryogenesis, and larval growth is achieved by increasing cell size rather than additional proliferation. Thus, all the cells that will comprise the larval body wall musculature and epithelia are in place when dendrite outgrowth begins. To simultaneously visualize growth of class IV dendrites and epithelial cells, a protein trap line was used that directs GFP expression in epithelial cells and outlines their borders (Armadillo::GFP, adherens junctions, or Neuroglian::GFP, septate junctions) in combination with ppk-GAL4 driving expression of mCD8-RFP in class IV neurons. Using these markers, growth of class IV dendrites and epithelial cells was monitored throughout embryonic/larval stages (Parrish, 2009).

    Epithelial cells grow at a nearly constant rate over the time course. Likewise, the class IV neuron soma grows at a relatively constant rate. In contrast, the dendrite growth is biphasic. Initially, class IV dendrite growth outpaces growth of epithelial cells and the larva as a whole between 16 hr and 48 hr AEL, the timeframe in which class IV dendrites establish tiling. Dendrite growth slows as class IV dendrite arbors achieve complete body wall coverage, and from 48 hr to 120 hr AEL class IV dendrites grow in proportion to larval growth at a rate comparable to that of epithelial cells. This late dendrite growth will be referred to as scaling growth of dendrites (a phenomenon unrelated to synaptic scaling) to reflect the physical scaling of dendrite arbors as they grow precisely in proportion to surrounding cells and the larva as a whole in order to maintain proper coverage of the receptive field (Parrish, 2009).

    To determine whether scaling growth is a general property of da neurons, dendrite growth was monitored in class I and class III da neurons, two additional morphologically distinct classes of da neurons, using the coverage index metric introduced above. Like class IV neurons, dendrites of class I and III neurons rapidly establish coverage of a characteristic region of the body wall and subsequently maintain their coverage by expanding their dendrite arbors in precise proportion to larval growth. Class III neurons cover their territory in the same timeframe as class IV neurons, first establishing receptive field coverage at about 48 hr AEL. In contrast, class I neurons covered their characteristic territory by 24 hr AEL. Thus, temporally distinct signals may regulate scaling of dendrite growth in class I and class III/IV neurons. Nevertheless, scaling growth of dendrites seems to be a general feature of da neuron development (Parrish, 2009).

    Based on the fidelity of dendrite coverage in class IV neurons, a focus was placed on these neurons for studies of dendrite scaling. The finding that class IV dendrites have a rapid growth phase during establishment of tiling and a scaling phase with slower dendrite growth to maintain tiling suggests that some signal(s) attenuate dendrite growth following establishment of tiling, synchronizing growth of class IV dendritic arbors with growth of surrounding tissue. Attempts were therefore made to characterize the signaling that underlies dendrite scaling (Parrish, 2009).

    To test the capacity of dendrite scaling, the effects were examined of mutations that alter the dimensions of larvae at different developmental states on class IV dendrite growth. Alleles were chosen that survive until at least the second larval instar, allowing monitoring of dendrite coverage by class IV neurons at a time when they should have already established tiling. Overall, 35 mutant alleles were screened that cause a range of defects in larval size, shape, and growth rate. Notably, class IV dendrites properly covered the receptive field in nearly all of these mutants, accommodating a broad range of receptive field areas (ranging from 10% of wild-type [WT] in chico mutants to 120% of WT in giant [gt] mutants) and shapes. Dendrites also scaled properly in mutants defective in developmental rate, for example maintaining proper receptive field coverage in b6-22 mutants that develop slowly and persist as second instar larvae or in broad (br) mutants that persist as third instar larvae for days or even weeks. Taken together, these results demonstrate the robustness of dendrite scaling growth in class IV neurons (Parrish, 2009).

    Among the few mutants that had any effect on scaling growth of dendrites, the ban mutant had the most severe dendrite overgrowth phenotype observed, with the first sign of larval growth defects at 72 hr AEL. It was reasoned that ban might be required for dendrite scaling but not earlier aspects of dendrite development, and the remainder of the study focused on the role of ban in dendrite scaling. Notably, ban encodes a miRNA and might represent a regulatory node for scaling of dendrite growth since miRNAs likely regulate expression of 100 or more target genes (Parrish, 2009).

    Dendrites of individual class IV neurons occupy a larger proportion of the body wall in ban mutant third instar larvae. At 96 hr AEL, ddaC class IV neurons in ban mutants have a mean coverage index of 1.22, meaning that the receptive field of the average ddaC dendrite in ban mutant larvae is 122% of the size of the dorsal hemisegment that harbors the neuron. Thus, dendrites in ban mutants promiscuously cross boundaries that are observed by dendrites of WT neurons. For example, fewer than two dendrite branches cross the midline for a given WT class IV neuron, whereas more than 18 dendrite branches cross the midline in ban mutants. The exuberant growth of dendrites in ban mutants is manifest throughout the arbor, not just at the boundaries. However, although a coverage index of >1 is seen for ban mutant, no significant tiling defect is seen because branches that cross normal boundaries still avoid dendrites of neighboring class IV neurons. In addition to these defects in dendrite coverage, class IV neurons in ban mutants show significant increases in the number of dendrites, the density of dendrites, and overall dendrite length (data not shown). However, increased terminal dendrite branching is not sufficient to increase receptive field coverage. Several other mutants have been described that increase terminal dendrite branching in class IV neurons, and none of these mutants cause an overall increase in the size of the dendritic field. For example, furry (fry) mutations cause a 100% increase in the number/density of terminal dendrites without an accompanying increase in coverage index at 96 hr AEL. Likewise, overexpression of the small GTPase Rac drastically increases terminal dendrite branching but reduces receptive field coverage (Parrish, 2009).

    The dendrite growth defects in ban mutants could reflect increased dendrite growth from early stages of development or defects specific to the scaling phase of dendrite growth. To distinguish between these possibilities, dendrite growth was monitored over a developmental time course, focusing on the coverage index and midline crossing events as metrics for growth of the dendrite arbor as a whole. Importantly, class IV dendrites in ban mutants are indistinguishable from WT during the early, rapid growth phase (through 48 hr AEL) as measured by coverage index, midline crossing events, and total dendrite branch number. However, beginning at 72 hr AEL, progressively more severe defects are noted in the coverage index and a greater number of midline crossing events in ban mutants. This late-onset exuberant dendrite growth demonstrates that ban is not causing a general growth defect since ban is dispensable for establishment of dendrite coverage. Whereas a generalized defect in dendrite growth, as seen in dendritic arbor reduction (dar; mutations that lead to defective dendritic arbors but normal axonal projections), would affect both the early (isometric) and late (scaling) phases of growth, mutations that specifically affect the scaling growth of dendrites would be dispensable for the early, rapid growth of dendritic fields. This is precisely what is seen for ban mutants. Therefore, ban is specifically required for scaling of dendrite arbors, potentially by affecting growth-inhibitory signals that normally restrict dendrite growth (Parrish, 2009).

    To confirm that loss of ban causes these phenotypes, the following experiments were conducted. First, whereas heterozygosity for a ban null allele or deficiencies that span the ban locus show no obvious defects in dendrite scaling, placing ban mutations in trans to a deficiency that spans the locus, but not a nearby deficiency that does not span the ban locus, recapitulates the dendrite defects described above. Second, the ban mutant dendrite defects can be fully rescued by a ban genomic rescue transgene but not a genomic transgene in which the ban locus has been deleted. Therefore, disrupting ban function is sufficient to cause defects in scaling growth of dendrites (Parrish, 2009).

    Next whether ban is required for scaling growth of dendrites was tested in other classes of da neurons. Both class I and class III neurons establish proper dendrite coverage in ban mutants. However, class III dendrites are defective in scaling of dendrite growth in ban mutants, showing a significant increase in dendrite coverage after 48 hr AEL. In contrast, larval class I dendrites show no obvious defects in dendrite coverage in ban mutants, demonstrating that ban is not required for scaling in class I neurons. The onset of scaling growth of dendrites differs by 24 hr in class I and class III/IV neurons, thus different scaling signals may operate at the two time points with ban required for the scaling growth signal for class III/IV neurons that tile (Parrish, 2009).

    Next, time-lapse microscopy of single neurons was conducted to characterize the cellular basis of the ban mutant phenotype. Single class IV neurons were imaged from time-matched WT or ban mutant larvae at 24 hr intervals beginning at 72 hr AEL, just after the ban phenotype is first apparent. Dynamics were monitored of every terminal dendrite that could be unambiguously followed through the time course and dendrite growth, initiation of new dendrites, dendrite retraction, and branch loss were measured. For each of these categories, ban mutants differed from WT controls, exhibiting significantly more dendrite growth and branch initiation and significantly less dendrite retraction and branch loss. Therefore, stabilization of existing dendrites, increased dendrite growth, and increased addition of new dendrites all contribute to the defect in dendrite scaling growth of the ban mutant (Parrish, 2009).

    Time-lapse studies suggest that signals normally restricting dendrite growth are largely absent in ban mutants. Attempts were made to verify this hypothesis using laser ablation assays. Previous studies showed that, following embryonic ablation of a class IV neuron, dendrites of neighboring neurons grow exuberantly to invade the unoccupied territory of the ablated neuron, with the ability of dendrites to invade unoccupied territory progressively restricted in older larvae. It was therefore important to determine whether the timing of this restricted growth potential correlates with the onset of scaling of dendrite growth and whether ban is required for restriction of the dendrite growth potential (Parrish, 2009).

    Consistent with prior reports, ablating a class IV neuron at 24 hr AEL led to extensive invasion by dendrites of neighboring neurons, with 55% of the unoccupied territory covered by neighboring neurons 48 hr postablation. This ability of dendrites to grow into unoccupied territory was severely attenuated 1 day later, with dendrites of neighboring neurons invading only 23% of the unoccupied territory after ablation of a class IV neuron at 48 hr AEL . The extent of invasion was even further reduced when neurons were ablated at 72 hr AEL. Therefore, the ability of dendrites to grow beyond their normal boundaries to invade unoccupied territory is severely restricted during larval development at a time coincident with the onset of scaling of dendrite growth (Parrish, 2009).

    If the restriction of dendrite growth potential in larvae is caused by scaling signals that limit dendrites to growth in proportion to body wall growth, the majority of invading activity by neighboring dendrites should be present before scaling growth ensues at 48 hr AEL. To test this prediction, class IV neurons were ablated at 24 hr AEL and invasion activity was monitored at 24 hr intervals over the next 72 hr. By 48 hr AEL, dendrites of neighboring neurons had invaded unoccupied territory, and the extent of invasion was not noticeably increased at later time points. Instead, the entire dendrite arbor of class IV neurons, including the portion that invaded unoccupied territory, scaled with larval growth after 48 hr AEL. Thus, the receptive field that is established by 48 hr AEL is maintained by scaling of dendrite growth, even in cases in which dendrites establish aberrant body wall coverage. The signals responsible for dendrite scaling growth are likely distinct from the homotypic repulsion required to establish tiling as ablation of all neighboring same-type neurons does not potentiate the ability of a class IV neuron to invade unoccupied territory. Additionally, dendrites of class I da neurons, which do not rely on homotypic repulsion to establish their coverage, also exhibit scaling growth (Parrish, 2009).

    As described above, dendrite coverage is properly established in ban mutants. Importantly, unlike WT controls, following ablation at 48 hr AEL, dendrites in ban mutants extensively fill unoccupied space, with dendrites in ban mutants invading unoccupied territory just as efficiently as dendrites in WT controls ablated at 24 hr AEL. Therefore, the receptive field boundaries of class IV neurons have not been fixed in ban mutants at 48 hr AEL. Dendrites in ban mutants invade unoccupied territory more efficiently than WT controls at later time points as well. Thus, either the growth-inhibitory scaling signal is lost or dendrites are refractory to the signal in ban mutants (Parrish, 2009).

    To test whether machineries for dendritic tiling contribute to the progressive reduction of a dendrite's ability to invade vacant territories, mutations of fry, which encodes a gene required for establishment of dendritic tiling and of extra sex combs (esc) and salvador (sav), which function in a common pathway to regulate stability of terminal dendrites and, consequently, maintenance of dendrite coverage, were examined for effects on dendrite invasion following neuron ablation. Unlike mutations in ban, mutations in fry, esc, or sav had no effect on the ability of dendrites to invade unoccupied territory. Moreover, consistent with the scaling signal functioning in a distinct pathway, double-mutant combinations of ban with fry or esc showed additive phenotypes. Thus, ban exerts its effects on scaling of dendrite growth independently of known pathways for establishment and maintenance of dendrite coverage (Parrish, 2009).

    To further characterize the signaling required for scaling growth of dendrites, it was of interest to determine where ban functions to regulate scaling. First, whether ban is expressed in neurons, surrounding cells, or both, was examined by using a miRNA activity sensor as a reporter for ban expression in third instar larvae. A control sensor directs ubiquitous expression of GFP, including robust GFP expression in muscle, epithelial cells, and sensory neurons. The ban sensor contains two ban binding sites in the 3'UTR of the transgene, hence GFP expression is attenuated in cells that express ban. Unlike the control sensor, very little, if any, GFP expression was detected in third instar muscle cells, epithelial cells, or sensory neurons using several independent transgenic fly lines with distinct insertions of the ban sensor. Significant attenuation of the ban sensor was first observ ed in larval muscle, epithelium, and PNS neurons between 48 and 72 hr AEL, precisely at the time when dendrite defects were first observed in ban mutants, suggesting that ban activity is more pronounced during this period than at earlier time points. Notably, the attenuation of the ban sensor was dependent on ban activity, as shown by the persistent, ubiquitous expression of the sensor in ban mutant larvae. Thus, ban is likely expressed in the muscle, epithelium, and PNS neurons and may be required in any of these cell types for scaling of dendrite growth (Parrish, 2009).

    To determine whether ban is required cell-autonomously for dendrite scaling, MARCM was used to generate single neuron clones homozygous for a ban mutation in a heterozygous background. ban activity was effectively dampened in MARCM clones, as indicated by derepression of the ban sensor in the clones. However, loss of ban function had no significant effect on dendrite coverage of class IV neurons. Time-lapse analysis of ban mutant class IV MARCM clones revealed no defects in dendrite coverage at any time during larval development. Furthermore, ban is dispensable in other da neurons for dendrite scaling growth. Thus, ban function in sensory neurons is dispensable for scaling growth of dendrites (Parrish, 2009).

    Although scaling of dendrite growth proceeds normally, there is some reduction of overall dendrite length and the number of dendrite branches in ban mutant class IV clones. Therefore, ban likely acts cell-autonomously to promote dendrite growth and nonautonomously to limit dendrite Taking advantage and ensure proper scaling (Parrish, 2009).

    A genetic rescue assay was used to test the ability of transgenic expression of ban in different tissues to rescue the dendrite growth defects of ban mutants. Consistent with MARCM results, neuronal expression of ban, using either panneuronal or PNS-specific Gal4 drivers, was not sufficient to rescue the scaling growth defect of ban mutants. Thus, ban likely functions nonautonomously in nonneuronal cells to regulate scaling of da neuron dendrite growth. Moreover, expression of ban in muscle alone could not ameliorate the dendrite defects of ban mutants. Remarkably, every time ban expression was rescued in epithelial cells, significant suppression of the exuberant dendrite growth of ban mutants was found. The three epithelial Gal4 driver lines caused reductions of dendrite growth that correlated with Gal4 expression levels in epithelial cells: arm-Gal4 caused the greatest reduction in dendrite growth and had the strongest epithelial expression, whereas twi-Gal4 displayed the lowest activity and drove epithelial Gal4 expression at the lowest level. Taking advantage of the temperature-sensitive nature of Gal4 activity, rescue activity of each epithelial Gal4 line was monitored over a graded temperature series (18°C to 29°C) and it was found that, for each driver, rescue activity was directly proportional to expression level. Therefore, epithelial ban expression is sufficient to suppress the exuberant dendrite growth of ban mutants, and the extent of dendrite growth inhibition varies with the level of ban expression in epithelial cells (Parrish, 2009).

    Epithelial expression of twi-Gal4 was first apparent in larval stages, suggesting that postembryonic expression of ban in epithelia is sufficient for proper scaling of dendrite growth. Given that dendrite defects in ban mutants first appear after 48 hr AEL, it was asked whether late expression of ban would suffice for dendrite scaling. To examine the temporal requirement for ban function, a heat-shock-inducible Gal4 driver was used to express ban during larval development. Indeed, inducing ban expression at 48 hr AEL was sufficient to rescue the dendrite defects of ban mutants. These findings reinforce the notion that ban is dispensable for early aspects of dendrite development (Parrish, 2009).

    Resupplying ban in tissues known to regulate larval growth, such as the fat body, prothoracic (PTTH) gland, or insulin-producing cells (IPCs), had no measurable effect on dendrite growth in ban mutants. Moreover, ablation of each of these tissues mediated by a reaper transgene caused larval growth defects without obvious dendrite growth defects. Thus, ban function in the fat body, PTTH gland, or IPCs is not sufficient to modulate scaling of dendrite growth. Altogether, these results suggest that epithelial cells are likely the major functional sites for ban in regulation of PNS dendrite scaling (Parrish, 2009).

    Because ban expression in epithelial cells affects scaling growth of dendrites in a dose-dependent fashion via a mechanism that likely involves growth-inhibitory signals, it was of interest to see whether ectopic epithelial expression of ban in a WT background could further inhibit dendrite growth and thus disrupt scaling of dendrite arbors. Indeed, overexpression of ban in epithelial cells resulted in a severe reduction in dendrite growth and induced striking defects in the pattern of dendrite growth over epithelial cells, with terminal dendrites appearing to wrap around epithelial cells. Consistent with ban dosage in epithelial cells regulating the strength of dendrite growth-inhibitory signals, epithelial overexpression of ban induced more robust inhibition of dendrite growth at higher temperatures (which lead to higher levels of transgene expression) (Parrish, 2009).

    Since ban expression in epithelial cells is sufficient to ensure proper scaling, it was of interest to address whether ban function in epithelial cells is necessary for scaling of dendrite growth. To this end, MARCM was used to generate ban mutant epithelial cell clones. Although it was not possible to address the contribution of epithelial ban to scaling of the entire dendrite arbor using this approach (it was only possible to generate one to four cell epithelial clones), the pattern was monitored of dendrite growth over ban mutant or WT control epithelial clones. Class IV dendrites grow extensively over epithelial cells, with multiple dendrite branches often coursing over a single epithelial cell. The epithelial nucleus was used as a landmark and dendrite growth was monitored over the epithelial cell surface shadowed by the nucleus. Although the gross morphology of epithelial cells was not obviously affected in ban mutant clones, the propensity of class IV dendrites to grow into the region shadowed by the epithelial nucleus was significantly increased for ban mutant epithelial clones when compared to WT controls or ban heterozygous epithelial cells. Therefore, ban is required in epithelial cells to ensure proper dendrite growth and placement over epithelial cells (Parrish, 2009).

    To gain insight into the molecular mechanism underlying dendrite scaling, a platform was developed for microarray-based expression profiling of dissociated, FACS-isolated PNS neurons or epithelial cells. Akt and numerous other candidate genes were identified that were deregulated in PNS neurons and/or epithelial cells of ban mutant larvae. Because Akt is a well-established regulator of growth, including dendrite growth in mammalian hippocampal neurons, whether ban regulates Akt as part of the scaling program was investigated (Parrish, 2009).

    Microarray experiments found that Akt expression was increased in neurons but reduced in epithelial cells of ban mutants relative to WT controls. By monitoring Akt levels in lysates of larval fillets composed mostly of muscle and epithelial cells, it was found that in the absence of ban function Akt protein levels were substantially reduced. Furthermore, Akt activity was substantially reduced as shown by reductions in active, phosphorylated Akt and phosphorylated S6K, a downstream reporter of Akt activity. Therefore, Akt expression and activity are substantially reduced in ban mutant larval lysates, likely reflecting reduced Akt function in muscle/epithelia (Parrish, 2009).

    Next larval fillets were immunostained to determine whether ban influences Akt protein levels in the PNS. In WT controls, Akt is detectible only at low levels in the soma or dendrites of the PNS. By contrast, in ban mutants Akt is highly expressed in the PNS and is detectible in axons, the soma, and dendrites. Similarly, phosphorylated Akt is barely detectible in the larval PNS of WT controls but is present at high levels in the PNS of ban mutants. Therefore, ban regulates Akt expression and activity in the larval PNS (Parrish, 2009).

    To test whether this effect on Akt levels reflects a neuronal requirement for ban, Akt expression levels were monitored in ban mutants in which ban expression is resupplied under the control of twist-Gal4, an experimental condition that rescues both the dendrite scaling defect and larval size defect of ban mutants. It was found that ban nonautonomously regulates Akt levels in da neurons since nonneuronal expression of ban (twist-Gal4) is sufficient to dampen the ectopic neuronal Akt expression normally seen in ban mutants. Therefore, ban likely functions in epithelia to regulate signals that influence Akt expression and activity in neurons (Parrish, 2009).

    Finally, it was of interest to determine whether Akt function in class IV neurons is important for scaling of dendrite growth. Based on expression data, it was predicted that increasing Akt expression/activity in class IV neurons should cause a scaling defect similar to what is seen in ban mutants. Indeed, ectopic expression of Akt, or a constitutively active form of PI3 kinase (PI3k) that leads to activation of Akt, caused a significant increase in dendrite coverage, similar to ban mutants. Conversely, antagonizing Akt activity in class IV neurons by overexpressing Pten, a PIP3 phosphatase that functions as an inhibitor of Akt activity, by knocking down Akt expression via RNAi in class IV neurons, or by generating Akt null mutant class IV neuron MARCM clones caused a significant reduction in dendrite coverage. Therefore, Akt plays a critical role in regulating dendrite coverage (Parrish, 2009).

    If increased neuronal Akt activity underlies the dendrite defects in ban mutants, then antagonizing neuronal Akt activity should suppress the dendrite overgrowth in ban mutants. This hypothesis was tested with the following three experiments. First, RNAi was used to knock down Akt expression in class IV neurons of ban mutant larvae. On its own, Akt(RNAi) causes a reduction in dendrite growth and overall coverage of the receptive field, and this phenotype is epistatic to the dendrite overgrowth seen in ban mutants. Similarly, Pten was overexpressed in class IV neurons of ban mutant larvae and it was found that the Pten-mediated reduction in dendrite coverage is epistatic to the dendrite overgrowth seen in ban mutants. Finally, class IV neurons was ablated in ban mutants in the absence or presence of neuron-specific Akt RNAi and it was found that reducing neuronal Akt expression blocks the exuberant dendrite invasion activity of ban mutants. Altogether, these results strongly suggest that ban functions in epithelial cells to regulate neuronal expression/activation of Akt, and deregulation of Akt leads to the dendrite growth defects of ban mutants (Parrish, 2009).

    This study has shown that nonautonomous signals coordinate growth of dendrites with the growth of their substrate and the body as a whole. Dendrites of many types of neurons cover characteristic receptive fields, and growth of the dendrite arbor in synchrony with the receptive field in a process, referred to as scaling growth of dendrites, allows a neuron to maintain proper dendrite coverage of the receptive field. Thus, scaling growth of dendrites is likely a general mechanism to ensure fidelity of dendrite coverage (Parrish, 2009).

    Dendrites of class IV neurons cover their receptive field before larval growth is complete and must maintain this coverage as the larva grows. Two properties distinguish the scaling phase of dendrite growth from the early dendrite growth when the neuron establishes receptive field coverage. During the scaling phase of growth, the dendrite arbor grows precisely in proportion to receptive field expansion (which is often achieved by animal growth). Moreover, dendrite growth is constrained by boundaries delineated when the dendrite arbor first covers the receptive field. Thus, although dendrites continue to grow, growth occurs only to maintain proportional coverage of the receptive field (Parrish, 2009).

    Dendrites of Drosophila da neurons exhibit a biphasic growth profile: dendrites establish coverage of their receptive field via an early, rapid growth phase and maintain this coverage via a late scaling growth phase in which dendrites grow in proportion with epithelial cells and the animal as a whole. The miRNA ban acts in the second phase to enable scaling of dendrite growth in da neurons, ensuring that dendrites maintain proper body wall coverage. Loss of ban disrupts epithelial-derived signaling that normally modulates dendrite growth, and, as a result, dendrites remain in the 'rapid growth' phase, extending beyond their normal territories. This phenotype is reminiscent of the heterochronic phenotypes of C. elegans lin-4 and let-7 mutants in that an early developmental phase is inappropriately reiterated during a later phase (Parrish, 2009).

    How broadly do miRNAs regulate developmental progressions in the nervous system? Although the developmental roles of vertebrate miRNAs have been somewhat elusive because of the vast number of miRNA-encoding genes, several studies suggest that miRNAs may serve highly specialized roles in regulating developmental transitions in neuron morphogenesis. For example, completely abrogating miRNA function causes robust defects in neuron morphogenesis but not specification in zebrafish, consistent with miRNAS regulating late aspects of neuronal differentiation. Likewise, miRNAs miR9a* and miR-124 regulate the switch in subunit composition of chromatin remodeling complexes as neural progenitors differentiate into neurons in mice. Additionally, a number of miRNAs function primarily at a very late step of neuron development to regulate activity-dependent dendrite growth and synaptic plasticity. For example, neuronal activity antagonizes miR-134, which normally inhibits growth of dendritic spines, and promotes expression of mir-132, which promotes dendritic plasticity (Parrish, 2009).

    Although ban is known to regulate growth in proliferating tissues in Drosophila, ban-mediated regulation of dendrite growth likely represents a distinct mode of growth control by ban for the following reasons. First, previous studies focused on autonomous regulation of tissue growth by ban. In contrast, ban acts nonautonomously to regulate scaling of dendrite growth. Second, prior studies of ban function focused on imaginal discs where growth is achieved by increasing cell number rather than cell size. By contrast, dendrite scaling involves ban-mediated regulation of growth in differentiated, postmitotic cells. Likewise, postmitotic expression of ban in the larval eye disc can also regulate cell size. Third, ban functions downstream of the tumor suppressor kinase Hippo to control proliferation, with Hippo activating the transcription factor Yorkie, which in turn activates ban expression. Whereas hippo is required cell-autonomously for establishment and maintenance of dendrite tiling, yorkie is dispensable for dendrite growth. As to the cell-nonautonomous function of ban in dendrite scaling, Hippo is not required. Although these findings suggest that ban regulates growth of proliferating and differentiated tissues by different means, it is possible that in both scenarios ban is antagonizing expression of growth-inhibitory factors, possibly even the same factors, and removing growth inhibition has different consequences on proliferative and differentiated tissues (Parrish, 2009).

    It is proposed that ban positively regulates an epithelial-derived signal that modulates neuronal Akt expression and activity to influence dendrite growth. Several observations suggest that the signal acts over a short range, possibly even via direct adhesive interactions between dendrites and epithelia or the underlying matrix. First, ban overexpression in epithelial cells but not in muscle influences growth of dendrites. Second, removing ban function from epithelial cell clones influences the distribution of dendrites over the clone but not over adjacent WT epithelial cells. Third, in addition to inhibiting the overall rate of dendrite growth, overexpressing ban in epithelial cells induces exaggerated “wrapping” of epithelial cells by terminal dendrites. Since these signals appear to preferentially regulate dendrite growth during the scaling phase, ban may modulate dendrite/epithelial adhesion during the scaling phase of dendrite development (Parrish, 2009).

    Morphologically distinct classes of da neurons establish type-specific dendritic coverage of the body wall and maintain this coverage by means of dendrite scaling as larvae grow. However, arbors of different da neurons develop at different rates, with class I dendrites establishing their coverage 1 day earlier than class III and class IV dendrites. Mutations in ban disrupt scaling of dendrite growth in class III and class IV but not class I neurons, suggesting that different signals regulate dendrite scaling in distinct types of neurons. Thus, temporally distinct signals or temporally restricted sensitivity to the signals may ensure that different neurons maintain appropriate coverage of their receptive field (Parrish, 2009).

    Drosophila Valosin-containing protein is required for dendrite pruning through a regulatory role in mRNA metabolism

    The dendritic arbors of the larval Drosophila peripheral class IV dendritic arborization neurons degenerate during metamorphosis in an ecdysone-dependent manner. This process-also known as dendrite pruning-depends on the ubiquitin-proteasome system (UPS), but the specific processes regulated by the UPS during pruning have been largely elusive. This study shows that mutation or inhibition of Valosin-Containing Protein (VCP; termed TER94 by FlyBase), a ubiquitin-dependent ATPase whose human homolog is linked to neurodegenerative disease, leads to specific defects in mRNA metabolism and that this role of VCP is linked to dendrite pruning. Specifically, it was found that VCP inhibition causes an altered splicing pattern of the large pruning gene Molecule interacting with CasL and mislocalization of the Drosophila homolog of the human RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). These data suggest that VCP inactivation might lead to specific gain-of-function of TDP-43 and other RNA-binding proteins. A similar combination of defects is also seen in a mutant in the ubiquitin-conjugating enzyme ubcD1 (Effete) and a mutant in the 19S regulatory particle of the proteasome, but not in a 20S proteasome mutant. Thus, these results highlight a proteolysis-independent function of the UPS during class IV dendritic arborization neuron dendrite pruning and link the UPS to the control of mRNA metabolism (Rumpf, 2014).

    To achieve specific connections during development, neurons need to refine their axonal and dendritic arbors. This often involves the elimination of neuronal processes by regulated retraction or degeneration, processes known collectively as pruning. In the Drosophila, large-scale neuronal remodeling and pruning occur during metamorphosis. For example, the peripheral class IV dendritic arborization (da) neurons specifically prune their extensive larval dendritic arbors, whereas another class of da neurons, the class III da neurons, undergo ecdysone- and caspase-dependent cell death. Class IV da neuron dendrite pruning requires the steroid hormone ecdysone and its target gene SOX14, encoding an HMG box transcription factor. Class IV da neuron dendrites are first severed proximally from the soma by the action of enzymes like Katanin-p60L and Mical that sever microtubules and actin cables, respectively. Later, caspases are required for the fragmentation and phagocytic engulfment of the severed dendrite remnants . Another signaling cascade known to be required for pruning is the ubiquitin-proteasome system (UPS). Covalent modification with the small protein ubiquitin occurs by a thioester cascade involving the ubiquitin-activating enzyme Uba1 (E1), and subsequent transfer to ubiquitin-conjugating enzymes (E2s) and the specificity-determining E3 enzymes. Ubiquitylation of a protein usually leads to the degradation of the modified protein by the proteasome, a large cylindrical protease that consists of two large subunits, the 19S regulatory particle and the proteolytic 20S core particle. Several basal components of the ubiquitylation cascade—Uba1 and the E2 enzyme ubcD1—as well as several components of the 19S subunit of the proteasome have been shown to be required for pruning, as well as the ATPase associated with diverse cellular activities (AAA) ATPase Valosin-Containing Protein (VCP) (CDC48 in yeast, p97 in vertebrates, also known as TER94 in Drosophila), which acts as a chaperone for ubiquitylated proteins. Interestingly, autosomal dominant mutations in the human VCP gene cause hereditary forms of ubiquitin-positive frontotemporal dementia (FTLD-U) and amyotrophic lateral sclerosis (ALS). A hallmark of these diseases is the occurrence of both cytosolic and nuclear ubiquitin-positive neuronal aggregates that often contain the RNA-binding protein TAR-DNA-binding protein of 43 kilo-Dalton (TDP-43). It has been proposed that ubcD1 and VCP promote the activation of caspases during dendrite pruning via degradation of the caspase inhibitor DIAP1. However, mutation of ubcD1 or VCP inhibit the severing of class IV da neuron dendrites from the cell body, whereas in caspase mutants, dendrites are still severed from the cell body, but clearance of the severed fragments is affected. This indicates that the UPS must have additional, as yet unidentified, functions during pruning (Rumpf, 2014).

    This study further investigated the role of UPS mutants in dendrite pruning. vcp mutation was shown to lead to a specific defect in ecdysone-dependent gene expression, as VCP is required for the functional expression and splicing of the large ecdysone target gene molecule interacting with CasL (MICAL). Concomitantly, mislocalization of Drosophila TDP-43 and up-regulation of other RNA-binding proteins were observed, and genetic evidence suggests that these alterations contribute to the observed pruning defects in VCP mutants. Defects in MICAL expression and TDP-43 localization are also induced by mutations in ubcD1 and in the 19S regulatory particle of the proteasome, but not by a mutation in the 20S core particle, despite the fact that proteasomal proteolysis is required for dendrite pruning, indicating the requirement for multiple UPS pathways during class IV da neuron dendrite pruning (Rumpf, 2014).

    Class IV da neurons have long and branched dendrites at the third instar larval stage. In wild-type animals, these dendrites are completely pruned at 16-18 h after puparium formation (h APF). VCP mutant class IV da neurons were generated by the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique for clonal analysis. Mutant vcp26-8 class IV da neurons displayed strong pruning defects and retained long dendrites at 16 h APF. Expression of an ATPase-deficient dominant-negative VCP protein (VCP QQ) under the class IV da neuron-specific driver ppk-GAL4 recapitulated the pruning phenotype and also led to the retention of long and branched dendrites at 16 h APF. VCP inhibition also causes defects in class III da neuron apoptosis. This combination of defects in both pruning and apoptosis is reminiscent of the phenotypes caused by defects in ecdysone-dependent gene expression. Indeed, overexpression of the transcription factor Sox14, which induces pruning genes, led to a nearly complete suppression of the pruning phenotype caused by VCP QQ. This genetic interaction suggested that VCP might be required for the expression of one or several ecdysone target genes during pruning (Rumpf, 2014).

    How could VCP be linked to Sox14? The suppression of the vcp mutant phenotype by Sox14 overexpression could be achieved in one of several ways. Sox14 could be epistatic to VCP -- that is, VCP could be required for functional Sox14 expression -- and this effect would be mitigated by Sox14 overexpression. However, VCP could also be required for the expression of one or several Sox14 target genes, and enhanced Sox14 expression could overcome this requirement either via enhanced induction of one or several particular targets or via enhanced induction of other pruning genes, in which case Sox14 would be a bypass suppressor of VCP QQ. To distinguish between these possibilities, the effects were assessed of VCP inhibition on the expression of known genes in the ecdysone cascade required for pruning in class IV da neurons. Class IV da neuron pruning is governed by the Ecdysone Receptor B1 (EcR-B1) isoform, which in turn directly activates the transcription of Sox14 and Headcase (Hdc), a pruning factor of unknown function. Sox14, on the other hand, activates the transcription of the MICAL gene encoding an actin-severing enzyme. In immunostaining experiments, VCP QQ did not affect the expression of EcR-B1, Sox14, or Hdc at the onset of the pupal phase. However, the expression of Mical was selectively abrogated in class IV da neurons expressing VCP QQ, or in vcp26-8 class IV da neuron MARCM clones . These data indicated that VCP might affect dendrite pruning by regulating the expression of the Sox14 target gene Mical, indicating that Sox14 might act as a bypass suppressor of VCP QQ (Rumpf, 2014).

    How could VCP inhibition suppress Mical expression? To answer this question, whether Mical mRNA could still be detected in class IV da neurons expressing VCP QQ was assessed. To this end, enzymatic tissue digestion and FACS sorting were used to isolate class IV da neurons from early pupae (1-5 h APF). Total RNA was then extracted from the isolated neurons, and the presence of Mical mRNA expression was assessed by RT-PCR, using control samples or samples from animals expressing VCP QQ under ppk-GAL4. The Mical gene is large (~40 kb) and spans multiple exons that are transcribed to yield a ~15 kb mRNA. To detect Mical cDNA, primer pairs spanning several exons were used for two different regions of Mical mRNA, exons 14-16 and exons 8-12. [MICAL is on the (-) strand, but the exon numbering denoted by Flybase follows the direction of the (+) strand. Therefore, exons 14-16 are upstream of exons 8-12, and the latter are closer to the 3' end of MICAL mRNA.] MICAL mRNA was detectable upon VCP inhibition in these extracts with a primer pair spanning exons 14-16. The second primer pair spanning exons 8-12 also detected MICAL mRNA in both samples, but the RT-PCR product from the VCP QQ-expressing class IV da neurons had a larger molecular weight. Sequencing of the PCR products indicated that MICAL mRNA from VCP QQ-expressing class IV da neurons contained exon 11, which was not present in Mical mRNA from the control sample. Exon 11 is absent from all predicted MICAL splice isoforms except for a weakly supported isoform designated 'Mical-RM'. It introduces a stop codon into MICAL mRNA that would lead to the truncation of the C-terminal 1,611 amino acids from Mical protein. This portion of Mical protein contains several predicted protein interaction domains such as a proline-rich region, a coiled-coil region with similarity to Ezrin/Radixin/Moesin (ERM) domains, and a C-terminal PDZ-binding motif, and is required for the interaction between Mical and PlexinA. In addition, the truncated region contains the epitope for the antibody used in the immunofluorescence experiments, thus explaining the observed lack of Mical expression upon VCP inhibition. Given that a mutant of Mical with a smaller C-terminal truncation (compared with the one induced by VCP inhibition) was not sufficient to rescue the class IV da neuron dendrite pruning defect of mical mutants, disruption of VCP function likely results in expression of a truncated Mical protein without pruning activity. Taken together, these data suggest that the observed defect in MICAL mRNA splicing contributes significantly to the pruning defects of VCP mutants (Rumpf, 2014).

    How is VCP linked to alternative splicing of MICAL mRNA? A plausible mechanism for the control of an alternative splicing event would be the modulation of specific (pre)mRNA-binding proteins. VCP has recently been linked to several RNA-binding proteins: human autosomal dominant VCP mutations cause frontotemporal dementia or ALS with inclusion bodies that contain aggregated human TDP-43; a genetic screen in Drosophila identified the RNA-binding proteins Drosophila TDP-43, HRP48, and x16 as weak genetic interactors of the dominant effects of VCP disease mutants; and HuR (a human homolog of the neuronal Drosophila RNA-binding protein elav) was recently shown to bind human VCP. Of these, TDP-43 and also elav have been linked to alternative splicing in various model systems, including Drosophila. Therefore this study used available specific antibodies to assess the levels and distribution of Drosophila TDP-43 (hereafter referred to as TDP-43) and elav. TDP-43 has previously been shown to localize to the nucleus in Drosophila motoneurons and mushroom body Kenyon cells. Surprisingly, TDP-43 was largely localized to the cytoplasm in class IV da neurons, where it was enriched in a punctate pattern around the nucleus, with only a small fraction also detectable in the nucleus, a localization pattern that could be reproduced with transgenic N-terminally HA-tagged TDP-43. Elav is a known nuclear marker for Drosophila neurons; in class IV da neurons, it was somewhat enriched in nuclear punctae. The effects of VCP inhibition on these two RNA-binding proteins was assessed. Elav localization did not change notably upon VCP QQ expression. Strikingly, TDP-43 became depleted from the cytoplasm of class IV da neurons and relocalized to the nucleus upon VCP QQ expression. Closer inspection revealed that TDP-43 in VCP-inhibited neurons was now enriched in nuclear dots that often also exhibited increased elav staining. The relocalization of TDP-43 from the cytoplasm to the nucleus was also observed in vcp26-8 mutant class IV da neuron MARCM clones. Importantly, quantification and normalization of TDP-43 levels showed that VCP inhibition did not alter the absolute levels of TDP-43, suggesting that the observed effects were not a consequence of a defect in TDP-43 degradation. In fact, the only manipulation that resulted in a mild but significant increase in TDP-43 levels -- but without a change in localization -- was the expression of an RNAi directed against the autophagy factor ATG7, perhaps reflecting the degradation of cytoplasmic RNA granules through the autophagy pathway (Rumpf, 2014).

    It was next asked whether manipulation of TDP-43 would affect class IV da neuron dendrite pruning. A previously characterized TDP-43 mutant, TDP-43 Q367X (28-128">28), did not display pruning defects, but overexpression of TDP-43 led to strong dendrite pruning defects at 16 h APF. In support of the hypothesis that TDP-43 acts in the same or a similar pathway as VCP during dendrite pruning, it was also found that a more weakly expressed TDP-43 transgene (UAS-TDP-43weak) and VCP A229E, a weakly dominant-active VCP allele corresponding to a human VCP disease mutation, exhibited a synergistic inhibition of pruning when coexpressed. Interestingly, manipulation of elav gave very similar results as with TDP-43: elav down-regulation by RNAi did not affect pruning, but elav overexpression led to highly penetrant pruning defects (Rumpf, 2014).

    To exclude the possibility that the pruning defects induced by TDP-43 or elav overexpression were due to long-term expression and aggregation of RNA-binding proteins, TDP-43 and elav overexpression was also induced acutely (24 h before the onset of pupariation). Pruning was still inhibited in these cases. Also, overexpression of several other RNA-binding proteins did not cause pruning defects, with two exceptions: a UAS-carrying P-element in the promotor of the adjacent x16 and HRP48 genes caused a strong pruning defect when expression was induced in class IV da neurons, and levels of a GFP protein trap insertion into the x16 gene were also markedly increased in class IV da neurons expressing VCP QQ, possibly indicating a role for VCP in x16 degradation. In further support of an involvement of VCP with RNA-binding proteins during neuronal pruning processes, it was also found that VCP is required for mushroom body γ neuron axon pruning and induces the accumulation of Boule, an RNA-binding protein that had previously been shown to inhibit γ neuron axon pruning when overexpressed. Thus, the data suggest that VCP regulates a specific subset of RNA-binding proteins and that this regulatory role of VCP is associated with its role in pruning (Rumpf, 2014).

    As VCP is an integral component of the UPS, it was next asked whether the role of VCP in MICAL regulation and TDP-43 localization was also dependent on ubiquitylation and/or the proteasome. To address this question, Mical levels and TDP-43 distribution was assessed in UPS mutants with known pruning defects. An ubiquitylation enzyme known to be required for pruning is the E2 enzyme ubcD1. When TDP-43 localization was assessed in larval ubcD1D73 mutant class IV da neurons, TDP-43 was again localized to the nucleus in these cells. Furthermore, a pronounced reduction of Mical expression in ubcD1D73 mutant class IV da neurons was noted during the early pupal stage, indicating that ubiquitylation through ubcD1 is involved in the regulation of TDP-43 localization and Mical expression (Rumpf, 2014).

    TDP-43 localization and Mical expression were assessed in proteasome mutants. A previously characterized mutant in the Mov34 gene encoding the 19S subunit Rpn8 was used. TDP-43 was again relocalized to the nucleus in Mov34 mutant class IV da neurons, and Mical expression was absent from Mov34 mutant class IV da neurons at 2 h APF. To rigorously address whether proteasomal proteolysis was also required for TDP-43 localization and Mical expression, the effect was assessed of Pros261, a previously characterized mutation in the 20S core particle subunit Prosβ6. In contrast to Mov34 mutant class IV da neurons, Pros261 mutant class IV da neurons displayed cytoplasmic TDP-43 localization, and robust Mical expression was detected in these neurons at 2 h APF. Thus, although ubiquitylation and the 19S proteasome are both required for Mical expression and normal TDP-43 localization, proteolysis through the 20S core particle of the proteasome is not. Importantly, Pros261 MARCM class IV da neurons showed strong dendrite pruning defects at 16 h APF, as did expression of RNAi constructs directed against subunits of the 20S core particle (Rumpf, 2014).

    These data indicate that there must be several ubiquitin- and proteasome-dependent pathways that are required for dendrite pruning: one pathway requires ubcD1, VCP, and the 19S regulatory particle of the proteasome, but not the 20S core particle. This pathway regulates MICAL expression. A second UPS pruning pathway does depend on proteolysis through the 20S core. In an E3 ubiquitin ligase candidate screen, cul-1/lin19 was identified as a pruning mutant. Cul-1 encodes cullin-1, a core component of a class of multisubunit ubiquitin ligases known as SCF (for Skp1/Cullin/F-box) ligases. Class IV da neurons mutant for cul-1 or class IV da neurons expressing an RNAi construct directed against cul-1 had not pruned their dendrites at 16 h APF. However, unlike with VCP, ubcD1, and Mov34, cul-1 mutation did not affect Mical expression at 2 h APF, indicating that cullin-1 is not a component of the VCP-dependent UPS pathway involved in splicing and might thus be a component of a proteolytic UPS pathway. In support of this notion, a recent report independently identified cul-1 as a pruning mutant and associated it with protein degradation (Rumpf, 2014).

    It has been proposed that the E2 enzyme ubcD1 and VCP would act to activate caspases during pruning. However, the dendrite pruning defects caused by those UPS mutants are much stronger than the phenotypes caused by caspase inactivation, which mostly causes a delay in the phagocytic uptake of severed dendrites by the epidermis. Although it cannot be excluded that ubcD1 and VCP contribute to caspase activation during pruning, the new mechanism proposed in this study -- control of RNA-binding proteins and MICAL expression -- likely makes a much stronger contribution to the drastic pruning phenotypes of UPS mutants (Rumpf, 2014).

    How precisely do VCP, ubcD1, and the 19S proteasome contribute to MICAL expression? The data indicate that VCP inhibition causes missplicing of MICAL mRNA that likely leads to the expression of an inactive Mical protein variant. At the same time, VCP inhibition leads to the mislocalization of TDP-43, and possibly the dysregulation of a number of other RNA-binding proteins. The fact that these phenotypes correlate in the vcp, ubcD1, and Mov34 mutants gives a strong indication that they are related. TDP-43 had previously been identified as a suppressor of the toxicity induced by a weak VCP disease allele in the Drosophila eye. In class IV da neurons, reducing the amounts of TDP-43 (with a deficiency) or elav (by RNAi) did not ameliorate the pruning defect induced by VCP inhibition. Therefore, the possibility cannot be excluded that the two proteins act in parallel rather than in an epistatic fashion. As VCP has been shown to remodel protein complexes that contain ubiquitylated proteins and is structurally similar to the 19S cap, it is interesting to speculate that VCP and the 19S cap might alter the subunit composition of ubiquitylated TDP-43-containing complexes of RNA-binding proteins, and that this activity—rather than a direct action on TDP-43 (or maybe also elav) alone—might lead to both MICAL missplicing and TDP-43 mislocalization (Rumpf, 2014).

    Interestingly, autosomal dominant mutations in human VCP cause frontotemporal dementia and ALS, a hallmark of which is the formation of aggregates that contain TDP-43. Most of these aggregates are cytoplasmic (and contain TDP-43 that has relocalized from the nucleus to the cytoplasm), but VCP mutations also induce TDP-43 aggregation in the nucleus, a situation that might be similar to the situation caused by VCP inhibition in class IV da neurons. Although human VCP disease mutations have been proposed to act as dominant-active versions of VCP with enhanced ATPase activity, both the disease allele and the dominant-negative ATPase-dead VCP QQ mutant cause class IV da neuron pruning defects and TDP-43 relocalization to the nucleus of class IV da neurons and therefore act in the same direction. It is thought that VCP can only bind substrates when bound to ATP, and will release bound substrates upon ATP hydrolysis. Thus, it is conceivable that the phenotypic outcome of inhibiting the ATPase (no substrate release) should be similar to that of ATPase overactivation (reduced substrate binding or premature substrate release): in both cases, a substrate protein complex would not be properly remodeled (Rumpf, 2014).

    Taken together, these results indicate the existence of a nonproteolytic function of VCP and the UPS in RNA metabolism and highlight its importance during neuronal development (Rumpf, 2014).

    Wnt5 and drl/ryk gradients pattern the Drosophila olfactory dendritic map

    During development, dendrites migrate to their correct locations in response to environmental cues. The mechanisms of dendritic guidance are poorly understood. Recent work has shown that the Drosophila olfactory map is initially formed by the spatial segregation of the projection neuron (PN) dendrites in the developing antennal lobe (AL). This study reports that between 16 and 30 h after puparium formation, the PN dendrites undergo dramatic rotational reordering to achieve their final glomerular positions. During this period, a novel set of AL-extrinsic neurons express high levels of the Wnt5 protein and are tightly associated with the dorsolateral edge of the AL. Wnt5 forms a dorsolateral-high to ventromedial-low pattern in the antennal lobe neuropil. Loss of Wnt5 prevents the ventral targeting of the dendrites, whereas Wnt5 overexpression disrupts dendritic patterning. Drl/Ryk, a known Wnt5 receptor, is expressed in a dorsolateral-to-ventromedial (DL > VM) gradient by the PN dendrites. Loss of Drl in the PNs results in the aberrant ventromedial targeting of the dendrites, a defect that is suppressed by reduction in Wnt5 gene dosage. Conversely, overexpression of Drl in the PNs results in the dorsolateral targeting of their dendrites, an effect that requires Drl's cytoplasmic domain. It is proposed that Wnt5 acts as a repulsive guidance cue for the PN dendrites, whereas Drl signaling in the dendrites inhibits Wnt5 signaling. In this way, the precise expression patterns of Wnt5 and Drl orient the PN dendrites allowing them to target their final glomerular positions (Wu, 2014).

    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. 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. Developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. 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 (Brierley, 2009).

    Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. The architecture of dendrites and the role they play in establishing connectivity within maps has been somewhat overlooked. Classic cell-labelling studies in the moth Manduca sexta revealed that the dendrites of motoneurons are topographically organized to reflect their site of innervation in the bodywall. More recent work by Landgraf and colleagues has demonstrated that motoneurons in Drosophila embryos generate a detailed dendritic (myotopic) map of body wall muscles within the CNS. Alongside these data, studies on the architecture of the spinal cord also suggest that similar design principles may play a role in organizing information in vertebrate motor systems. How such dendritic maps are built is still largely unknown. This study describes the role dendritic targeting plays in constructing a myotopic map and the molecular mechanisms that control it (Brierley, 2009).

    The majority of leg motoneurons in a fly are born postembryonically and most of those are derived from a single neuroblast lineage, termed lineage 15. Perhaps the most striking feature of this lineage is its birth-order-based pattern of innervation along the proximo-distal axis of the leg. Using mosaic analysis, the sequential production was observed of four neuronal subtypes during larval life, each elaborating stereotyped axonal and dendritic projections in the adult. The axon of the first-born neuron innervates a muscle in the body wall and subsequent neurons innervate more distal targets in the leg. This organization has also been reported by Baek (2009) (Brierley, 2009).

    This birth-order-based peripheral pattern of lineage 15 is mirrored in the CNS, where dendrites generate a stereotyped anatomical organization. Dendrites of early-born cells span medial to lateral territories, whereas late-born cells elaborate dendrites in the lateral neuropil and cells born between these times occupy intermediate territories. The sequential production of neuronal subtypes by neural precursor cells is a common mechanism for generating a diversity of circuit components. A similar birth-order-based specification of axonal and dendritic projection patterns has previously been described for projection neurons in the fly's olfactory system (Brierley, 2009).

    The data reveal the existence of a myotopic map in the adult fly and supports the proposition that dendritic maps are a common organizing principle of all motor systems. Mauss (2009) also reveal a map in the embryonic CNS of Drosophila, where the dendrites of motoneurons are organized along the medio-lateral axis of the neuropil representing dorsoventral patterns of innervation in the body wall muscles (Brierley, 2009).

    How are dendritic maps built? The myotopic map seen in the leg neuropil could be generated by two distinctly different mechanisms. Neurons could elaborate their dendrites profusely across a wide field and then remove branches from inappropriate regions or, alternatively, they could target the growth of dendrites into a distinct region of neuropil throughout development. Both mechanisms can generate cell-type-specific projection patterns as seen in the vertebrate retina. To reveal which mechanism is deployed in the leg motor system of Drosophila, single-cell clones of motoneuron subtypes generated by heatshocks at 48 and 96 h AH were imaged, since their final dendritic arborizations cover clearly distinct territories within the map. The dendrites of both elaborate branches only in territories where the mature arborizations eventually reside, which strongly supports the notion that this myotopic map is generated by targeting and not large-scale branch elimination. Importantly, this developmental timeline also revealed that the motoneurons elaborate their dendrites synchronously, regardless of the birth date of the cell. This observation suggests that a 'space-filling/occupancy based' model, where later-born neurons are excluded from medial territories by competitive interactions is unlikely. Similarly, heterochronic mechanisms where different members of the lineage experience different signalling landscapes due to differences in the timing of outgrowth are not likely either. With synchronous outgrowth dendrites experience the same set of extracellular signals, suggesting that the intrinsic properties of cells, defined by their birth order, may be more important for the generation of subtype-specific projections. Such intrinsic properties could include cell-cell recognition systems such as adhesion molecules, e.g., Dscams or classical guidance receptors, that could interpret extracellular signals. In the Drosophila embryo motoneurons also use dendritic targeting to generate a myotopic map (Brierley, 2009).

    It is emerging that dendrites are guided by the same molecules that control axon pathfinding. The medio-lateral organization of leg motoneuron dendrites within the leg neuropil prompted an investigation as to whether the midline signalling molecules Slit and Netrin and their respective receptors Roundabout and Frazzled could be involved in targeting growth to specific territories (Brierley, 2009).

    Using mosaic analysis it was found that both the 48 and 96 h AH motoneuron subtypes require Robo to generate their appropriate shape and position within the medio-lateral axis. When Robo was removed from the 48 h AH subtype the mean centre of arbor mass was shifted toward the midline. The dendrites of 96 h AH neurons showed a shift in distribution in the absence of Robo but still failed to reach the midline, suggesting that only part of this cell's targeting is due to repulsive cues mediated by the Robo receptor. It was predicted that if Robo levels played an instructive role in dendrite targeting it would be possible to shift dendrites laterally by cell autonomously increasing Robo. This was found to be the case in both subtypes. Taken together these data suggest that differences in the level of Robo signalling may provide a mechanism by which Slit could be differentially interpreted to allow subtype-specific targeting along the medio-lateral axis (Brierley, 2009).

    The Robo receptor is part of a larger family of receptors that includes Robo2 and Robo3. This family of receptors have been found to be important for targeting axons to the appropriate longitudinal pathway in the embryonic CNS. Comm plays a key role in allowing contralaterally projecting neurons to cross the midline, and its ectopic expression (CommGOF) is known to robustly knock down Robo and Robo2 and 3. Comm was cell autonomously expressed in both lineage 15 subtypes and shifts to the midline were found in both 48 and 96 h AH neurons. For the 48 h AH neurons, Robo LOF data and CommGOF data are comparable, suggesting that Robo alone plays a major role in the positioning dendrites of these cells. In contrast, in the 96 h AH subtype RoboLOF and CommGOF effects were found to be significantly different, suggesting that the 96 h AH subtype may not only use the Robo receptor but additional Robos as well. Knockdown of Slit also supports this idea, as the branches of late-born neurons were occasionally found reaching the midline, something that was never see in RoboLOF clones. Thus, one way of establishing differences in the medio-lateral position could be through a dendritic “Robo code” where early-born cells express Robo and late-born cell express multiple Robo receptors (Brierley, 2009).

    With Netrin being expressed in the midline cells during the pupal-adult transition it was asked whether attractive Netrin-Fra signalling could also contribute to positioning dendrites in the leg neuropil. When Fra was removed from the 48 h AH subtype it was found that the arborization was shifted laterally, whereas removing it from the 96 h AH subtype had little effect, and neither did the removal of Netrin A and B from the midline, suggesting that Netrin-Fra signalling may not play a role in dendritic targeting in the later-born cell. It may be that Fra is expressed in early-born cells within the lineage and then down-regulated, although it cannot be excluded that Netrin-Fra signalling was masked by the repulsion from Slit-Robo signalling. These data are consistent with Fra being a major player in targeting the dendrites of the 48 h AH cell. The fact that both Fra and Robo are required for normal morphogenesis of 48 h AH neurons raises the possibility that members of lineage 15 could use a 'push-pull' mechanism for positioning their dendrites, where the blend of receptors within a cell dictates the territory within the map that they will innervate (Brierley, 2009).

    How could such subtype-specific blends of receptors be established? A number of studies have revealed that spatial codes of transcription factors are important for specifying the identity of motoneuron populations. Within lineage 15 it is possible that temporal, rather than spatial, transcription factor codes are important for regulating the blend of guidance receptors. A number of molecules have been identified that control the sequential generation of cell types within neuroblast lineages. Chief amongst these are a series of transcription factors that include Hunchback, Krüppel, Pdm, Castor and Seven-up. These temporal transcription factors are transiently expressed within neuroblasts and endow daughter neurons with distinct “temporal identities”. Castor and Seven-up are known to schedule transitions in postembryonic lineages, regulating the neuronal expression of BTB-POZ transcription factors Chinmo and Broad. It is possible that the temporal transcription factors Broad and Chinmo could control the subtype-specific expression of different Robo receptors or the Netrin receptor Frazzled in leg motoneurons. There is a precedent for this in the Drosophila embryo, where motoneuron axon guidance decisions to distal (dorsal) versus proximal (ventral) targets are orchestrated by Even-Skipped, a homeobox transcription factor, which in turn controls the expression of distinct Netrin receptor combinations (Brierley, 2009).

    Studies focusing on the growth of olfactory projection neuron dendrites in Drosophila reveal that they elaborate a glomerular protomap prior to the arrival of olfactory receptor neurons suggesting that target/partner-derived factors may not be necessary for establishing coarse patterning of synaptic specificity. The global nature of the signals describe in this study and their origin in a third-party tissue is a fundamentally different situation to that where target-derived factors instruct partner cells, such as presynaptic amacrine cells signalling to retinal ganglion cell dendrites in the zebrafish retina. Furthermore, although this study shows that Slit and Netrin control the positioning of dendrites across the medio-lateral axis of the CNS, it may be that other similar guidance signals are important for patterning dendrites in other axes. There is a striking conservation of the molecular mechanisms that build myotopic maps in the embryo and pupae. Understanding the similarities and differences between these myotopic maps, from an anatomical, developmental, and functional perspective, may give insight into the evolution of motor systems and neural networks in general (Brierley, 2009).

    This study found that individual leg motoneurons that lacked Robo signalling appeared to have more complex dendritic arborizations. The working hypothesis, that dendrites invaded medial territories because of a failure of Slit-Robo guidance function, did not take into account the possibility that cells may generate more dendrites due to a change in a cell-intrinsic growth program. Thus the changes seen in dendrite distribution relative to the midline could formally be a result of 'spill-over' from that increase in cell size/mass. To determine whether this was the case larger cells were generated by activating the insulin pathway in single motoneurons. It was found the dendrites of these 'large cells' remained within their normal neuropil territory, supporting the idea that the removal of Robo-Slit signalling results in a disruption in guidance, not growth. These data underline the fundamental importance of midline signals in controlling the spatial coordinates that these motoneuron dendrites occupy, i.e., that a neuron twice the size/mass of a wild-type cell is still marshalled into the same volume of neuropil (Brierley, 2009).

    When the image stacks were reconstructed to look at the distribution of the dendrites in the dorso-ventral axis, it was found that the apparent increase in size was in fact a redistribution of the dendrites from ventral territories into more dorsal medial domains. This was unexpected and suggests that changes in midline signalling can also impact the organization of dendrites in the dorso-ventral axis. So CommGOF 96 h AH neurons may not only encounter novel synaptic inputs by projecting into medial territories, but they may also lose inputs from the ventral domains of neuropil they have vacated. These observations suggest that motoneurons within lineage 15 have a fixed quota of dendrites and where it is distributed in space depends on cell-intrinsic blends of guidance receptors. Taken together these data support the idea that growth and guidance mechanisms are genetically separable programs. In identified embryonic motoneurons where Slit-Robo and Netrin-Fra signalling has been disrupted, quantitative analysis reveals dendrites also show no measurable difference in their total number of branch tips or length (Mauss, 2009). Moreover, recent computational studies in larger flies reveal that dendritic arborizations generated by the same branching programs can generate very different shapes depending on how their 'dendritic span' restricted within the neuropil. Previous work in both vertebrates and Drosophila has shown that a loss of Slit-Robo signalling results in a reduction in dendrite growth and complexity, but this study found no evidence to support this (Brierley, 2009).

    Neural maps and synaptic laminae are universal features of nervous system design and are essential for organizing and presenting synaptic information. How the appropriate pre- and postsynaptic elements within such structures are brought together remains a major unanswered question in neurobiology. Studies in recent years have shown that neural network development involves both hardwired molecular guidance mechanisms and activity-dependent processes; the relative contribution that each makes is still unclear. Work on the spinal cord network of Xenopus embryos revealed that seven identifiable neuron subtypes can establish connections with one another and that the key predictor of connectivity was their anatomical overlap. This could be interpreted to mean that connectivity is promiscuous and that the major requirement for the generation of synaptic specificity is the proximity of axons and dendrites. This is particularly interesting in light of the current dendrite targeting data and the observation that both sensory neurons and interneurons in Drosophila use the same midline cues to position their pre-synaptic terminals in the CNS. Moreover, a recent study has shown that Semaphorins control the positioning of axons within the dorso-ventral axis. Taken together these observations suggest that during development the coordinated targeting of both pre- and postsynaptic elements into the same space using global, third-party guidance signals could provide a simple way of establishing the specificity of synaptic connections within neural networks. This idea is akin to 'meeting places' such as the traditional rendezvous underneath the four-sided clock at Waterloo railway station where two interested parties organize to meet. Understanding how morphogenetic programs contribute to the generation of synaptic specificity is likely to be key to solving the problem of neural network formation (Brierley, 2009).

    Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites

    In many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, this system was used to understand how cells can maintain dynamic arrays of polarized microtubules. It was found that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, it was shown that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. It is concluded that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. It is proposed that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules (Mattie, 2010).

    Using Drosophila dendrites as a model system, this study demonstrates that growing microtubule plus ends almost always turn toward the cell body at branch points and that they track stable microtubules through branches. Kinesin-2, EB1, and APC are all required for maintaining microtubule polarity and are linked in an interaction network (Mattie, 2010).

    On the basis of these results, a model for directed growth of microtubules in dendrites is proposed. Apc2 most likely contains a branch localization signal because Apc2-GFP localizes well to dendrite branches even when overexpressed. Localization of Apc2 to dendrite branch points can recruit Apc to the branch. Apc can interact with the Kap3 subunit of kinesin-2 and so could increase concentration of the motor near the branch. EB1-GFP does not concentrate at branches, therefore it is proposed that a growing microtubule plus end coated with EB1 is transiently linked, through the interaction between Apc and the EB1 tail, to kinesin-2 as it passes through the branch. SxIP motifs in Apc and Klp68D could also contribute to this interaction. Because both Kap3 and the SxIP motif in Klp68D are in the kinesin-2 tail, the motor domain would be free to walk along a nearby stable microtubule toward the plus end and cell body (Mattie, 2010).

    Even a very brief application of force pulling the growing microtubule toward the cell body should be sufficient to steer growth toward the cell body. Once the tip of the microtubule turns, growth should be constrained by the dendrite walls. The association of the growing plus end with a stable microtubule would probably only need to be maintained over a distance of a micron. This model is consistent with the observations that kinesin-2 has shorter run lengths than kinesin-1 and that individual EB1 interactions with the microtubule plus end persist for less than a second (Mattie, 2010).

    Observations of plus-end behavior in vivo also favor a model in which only transient interactions of the motor and microtubule plus end are involved; frequently plus ends are seen turning sharply. Stable microtubules do not accommodate such sharp turns, so the sharp turns of plus ends are not likely to occur while the plus end is tracking along a stable microtubule. Instead, they probably represent a switch from a freely growing plus end to one that is following a track (Mattie, 2010).

    Directed growth of microtubules at dendrite branch points allows dendrites to maintain uniform minus-end-out polarity despite continued microtubule remodeling. This mechanism is also probably necessary to establish uniform microtubule polarity in branched dendrites, but it probably cannot account for minus-end-out polarity on its own. It is hypothesized that directed microtubule growth is used in concert with an unidentified mechanism to control microtubule polarity in dendrites. Transport of oriented microtubule pieces has been proposed to contribute to axon microtubule polarity, and a similar mechanism could play a role in dendrites. For example, a kinesin anchored to the cell cortex by its C terminus could shuttle minus-end-out microtubule seeds into dendrites. Alternatively, polarized microtubule nucleation sites could be localized within dendrites. Identifying directed growth as one mechanism that contributes to dendrite microtubule polarity should facilitate identification of the missing pieces of this puzzle (Mattie, 2010).

    Because kinesin-2, APC, EB1, and polarized microtubules are found in many cell types, directed growth of microtubules along stable microtubule tracks could be very broadly used for maintaining microtubule polarity. This is a newly identified function for both the +TIP proteins and kinesin-2. APC has previously been localized to junctions between a microtubule tip and the side of another microtubule at the cortex of epithelial cells, and sliding of microtubule tips along the side of microtubules was also seen in this system, but there was no association with a particular direction of movement or overall microtubule polarity (Mattie, 2010).

    Kinesin-2 has previously been shown to be enriched in the tips of growing axons in cultured mammalian neurons, and it is possible that they could mediate tracking of growing microtubules along existing microtubule tracks in the growth cone. In fact, this type of microtubule tracking behavior has been observed in axonal growth cones under low-actin conditions. Thus, directed growth of microtubules could also be important during axon outgrowth. Indeed, the same principle of directed growth by a motor protein connected to a growing microtubule plus end could be involved in aligning microtubules in many circumstances (Mattie, 2010).

    The Me31B DEAD-box helicase localizes to postsynaptic foci and regulates expression of a CaMKII reporter mRNA in dendrites of Drosophila olfactory projection neurons

    mRNP granules at adult central synapses are postulated to regulate local mRNA translation and synapse plasticity. However, they are very poorly characterized in vivo. This study presents early observations and characterization of candidate synaptic mRNP particles in Drosophila olfactory synapses; one of these particles contains a widely conserved, DEAD-box helicase, Me31B. In Drosophila, Me31B is required for translational repression of maternal and miRNA-target mRNAs. A role in neuronal translational control is primarily suggested by Me31B's localization, in cultured primary neurons, to neuritic mRNP granules that contain: (1) various translational regulators; (2) CaMKII mRNA; and (3) several P-body markers including the mRNA hydrolases, Dcp1, and Pcm/Xrn-1. In adult neurons, Me31B localizes to P-body like cytoplasmic foci/particles in neuronal soma. In addition it is present to synaptic foci that may lack RNA degradative enzymes and localize predominantly to dendritic elements of olfactory sensory and projection neurons (PNs). MARCM clones of PNs mutant for Me31B show loss of both Me31B and Dcp1-positive dendritic puncta, suggesting potential interactions between these granule types. In PNs, expression of validated hairpin-RNAi constructs against Me31B causes visible knockdown of endogenous protein, as assessed by the brightness and number of Me31B puncta. Knockdown of Me31B also causes a substantial elevation in observed levels of a translational reporter of CaMKII, a postsynaptic protein whose mRNA has been shown to be localized to PN dendrites and to be translationally regulated, at least in part through the miRNA pathway. Thus, neuronal Me31B is present in dendritic particles in vivo and is required for repression of a translationally regulated synaptic mRNA (Hillebrand, 2010).

    The Me31B/Dhh1p/DDX6/CGH-1 class of DEAD box helicases is associated with many different kinds of mRNP aggregates, including maternal RNA storage granules, P-bodies, stress granules, as well as various granule subtypes observed during C. elegans germline development. In addition it is required for the assembly of P-bodies in yeast, Drosophila and mammalian cells and as well for the formation of stress granules in mammals. For these reasons, the punctate distribution of Me31B in postsynaptic dendrites is likely to indicate its presence in a specific type of synaptic mRNP particle. However, unlike Me31B-positive particles described in neurites of cultured Drosophila neurons, synaptic Me31B foci do not appear to contain the RNA hydrolases Dcp1 and Pcm/Xrn-1. Thus, they may be a distinct class of particle, which localize preferentially to postsynaptic dendrites. These represent early images of candidate mRNA storage particles at synapses in vivo. A paucity of antibodies and the challenging nature of such high-resolution immunocytochemistry in whole brain tissue has so far made it difficult to more completely characterize other components of synaptic Me31B particles as well as to establish whether Dcp1, Pcm, and Stau coexist on the same or different particle in the adult brain. Indeed even the conclusion that Me31B particles constitute a separate class must be qualified by the possibility that the visualization of two apparently distinct particle types arises from an artifact of incomplete antibody penetration into the neuropil (Hillebrand, 2010).

    It is possible that synaptic Me31B particles could be analogous to recently described granules in the C. elegans germline, which contain translationally controlled mRNAs and CGH-1/Me31B but exclude decapping enzymes and the P-body protein PATR1/PAT1. Immunoprecipitation and further colocalization studies suggest that these granules can also contain PAB-1, ATX-2, or TIA-1, markers of stress granules, which in other systems, contain translation initiation factors together with mRNAs stalled in translational initiation. Thus, it is conceivable that Me31B/CGH-1-containing storage particles contain mRNAs stored in a stress-granule like state, in which the resident mRNAs are available for rapid activation (Hillebrand, 2010).

    The potential separation of storage and degradative particles leads to an attractive model in which individual mRNAs may transition from being available for translational activation in a storage granule, to being targeted for degradation in a P-body like particle. This is supported as well by observations in dendrites of cultured mammalian neurons where a distinct class of RNPs contain the degradative enzyme Xrn1, which is excluded from RNPs supposedly involved in storage (Hillebrand, 2010).

    At synapses, a transition between storage and degradation particles may occur by three, non-exclusive, candidate mechanisms: (1) by the remodeling of a storage mRNP to a degradative one through protein exchange; (2) by the initial exit of mRNA from the storage RNP to a translating pool, followed by its subsequent targeting to a degradative particle; or (3) the fusion of the two particles. Recent studies in Drosophila provide a possible mechanism by which a change of proteins in RNP complexes could alter its function. Two related proteins of the Lsm-family, Enhancer of Decapping 3 (EDC3), which is implicated to play a role in mRNA decay, and Trailer Hitch (Tral), which supposedly is involved in mRNA repression, interact at the same domain with the Me31B protein. This suggests that the function of Me31B complexes might be determined by the interaction with specific binding partners. Some support for the second model is provided by the observation that the synaptically localized Arc mRNA is targeted for degradation after its translation is induced by synaptic activity and also by the observation that RCK-positive particles in dendrites of cultured hippocampal neurons are transiently disassembled following BDNF stimulation. Further studies are required to understand how, when, and even whether these transitions of mRNA state occur in synapses and other biological contexts (Hillebrand, 2010).

    Together with many analogous studies in yeast and mammalian cells, previous observations in Drosophila that Me31B is a repressor of maternal mRNA translation, a component of a repression pathway mediated by the bantam microRNA, and a repressor of growth of terminal dendrites, has led to a strong model that Me31B is a translational repressor protein. In contrast, recent studies in C. elegans and P. falciparum have shown that Me31B orthologs, CGH-1 and DOZI, associate with specific mRNAs and protects them from degradation (Hillebrand, 2010).

    The observations in neurons indicate a function for Me31B in repressing translation of a miRNA regulated, dendritically localized reporter mRNA in vivo. This is consistent with two related lines of data. First, it is consistent with the known function for Me31B in repression of miRNA-target genes in Drosophila wing imaginal cells as well as for its human homolog RCK in mammalian cultured cells. Second, the correlation observed between loss of synaptic Dcp1 puncta and upregulation of the CaMKII reporter, is consistent with observations in hippocampal cultured cells, where observed disassembly of 'dendritic P-bodies' induced by synaptic stimulation has been proposed to underlie the temporally coincident translation of localized mRNAs (Hillebrand, 2010).

    Thus, this study suggestd a simple model in which neuronal Me31B, as well as its homologs in other metazoa, mediates the formation of synaptic mRNP particles that contain locally repressed mRNAs. And that synaptic stimulation-induced disassembly of these particles is one aspect of the mechanism of local translational control (Hillebrand, 2010).

    One key goal of future studies will be to understand the composition and dynamics of dendritic mRNPs in vivo. This will be aided by genetic techniques to replace endogenous translational control molecules with genetically encoded, fluorescently tagged variants that retain functional and localization patterns of the endogenous proteins. When coupled with procedures to induce local protein synthesis in dendrites, such reagents will allow analysis of functionally relevant particle dynamics in vivo. In addition, by eliminating the need for antibodies whose use may be associated with artifacts of inclusion and exclusion, such reagents may provide more direct insight into the real nature of synaptic mRNPs in vivo (Hillebrand, 2010).

    A second goal is to understand the mechanism by which Me31B regulates the expression of CaMKII reporter levels in vivo. Although Me31B has been shown to be required for the miRNA pathway it is also required for other forms of translational repression, for example in S. cerevisiae that does not have miRNAs. Similarly, although the reporter used in this study is miRNA regulated, the same UTR also has binding sites for translational regulators that may operate independently of miRNAs. Thus, important and linked goals of future studies are to understand mechanisms by which the CaMKII UTR is regulated in dendrites and how Me31B engages with these mechanisms of neuronal translational control (Hillebrand, 2010).

    The chromatin remodeling factor Bap55 functions through the TIP60 complex to regulate olfactory projection neuron dendrite targeting

    The Drosophila olfactory system exhibits very precise and stereotyped wiring that is specified predominantly by genetic programming. Dendrites of olfactory projection neurons (PNs) pattern the developing antennal lobe before olfactory receptor neuron axon arrival, indicating an intrinsic wiring mechanism for PN dendrites. These wiring decisions are likely determined through a transcriptional program. This study found that loss of Brahma associated protein 55 kD (Bap55) results in a highly specific PN mistargeting phenotype. In Bap55 mutants, PNs that normally target to the DL1 glomerulus mistarget to the DA4l glomerulus with 100% penetrance. Loss of Bap55 also causes derepression of a GAL4 whose expression is normally restricted to a small subset of PNs. Bap55 is a member of both the Brahma (BRM) and the Tat interactive protein 60 kD (Tip60) ATP-dependent chromatin remodeling complexes. The Bap55 mutant phenotype is partially recapitulated by Domino and Enhancer of Polycomb mutants, members of the TIP60 complex. However, distinct phenotypes are seen in Brahma and Snf5-related 1 mutants, members of the BRM complex. The Bap55 mutant phenotype can be rescued by postmitotic expression of Bap55, or its human homologs BAF53a and BAF53b. These results suggest that Bap55 functions through the TIP60 chromatin remodeling complex to regulate dendrite wiring specificity in PNs. The specificity of the mutant phenotypes suggests a position for the TIP60 complex at the top of a regulatory hierarchy that orchestrates dendrite targeting decisions (Tea, 2011).

    The stereotyped organization of the Drosophila olfactory system makes it an attractive model to study wiring specificity. The first olfactory processing center is the antennal lobe, a bilaterally symmetric structure at the anterior of the Drosophila brain. It is composed of approximately 50 glomeruli in a three-dimensional organization. Each olfactory projection neuron (PN) targets its dendrites to one of those glomeruli to make synaptic connections with a specific class of olfactory receptor neurons. Each PN sends its axon stereotypically to higher brain centers (Tea, 2011).

    During development, the dendrites of PNs pattern the antennal lobe prior to axons of olfactory receptor neurons. The specificity of PN dendrite targeting is largely genetically pre-determined by the cell-autonomous action of transcription factors, several of which have been previously described. Furthermore, chromatin remodeling factors have been shown to play an important role in PN wiring (Tea, 2010), although very little is currently known about their specific functions. This study reports a genetic screen for additional factors that regulate PN dendrite wiring specificity; Brahma associated protein 55 kD (Bap55) was identified as a regulator of PN dendrite wiring specificity as part of the TIP60 chromatin remodeling complex (Tea, 2011).

    Bap55 is an actin-related protein, the majority of which physically associates with the Brahma (BRM) chromatin remodeling complex in Drosophila embryo extracts. There are two distinct BRM complexes: BAP (Brahma associated proteins; homologous to yeast SWI/SNF) and PBAP (Polybromo-associated BAP; homologous to yeast RSC), both of which contain Brahma, Bap55, and Snf5-Related 1 (Snr1). The human homologs of the BAP and PBAP complexes are called the BAF (Brg1 associated factors) and PBAF (Polybromo-associated BAF) complexes, respectively. The BRM/BAF complexes are members of the SWI/SNF family of ATP-dependent chromatin-remodeling complexes, and have been shown to both activate and repress gene transcription, in some cases, of the same gene (Tea, 2011).

    In experiments purifying proteins in complex with tagged Drosophila Pontin in S2 cells, Bap55 was also co-purified as a part of the TIP60 complex, as determined by mass spectrometry. The TIP60 histone acetyltransferase complex has been shown to be involved in many processes, including both transcriptional activation and repression. The complex contains many components, including Bap55, Domino (Dom), and Enhancer of Polycomb (E(Pc)). Dom, homologous to human p400, is the catalytic DNA-dependent ATPase; its ATPase domain is highly similar to Drosophila Brahma and human BRG1 ATPase domains. E(Pc) is homologous to human EPC1 and EPC2 and is an unusual member of the Polycomb group; it does not exhibit homeotic transformations on its own, but rather enhances mutations in other Polycomb group genes (Tea, 2011).

    This study provides evidence that Bap55 functions as a part of the TIP60 complex rather than the BRM complex in postmitotic PNs to control their dendrite wiring specificity (Tea, 2011).

    To further understanding of dendrite wiring specificity in Drosophila olfactory PNs, a MARCM-based forward genetic screen was performed using piggyBac insertional mutants. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous background, permitting the study of essential genes in mosaic animals. In this screen, GH146-GAL4 was used to label a single PN born soon after larval hatching, which in wild-type (WT) animals always projects its dendrites to the dorsolateral glomerulus DL1 in the posterior of the antennal lobe. The DL1 PN also exhibits a stereotyped axon projection, forming an L-shaped pattern in the lateral horn, with additional branches in the mushroom body calyx. A mutant, called LL05955, was identified in which DL1 PNs mistargeted to the dorsolateral glomerulus DA4l in the anterior of the antennal lobe. This phenotype is strikingly specific, with 100% penetrance. Arborization of mutant axons, however, was not obviously altered. The piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. LL05955 is inserted into the coding sequence of Bap55, encoding a homolog of human BAF53a and BAF53b. Precise excision of the piggyBac insertion reverted the dendrite mistargeting phenotype, confirming that disruption of the Bap55 gene causes the dendrite mistargeting (Tea, 2011).

    In addition to causing DL1 mistargeting, Bap55 mutants also display neuroblast clone phenotypes. In WT, GH146-GAL4 can label three distinct types of PN neuroblast clones generated in newly hatched larvae. Two of these clones, the anterodorsal neuroblast clone and the lateral neuroblast clone, possess cell bodies that lie dorsal or lateral to the antennal lobe, respectively. PNs from these two lineages project their dendrites to stereotyped and nonoverlapping subsets of glomeruli in the antennal lobe. The third type of clone, the ventral neuroblast clone, has cell bodies that lie ventral to the antennal lobe and dendrites that target throughout the antennal lobe due to the inclusion of at least one PN that elaborates its dendrites to all glomeruli (Tea, 2011).

    In Bap55-/- PNs, anterodorsal neuroblast clones display a mild reduction in cell number, and their dendrites are abnormally clustered in the anterior dorsal region of the antennal lobe, including the DA4l glomerulus. Lateral neuroblast clones display a severe reduction in cell number, and the remaining dendrites are unable to target to many glomeruli throughout the antennal lobe. Ventral neuroblast clones display a mild reduction in cell number and a reduced dendrite mass throughout the antennal lobe. During development, the lateral neuroblast first gives rise to local interneurons before switching to produce PNs; in mutants affecting cell proliferation, this property of the lateral neuroblast displays as a severe reduction in GH146-labeled PNs. The severely reduced cell number in Bap55 mutants suggests that Bap55 is essential for neuroblast proliferation or neuronal survival. In the anterodorsal and ventral neuroblasts, PN numbers are not drastically changed; thus, the phenotypes indicate that Bap55 is important for dendrite targeting in multiple classes of PNs (Tea, 2011).

    In WT, Mz19-GAL4 labels a subset of the GH146-GAL4 labeling pattern. It labels a small number of PNs derived from two neuroblasts, which can be clearly identified in WT clones generated in newly hatched larvae. Anterodorsal neuroblast clones target their dendrites to the VA1d glomerulus, as well as the DC3 glomerulus residing immediately posterior to VA1d (not easily visible in confocal stacks). Lateral neuroblast clones target all dendrites to the DA1 glomerulus. Unlike GH146-GAL4, WT Mz19-GAL4 never labels ventral neuroblast clones because it is not normally expressed in those cells (Tea, 2011).

    In Bap55 mutant PN clones, however, Mz19-GAL4 labels additional PNs in anterodorsal, lateral, and ventral clones compared to their WT counterparts. This phenotype suggests that some Mz19-negative PNs now express Mz19-GAL4. In anterodorsal clones, Mz19-GAL4 labels additional cells, although not as many as are labeled by GH146-GAL4. The PNs also mistarget their dendrites to the anterior antennal lobe, similar to mutant GH146-GAL4 anterodorsal neuroblast clones. WT lateral neuroblast clones normally contain GH146-positive PNs and GH146-negative local interneurons. In Bap55-/- lateral neuroblast clones, Mz19-GAL4 predominantly labels local interneurons that send their processes to many glomeruli throughout the antennal lobe and do not send axon projections to higher brain centers. Lateral clones also show ectopic PN labeling with a lower frequency. The Bap55 mutant appears to cause derepression of Mz19-GAL4, resulting in labeled local interneurons. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in Bap55 mutants. This further indicates a derepression of the Mz19-GAL4 labeling pattern (Tea, 2011).

    Unlike GH146-GAL4, WT Mz19-GAL4 never labels single cell clones when clone induction is performed in newly hatched larvae. This is because Mz19-GAL4 is not expressed in the DL1 PN, the only GH146-positive cell generated during this heat shock time of clone generation. However, in Bap55 mutant PN clones, Mz19-GAL4 ectopically labels single cell anterodorsal PN clones targeting to the DA4l glomerulus, which show an L-shaped pattern in the lateral horn with branches in the mushroom body calyx, similar to GH146-GAL4 labeling. The simplest interpretation is that this compound phenotype reflects first a derepression of Mz19-GAL4 in the DL1 PN, and second a DL1 to DA4l mistargeting phenotype in Bap55 mutants (Tea, 2011).

    To test whether Bap55 functions in the neuroblast or postmitotically in PNs, GH146-GAL4, which expresses only in postmitotic PNs, was used to express UAS-Bap55 in a Bap55-/- single cell clone. The dendrite mistargeting phenotype was shown to be rescued to the WT DL1 glomerulus and it is concluded that Bap55 functions postmitotically to regulate PN dendrite targeting. The axon phenotype remains the stereotypical L-shaped pattern (Tea, 2011).

    The Drosophila Bap55 protein is 70% similar and 54% identical to human BAF53a and 71% similar and 55% identical to human BAF53b. BAF53a and b are 91% similar and 84% identical to each other. Using GH146-GAL4 to express human BAF53a or b in a Bap55-/- single cell clone, it was found that the human homologs can effectively rescue the Bap55-/- dendrite mistargeting phenotype. Interestingly, both also cause the de novo DM6 dendrite and ventral axon mistargeting phenotypes in 6 out of 19 cases for BAF53a and 2 out of 32 cases for BAF53b. Thus, human BAF53a and b can largely replace the function of Drosophila Bap55 in PNs (Tea, 2011).

    To address whether Bap55 functions as a part of the BRM complex in PN dendrite targeting, two additional BRM complex mutants were tested for their PN dendrite phenotypes. First, Brahma (brm), the catalytic ATPase subunit of the BRM complex, which is required for the activation of many homeotic genes in Drosophila, was tested. Null mutations have been shown to decrease cell viability and cause peripheral nervous system defects. RNA interference knockdown of brm in embryonic class I da neurons revealed dendrite misrouting phenotypes, although not identical to the Bap55 phenotype. The human homologs of brm, BRM and BRG1 (Brahma-related gene-1), both have DNA-dependent ATPase activity. Inactivation of BRM in mice does not yield obvious neural phenotypes, but inactivation of BRG1 in neural progenitors results in reduced proliferation. BRG1 is likely to be required for various aspects of neural development, including proper neural tube development (Tea, 2011).

    In PNs, brm mutants displayed anterodorsal single cell clone mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone differing from the next. This is in contrast to the highly stereotyped DA4l mistargeting of Bap55 mutants. brm-/- neuroblast clones also displayed phenotypes where dendrites make small, meandering projections throughout the antennal lobe, which does not resemble the Bap55-/- phenotype. They additionally exhibit a perturbed cell morphology phenotype, which is markedly more severe than the Bap55 mutant phenotype (Tea, 2011).

    Next, Snr1, a highly conserved subunit of the BRM complex, was tested. It is required to restrict BRM complex activity during the development of wing vein and intervein cells and functions as a growth regulator. Its human homolog, SNF5, is strongly correlated with many cancers, yet little is known about its specific role in neurons (Tea, 2011).

    In PNs, Snr1 mutants displayed similar phenotypes to brm mutants. The single cell clones displayed mistargeting to non-stereotyped glomeruli throughout the antennal lobe, with each clone demonstrating a unique phenotype. The neuroblast clones exhibited small meandering dendrites throughout the antennal lobe, which also showed extremely perturbed cell morphology. These results, especially the non-sterotyped single cell clone phenotypes, indicate that the BRM complex functions differently from Bap55 in controlling PN dendrite targeting (Tea, 2011).

    brm and Snr1 mutants were further analyzed with Mz19-GAL4 to determine whether their phenotypes resembled the Bap55 mutant phenotype of derepression. It was not possible to generate any labeled clones, indicating one of three possibilities: increased cell death, severe cell proliferation defects, or repression of the Mz19-GAL4 labeling pattern. In any of the three cases, the phenotype does not resemble the Bap55-/- mutant phenotype of abnormal activation of Mz19-GAL4 in single cell or neuroblast clones, indicating that the BRM complex functions differently from Bap55 in PNs (Tea, 2011).

    In the same screen in which the Bap55 mutation was identified, LL05537, a mutation in dom that resulted in a qualitatively similar phenotype to Bap55 mutants was identified. dom-/- DL1 PNs split their dendrites between the posterior glomerulus DL1 and the anterior glomerulus DA4l. Their axons exhibit a WT L-shaped pattern in the lateral horn (Tea, 2011).

    The LL05537 allele contains a piggyBac insertion in an intron just upstream of the translation start of dom. Because the piggyBac insertion contains splice acceptor sites and stop codons in all three coding frames, this allele likely disrupts all isoforms of dom. Similarly to Bap55, the piggyBac insertion site was identified using inverse PCR and Splinkerette PCR. Precise excision of the piggyBac insertion reverted the dendrite targeting phenotype, confirming that disruption of the dom gene causes the dendrite mistargeting. In addition, a BAC transgene that contains the entire dom transcriptional unit rescued the dom-/- mutant phenotypes (Tea, 2011).

    Dom is the catalytic DNA-dependent ATPase of the TIP60 complex and has been shown to contribute to a repressive chromatin structure and silencing of homeotic genes. Dom is a member of the SWI/SNF family and its ATPase domain is highly similar to the Drosophila Brahma and human BRG1 ATPase domains. The human homolog of Dom is p400, which is important for regulating nucleosome stability during repair of double-stranded DNA breaks and an important regulator of embryonic stem cell identity (Tea, 2011).

    To determine whether Bap55 and Dom genetically interact, UAS-Bap55 was expressed in a dom-/- PN. This manipulation did not significantly alter the dendrite phenotype. The axon branching pattern also was not altered (Tea, 2011).

    Another component of the TIP60 complex, E(Pc), was also examined. In Drosophila, E(Pc) is a suppressor of position-effect variegation and heterozygous mutations in E(Pc) result in an increase in homologous recombination over nonhomologous end joining at double-stranded DNA breaks. Following ionizing radiation, heterozygous animals also exhibit higher genome stability and lower incidence of apoptosis. Yet little is known about its role in neurons (Tea, 2011).

    In this study, it was found that E(Pc)-/- DL1 PN dendrites also mistarget to the anterior glomerulus DA4l and exhibit the stereotyped L-shaped axon pattern in the lateral horn. A BAC transgene that contains the entire E(Pc) transcription unit rescued the E(Pc) mutant phenotypes. To determine whether Bap55 and E(Pc) genetically interact, UAS-Bap55 was expressed in an E(Pc)-/- DL1 PN. This manipulation caused the dendrites to split between the DA4l and DM6 glomeruli, and resulted in axons targeting ventrally to the lateral horn (Tea, 2011).

    Neuroblast clones mutant for dom also exhibit dendrite mistargeting phenotypes to inappropriate glomeruli throughout the antennal lobe. Anterodorsal and lateral neuroblast clones show a very mild reduction in cell number and their dendrites do not target to the full set of proper glomeruli. Ventral neuroblast clones, when compared to WT, exhibit incomplete targeting throughout the antennal lobe (Tea, 2011).

    Further analysis of dom mutants by labeling with Mz19-GAL4 revealed the same derepression as in Bap55 mutants. dom mutant Mz19-GAL4 PN clones also label anterodorsal, lateral, and ventral neuroblast clones with phenotypes similar to GH146-GAL4 labeled neuroblast clones. In anterodorsal and lateral neuroblast clones, Mz19-GAL4 labels a large number of PNs that target to many glomeruli throughout the antennal lobe, although the cell number is smaller than GH146-GAL4 labeling. Ventral neuroblast clones are never labeled in WT Mz19-GAL4, yet are labeled in dom mutants. Mz19-GAL4 also labels single cell clones that split their dendrites between the DA4l and DL1 glomeruli and form the stereotypical L-shaped axon pattern in the lateral horn. As in Bap55 mutants, this compound phenotype likely results from ectopic labeling of a DL1 PN, which further mistargets to DA4l (Tea, 2011).

    The E(Pc) phenotypes in GH146 and Mz19-GAL4 labeled neuroblast clones, as well as Mz19-GAL4 labeled single cell clones, displayed similar phenotypes to dom as described above. The phenotypic similarities in single cell clone dendrite mistargeting and derepression of a PN-GAL4 in mutations that disrupt Bap55, dom and E(Pc) strongly suggest that these three proteins act together in the TIP60 complex to regulate PN development (Tea, 2011).

    This study has demonstrated a similar role for three members of the TIP60 complex in olfactory PN wiring. The TIP60 complex plays a very specific role in controlling dendrite wiring specificity, with a precise mistargeting of the dendrite mass in Bap55, dom, and E(Pc) mutants. This specific DL1 to DA4l mistargeting phenotype has only been seen in these three mutants, out of approximately 4,000 other insertional and EMS mutants screened, supporting the conclusion that the TIP60 complex has a specific function in controlling PN dendrite targeting. TIP60 complex mutants show discrete glomerular mistargeting, rather than randomly distributed dendrite spillover to different glomeruli. In contrast, perturbation of individual cell surface receptors often leads to variable mistargeted dendrites that do not necessarily obey glomerular borders, possibly reflecting the combinatorial use of many cell surface effector molecules. Even transcription factor mutants yield variable phenotypes. Interestingly, BRM complex mutants yield non-stereotyped phenotypes in PNs. No stereotyped glomerular targeting was seen for brm or Snr1 mutant dendrites; each PN spreads its dendrites across different glomeruli. These data suggest that different chromatin remodeling complexes play distinct roles in regulating neuronal differentiation. The uni- or bi-glomerular targeting to specific glomeruli implies that the TIP60 complex sits at the top of a regulatory hierarchy to orchestrate an entire transcriptional program of regulation (Tea, 2011).

    This study suggests a function for Bap55 in Drosophila olfactory PN development as a part of the TIP60 complex rather than the BRM complex. Another possibility could be that Bap55 also serves as the interface between the BRM and TIP60 complexes. While loss of core BRM complex components results in a more general defect, loss of Bap55 could specifically disrupt interactions with the TIP60 complex but maintain other BRM complex functions, causing a more specific targeting phenotype mimicking loss of TIP60 complex components (Tea, 2011).

    Interestingly, both human BAF53a and b can significantly rescue the Bap55-/- phenotype. Though in mammals BAF53a is expressed in neural progenitors and BAF53b is expressed in postmitotic neurons, they can perform the same postmitotic function in Drosophila PNs. Further, both can function with the TIP60 complex in PNs to regulate wiring specificity. These data suggest that the functions for BAF53a and b (in neural precursors and postmitotic neurons, respectively) diverge after the evolutionary split between vertebrates and insects (Tea, 2011).

    The discrete glomerular states of the mistargeting phenotypes may suggest a role for the TIP60 complex upstream of a regulatory hierarchy determining PN targeting decisions. It is possible that disrupting various components changes the composition of the complex. Additionally, overexpression of Bap55 in various mutant backgrounds might alter the sensitive stoichiometry of the TIP60 complex, resulting in targeting to different but still distinct glomeruli (Tea, 2011).

    Several mutants have been identified that cause DL1 PNs to mistarget to areas near the DM6 glomerulus (Tea, 2010). Interestingly, WT DM6 PNs have the most similar lateral horn axon arborization pattern to DL1 PNs. It is hypothesized that the transcriptional code for DM6 is similar to that of DL1, which is at least partially regulated by the TIP60 complex. The genes described in this manuscript are the only mutants that have yielded specific DA4l mistargeting to date. It is possible that the targeting 'code' for DA4l, DL1, and DM6 may be most similar, such that perturbation of the TIP60 complex might result in reprogramming of dendrite targeting. PNs have previously been shown to be pre-specified by birth order. Yet DA4l is born in early embryogenesis, DL1 is born in early larva, and DM6 is born in late larva. This implies that the TIP60 transcriptional code does not correlate with PN birth order. The mechanisms by which the TIP60 complex specifies PN dendrite targeting remain to be determined (Tea, 2011).

    This study has characterize PN phenotypes of mutants in the BRM and TIP60 complexes, with a focus on Bap55, which is shared by the two complexes. The TIP60 complex was found to play a very specific role in regulating PN dendrite targeting; mutants mistarget from the DL1 to the DA4l glomerulus. This specific mistargeting phenotype suggests that TIP60 controls a transcriptional program important for making dendrite targeting decisions (Tea, 2011).

    The RhoGEF trio functions in sculpting class specific dendrite morphogenesis in Drosophila sensory neurons

    As the primary sites of synaptic or sensory input in the nervous system, dendrites play an essential role in processing neuronal and sensory information. Moreover, the specification of class specific dendrite arborization is critically important in establishing neural connectivity and the formation of functional networks. Cytoskeletal modulation provides a key mechanism for establishing, as well as reorganizing, dendritic morphology among distinct neuronal subtypes. While previous studies have established differential roles for the small GTPases Rac and Rho in mediating dendrite morphogenesis, little is known regarding the direct regulators of these genes in mediating distinct dendritic architectures. This study demonstrates that the RhoGEF Trio is required for the specification of class specific dendritic morphology in dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS). Trio is expressed in all da neuron subclasses and loss-of-function analyses indicate that Trio functions cell-autonomously in promoting dendritic branching, field coverage, and refining dendritic outgrowth in various da neuron subtypes. Moreover, overexpression studies demonstrate that Trio acts to promote higher order dendritic branching, including the formation of dendritic filopodia, through Trio GEF1-dependent interactions with Rac1, whereas Trio GEF-2-dependent interactions with Rho1 serve to restrict dendritic extension and higher order branching in da neurons. Finally, it was shown that de novo dendritic branching, induced by the homeodomain transcription factor Cut, requires Trio activity suggesting these molecules may act in a pathway to mediate dendrite morphogenesis. Collectively, these analyses implicate Trio as an important regulator of class specific da neuron dendrite morphogenesis via interactions with Rac1 and Rho1 and indicate that Trio is required as downstream effector in Cut-mediated regulation of dendrite branching and filopodia formation (Iyer, 2012).

    This analysis demonstrates that Trio functions in promoting and refining class specific dendritic arborization patterns via GEF1- and GEF2-dependent interactions with Rac1 and Rho1, respectively. It was also demonstrated that Trio is required in mediating Cut induced effects on dendritic branching and filopodia formation suggesting that these molecules may operate in a common pathway to direct dendritic morphogenesis. Giniger and colleagues (NINDS/NIH) have likewise been investigating Trio function in da neurons via a non-overlapping, complementary experimental approach, and that they arrived at conclusions regarding Trio function largely consistent with those reported in this study (Iyer, 2012).

    Previous studies have demonstrated that Trio functions via its GEF1 domain in mediating the regulation of axon morphogenesis by modulating Rac1 activity, however much less is known regarding the potential in vivo functional role(s) of the Trio GEF2 domain. Intriguingly, a previous study demonstrated that trio mutant neuroblast clones display a neurite overextension phenotype from the dendritic calyx region of the mushroom body which strongly resembled the dendrite-specific overextension phenotype observed in RhoA mutant mushroom body clones suggesting that RhoA/Rho1 activation may be required for restricting dendritic extension. In Drosophila da neurons, trio loss-of-function analyses reveal a reduction in dendritic branching in three distinct da neuron subclasses (class I, III, and IV), indicating a functional role for Trio in promoting dendritic branching. However, class specific differences are observed with Trio gain-of-function studies in which Trio overexpression in class I neurons increases dendritic branching, whereas in class III neurons there is no change in overall dendritic branching, but rather a redistribution of branches, and in class IV there is a reduction in overall dendritic branching. The basis for these differences appear to lie in the observation that refinement of dendritic branching in da neurons is subject to the opposing roles of Rac1 and Rho1 activation via Trio-GEF1 and Trio-GEF2, respectively, where Trio-GEF1 activity promotes higher order dendritic branching, whereas Trio-GEF2 activity restricts higher order branching and also limits overall dendritic length/extension (Iyer, 2012).

    One of the key distinctions between class I versus class III and IV neurons relates to inherent differences in normal dendritic branching complexity and the relative roles of dynamic actin cytoskeletal based processes in these neurons which are known to mediate higher order branching including the dendritic filopodia of class III neurons and fine terminal branching in class IV neurons, whereas the class I neurons do not normally exhibit this degree of higher order branching and are predominantly populated by stable, microtubule-based primary and secondary branches. As such, Trio overexpression in these distinct subclasses may yield different effects on overall dendritic branching morphology based upon the normal distribution of actin cytoskeleton within these subclasses leading to unique effects on class specific dendritic architecture. Both loss-of-function and gain-of-function results support this hypothesis as the predominant effects are restricted to actin-rich higher order branching, whereas the primary branches populated by microtubles are relatively unaffected. This is further supported by the demonstration that trio knockdown suppresses Cut induced formation of actin-rich dendritic filopodia. Moreover, phenotypic analyses revealed that co-expression of Cut and Trio-GEF1 synergistically enhance dendritic branching in class I neurons likely due to increased activation of Rac1, whereas co-expression of Cut and Trio-GEF2 lead primarily to increased dendritic extension likely due to increased activation of Rho1. Thus, Trio mediated regulation of Rac1 and/or Rho1 signaling has the potential for sculpting dendritic branching and outgrowth/extension depending upon the combinatorial and opposing effects of Rac1 and Rho1 (Iyer, 2012).

    In contrast to Cut, which has been shown to be differentially expressed in da neuron subclasses and exert distinct effects on class specific dendritic arborization, this study has demonstrated that Trio is expressed in all da neuron subclasses and can exert distinct effects on class specific dendritic branching. For example, in all subclasses examined, loss-of-function analyses indicate Trio is required to promote dendritic branching and yet individual subclasses exhibit strikingly distinct dendritic morphologies. These results suggest that Trio is generally required in each of these subclasses to regulate branching, however alone is insufficient to drive these class specific morphologies solely via activation of Rac1 and/or Rho1 signaling. One logical hypothesis is that differential expression of RhoGAP family members in distinct da neuron subclasses may work in concert with Trio to refine class specific morphologies. The potential for combinatorial activity between Trio and various RhoGAPs is significant given that 20 RhoGAPs have been defined in the Drosophila genome. For example, given that class I da neurons exhibit a simple branching morphology which becomes more complex when Trio or Trio-GEF1 domains are overexpressed, perhaps there is higher expression of Rac-inactivating GAPs in class I neurons that function in limiting dendritic branching, whereas in the more complex class III or IV da neurons, there may be lower expression of RacGAPs. Since overexpression of Trio-GEF2 reduces dendritic branching complexity in all three da neuron subclasses analyzed, it might be predicted that Rho1 activation limits dendritic branching and that therefore the expression of RhoGAPs may be modulated to facilitate branching in class III and IV neurons relative to class I neurons. In concert, differential expression of RacGAPs and RhoGAPs together with the uniform expression of Trio in all da neuron subclasses could potentially account for differential levels of activation/inactivation of Rac1 and/or Rho1 in individual subclasses and thereby influence overall class specific dendritic architecture (Iyer, 2012).

    In support of this hypothesis, class-specific microarray analyses conducted in class I, III, and IV da neurons indeed reveal differential gene expression levels for most of the 20 known RhoGAP family members at a class-specific level. These expression analyses reveal one trend whereby select RhoGAP encoding genes are upregulated in the more complex class III and IV da neurons relative to the simple class I da neurons, whereas select RacGAP encoding genes are downregulated in complex neurons relative to simple neurons. Moreover, it is known that individual RhoGAPs display differential specificities for Rac, Rho and Cdc42 in vivo, such that a given RhoGAP may function in activating one or more of these small G proteins thereby increasing the potential for fine-tuning activation levels of a particular G protein at a class specific level. Furthermore recent studies provide direct evidence of the importance of RhoGAP family members in regulating da neuron dendritic morphogenesis. Analyses of the tumbleweed (tum) gene, which encodes the GTPase activating protein RacGAP50C, demonstrate that tum mutants display excessive da neuron dendritic branching. The dendritic phenotype observed in tum mutant da neurons is strikingly similar to that observed with Trio-GEF1 overexpression which also leads to excessive dendritic branching. Together these data suggest that Trio-GEF1 functions in activating Rac1 to promote dendritic branching whereas Tum/RacGAP50C function in inactivating Rac1 via its GTPase activity and thereby limit dendritic branching. In contrast, mutant analyses of the RhoGAP encoding gene, crossveinless-c, whose target in da neurons is the Rho1 small G protein, reveal defects in directional growth of da neuron dendrites. These results indicate that Crossveinless-C is required to inactivate Rho1 in order to promote directional dendritic growth and further suggest that a failure to inactivate Rho1 leads to restricted dendritic growth consistent with the phenotypes observed with Trio-GEF2 overexpression in all da neuron subclasses examined. These results, together with those presented herein, suggest that potential combinatorial activity of Trio and RhoGAP family proteins may converge in shaping the class specific dendritic architecture. Ultimately, future functional studies will be required to validate this hypothesis (Iyer, 2012).

    While previous studies have revealed Trio acts in concert with Abl and Ena in coordinately regulating axon guidance, the same regulatory relationship does not appear to operate in da neuron dendrites as Abl has been shown to function in limiting dendritic branching and the formation of dendritic filopoda, whereas both Ena functions in promoting dendritic branching. This study demonstrates that Trio functions in promoting dendritic branching, consistent with Ena activity, but in da neuron dendrites works in an opposite direction to Abl. These findings suggest that, at least in da neuron dendrites, Trio may operate in either an Abl-independent pathway or that Trio and Abl may exhibit a context dependent regulatory interaction that is distinctly different in dendrites versus axons (Iyer, 2012).

    Golgi outposts shape dendrite morphology by functioning as sites of acentrosomal microtubule nucleation in neurons

    Microtubule nucleation is essential for proper establishment and maintenance of axons and dendrites. Centrosomes, the primary site of nucleation in most cells, lose their function as microtubule organizing centers during neuronal development. How neurons generate acentrosomal microtubules remains unclear. Drosophila dendritic arborization (da) neurons lack centrosomes and therefore provide a model system to study acentrosomal microtubule nucleation. This study investigated the origin of microtubules within the elaborate dendritic arbor of class IV da neurons. Using a combination of in vivo and in vitro techniques, it was found that Golgi outposts can directly nucleate microtubules throughout the arbor. This acentrosomal nucleation requires gamma-tubulin and CP309, the Drosophila homolog of AKAP450, and contributes to the complex microtubule organization within the arbor and dendrite branch growth and stability. Together, these results identify a direct mechanism for acentrosomal microtubule nucleation within neurons and reveal a function for Golgi outposts in this process (Ori-McKenney, 2012).

    Microtubules are organized into dynamic arrays that serve as tracks for directed vesicular transport and are essential for the proper establishment and maintenance of neuronal architecture. The organization and nucleation of microtubules must be highly regulated in order to generate and maintain such complex arrays. Nucleating complexes, in particular, are necessary because spontaneous nucleation of new tubulin polymers is kinetically limiting both in vivo and in vitro. Gamma(Γ)-tubulin is a core component of microtubule organization centers and has a well-established role in nucleating spindle and cytoplasmic microtubules. Previous studies have proposed that in mammalian neurons, microtubules are nucleated by γ-tubulin at the centrosome, released by microtubule severing proteins, and then transported into developing neurites by motor protein. Indeed, injection of antibodies against γ-tubulin or severing proteins inhibited axon outgrowth in neurons cultured for one day in vitro (DIV1) (Ori-McKenney, 2012).

    However, proper neuron development and maintenance may not rely entirely on centrosomal sites of microtubule nucleation. Although the centrosome is the primary site of microtubule nucleation at DIV2, it loses its function as a microtubule-organizing center during neuronal development. In mature cultured mammalian neurons (DIV 11-12), γ-tubulin is depleted from the centrosome, and the majority of microtubules emanate from acentrosomal sites. In Drosophila dsas-4 mutants that lack centrioles, organization of eye-disc neurons and axon outgrowth are normal in third-instar larvae. Within the Drosophila peripheral nervous system (PNS), although dendritic arborization neurons contain centrioles, they do not form functional centrosomes, and laser ablation of the centrioles does not perturb microtubule growth or orientation (Nguyen, 2011). These results indicate that acentrosomal generation of microtubules contributes to axon development and neuronal polarity. How and where acentrosomal microtubule nucleation may contribute to the formation and maintenance of the more complex dendrites, and what factors are involved in this nucleation is unknown. Dendritic arborization (da) neurons provide an excellent system for investigating these questions. They are a subtype of multipolar neurons in the PNS of Drosophila melanogaster which produce complex dendritic arrays and do not contain centrosomes. Based on their patterns of dendrite projections, the da neurons have been grouped into four classes (I-IV) with branch complexity and arbor size increasing with class number. Class IV da neurons are ideal for studying acentrosomal microtubule nucleation because they have the most elaborate and dynamic dendritic arbor, raising intriguing questions about the modes of nucleation for its growth and maintenance (Ori-McKenney, 2012).

    One potential site of acentrosomal microtubule nucleation within these neurons is the Golgi complex. A number of studies have shown that the Golgi complex can nucleate microtubules in fibroblasts. Although, in these cell types, the Golgi is tightly coupled to the centrosome, it does not require the centrosome for nucleation. It does, however, require γ-tubulin, the centrosomal protein AKAP450, and the microtubule binding proteins CLASPs. When the Golgi is fragmented upon treatment with nocodazole, the dispersed Golgi ministacks can still promote microtubule nucleation, indicating that these individual ministacks contain the necessary machinery for nucleation (Ori-McKenney, 2012 and references therein).

    In both mammalian and Drosophila neurons, the Golgi complex exists as Golgi stacks located within the soma and Golgi outposts located within the dendrites. In cultured mammalian hippocampal neurons, these Golgi outposts are predominantly localized in a subset of the primary branches; however, in Drosophila class IV da neurons, the Golgi outposts appear throughout the dendritic arbor, including within the terminal branches (Ye, 2007). The Golgi outposts may provide membrane for a growing dendrite branch, as the dynamics of smaller Golgi outposts are highly correlated with dendrite branching and extension. However, the majority of larger Golgi outposts remains stationary at dendrite branchpoints and could have additional roles beyond membrane supply. It is unknown whether Drosophila Golgi outposts contain nucleation machinery similar to mammalian Golgi stacks. Such machinery could conceivably support microtubule nucleation within the complex and dynamic dendritic arbor. This study identifies a direct mechanism for acentrosomal microtubule nucleation within the dendritic arbor and reveal a role for Golgi outposts in this process. Golgi outposts contain both γ-tubulin and CP309, the Drosophila homolog of AKAP450, both of which are necessary for Golgi outpost-mediated microtubule nucleation. This type of acentrosomal nucleation contributes not only to the generation of microtubules at remote terminal branches, but also to the complex organization of microtubules within all branches of the dendritic arbor. Golgi outposts are therefore important centers of acentrosomal microtubule nucleation, which is necessary to establish and maintain the complexity of the class IV da neuronal arbor (Ori-McKenney, 2012).

    This study has addressed how microtubules are organized and nucleated within the complex arbor of class IV da neurons and how essential these processes are for dendrite growth and stability. Microtubule organization within different subsets of branches in da neurons must require many levels of regulation. This study has identified the first direct mechanism for acentrosomal microtubule nucleation within these complex neurons and has uncovered a role for Golgi outposts in this process. The data are consistent with the observation that pericentriolar material is redistributed to the dendrites in mammalian neurons (Ferreira, 1993) and that γ-tubulin is depleted from the centrosome in mature mammalian neurons (Stiess, 2010). This suggests that the Golgi outposts may be one structure involved in the transport of centriole proteins such as γ-tubulin and CP309. This study found that microtubule nucleation from these Golgi outposts correlates with the extension and stability of terminal branches, which is consistent with the observation that EB3 comet entry into dendritic spines accompanies spine enlargement in mammalian neurons (Jaworski, 2009). It is striking that microtubule organization in shorter branches, but not primary branches, mimics the organization in mammalian dendrites, with a mixed microtubule polarity in the secondary branches and a uniform plus end distal polarity in the terminal branches. Kinesin-2 and certain +TIPS are necessary for uniform minus end distal microtubule polarity in the primary dendrites of da neurons. Golgi outpost mediated microtubule nucleation could also contribute to establishing or maintaining this polarity both in the terminal branches and in the primary branches. It will be of interest to identify other factors that may be involved in organizing microtubules in different subsets of branches in the future (Ori-McKenney, 2012).

    In vivo and in vitro data support a role for Golgi outposts in nucleating microtubules at specific sites within terminal and primary branches. However, it is noted that not all EB1 comets originate from Golgi outposts, indicating other possible mechanisms of generating microtubules. One potentially important source of microtubules is the severing of existing microtubules by such enzymes as katanin and spastin, both of which are necessary for proper neuronal development. It is likely that both microtubule nucleation and microtubule severing contribute to the formation of new microtubules within the dendritic arbor; however, the current studies suggest that Golgi-mediated nucleation is especially important for the growth and maintenance of the terminal arbor. In γ-tubulin and CP309 mutant neurons, the primary branches contain a similar number of EB1 comets, but only a small fraction of the terminal branches still contain EB1 comets. This result indicates that severing activity or other sources of nucleation may suffice for microtubule generation within the primary branches, but γ-tubulin mediated nucleation is crucial in the terminal branches. As a result, the terminal branch arbor is dramatically reduced by mutations compromising the γ-tubulin nucleation activity at Golgi outposts (Ori-McKenney, 2012).

    It is important to note that Golgi outposts are present in the dendrites, but not in the axons of da neurons; thus, this mode of nucleation is dendrite specific and likely contributes to the difference in microtubule arrays in axons and dendrites. While the axon is one long primary branch with uniform microtubule polarity, the dendrite arbor is an intricate array of branches where microtubule polarity depends on branch length. Therefore, this more elaborate branched structure may have evolved a variety of nucleation mechanisms, including Golgi outpost nucleation and microtubule severing. Intriguingly, in da neurons lacking cytoplasmic dynein function, the Golgi outposts are mislocalized to the axon, which appears branched and contains microtubules of mixed polarity (Zheng, 2008). It is speculated that in these mutants, Golgi-mediated microtubule nucleation within the axon is contributing to the mixed microtubule orientation and formation of ectopic dendrite-like branches (Ori-McKenney, 2012).

    Only a subpopulation of Golgi outposts could support microtubule nucleation both in vivo and in vitro. The results show that Golgi outpost mediated microtubule nucleation is restricted to stationary outposts and dependent upon γ-tubulin and CP309, but why some outposts contain these proteins while others do not is unknown. γ-tubulin and CP309 could be recruited to the Golgi outposts in the cell body and transported on the structure into the dendrites, or they could be recruited locally from soluble pools throughout the dendritic arbor. Golgi outposts are small enough to be trafficked into terminal branches that are 150-300 nm in diameter, and therefore may provide an excellent vehicle for transporting nucleation machinery to these remote areas of the arbor. It will be interesting to determine how these nucleation factors are recruited to the Golgi outposts (Ori-McKenney, 2012).

    It has been previously shown that GM130 can recruit AKAP450 to the Golgi complex, but whether the first coiled-coil domain of the Drosophila AKAP450 homolog, CP309, can also bind GM130 is unknown. Interestingly, this study has observed that predominantly stationary Golgi outposts correlated with EB1 comet formation, indicating that this specific subpopulation may contain γ-tubulin and CP309. What other factors may be necessary to properly position the Golgi outposts at sites such as branchpoints, and how this is achieved will be a fascinating direction for future studies (Ori-McKenney, 2012).

    Whether the acentrosomal microtubule nucleation uncovered in this study also occurs in the dendrites of mammalian neurons is a question of great interest. Golgi outpost distribution in cultured hippocampal neurons is significantly different than that in da neurons, and hippocampal neurons do not form as elaborate arbors as da neurons. However, other types of mammalian neurons form much more complex dendritic arbors and may conceivably require acentrosomal nucleation for the growth and perpetuation of the dendrite branches (Ori-McKenney, 2012).

    This study provides the first evidence that Golgi outposts can nucleate microtubules at acentrosomal sites in neurons, shedding new light on the longstanding question about the origin of the microtubule polymer in elongated neuronal processes. This source of nucleation contributes to the complex organization of microtubules within all branches of the neuron, but is specifically necessary for terminal branch development. It is thus conclude that acentrosomal microtubule nucleation is essential for dendritic branch growth and overall arbor maintenance of class IV da neurons, and that Golgi outposts are important nucleation centers within the dendritic arbor (Ori-McKenney, 2012).

    Centrosomin represses dendrite branching by orienting microtubule nucleation

    Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

    Neurons primarily receive inputs through their dendrite arbors. The shape and complexity of the dendrite arbor, which is elaborated during differentiation, enables the neuron to properly cover its receptive field and establishes the positions of inputs into the arbor. Disruptions to dendritic branching can precipitate intellectual disability and psychiatric disorders (Yalgin, 2015).

    Arbor morphology is regulated for each neuron class to support its structural and functional requirements3; it is genetically encoded, being linked to class specification by transcriptional programs. For example, in Drosophila, the single unbranched dendrite of external sensory neurons is specified over an alternative multipolar dendritic arborization (da) neuron fate by the Prdm transcription factor Hamlet. Similarly, the proneural transcription factor Ngn2 regulates multiple aspects of pyramidal neuron development in the mammalian cortex, including the specification of a characteristic apical dendrite, whereas Cux1, Cux2 and SatB2 link dendrite development to cortical layer-specific developmental program (Yalgin, 2015).

    Dendrite development is controlled in a neuron class-specific manner to create differences in arbor morphology and complexity. Class-specific dendrite targeting is regulated via the activity of transmembrane adhesion proteins. For example, in C. elegans, class-specific expression patterns of the transcription factors MEC-3, AHR-1 and ZAG-1 regulate the morphology of mechanosensory neurons, and MEC-3 promotes differential expression of the Claudin-like membrane protein HPO-30 to enable lateral branch stabilization. Drosophila da neurons exist in four classes, of which class I neurons express Abrupt (Ab), which defines their simple arbor shape, and class IV express Knot and Cut, which together promote the complex morphology of this class. The EGF-repeat factor Ten-m is co-regulated by both Knot and Ab to control the direction of branch outgrowth in both class I and IV neurons (Yalgin, 2015).

    Contrasting activities of Knot, Cut and Ab in da neurons emphasize that altering dendrite branching is fundamental for regulating arbor complexity. Knot and Cut promote branch formation; conversely, Ab represses branch formation. Little is understood about how modulatory control over branching is achieved (Yalgin, 2015).

    Microtubules polymerize via the addition of Tubulin dimers, primarily at the plus end. In axons, microtubules polymerize in an anterograde direction, providing a protrusive force for outgrowth. Microtubule polymerization also drives axon branch formation, as precursors only transform into branches after microtubule invasion. Mature dendrites have a predominantly minus-ends-out microtubule array, nevertheless recent studies have identified that anterograde microtubule polymerization events can initiate or extend branches, or modulate the size of dendritic spines. In addition, the re-initiation of dendrite growth and branch formation following injury uses upregulation of microtubule polymerization and its polarization in the anterograde direction (Yalgin, 2015).

    This study examined whether class-specific transcription factors regulate branch promotion and repression by controlling microtubule organization during arbor development. In da sensory neurons, microtubule nucleation and polarity can be assayed in vivo using transgenic markers. Using genetic manipulation of class I and class IV da neurons, this study found that Ab controls class-specific differences in the localization of microtubule minus-end-directed markers in the da neuron arbor. By assaying Ab-mediated changes in the expression of a set of candidate microtubule regulators and using chromatin immunoprecipitation (ChIP), this study identified Cnn (Centrosomin) as an effector of Ab action. Cnn-centered control mechanisms, analogous to those that cluster microtubule nucleation events to create the mitotic spindle, are used in growing dendrites to regulate branching and to create class-specific arbor complexity (Yalgin, 2015).

    Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

    Neuronal dendrite branching is fundamental for building nervous systems. Branch formation is genetically encoded by transcriptional programs to create dendrite arbor morphological diversity for complex neuronal functions. In Drosophila sensory neurons, the transcription factor Abrupt represses branching via an unknown effector pathway. Targeted screening for branching-control effectors identified Centrosomin, the primary centrosome-associated protein for mitotic spindle maturation. Centrosomin repressed dendrite branch formation and was used by Abrupt to simplify arbor branching. Live imaging revealed that Centrosomin localized to the Golgi cis face and that it recruited microtubule nucleation to Golgi outposts for net retrograde microtubule polymerization away from nascent dendrite branches. Removal of Centrosomin enabled the engagement of wee Augmin activity to promote anterograde microtubule growth into the nascent branches, leading to increased branching. The findings reveal that polarized targeting of Centrosomin to Golgi outposts during elaboration of the dendrite arbor creates a local system for guiding microtubule polymerization (Yalgin, 2015).

    Dynein-Dependent Transport of nanos RNA in Drosophila Sensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

    Intracellular mRNA localization is a conserved mechanism for spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity. This study investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites. Dynein motor machinery components were shown to mediate transport of nos mRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar function in da neurons for formation and transport of nos RNP particles. nos was shown to regulate neuronal function, most likely independently of its dendritic localization and function in morphogenesis. These results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts (Xu, 2013).

    This study has combined a method that allows live imaging of mRNA in intact Drosophila larvae with genetic analysis to investigate the mechanism underlying transport of nos mRNA in class IV da neurons. Live imaging over the short time periods allowed has provided a snapshot into the steady-state behavior of nos*RFP particles in the proximal dendrites of mature da neurons. The results indicate that anterograde transport of nos RNP particles into and within da neuron dendrites is mediated by dynein and is consistent with the minus-end out model for microtubule polarity in the proximal dendrites of da neurons (Zheng, 2008). This model predicts that bidirectional trafficking would be mediated by opposite polarity motors and the predominance of retrograde movement of nos*RFP particles when dynein function is partially compromised is consistent with this. Moreover, Rab-5 endosomes, whose accumulation in class IV da neuron dendrites is dynein-dependent, also exhibit bidirectional movement (Satoh, 2008), suggesting that different cargos may use similar dendritic transport strategies. Unfortunately, the severe defects caused by loss of kinesin have thus far hampered confirmation of a role for kinesin in these events (Xu, 2013).

    The observed bidirectional movement of nos RNP particles resembles the constant bidirectional transport observed for dendritic mRNAs near synapses in hippocampal neurons. In contrast to da neurons, proximal dendrites of mammalian neurons have mixed microtubule polarity so that bidirectional trafficking could be mediated by a single motor that switches microtubules or by switching between the activities of plus-end and minus-end motors. The association of kinesin with neuronal RNP granule components and inhibition of CaMKIIα RNA transport by dominant-negative inhibition of kinesin has implicated kinesin as the primary motor for dendritic mRNA transport. However, a recent study showed that dynein mediates unidirectional transport of vesicle cargoes into dendrites of cultured hippocampal neurons as well as bidirectional transport within the dendrites. Whether dynein plays a role in RNP particle transport in mammalian dendrites as it does in Drosophila neurons remains to be determined (Xu, 2013).

    Despite its prevalence, the role of bidirectional motility is not yet clear. A recently proposed 'sushi belt' model suggests that neuronal RNP particles traffic back and forth along the dendrite until they are recruited by an active synapse and disassembled for translation (Doyle, 2011). Although da neuron dendrites do not receive synaptic input, this continual motility may provide a reservoir of nos mRNA that can be rapidly mobilized for translation locally in response to external signals that regulate dendrite branching (Xu, 2013).

    These studies have shown that nos mRNA can be adapted for different localization mechanisms depending on cellular context: diffusion/entrapment in late oocytes that lack a requisite polarized microtubule cytoskeleton and microtubule-based transport during germ cell formation in the embryo and in class IV da neurons. Surprisingly, Rump and Osk are specifically required for nos localization in both oocytes and da neurons, suggesting that they function in the assembly or recognition of a fundamental nos RNP that can be adapted to both means of localization. However, because it is not possible to distinguish individual particles within the cell body, the possibility cannot be ruled out that Rump and/or Osk mediate coupling of nos RNP particles to dynein motors rather than particle formation. Within the germ plasm, nos associates with Vasa (Vas), a DEAD-box helicase, and is transported together with Vas into germ cells. Although dendritic branching complexity is reduced in vas mutants, no effect on dendritic localization of nos RNP particles was detected, suggesting that only a subset of germ plasm components are shared by neuronal localization machinery. A role for osk in learning and memory was proposed based on the isolation of an enhancer trap insertion upstream of osk in a screen for mutants with defective long-term memory, but osk function in memory formation has not been directly tested. Notably, however, a recent study showed that the osk ortholog in the cricket Gryllus bimaculatus functions in development of the embryonic nervous system rather than in germ cell formation. Thus, the ancestral function of osk appears to be in neural development, whereas its role in germ plasm formation is a later adaptation in higher insects. The results showing that Osk protein function is not limited to Dipteran germ plasm organization but also plays an important role in neuronal development and function supports this idea (Xu, 2013).

    The data indicate that the Nos/Pum complex is not only required for da neuron morphogenesis, but also for nociceptive function. However, nociception does not appear to require local function of Nos/Pum in the dendrites and reduced dendritic branching does not necessarily correlate with a deficit in nociception. These results suggest that morphogenesis and function are regulated separately and that Nos/Pum plays a second role in regulating the somatic translation of proteins required for the nociceptive response. Systematic identification of Nos/Pum targets will be essential to further investigate these different roles (Xu, 2013).

    GM130 is required for compartmental organization of dendritic Golgi outposts

    Golgi complexes (Golgi) play important roles in the development and function of neurons. Not only are Golgi present in the neuronal soma (somal Golgi), they also exist in the dendrites as Golgi outposts. Previous studies have shown that Golgi outposts serve as local microtubule-organizing centers and secretory stations in dendrites. It is unknown whether the structure and function of Golgi outposts differ from those of somal Golgi. This study show sin Drosophila that, unlike somal Golgi, the biochemically distinct cis, medial, and trans compartments of Golgi are often disconnected in dendrites in vivo. The Golgi structural protein Golgi matrix protein 130 kD ortholog (GM130) is responsible for connecting distinct Golgi compartments in soma and dendritic branch points, and the specific distribution of GM130 determines the compartmental organization of dendritic Golgi in dendritic shafts. It was further shown that compartmental organization regulates the role of Golgi in acentrosomal microtubule growth in dendrites and in dendritic branching. This study provides insights into the structure and function of dendritic Golgi outposts as well as the regulation of compartmental organization of Golgi in general (Zhou, 2014).

    Dendritic Golgi outposts have been previously shown to participate in the patterning of dendritic branches of Drosophila da neurons. To test whether Golgi compartmental organization might contribute to dendritic branching, Golgi compartmental organization was examined in several mutants with dendritic branching defects. Loss-of-function mutations of the transcription factor dar1, which reduces dendritic branching in class III da (C3 da) neurons (Ye, 2011) led to a decrease in the percentage of multicompartmental Golgi in dendrites without affecting the Golgi in the soma and branch points. In contrast, overexpression of Knot, a transcription factor known to increase dendritic growth, did not change the compartmental organization in dendrites. These results suggest that certain regulators of dendritic branching and growth may act by regulating the compartmental organization of dendritic Golgi outposts (Zhou, 2014).

    A recent study suggests that dendritic Golgi outposts regulate dendritic branching by functioning as acentrosomal nucleation sites for microtubules. In light of the current findings that the Golgi outposts comprise two populations, one with single compartments and the other with multiple compartments, it was asked whether the structural organization of Golgi outposts regulates microtubule growth. Microtubule growth was examined in da neurons in vivo by time-lapse imaging of EB1-GFP. EB1 binds to growing microtubule plus ends and moves in a way that resembles comets (hence termed 'EB1-GFP comets') as microtubules grow. The association of microtubule growth initiation with multicompartment outposts was compared to that with single-compartment outpost by live imaging the presence of ManII-EBFP and GalT-TagRFP together with EB1-GFP in wild-type da neurons. The number of microtubule initiation events associated with the Golgi outposts containing both medial- and trans-Golgi compartments was significantly greater than the events associated with single-compartment outposts. Consistent with the result that Golgi outposts at branch points contain multiple compartments, branch points also initiated more microtubule growth than the single- compartment outposts in dendritic shafts. In the dendritic shafts, 57.8% of microtubule growth initiation sites were associated with Golgi outposts containing both medial and trans compartments, compared to 8.9% for medial- only and 13.3% for trans- only outposts. 20% of dendritic microtubule growth initiation sites were either not associated with dendritic Golgi outposts or associated with Golgi outposts that were below detection sensitivity. These results raised the possibility that connecting multiple Golgi compartments promotes microtubule growth in vivo. Because introducing dGM130 into dendrites connects the cis, medial, and trans compartments of dendritic Golgi, the number of initiation events of microtubule growth in the dendrites were compared between control and dGM130-overexpressing neurons. The number of microtubule growth initiation events was significantly increased in the dendritic shafts of neurons overexpressing dGM130 compared to control. The increase was largely due to events associated with dGM130-containing multicompartment Golgi outposts. In dGM130 null mutant neurons, microtubule growth initiation events were reduced in distal dendrites. Because Golgi compartments in dGM130-deficient neurons were dispersed in branch points, microtubule growth initiation at these branch points was examined (Zhou, 2014).

    Consistently, microtubule growth initiation at dendritic branch points was dramatically suppressed by dGM130 mutations. In contrast, loss of dGMAP, another Golgi structural protein, did not affect microtubule growth initiation. Taken together, these results suggest that the dGM130-mediated compartmental organization of dendritic Golgi outposts regulates microtubule growth in dendrites (Zhou, 2014).

    The role of dGM130 was also assessed in dendritic branching was also assessed. The total number of dendritic branch points as well as the number of higher-order branches (fourth order and up) was significantly reduced in dGM130 mutant class III da neurons but increased in dGM130-overexpressing neurons of the same type. These results suggest that dGM130, and possibly compartmental organization of Golgi outposts, is a factor that determines the number of higher- order dendritic branches (Zhou, 2014).

    The mechanism underlying the microtubule growth regulated by dGM130 is currently unclear. dGM130 might regulate microtubule growth through three different mechanisms. First, different Golgi compartments may each serve a unique role in microtubule nucleation. Thus, multicompartment Golgi serve as a functional scaffold for the microtubule nucleation machinery. Second, GM130, rather than multicompartment Golgi, may be responsible for initiating microtubule growth. Third, it is also possible that dGM130 and compartmental organization of Golgi regulates microtubule growth indirectly through other Golgi functions such as membrane trafficking. Previous studies on mammalian hippocampal neurons have shown that ribbon-like Golgi stacks that are disconnected from somal Golgi and are positive for GM130 are located only in the soma and proximal dendrites. This has led to the speculation that Golgi outposts might exist only in proximal dendrites. However, this speculation contradicts the proposal that membrane proteins are synthesized locally at synapses in distal dendrites and ultrastructural and immunofluorescence studies showing the presence of membranous organelles positive for Golgi markers. The findings described in this study reconcile this contradiction by showing that Golgi in the soma and those in the dendrites assume different compartmental organizations (Zhou, 2014).

    Coordinate control of terminal dendrite patterning and dynamics by the membrane protein Raw

    The directional flow of information in neurons depends on compartmentalization: dendrites receive inputs whereas axons transmit them. Axons and dendrites likewise contain structurally and functionally distinct subcompartments. Axon/dendrite compartmentalization can be attributed to neuronal polarization, but the developmental origin of subcompartments in axons and dendrites is less well understood. To identify the developmental bases for compartment-specific patterning in dendrites, a screen was carried out for mutations that affect discrete dendritic domains in Drosophila sensory neurons. From this screen, mutations were identified that affected distinct aspects of terminal dendrite development with little or no effect on major dendrite patterning. Mutation of one gene, raw, affected multiple aspects of terminal dendrite patterning, suggesting that Raw might coordinate multiple signaling pathways to shape terminal dendrite growth. Consistent with this notion, Raw localizes to branch-points and promotes dendrite stabilization together with the Tricornered (Trc) kinase via effects on cell adhesion. Raw independently influences terminal dendrite elongation through a mechanism that involves modulation of the cytoskeleton, and this pathway is likely to involve the RNA-binding protein Argonaute 1 (AGO1), as raw and AGO1 genetically interact to promote terminal dendrite growth but not adhesion. Thus, Raw defines a potential point of convergence in distinct pathways shaping terminal dendrite patterning (Lee, 2015).

    Although the concept of positional information was first applied to embryonic development, intracellular positional information governs morphogenesis of individual cells as well. For example, positioning the nucleus at the cell center and growth zones at the cell periphery depends on positional information from the microtubule cytoskeleton in Schizosaccharomyces pombe. Several lines of evidence support the existence of distinct subcompartments in axons and dendrites, but the forms of intracellular positional information and the coordinate systems that guide the development of these subcompartments have not been extensively characterized. Results from this screen and other studies suggest that at least two types of positional information govern C4da dendrite patterning. First, terminal branch distribution along the proximal-distal axis depends on microtubule-based processes; perturbing microtubule-based transport leads to a distal-proximal shift in the distribution of terminal dendrites in C4da arbors. Interestingly, modulating the activity of the F-actin nucleator Spire also affects terminal dendrite positioning along the proximal-distal axis, suggesting that multiple pathways contribute to the fidelity of branch placement. Second, terminal dendrites rely on dedicated programs that may act locally to regulate terminal dendrite patterning. The observation that different pathways regulate different aspects of terminal dendrite development suggests that multiple signaling systems exist for the local control of dendrite growth (Lee, 2015).

    This study identified raw as a key regulator of terminal dendrite patterning. raw encodes a membrane protein that accumulates at branch-points and coordinately regulates terminal dendrite adhesion/stability via a pathway that involves Trc and terminal dendrite elongation via a pathway that is likely to involve cytoskeletal remodeling and AGO1. Raw therefore provides a potential point of integration for external signals that regulate these downstream growth programs. These pathways could be responsive to the same signal -- for example, Raw association with an extracellular ligand or a co-receptor -- or could be spatially/sequentially segregated. Identification of additional raw-interacting genes should help clarify the architecture of these signaling pathways (Lee, 2015).

    Raw regulates cell-cell signaling, and in gonad morphogenesis Raw modulates Cadherin-based interactions between somatic gonadal precursor cells and germ cells, in part by localizing Armadillo to the cell surface. Likewise, the data support a role for Raw in promoting Trc activation by localizing Trc to the plasma membrane. Thus, one plausible model for Raw function in dendrite development is that it interacts with an extracellular signal, which might be a component of the ECM or a cell surface protein on epithelial cells, and signals together with a co-receptor to stimulate downstream pathways for adhesion and cytoskeletal remodeling. Several analogous signaling systems involving interactions with the epidermis that influence terminal dendrite or sensory axon patterning have been described, but how many of these signaling systems are at work in a given neuron, and how Raw interfaces with other signaling pathways, remain to be determined (Lee, 2015).

    Although Raw has no obvious vertebrate counterpart, stretches of the ECD bear similarity to mucins and leucine-rich repeat proteins, one of which might serve an analogous function. Moreover, components of both downstream signaling pathways that this study identified are conserved in vertebrates and play known roles in dendrite patterning, including roles in the local control of dendrite growth: the Trc orthologs NDR1/2 (STK38/STK38L) regulate aspects of dendrite branch and spine morphogenesis, and Argonaute proteins mediate miRNA-mediated control of dendrite patterning, in part through local effects on translation. Additionally, dendrites contain structures related to P-granules, and Argonaute proteins may influence local translation in P-granules as well. Thus, versions of the Raw-regulated signaling pathways might control terminal dendrite patterning in vertebrates (Lee, 2015).

    Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

    During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. This study shows that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, it was shown that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. These findings reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance (Han, 2014).

    Removal of nonfunctional or damaged tissues is an important biological process during tissue remodeling or repair. This study shows that, for Drosophila class IV da neurons in the periphery, degenerating dendrites in both dendrite pruning and injury models are removed by neighboring epithelial cells rather than professional phagocytes. By developing multiple dendritic markers that label phagosomes differentially, the clearance of degenerating dendrites was established as an in vivo model to study phagocytosis. With these tools, key players in engulfment and phagosome maturation were analyzed, and roles of the CD36 family members Crq and Dsb were elucidated. This study further reveals that, as phagocytes, epidermal cells actively participate in not only the removal but also the fragmentation of degenerating dendrites (Han, 2014).

    Professional phagocytes such as macrophages in vertebrates and plasmatocytes in Drosophila dispose the majority of apoptotic cells in development, as well as invading microorganisms during infection. However, nonprofessional phagocytes may take charge when macrophages or other professional phagocytes are absent or cannot easily access cell corpses, as in apoptosis of rat lens cells, follicular atresia, and degeneration of Drosophila egg chambers induced by protein deprivation. This scenario does not apply to Drosophila da neuronal dendrites, which are exposed to circulating plasmatocytes in the hemolymph and sessile plasmatocytes clustered around da neuron somas. Indeed, previous observation of dendrite debris engulfment by plasmatocytes during dendrite pruning has led to the conclusion that plasmatocytes clear pruned dendrites. The current finding that clearance of degenerating dendrites is mainly carried out by epidermal epithelial cells demonstrates that nonprofessional phagocytes are not just a substitute for professional phagocytes in their absence. Rather, plasmatocytes and epidermal cells probably carry out different functions reflecting specialization of cellular functions. The removal of pruned dendrites by Drosophila epidermal cells perhaps can be seen as a parallel to the clearance of photoreceptor outer segments by retinal pigment epithelial cells; in both cases epithelial cells maintain homeostasis of the nervous system as part of their physiological functions. The observation that epidermal cells are also responsible for clearing injured dendrites indicates that the same cellular mechanism is also used to cope with perturbations in the peripheral nervous system (Han, 2014).

    Epithelial cells may profoundly influence the development of dendritic arbors of da neurons. During larval development, growing epithelial cells signal to the dendritic arbors so they can grow proportionally to epithelial cells in order to maintain the same coverage of receptive fields of the sensory neurons, a phenomenon known as dendritic scaling. Epithelial cells also contribute to the patterning of dendritic arbors of da neurons by tethering dendrites to the 2D space of the extracellular matrix so that dendrites have to avoid sister dendrites from the same neuron (self-avoidance) or dendrites from neighboring like-neurons (tiling). The finding that epithelial cells mediate the clearance of degenerating dendrites substantially adds to the growing list of dendrite properties regulated by epithelial cells (Han, 2014).

    The vertebrate CD36 family members CD36 and scavenger receptor class B type I (SR-BI) mediate phagocytosis of apoptotic cells and microbial pathogens in vitro. The Drosophila CD36 family member Crq is required for efficient phagocytosis of cell corpses in embryos and mediates binding of apoptotic cells by in vitro cultured cells, leading to its proposed role as a receptor for apoptotic cells. This study shows that in epithelial cells crq is required for phagosome maturation but not for the engulfment of degenerating dendrites. As loss of crq does not completely abolish the engulfment of apoptotic cells in the embryo, it is possible that the cell-corpse clearance defect in crq mutant embryos may be a consequence of blocked phagosome maturation. An alternative possibility is that Crq may be required for engulfment and/or phagosome maturation of apoptotic cells by embryonic macrophages but only required for phagosome maturation of pruned or injured dendrites by epithelial cells. This could be due to the fact that macrophages have to actively search for and bind apoptotic cells, while epithelial cells engulf neighboring debris. Further experiments will be needed to determine whether Crq also plays a role in phagosome maturation during phagocytosis by macrophages (Han, 2014).

    Loss of Crq function resulted in the fusion of dendrite-derived phagosomes accompanied with a failure of degradation of phagosome contents, most likely due to inefficient delivery of degradation machineries to late phagosomes. As phagosomes normally acquire hydrolases and other phagolysosomal components by fusing with endosomes and lysosomes, it is hypothesized that Crq suppresses homotypic phagosome fusion to promote fusion between phagosomes and late endosomes/lysosomes. Homotypic phagosome fusion rarely happens during normal phagocytosis but is induced by infection of bacterial pathogens such as Helicobacter pylori and Chlamydia trachomatis; the ability of different strains of H. pylori to induce phagosome fusion correlates with the virulence and intracellular survival of these bacteria. Therefore, regulation of the balance between homotypic phagosome fusion and heterotypic fusion between phagosomes and late endosomes/lysosomes is probably critical for the degradation of internalized materials (Han, 2014).

    The Drosophila genome encodes fourteen CD36 family members. Besides the involvement of Crq in phagocytosis, another member Pes mediates mycobacteria infection. This study found that the CD36 family member Dsb regulates late stages of phagosome maturation. Interestingly, LIMP-2, the mammalian CD36 family member with the highest homology to Dsb, is an intrinsic lysosomal protein required for the degradation of Listeria in phagosomes. Dsb and LIMP-2 thus appear to have evolutionarily conserved functions in phagosome maturation (Han, 2014).

    Phagocytes not only clear cell corpses but may also engulf still-living cells and promote cellular degeneration in many contexts. This study shows that efficient degeneration of dendrites requires the coordination with phagocytic epithelial cells. One mechanism for such coordination is the Drpr-mediated recognition of degenerating dendrites by epidermal phagocytes that form actin-rich membrane structures wrapping around the dendrites to facilitate their fragmentation. In the postnatal mouse brain, microglia actively induce apoptosis of Purkinje cells by producing superoxide ions. It remains to be determined whether nonprofessional phagocytes such as epidermal cells also promote neurite degeneration by emitting diffusible agents (Han, 2014).

    Double-bromo and extraterminal (BET) domain proteins regulate dendrite morphology and mechanosensory function

    A complex array of genetic factors regulates neuronal dendrite morphology. Epigenetic regulation of gene expression represents a plausible mechanism to control pathways responsible for specific dendritic arbor shapes. By studying the Drosophila dendritic arborization (da) neurons, this study discovered a role of the double-bromodomain and extraterminal (BET) family proteins in regulating dendrite arbor complexity. A loss-of-function mutation in the single Drosophila BET protein encoded by female sterile 1 homeotic [fs(1)h] causes loss of fine, terminal dendritic branches. Moreover, fs(1)h is necessary for the induction of branching caused by a previously identified transcription factor, Cut (Ct), which regulates subtype-specific dendrite morphology. Finally, disrupting fs(1)h function impairs the mechanosensory response of class III da sensory neurons without compromising the expression of the ion channel NompC, which mediates the mechanosensitive response. Thus, these results identify a novel role for BET family proteins in regulating dendrite morphology and a possible separation of developmental pathways specifying neural cell morphology and ion channel expression. Since the BET proteins are known to bind acetylated histone tails, these results also suggest a role of epigenetic histone modifications and the 'histone code,' in regulating dendrite morphology (Bagley, 2014).

    Dendrites are the primary site of information input to neural circuits, and the shape of dendritic arbors influences the electrophysiological responses of neurons. Due to the existence of highly diverse morphologies among different neuronal subtypes, a question of the relationship between form and function arises: By understanding how the shape of a neuron is specified, it is possible to understand how morphology relates to neural function and how altered morphology relates to dysfunction (Bagley, 2014).

    Neurons can be defined by their physiology, morphology, and gene expression. Neuronal diversity is thought to arise from the combinatorial expression of genetic determinants. The dendritic arborization (da) sensory neurons of the Drosophila peripheral nervous system (PNS) constitute a powerful system to study genetic determinants of dendritic arbor morphology. In particular, the use of Drosophila genetic techniques to study the specification of stereotyped, subtype-specific dendritic arbor shapes resulted in the identification of multiple transcription factors, encoded by abrupt (ab), knot/collier (kn/col), spineless (ss), and cut (ct), which regulate dendritic arbor morphology. However, large-scale genomic analyses comparing the transcriptomes of various neural subtypes indicate a daunting amount of varied gene expression and implicate regulation by multiple transcription factors. Thus, a particular neuronal morphology is likely the result of coordination between multiple genomic programs (Bagley, 2014). Epigenetic modifications are one mechanism that could allow coordinated, genome-wide expression profiles. Chromatin is packaged into nucleosomes, where DNA nucleotides wrap an octamer of histone proteins. The chromatin structure can be altered through three main types of modifications, consisting of direct methylation of DNA nucleotides, post-translational histone-tail modifications such as acetylation and methylation, and ATP-dependent chromatin remodeling. ATP-dependent chromatin remodelers were first shown to regulate dendrite morphology when RNAi knockdown of brahma (brm)-associated protein 60kD (Bap60), Bap55, and the ATPase brm altered the dendritic arbors of class I da sensory neurons. In mammalian neurons, the neural-specific Brg/Brm-associated factor (BAF) complex (nBAF), which contains BAF53b and the ATPase Brg, regulates activity-dependent dendrite growth. In addition, the Drosophila BAF53a/b homolog Bap55 regulates dendritic targeting of olfactory projection neurons (PNs) (Bagley, 2014).

    The post-translational modification of histone tails involves three types of molecules: The 'writers' add methyl, acetyl, or phospho groups and consist of histone methyl transferase (HMT), histone acetyltransferase (HAT), and kinase enzymes. The 'erasers' remove these modifications and include demethylases (DMTs), histone deacetyltransferases (HDACs), and phosphatases. Finally, the 'readers' are scaffolding proteins that recognize and bind acetyl, methyl, or phosphate modifications to position the 'writer' and 'eraser' enzymes along with transcriptional machinery to the correct genomic position and thereby modify gene expression. The discovery that the Polycomb repressor complex, which binds methylated histone tails, regulates da sensory neuron dendrite morphology indicates a role of histone methylation in dendrite development and a notion supported by the recent finding that the chromodomain Y-like (CDYL) protein negatively regulates dendritic complexity (Qi, 2014). Regarding a role of histone acetylation in dendrite morphogenesis, both HDAC and HAT activities have been implicated in regulating dendrite morphology. Specifically, the Drosophila HDAC1/2 homolog Rpd3 regulates class I da sensory neuron morphology and olfactory PN dendritic targeting. In addition, HDAC2 suppresses dendritic spine density of hippocampal CA1 and dentate granule neurons. The HAT enzyme Pcaf also regulates class I da sensory neuron dendrite morphology. A different HAT enzyme, CREB-binding protein (CBP), regulates the developmental pruning of class IV da sensory neuron dendrites, and mutations in the human homolog CREBBP cause the mental retardation syndrome Rubenstein-Taybi. While these studies indicate a definite role of 'writers' and 'erasers' of histone modifications in regulating dendrite morphogenesis, the role of 'reader' scaffolding proteins associated with histone acetylation has not been thoroughly investigated (Bagley, 2014).

    Double-bromo and extraterminal (BET) domain-containing proteins bind acetylated histone tails (Umehara 2010a; Umehara 2010b) and modulate gene expression. In mice, mutations in one BET family member, BRD2, cause neural tube closure defects, behavioral abnormalities, and altered interneuron numbers. In addition, in certain human genomic population studies, mutations in BRD2 have been associated with juvenile myoclonic epilepsy and photosensitivity, which is frequently observed in idiopathic generalized epilepsies. In the current study, evidence is provided for a role of the Drosophila homolog of BRD2, encoded by female sterile 1 homeotic [fs(1)h], in regulating dendrite morphology and sensory function (Bagley, 2014).

    This study examined the role of fs(1)h in dendritic development. The effect of a loss-of-function allele [fs(1)h1112] was examined on the morphology of class III da sensory neurons in the Drosophila PNS. Overall, fs(1)h1112 causes a reduction in dendritic arbor complexity, most notably in the finer, higher-order branches. It was possible to partially rescue this reduced morphological complexity by reintroducing Drosophila Fsh-S or the human homolog (huBRD2) proteins. Furthermore, one aspect of the genetic mechanism of action for fs(1)h was found to be regulating the expression of ct (and possibly other genes in the pathway) in multiple da neuron subtypes as well as subtype-specific transcription factors, such as Abrupt for class I and Knot/Collier for class IV da neurons, which in turn affect subtype-specific dendrite development. The data show that fs(1)h regulates genetic pathways controlling dendritic arbor development but does not specify which ion channels are expressed. Finally, the results suggest that the subtype-specific spike morphology is important for an optimal response to relevant sensory stimuli in the mechanosensitive class III da neurons (Bagley, 2014).

    The development of a dendritic arbor involves multiple steps beginning with differentiation, where a neuronal precursor acquires a neural fate. Next, neurites begin to extend, and a neuron becomes polarized as neurites are designated as axon or dendrite. The immature axons and dendrites continue to grow as the neuron and the nervous system develop. Initially, the dendritic arbors are simple, with only a few primary dendrites, but as development progresses, the number of branches and overall arbor size increase. The terminal dendrite branches are dynamic throughout development, exhibiting growth, retraction, or stability. In addition, as the animal body size increases, the dendritic field area increases, and therefore a dendritic arbor must scale accordingly. Thus, dendritic development involves a complex plethora of processes, and dendritic morphology could be altered by affecting any of these processes. For instance, if the balance of dendrite dynamics is shifted such that retraction is greater than growth, then dendritic branching will become reduced over time. This appears to be the case in fs(1)h1112 mutants, since an increase was observed in retracting branches with no change in growth as well as a decrease in the proportion of stable branches in dendritic arbors of fs(1)h1112 mutant da neuron clones compared with wild-type clones. Alternatively, if scaling of the dendritic arbor is affected, the size of the dendritic arbor will become disproportionately small as the body size of the animal increases throughout development. This does not seem to occur in fs(1)h1112 mutants because the primary dendrites of fs(1)h1112 arbors exhibited growth throughout development, although at a delayed rate. Instead, the number of spikes in class III da neuron arbors was reduced early in development and remained reduced throughout development, probably due to the increased amount of dendritic branch retraction and reduced stability. Since the primary dendritic branches were not affected to a large degree by loss of fs(1)h function, it is concluded that the major role of fs(1)h in dendritic development is to regulate dendritic complexity at the level of higher-order dendritic spikes. Moreover, the data suggest that fs(1)h affects dendritic arbor complexity by modulating the dynamics of terminal dendritic branches (Bagley, 2014).

    In the da neurons, many molecules are known to regulate dendrite morphology. In particular, Ct, Ss, Ab, and Kn have been shown to regulate subtype-specific morphology of the four classes of da sensory neurons, and these proteins act in parallel genetic pathways. Moreover, the expression of Ct and Ss regulates class III da neuron spike morphology. This study observed a loss of Ct expression in fs(1)h1112 mutant class III da neuron clones, which suggests that fs(1)h regulates the induction or maintenance of Ct expression throughout class III da neuron development. However, reintroducing Ct expression to class III da neuron fs(1)h1112 clones did not rescue the nearly absent spike morphology. Therefore, the class III da neuron dendritic phenotype caused by loss of fs(1)h cannot be solely attributed to the loss of Ct protein. Since it is thought that Ct is a component of a genetic pathway responsible for subtype-specific dendritic arbor development, it is possible that fs(1)h regulates Ct expression as well as expression of genes necessary for the Ct pathway to affect dendritic morphology. Therefore, the relationship between ct and fs(1)h does not appear to be a linear pathway, and fs(1)h might regulate both upstream and downstream components of ct. These data indicating that fs(1)h is necessary for the Ct-induced overbranching and spike formation in class I da neuron dendrites support the idea that fs(1)h regulates the expression of downstream components of the Ct pathway, which are necessary for Ct-induced overbranching and spike formation. This hypothesis also explains why reintroducing Ct expression to fs(1)h1112 clones fails to rescue the dendrite phenotype. It is also known that Ct and Rac1 act synergistically to produce spike morphology. This study examined Rac1 overexpression in a fs(1)h1112 mutant background and found that Rac1 expression significantly rescued the loss of spikes in class III da neurons. However, Rac1-induced overbranching in class I da neurons was not affected by fs(1)h1112. Therefore, fs(1)h does not appear to regulate genes downstream from Rac1 but does regulate genes downstream from ct. Since these pathways are known to converge in order to regulate dendritic spike formation, the current data suggest that fs(1)h may be a crucial link between these two pathways. One possible scenario is that ct and Rac1 regulate parallel pathways, but ct may regulate the level of Rac1 expression such that increased Rac1 expression facilitates the formation of spikes. In this model, the results support the hypothesis that fs(1)h is necessary for the ct potentiation of Rac1 expression, which explains why increased expression of Rac1 with UAS-Rac1 causes a rescue of the class III da neuron dendritic phenotype in fs(1)h1112 mutants. Recent evidence indicates a role for reduced Rac1 expression in social defeat and depressive behavior in mice, possibly through regulating dendritic spine morphology (Golden, 2013). In these behavioral paradigms, reduced Rac1 expression occurred with altered epigenetic marks such that transcriptionally permissive histone H3 acetylation was reduced, while repressive histone H3 methylation was increased. Moreover, administering a class 1 HDAC inhibitor mitigated the reduced Rac1 expression. Thus, these data suggest that Rac1 expression can be regulated by histone acetylation. It is possible that epigenetic reader proteins, such as BET family proteins like fs(1)h, bind acetylated histone marks in the Rac1 promoter to recruit transcriptional machinery and in turn enhance Rac1 expression (Bagley, 2014).

    In addition, overexpression of UAS-Fsh-S in class I da neurons did not cause an overbranching phenotype similar to UAS-Ct. In fact, there was no alteration of class I morphology, suggesting that Fsh-S is not sufficient to induce necessary components of the ct pathway to alter dendrite morphology. However, overexpression of Fsh-S in class III and class IV da neurons did cause a decrease in dendritic spike numbers. These data indicate that dendrite morphology may be sensitive to the amount of Fsh-S expression, which was confirmed by modulating the amount of overexpression by reducing GAL4/UAS activity with lower temperature. This may explain why it is possible to achieve only a partial rescue of the fs(1)h1112 dendritic phenotype with UAS-Fsh-S expression and why overexpression causes a dendritic phenotype similar to the phenotype caused by loss of Fsh-S. In support of this expression level hypothesis, it was observed that Fsh-S overexpression can reduce Ct-induced branching in class I da neurons. Since BRD2 is known to be part of a protein complex (Denis 2006), it is possible overexpression causes a gain-of-function or dominant-negative effect by altering the availability of complex components (Bagley, 2014).

    Another possible explanation for the partial rescue of Fsh-S expression concerns the developmental timing of expression. Since these experiments were completed using MARCM, GAL80 is expressed until mitotic recombination occurs to generate the mutant clones. It is likely that GAL80 protein may persist for some time after the clones are formed, and the presence of GAL80 would block GAL4/UAS activity. Therefore, UAS-induced Fsh-S expression may occur at a delayed stage in embryonic development, which could produce a partial rescue. In actuality, a combination of both expression level and developmental timing probably explains the partial rescue of the fs(1)h1112 phenotype (Bagley, 2014).

    While this study focused on the role of fs(1)h in regulating class III da neuron dendrite morphology, phenotypes were observed in other classes of the da neurons as well as expression of Fsh-S in all da neuron classes. In fs(1)h1112 mutants, a loss of Ct expression was observed in all da neurons that normally express Ct (classes II, III, and IV), suggesting that fs(1)h regulates Ct expression broadly among different neural subtypes. A loss of the class I-specific transcription factor Ab and the class IV-specific transcription factor Kn/Col wer also observed. Thus, it appears that fs(1)h can regulate the expression of subtype-specific gene expression among various neuron subtypes. The loss of Ct or the loss of Kn/Col could explain the reduction in class IV da neuron dendritic arbor complexity, and this further illustrates the pleiotropic nature of the fs(1)h1112 phenotype. The loss of Ab from class I da neurons should produce an increase in dendritic complexity, but interestingly, this did not occur in fs(1)h1112 mutants. Thus, these results consistently suggest that fs(1)h is necessary for dendritic arbor complexity, probably by regulating the expression of many different genes. In this manner, fs(1)h could act as a necessary gate for the gene expression responsible for establishing dendritic complexity (Bagley, 2014).

    How can fs(1)h regulate gene expression? Histone modifications are a diverse set of post-translational modifications that produce a code whereby epigenetic reader proteins bind these modified histone tails with specificity for particular modifications, such as methylation, acetylation, or phosphorylation. Previous structural studies have shown that the bromodomains of BET family proteins form a hydrophobic pocket enveloping acetylated histone tails (Umehara 2010a; Umehara, 2010b). Moreover, histone acetylation is largely, but not exclusively, regarded as a mark for transcriptional activation. Therefore, fs(1)h may be required for transcriptional activation of gene expression, which has been shown in vitro with respect to Ubx (Chang, 2007). The current data suggest that fs(1)h is required for ct expression and is in agreement with the hypothesis that fs(1)h is a transcriptional activator. It is possible that expression of other genes in the ct pathway also depends on histone acetylation modifications for transcriptional activation, and this activation may require Fsh-S. This would explain the observed nonlinear genetic relationship between ct and fs(1)h. In addition, the results indicate a necessary, but not sufficient, role of fs(1)h in regulating gene expression. This may indicate that BET family proteins require histone acetylation marks to be established but that these scaffold reader proteins do not actively alter histone tail modifications (Bagley, 2014).

    Histone modifications, termed the histone code, vary among different cell types and constitute a genome-wide mechanism for coordinating gene expression programs. This is intriguing because fs(1)h contains bromodomains that require histone acetylation to be first established at specific genomic regions in order to influence transcription at these regions. The current results suggest BET family proteins as candidates for reading this histone code to allow the development of dendritic complexity. It is important to note that although many proteins are observed with altered expression in fs(1)h1112 mutant da neurons, some proteins were unaltered, such as the mechanosensitive ion channel NompC. Furthermore, even though the Ct-induced overbranching in class I da neurons was blocked by fs(1)h1112, the Ct-induced NompC expression was normal. These data indicate some specificity to the action of fs(1)h in regulating dendritic morphology but not ion channel specification. It is possible that epigenetic 'reader' proteins, such as the BET proteins, coordinate the activity of many genetic pathways but with relevance to a specific outcome, such as regulating dendritic arbor morphology. In this model, the epigenetic 'readers' provide coordination and specificity of genome-wide histone marks to regulate particular aspects of neural cell biology. Moreover, it is conceivable that the specific genes regulated by fs(1)h could vary among different cell types depending on the cell type-specific histone code. This is supported by the different effects of UAS-Fsh-S overexpression in class I versus class III and IV da neurons as well as the loss of expression of cell type-specific transcription factors (Ab and Kn/Col) in fs(1)h1112 mutants. Currently, there is no atlas of the histone code for individual neural subpopulations. However, as the technology for conducting these types of analyses improves for distinct cell populations, it is conceivable that future studies can provide an answer to how cell type-specific histone modifications affect neural subtype-specific dendritic arbor morphologies (Bagley, 2014).

    Finally, the results suggest that the specific morphological shape of the class III da neuron dendrites is important for their ability to appropriately respond to sensory stimuli. The results indicate that pathways regulating dendrite morphology, such as the ct pathway, are reduced in fs(1)h mutants, but other pathways involved in axon morphogenesis or cell type-specific physiology, such as NompC channel expression, remain active. Moreover, the number of spike protrusions correlates with the number of APs produced in response to a mechanosensitive stimulus. This was also observed in another study (Tsubouchi 2012) involving manipulation of the number of spiked protrusions through modulating Rac1 activity. In that study, the gentle touch response increases as spike numbers increase, causing elevated calcium activity detectable with GCaMP fluorescence imaging. Conversely, decreasing the spike numbers results in a decrease of the gentle touch response and calcium activity. One potential caveat to this study is that Rac1 can modulate many aspects of dendritic cell biology through modulating actin cytoskeletal dynamics, and therefore it is unclear whether manipulating Rac1 activity alters the electrophysiological properties or localization of ion channels such as NompC. The finding of a correlation between dendritic spike number and gentle touch/electrophysiological responses in fs(1)h mutant neurons with normal appearance of NompC expression implicates dendritic morphology in regulating touch sensitivity (Bagley, 2014).

    Interestingly, NompC is expressed in fs(1)h1112 mutants, and its distribution throughout the dendritic arbor resembles that of wild-type neurons. While nompC mutants lack a mechanosensory response, neurons lacking fs(1)h still respond to mechanical stimuli, but the magnitude of the response (number of APs) is reduced for a given stimulus intensity. At the behavioral level, this manifests as a reduced response to gentle touch. Therefore, the data suggest that the unique dendritic spike morphology of class III dendrites contributes to their mechanical sensitivity (Bagley, 2014).

    While various proteins involved in epigenetic regulation of gene expression have been implicated in dendrite morphogenesis, this study provides evidence that 'readers' of acetylated histone marks regulate dendrite morphology by demonstrating the involvement of BET family proteins in this process. Given the complexity of achieving a comprehensive view of molecularly defined neural subtypes, it is necessary to identify genome-wide mechanisms for molecular diversity that regulate dendritic morphology in order to further understand how morphological diversity is specified. Epigenetic regulators are an intriguing possibility in this endeavor, and future studies comparing gene expression profiles in mutants for regulators of histone modifications among neurons with varied morphologies may be one step forward in answering this fundamental question (Bagley, 2014).

    Engrailed alters the specificity of synaptic connections of Drosophila auditory neurons with the giant fiber

    A subset of sound-detecting Johnston's Organ neurons (JONs) in Drosophila melanogaster that express the transcription factors Engrailed (En) and Invected (Inv) form mixed electrical and chemical synaptic inputs onto the giant fiber (GF) dendrites. These synaptic connections are detected by trans-synaptic Neurobiotin (NB) transfer and by colocalization of Bruchpilot-short puncta. Misexpressing En postmitotically in a second subset of sound-responsive JONs causes them to form ectopic electrical and chemical synapses with the GF, in turn causing that postsynaptic neuron to redistribute its dendritic branches into the vicinity of these afferents. A simple electrophysiological recording paradigm was introduced for quantifying the presynaptic and postsynaptic electrical activity at this synapse, by measuring the extracellular sound-evoked potentials (SEPs) from the antennal nerve while monitoring the likelihood of the GF firing an action potential in response to simultaneous subthreshold sound and voltage stimuli. Ectopic presynaptic expression of En strengthens the synaptic connection, consistent with there being more synaptic contacts formed. Finally, RNAi-mediated knockdown of En and Inv in postmitotic neurons reduces SEP amplitude but also reduces synaptic strength at the JON-GF synapse. Overall, these results suggest that En and Inv in JONs regulate both neuronal excitability and synaptic connectivity (Pezier, 2014).

    Drosophila Hook-Related Protein (Girdin) is essential for sensory dendrite formation

    The dendrite of the sensory neuron is surrounded by support cells and is composed of two specialized compartments: the inner segment and the sensory cilium. How the sensory dendrite is formed and maintained is not well understood. Hook-related proteins (HkRP) like Girdin, DAPLE, and Gipie are actin-binding proteins, implicated in actin organization and in cell motility. This study shows that the Drosophila melanogaster single member of the Hook-related protein family, Girdin, is essential for sensory dendrite formation and function. Mutations in girdin were identified during a screen for fly mutants with no mechanosensory function. Physiological, morphological, and ultra-structural studies of girdin mutant flies indicate that the mechanosensory neurons innervating external sensory organs (bristles) initially form a ciliated dendrite that degenerates shortly after, followed by the clustering of their cell bodies. Importantly, it was observed that Girdin is expressed transiently during dendrite morphogenesis in three previously unidentified actin-based structures surrounding the inner segment tip and the sensory cilium. These actin structures are largely missing in girdin. Defects in cilia are observed in other sensory organs such as those mediating olfaction and taste, suggesting that Girdin has a general role in forming sensory dendrites in Drosophila. These suggest that Girdin functions temporarily within the sensory organ and that this function is essential for the formation of the sensory dendrites via actin structures (Ha, 2015).

    The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons
    Dendrites exhibit enormous diversity in form and can differ in size by several orders of magnitude even in a single animal. However, whether neurons with large dendrite arbors have specialized mechanisms to support their growth demands is unknown. To address this question, a genetic screen was conducted for mutations that differentially affected growth in neurons with different-sized dendrite arbors. From this screen, a mutant was identified that selectively affects dendrite growth in neurons with large dendrite arbors without affecting dendrite growth in neurons with small dendrite arbors or the animal overall. This mutant disrupts a putative amino acid transporter, Pathetic (Path), that localizes to the cell surface and endolysosomal compartments in neurons. Although Path is broadly expressed in neurons and nonneuronal cells, mutation of path impinges on nutrient responses and protein homeostasis specifically in neurons with large dendrite arbors but not in other cells. Altogether, these results demonstrate that specialized molecular mechanisms exist to support growth demands in neurons with large dendrite arbors and define Path as a founding member of this growth program (Lin, 2015).

    Functions of the SLC36 transporter Pathetic in growth control

    Neurons exhibit extreme diversity in size, but whether large neurons have specialized mechanisms to support their growth is largely unknown. The SLC36 amino acid transporter Pathetic (Path) has been identified as a factor required for extreme dendrite growth in neurons. Path is broadly expressed, but only neurons with large dendrite arbors or small neurons that are forced to grow large require path for their growth. To gain insight into the basis of growth control by path, this study generated additional alleles of path and further examined the apparent specificity of growth defects in path mutants. Prior finding that loss of path function imposes an upper limit on neuron growth was conformed, and additionally it was found that path likely limits overall neurite length rather than dendrite length alone. Using a GFP knock-in allele of path, additional tissues were identified where path likely functions in nutrient sensing and possibly growth control. Finally, it was demonstrated that path regulates translational capacity in a cell type that does not normally require path for growth, suggesting that path may confer robustness on growth programs by buffering translational output. Altogether, these studies suggest that Path is a nutrient sensor with widespread function in Drosophila (Lin, 2016).

    The Kruppel-like factor Dar1 determines multipolar neuron morphology

    Neurons typically assume multipolar, bipolar, or unipolar morphologies. Little is known about the mechanisms underlying the development of these basic morphological types. This study shows that the Kruppel-like transcription factor Dar1 determines the multipolar morphology of postmitotic neurons in Drosophila. Dar1 is specifically expressed in multipolar neurons and loss of dar1 gradually converts multipolar neurons into the bipolar or unipolar morphology without changing neuronal identity. Conversely, misexpression of Dar1 or its mammalian homolog in unipolar and bipolar neurons causes them to assume multipolar morphologies. Dar1 regulates the expression of several dynein genes and nuclear distribution protein C (nudC), which is an essential component of a specialized dynein complex that positions the nucleus in a cell. These genes were shown to be required for Dar1-induced multipolar neuron morphology. Dar1 likely functions as a terminal selector gene for the basic layout of neuron morphology by regulating both dendrite extension and the dendrite-nucleus coupling (Wang, 2015).

    Ramon y Cajal placed neurons into three major morphological types based on the number of dendrites connected to the soma (i.e., primary dendrites): unipolar, bipolar, and multipolar and this classification system is universally applicable to different species throughout evolution. Multipolar neurons, like mammalian pyramidal neurons, develop more than one primary dendrite. In contrast, bipolar neurons are defined as having a single primary dendrite that may (e.g., cerebellar Purkinje cells) or may not (e.g., photoreceptors) branch out into an elaborate dendritic arbor. Finally, unipolar neurons such as DRG neurons in vertebrates and the majority of CNS neurons in invertebrates extend a single primary neurite, which usually bifurcates into dendritic and axonal branches (Wang, 2015).

    Multipolar morphology separates the dendritic arbor into distinct fields around the soma, which has an impact, not only on the passive current spread and processing of electrical signals in the neuron), but also on the types of synaptic or sensory inputs that the neuron receives . In addition, the three basic morphologies of neurons are relevant to the distinct organizational principles used in both the nervous systems of different animal species and in different parts of a single nervous system. Although all three morphological types are found in different species throughout evolution, the majority of neurons in invertebrates are unipolar, whereas the majority of those in vertebrates are multipolar (Wang, 2015).

    In the insect CNS, unipolar neurons extend a single process from the soma to a synapse-enriched neuropil and then bifurcate into dendrites that arborize locally and an axon that typically projects to other neuropil areas or target tissues. Unipolar organization of neuronal processes allows the formation of synaptic connections away from the location of the neuronal cell body, so it is likely an alternative strategy for neuronal migration, which is rare in the insect CNS but common in the vertebrate CNS. Despite the importance of these fundamental organizations of neuronal processes, very little progress has been made toward understanding the molecular and cellular programs that lead postmitotic neurons to develop multipolar, bipolar, or unipolar morphologies since their description a century ago (Wang, 2015).

    This study shows that the transcription factor Dar1 determines the multipolar morphology of postmitotic neurons in Drosophila. Dar1 is selectively expressed in postmitotic multipolar neurons and is required for these neurons to assume the multipolar morphology. Ectopic expression in unipolar or bipolar neurons leads to multipolar morphology. Dar1 regulates the expression of several dynein genes and nudC, which is an essential component of a specialized dynein complex that positions the nucleus in a cell. It is further shown that this evolutionarily conserved complex is required for multipolar morphology of neurons. These results suggest that dar1 likely functions as a terminal selector gene for the basic layout of neuron morphology (Wang, 2015).

    The universal morphological organization of neuronal dendrites and axons-in the form of the unipolar, bipolar, and multipolar morphologies-is important for information processing in neurons and for the wiring of neural circuits. It has generally been assumed that the formation of the basic morphological types of neurons is determined by the number of dendrites growing out from the cell body (the 'outgrowth model'). This study shows that this model alone is insufficient to explain the formation of multipolar morphology. Nuclear positioning is introduced as a factor in determining the multipolar neuron morphology, and it is proposed that Dar1 determines multipolar morphology by regulating both dendrite extension and primary dendrite-nucleus coupling (Wang, 2015).

    A novel, instructive role is reported for Dar1 in determining the multipolar morphology of postmitotic neurons without changing cell fate. First, despite the dendritic defects, axon morphology (Ye, 2011) and targeting are unchanged in dar1 mutant neurons. Second, ectopic expression of Dar1 in postmitotic neurons leads to supernumerary primary dendrites. Third, the remaining dendrites in dar1-/- neurons still follow the branching pattern assumed by wild-type neurons (Ye, 2011). Fourth, dar1 mutations do not affect the expression of neuron type-specific markers. Based on extensive studies in C. elegans, Hobert proposed the concept of terminal selector genes (Hobert, 2008). A terminal selector gene is required for determining specific aspects of a neuron's identity by regulating the expression of genes responsible for these characteristics such as those encoding neurotransmitter receptors, enzymes in a neurotransmitter synthesis pathway, and structural proteins. Loss of a terminal selector gene results in the loss of a specific aspect of the neuron type without affecting the overall neuronal identity. Dar1 plays such a function in the basic layout of neuronal morphology and thus is likely a 'terminal selector gene' for neuronal morphology (Wang, 2015).

    Based on the findings in this study, it is proposed that generating neuronal multipolar morphology requires, not only dendritic extension, but also a coupling mechanism between the nucleus and the dendrites. Dar1 promotes both dendritic growth and dendrite-nucleus coupling. Therefore, its misexpression converts unipolar neurons into neurons with multipolar morphology. The results presented in this study raise the interesting possibility that a specialized dynein complex in multipolar neurons with components that are transcriptionally regulated by Dar1 couples the nucleus with the primary dendrites. If the primary dendrite-nucleus coupling is weakened, then the nucleus may move to a different location in relation to the dendrites and axons. It is speculated that there might also be an active or passive force that pulls the nucleus toward the axon, opposing the force that couples the primary dendrites and the nucleus. Consistent with this model, the remaining single primary dendrites of all bipolar-shaped da neurons-caused by loss of dar1, reduced functions of dynein, or nuclear positioning complex-are those that project in the direction opposite the axon. This observation again rules out the possibility that the reduction in number of primary dendrites of dar1-/- da neurons is the result of reduction in dendrite growth. If that were the case, then the remaining single primary dendrites would likely project in random directions and not solely away from the axon. Further studies are needed to determine the cellular and molecular basis of the dendrite-nucleus coupling (Wang, 2015).

    Several prior studies have demonstrated that neurons switch between different morphological types during development. These observations suggest that the acquisition of basic morphological types in many neurons includes intermediate morphologies with nucleus-primary dendrite relationships that are different from those seen in the mature neurons. It will be interesting to investigate whether the activity of the nuclear positioning complex and its regulators play a role in these developmental changes in morphological type (Wang, 2015).

    In summary, this study offers a novel model for understanding the establishment of the three basic morphological types of neurons. Starting from genetic analysis of the KLF transcription factor Dar1, the study not only uncovers an instructive factor that determines the multipolar morphology of neurons, but also provide a mechanistic model showing that the position of the nucleus is critical for establishing multipolar neuron morphology. This study also demonstrates that the basic morphological types are determined by intrinsic molecular mechanisms in postmitotic neurons rather than in precursor cells. The model presented in this study may also be applied to explaining the changes in basic morphological type during neuron development. This study therefore opens the door for a unifying theory of basic structural organization in neurons (Wang, 2015).

    Spindle-F is the central mediator of Ik2 kinase-dependent dendrite pruning in Drosophila sensory neurons

    During development, certain Drosophila sensory neurons undergo dendrite pruning that selectively eliminates their dendrites but leaves the axons intact. How these neurons regulate pruning activity in the dendrites remains unknown. This study identifies a coiled-coil protein Spindle-F (Spn-F) that is required for dendrite pruning in Drosophila sensory neurons. Spn-F acts downstream of IKK-related kinase Ik2 in the same pathway for dendrite pruning. Spn-F exhibits a punctate pattern in larval neurons, whereas these Spn-F puncta become redistributed in pupal neurons, a step that is essential for dendrite pruning. The redistribution of Spn-F from puncta in pupal neurons requires the phosphorylation of Spn-F by Ik2 kinase to decrease Spn-F self-association, and depends on the function of microtubule motor dynein complex. Spn-F is a key component to link Ik2 kinase to dynein motor complex, and the formation of Ik2/Spn-F/dynein complex is critical for Spn-F redistribution and for dendrite pruning. These findings reveal a novel regulatory mechanism for dendrite pruning achieved by temporal activation of Ik2 kinase and dynein-mediated redistribution of Ik2/Spn-F complex in neurons (Lin, 2015)

    The precise assembly of neural circuits is crucial for the nervous system to function properly. The developing nervous systems often start with a primitive prototype, characterized by exuberant branches and excessive connections. Thus, further remodeling is required to refine the developing nervous systems to maturity. Neuronal pruning, one such remodeling mechanism, is a highly regulated self-destruct process that eliminates excessive neuronal branches in the absence of cell death. Pruning is widely observed in the nervous systems of both vertebrates and invertebrates, that not only ensures precise wiring during development, but also allows for adjustment of neuronal connections in response to injury and disease. Various studies have shown that defects in developmental pruning affect the function of the nervous systems in C. elegans and Drosophila. Moreover, a progressive loss of neurites far ahead of cell death is commonly observed in many neurodegenerative disorders. Thus, any dysregulation of pruning activity even at the level of individual neurons would bring catastrophic consequences to the nervous systems. Although the primary triggers for developmental pruning and pruning that ensues upon neuronal injury and disease are diverse, the downstream machinery that eliminates neuronal processes shared some common features. For example, microtubule disruption is the earliest cellular event observed in all types of pruning, and the ubiquitin-proteasome system is required in all circumstances (Lin, 2015)

    During Drosophila metamorphosis, substantial neuronal remodeling takes place in both the central and peripheral nervous systems. Most of the larval peripheral neurons die during metamorphosis, whereas few, including some class IV dendritic arborization (C4da) neurons, survive and undergo large-scale dendrite pruning. Dendrite pruning of the dorsal C4da neuron ddaC starts with severing of the proximal dendrites at 4-6 h APF (after puparium formation). Subsequently these disconnected dendrites become fragmented and eventually eliminated by the surrounding epidermal cells by 16-18 h APF. In contrast to the central brain mushroom body (MB) gamma neurons where both larval dendrites and axons are pruned during development, the peripheral C4da neurons specifically prune their dendrites keeping the axons intact. The molecular basis for how the pruning activity is confined to the dendrites of C4da neurons remains unknown. It was reasoned that molecular differences between dendrites and axons should be considered for such differential pruning activity in C4da neurons. It is known that microtubule polarity is different in the dendrites and axons of neurons, including in the Drosophila sensory neurons. For example, C4da neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body, but have an opposite polarity in their axons. This difference in microtubule polarity is essential for maintaining the proper function and compartmental identities of dendrites and axons, and might be an important determinant for spatially restricting pruning activity in the dendritic compartments of C4da neurons. Based on this assumption, some molecules are required to connect the pruning activity with the distinctive microtubule polarity of the dendrites in C4da neurons during dendrite pruning (Lin, 2015)

    Previous studies have shown that dendrite pruning in C4da neurons is initiated by the steroid hormone ecdysone and its heterodimeric receptors, ecdysone receptor B1 (EcR-B1) and Ultraspiracle (Usp). Through transcriptional regulation of sox14, ecdysone signaling activates the Sox14 target gene mical, which encodes a cytoskeletal regulator, to regulate dendrite pruning. A few other molecules mediating specific cellular activities have been shown to participate in dendrite pruning of C4da neurons, such as the ubiquitin-proteasome system, caspases, matrix metalloproteases, microtubule severing proteins and mediators of dendritic calcium transients. Previous studies identified Ik2 kinase, a homologue of vertebrate IKK-ε in Drosophila, that plays an essential role in dendrite pruning of pupal neurons, and further demonstrated that Ik2 is sufficient to induce precocious dendrite severing in larval neurons. Ik2 is the only known molecule sufficient to induce premature dendrite severing in larvae, reflecting a central role of Ik2 kinase in dendrite pruning. Therefore, this study aimed to elucidate the mechanism by which Ik2 kinase signaling is transduced and regulated in Drosophila sensory neurons during dendrite pruning (Lin, 2015)

    To elucidate the mechanism of Ik2 kinase signaling, candidate molecules were sought that mediate Ik2 signals during dendrite pruning. Several lines of evidence suggested that Spn-F, a coil-coiled protein, is a good candidate. Firstly, spn-F mutant flies showed defects in developing oocytes and bristles (Abdu, 2006), similar to the phenotypes observed in ik2 mutants (Shapiro, 2006). Secondly, Spn-F physically interacts with Ik2 (Dubin-bar, 2008). It implied that ik2 and spn-F may act in the same pathway during oogenesis and bristle morphogenesis, and the possibility has been raised that a similar pathway might also be involved in dendrite pruning of C4da neurons. This study demonstrates Spn-F plays a key role in linking Ik2 kinase to microtubule motor dynein complex for dendrite pruning. Spn-F acts downstream of Ik2 kinase in the same pathway for dendrite pruning. Spn-F is shown to displays a punctate pattern in larval neurons and these Spn-F puncta become dispersed in pupal cells. The redistribution of Spn-F from puncta is essential for dendrite pruning, and depends on the activity of Ik2 kinase and the function of microtubule motor dynein complex. These data also demonstrate that Spn-F not only links Ik2 to dynein motor complex, but also mediates the formation of Ik2/Spn-F/dynein complex, that is critical for Spn-F punctum disassembly and dendrite pruning (Lin, 2015)

    In addition to apoptosis, neurons have a second self-destruct program in their axons for axonal pruning during development and in response to neuronal injury and disorders. This study proposes a third self-destruct program, which is mediated by Ik2 kinase activity in Drosophila sensory neurons, specific for dendrite pruning. Ik2 is essential for dendrite severing in pupal C4da neurons (Lee, 2009), and currently is the only known molecule sufficient to cause precocious dendrite severing in larval cells (Lee, 2009), indicating that Ik2 activation must be regulated temporally. For temporal regulation, ecdysone signaling plays a key role in dendrite pruning (Williams, 2005; Lee, 2009). These studies show that no Ik2 activation is detected in pupal C4da neurons with impaired ecdysone signaling and thus places Ik2 kinase downstream of ecdysone signaling. Microarray studies have identified ik2 as one of the ecdysone/EcR up-regulated genes in brain MB γ neurons during axon pruning (Hoopfer, 2008). This suggests one possible mechanism where ecdysone/EcR regulates Ik2 activation through increasing ik2 expression in C4da neurons. Although Ik2 kinase activity is crucial for oogenesis and bristle morphogenesis (Shapiro, 2006; Otani, 2011), the activation mechanisms of Ik2 kinase in both processes remain unknown. Since pruning activity is considered as a self-destruct program, how to regulate this activity spatially in subcellular compartments within individual neurons is an intriguing issue to investigate. This study identifies Spn-F and cytoplasmic dynein complex as critical regulators of Ik2-mediated dendrite pruning activity in C4da neurons (Lin, 2015)

    It is known that endogenous Spn-F exhibits a punctate pattern in nurse cells (Abdu, 2006), consistent with the observation of punctate Spn-F-GFP in larval C4da neurons. The formation of Spn-F puncta in cells is through self-association, and does not depend on the integrity of microtubule network or the function of cytoplasmic dynein. Since Ik2 could form oligomers in cells, the interaction between Ik2 and Spn-F might also play a role in Spn-F puncta formation. Indeed, it was observed that SpnF-ΔCC3-GFP has normal interaction with either SpnF-ΔCC3 or full-length Spn-F, but formed fewer puncta than the wild type Spn-F-GFP did in larval neurons. Therefore, the Spn-F puncta formation could be attributed not only to Spn-F self-association, but also to Ik2/Spn-F interaction and Ik2 oligomerization (Lin, 2015)

    In larval C4da neurons, Ik2 kinase is inactive and associates with Spn-F as puncta in the cytosol. After puparium formation, Ik2 kinase becomes activated promptly and phosphorylates Spn-F in C4da neurons. This Ik2-dependent phosphorylation on Spn-F decreases Spn-F self-association, and subsequently the numbers and sizes of Spn-F puncta were reduced. One may question that protein degradation might contribute to decrease the numbers and sizes of Spn-F puncta in C4da neurons during dendrite pruning. It was known that Ik2 promotes caspase inhibitor DIAP1 degradation via proteasomes during the development of sensory organ precursors; therefore, Ik2 might promote Spn-F degradation in C4da neurons during dendrite pruning. However, Ik2 overexpression does not alter the protein level of Spn-F in either S2 or germline cells (Dubin-Bar, 2008). Thus, protein degradation by proteasomes is unlikely the mechanism leading to decreased Spn-F puncta after Ik2 activation. Since P-Ik2 signals were indistinguishable between wild-type and Dhc64C RNAi neurons, it is reasonable to presume that both Ik2 activation and Spn-F phosphorylation occur normally in dynein mutant neurons. No significant differences were found between the pruning defects of C4da neurons in spn-F mutants and that in spn-F mutants with Dhc-RNAi, and between the pruning phenotypes observed in Dhc mutants and that in Dhc mutants with ik2-RNAi. These findings further support that Ik2, Spn-F and dynein complex function together in the same pathway in dendrite pruning of C4da neurons. However, the finding of Spn-F puncta in mutant pupal neurons with impaired dynein function indicated that dynein is required for Spn-F redistribution after Ik2 activation. Furthermore, Spn-F remains punctate in S2 cells with Ik2 overexpression even after microtubule depolymerization and inhibition of dynein function, suggesting that dynein might redistribute Ik2/Spn-F complexes via transporting complexes toward the minus ends of microtubules in C4da neurons during dendrite pruning. The results in this study and studies in germline cells, (the fact that more Spn-F puncta accumulated in nurse cells with colchicine treatment and with Dhc mutation) (Abdu, 2006), favor the mechanism of protein redistribution for Spn-F punctum reduction in dendrite pruning of C4da neurons (Lin, 2015)

    It has been shown that during Drosophila bristle elongation, directional transport of activated Ik2 and of Spn-F to the bristle tips, where the microtubule minus ends are concentrated, requires the function of cytoplasmic dynein, and Spn-F acts as an adaptor to link Ik2 to dynein complexes. These are similar to the current findings that both Ik2 activation and dynein complex are essential for Spn-F redistribution, and Spn-F plays a central role in the formation of Ik2/Spn-F/dynein complex, which is crucial for Spn-F redistribution and for dendrite pruning in C4da neurons. However, the studies in bristle elongation indicating that spn-F acts upstream of ik2 (Otani, 2015) disagree with the current finding that ik2 acts upstream of spn-F in dendrite pruning. The discrepancy between the epistasis analyses of ik2 and spn-F in these two different processes might be due to different cell-type specific factors in these two types of cells that affect the morphological readouts in genetic studies. Moreover, this study demonstrated that Ik2-dependent phosphorylation of Spn-F decreases Spn-F self-association, promotes Spn-F redistribution, and finally leads to dendrite pruning in C4da neurons (Lin, 2015)

    The activated Ik2 signals accumulate at the microtubule minus ends in cells with polarized microtubule distribution, such as oocytes, follicle cells and bristles. This is consistent with the conclusion that dynein transports activated Ik2 toward microtubule minus ends in C4da neurons. Since Drosophila sensory neurons have polarized microtubules in their proximal dendrites predominantly with microtubule minus end pointing away from the cell body, these studies revealed a possible mechanism that Spn-F and minus-end directed motor dynein complex confine Ik2-dependent pruning activity to the somatodendritic compartments of C4da neurons. During Drosophila bristle elongation, the accumulation of endogenous Spn-F observed at the bristle tip, where the microtubule minus ends are enriched, led to an examination of Spn-F-GFP signals along the dendrites of C4da neurons during dendrite pruning. However, no enriched of Spn-F-GFP signals in the proximal dendrites, where dendrite severing is expected to occur, was observed, by live imaging during pruning. Previous studies (Lee, 2009) showed that microtubules are first disassembled in the proximal dendrites of C4da neurons during dendrite severing. This local disassembly of microtubules is suppressed in ik2 mutant neurons. Since the current genetic studies indicate that both ik2 and spn-F act in the same pathway of dendrite pruning, tests were performed to see whether local microtubule disassembly happens normally in spn-F mutants. Local breakage of microtubules found in the proximal dendrites of C4da neurons was also suppressed in spn-F RNAi mutants, suggesting that Spn-F, like Ik2, plays a role in dendrite severing that involves local microtubule disassembly. However, the molecular mechanisms by which activated Ik2 and Spn-F lead to dendrite severing in the proximal dendrites of C4da neurons will be an important question for future studies (Lin, 2015)

    It is known that there is no decrease in cell death in wing discs with ik2 knockdown and in ik2 mutant embryos (Kuranaga, 2006), indicating that the primary function of Ik2 is not involved in the apoptotic pathway during development. However, ectopic Ik2 activation by overexpression leads to cell death in fly compound eyes (Kuranaga, 2006) and in C4da neurons (Lee, 2009), suggesting that excessive Ik2 kinase signaling could trigger a crosstalk with signaling molecules in apoptotic pathways and result in apoptosis. It is known that Ik2 kinase regulates the nonapoptotic function of caspase through promoting DIAP1 degradation (Kuranaga, 2006). In a similar manner, the confinement of activated Ik2 kinase in the dendritic compartments might restrict the detected caspase activity in the degenerating dendrites after separating from the soma of C4da neurons during dendrite pruning. Therefore, this raises a possibility that de-regulation of pruning activity in neurons may trigger a crosstalk with molecules in apoptotic pathways and lead to undesired cell death during neuronal injury and disorders. Recently, a caspase cascade, including caspase 3 and 6, was identified in mice to play a role in developmental axon pruning and in sensory axon pruning after trophic factor withdrawal. Moreover, activated caspase 6 was detected in human patient brains of Alzheimer and Huntington diseases long before cell death, highlighting a critical role in regulating caspase activity in both diseases. Understanding the regulatory mechanisms that confine pruning activity into proper subcellular compartments of the neuron might provide molecular insights into the pathogenesis of neural disorders (Lin, 2015)

    Kinesin-2 and Apc function at dendrite branch points to resolve microtubule collisions

    In Drosophila neurons, kinesin-2, EB1 and Apc are required to maintain minus-end-out dendrite microtubule polarity, and it has been proposed they steer microtubules at branch points. Motor-mediated steering of microtubule plus ends could be accomplished in two ways: 1) by linking a growing microtubule tip to the side of an adjacent microtubule as it navigates the branch point (bundling), or 2) by directing a growing microtubule after a collision with a stable microtubule (collision resolution). Using live imaging to distinguish between these two mechanisms, this study found that reduction of kinesin-2 did not alter the number of microtubules that grew along the edge of the branch points where stable microtubules are found. However, reduction of kinesin-2 or Apc did affect the number of microtubules that slowed down or depolymerized as they encountered the side of the branch opposite to the entry point. These results are consistent with kinesin-2 functioning with Apc to resolve collisions. However, they do not pinpoint stable microtubules as the collision partner as stable microtubules are typically very close to the membrane. To determine whether growing microtubules were steered along stable ones after a collision, the behavior was analyzed of growing microtubules at dendrite crossroads where stable microtubules run through the middle of the branch point. In control neurons, microtubules turned in the middle of the crossroads. However, when kinesin-2 was reduced some microtubules grew straight through the branch point and failed to turn. It is proposed that kinesin-2 functions to steer growing microtubules along stable ones following collisions (Weiner, 2016).

    A genome-wide screen for dendritically localized RNAs identifies genes required for dendrite morphogenesis

    Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating asymmetric protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types and the significance of their localization, especially in the nervous system, are not known. This study used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, 55 EP-MS2 insertions were identified producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. It is proposed that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling (Misra, 2016).

    The Ret receptor regulates sensory neuron dendrite growth and integrin mediated adhesion

    Neurons develop highly stereotyped receptive fields by coordinated growth of their dendrites. Although cell surface cues play a major role in this process, few dendrite specific signals have been identified to date. An in vivo RNAi screen in Drosophila class IV dendritic arborization (C4da) neurons identified the conserved Ret receptor (Ret oncogene), known to play a role in axon guidance, as an important regulator of dendrite development. The loss of Ret results in severe dendrite defects due to loss of extracellular matrix adhesion, thus impairing growth within a 2D plane. Evidence is provided that Ret interacts with integrins to regulate dendrite adhesion via rac1. In addition, Ret is required for dendrite stability and normal F-actin distribution suggesting it has an essential role in dendrite maintenance. Novel functions are proposed for Ret as a regulator in dendrite patterning and adhesion distinct from its role in axon guidance (Soba, 2015).

    Accurate functional connectivity and sensory perception require proper development of the neuronal dendritic field, which ultimately determines the (sensory) input a specific neuron can receive and detect. Thus, coordinated dendrite growth and patterning is important for establishing the often complex, but highly stereotyped organization of receptive fields. Two of the organizing principles in dendrite development are self-avoidance and tiling. While self-avoidance describes the phenomenon of recognition and repulsion of isoneuronal dendritic branches, tiling refers to the complete yet non-redundant coverage of a receptive field by neighboring neurons of the same type. Both phenomena have been described in different systems across species including the mouse, zebrafish, medicinal leech, Caenorhabditis elegans, and Drosophila melanogaster (Soba, 2015).

    Dendritic patterning by self-avoidance, tiling, and other mechanisms is thought to be mediated by cell surface receptors and cell adhesion molecules (CAMs), which play a pivotal role in integrating environmental and cellular cues into appropriate growth and adhesion responses. Many such receptors, prominently Robo and Ephrin receptors, have well understood roles in axon guidance. Although some of these axonal cues including Robo/Slit play a role in dendrite development as well, dendritic surface receptors and their functions are not fully characterized to date. Recent efforts have yielded some progress in this area. Down's syndrome cell adhesion molecule (Dscam) has been shown to regulate dendrite self-avoidance in Drosophila. Studies on protocadherins have revealed that they play an important role in dendrite self-avoidance in mammals. In C. elegans, sax-7/L1-CAM and menorin (mnr-1) form a defined pattern in the surrounding hypodermal tissue to guide PVD sensory neuron dendrite growth via the neuronal receptor dma-1. However, given the complexity and stereotypy of dendritic arbors within individual neuronal subtypes, it is important to search for additional signals for directing dendrite growth (Soba, 2015).

    The Drosophila peripheral nervous system (PNS) has served as an excellent model which has helped to elucidate several molecular mechanisms regulating dendrite development. The larval PNS contains segmentally repeated dendritic arborization (da) neurons which have been classified as class I-IV according to their increasing dendritic complexity. All da neuron classes feature highly stereotyped sensory dendrite projections. Moreover, all da neurons exhibit self-avoidance behavior allowing them to develop their individual receptive fields without overlap. It has been demonstrated that all da neuron classes require Dscam for dendrite self-avoidance. In addition, the atypical cadherin flamingo and immunoglobulin super family (IgSF) member turtle might play a more restricted role in C4da neuron self-avoidance. Netrin and its receptor frazzled have also been shown to act in parallel to Dscam in class III da neurons ensuring their proper dendritic field size and location by providing an attractive growth cue which is counterbalanced by self-avoidance. For tiling, no surface receptor has been identified to date. However, the conserved hippo and tricornered kinases, and more recently the torc2 complex, have been implicated in C4da neuron tiling, as the loss of function of these genes results in iso- and hetero-neuronal crossing of dendrites (Soba, 2015).

    Recent work has further shown that dendrite substrate adhesion plays an essential role in patterning. Da neuron dendrites are normally confined to a 2D space through interaction with the epithelial cell layer and the extracellular matrix (ECM) on the basal side of the epidermis. 2D growth of da neuron dendrites requires integrins, as loss of the α-integrin mew (multiple edomatous wing) or ß-integrin mys (myospheroid) results in dendrites being freed from the 2D confinement due to detachment from the ECM. Thus, they can avoid dendrites by growing into the epidermis leading to 3D crossing of iso- and hetero-neuronal branches . Integrins are therefore essential to ensure repulsion-mediated self-avoidance and tiling mechanisms, which restrict growth of dendrites competing for the same territory. How integrins are recruited to dendrite adhesion sites and whether they cooperate with other cell surface receptors is unknown (Soba, 2015).

    To identify novel receptors required for generating complex, stereotypical dendritic fields, an in vivo RNAi screen was performed for cell surface molecules in C4da neurons. The Drosophila homolog of Ret (rearranged during transfection) was identified as a patterning receptor of C4da dendrites. Loss of Ret function in C4da neurons severely affects dendrite coverage, dynamics, growth, and adhesion. In particular, dendrite stability and 2D growth are impaired resulting in reduced dendritic field coverage and abnormal 3D dendrite crossing, respectively. These defects can be completely rescued by Ret expression in C4da neurons. It was further shown that Ret interaction with integrins is needed to mediate C4da dendrite-ECM adhesion, but not dendrite growth. These data suggest that Ret together with integrins acts through the small GTPase rac1, which is required for dendrite adhesion and 2D growth of C4da neuron dendrites as well. This study thus describes a novel role for the Ret receptor in dendrite development and adhesion by direct receptor crosstalk with integrins and its downstream signals (Soba, 2015).

    This study provides evidence that Ret is a regulator of dendrite growth and patterning of C4da neurons. Ret is a conserved receptor tyrosine kinase (RTK) expressed in the nervous system of vertebrates and D. melanogaster , and has been shown to have a number of important functions in nervous system development and maintenance: it regulates motor neuron axon guidance (Kramer, 2006), dopaminergic neuron maintenance and regeneration, and mechanoreceptor differentiation and projection to the spinal cord and medulla. Ret signaling is activated by binding to glial cell line derived neurotrophic factor (GDNF) family ligands and their high affinity co-receptors, the GDNF family receptors (GFRα). Ret also plays an important role in human development and disease as loss of function mutations of Ret lead to Hirschprung's disease displaying colonic aganglionosis due to defective enteric nervous system development. Conversely, Ret gain of function mutations are causal for autosomal dominant MEN2 (multiple endocrine neoplasia type 2) type medullary thyroid carcinoma (Soba, 2015 and references).

    Prior to this study, Ret has not been implicated in dendrite development. This study shows that Ret is required specifically for 2D growth of C4da neurons by regulating integrin dependent dendrite-ECM adhesion. Normally, C4da neuron dendrites are virtually always in contact with the ECM and the basal surface of the epithelium lining the larval cuticle, and thus tightly sandwiched between the two compartments. In both integrin and Ret mutants, dendrite-ECM adhesion is impaired. Ret and integrins can co-localize in dendrites and thus likely form a functional complex that could induce and maintain adhesion of dendrites to the ECM. Since Ret loss of function primarily leads to detached terminal dendrite branches, it is tempting to speculate that Ret might be required to recruit integrins to sites of growing dendrites to promote ECM interaction. This is supported by the colocalization of Ret and integrins on the dendrite surface. Their cooperative interaction could thus ensure proper adhesion of growing branches and, conversely, the fidelity of self-avoidance and tiling (Soba, 2015).

    These results also highlight the importance of integrating different guidance and adhesion cues to achieve precise neuronal patterning. This has so far only been studied in axon guidance in vivo. Interestingly, vertebrate Ret has been shown to cooperate with Ephrins to ensure high fidelity axon guidance in motor neurons by mediating attractive EphrinA reverse signaling (Kramer, 2006; Bonanomi, 2012). Similar mechanisms may conceivably be employed for growing dendrites, which also encounter a multitude of attractive, repulsive, and adhesive cues that have to be properly integrated. Besides pathways acting independently or in a parallel fashion, an emerging view is that receptors exhibit direct crosstalk to integrate incoming signals. So far, only parallel receptor pathways like Dscam and Netrin-Frazzled signaling in class III da neurons or Dscam/integrins have been identified co-regulating dendrite morphogenesis. The current data show that the Ret receptor and integrins integrate dendrite adhesion and growth by collaborative interaction of the two cell surface receptors. The molecular and genetic link between Ret and integrins suggests that in this case direct receptor crosstalk plays a major role in their function. How exactly these cell surface receptors cooperate and interact remains to be elucidated. Integrins have been shown to display extensive crosstalk with other signaling receptors, including RTKs. Although integrins are involved in adhesion of virtually all cell types, the underlying signaling and recruitment of integrins to sites of adhesion in vivo is complex and not completely understood. It has been suggested that integrin and growth factor receptor crosstalk can occur by concomitant signaling, collaborative activation, or direct activation of associated signaling pathways. For example, matrix-bound VEGF can induce complex formation between VEGFR2 and β1-integrin with concomitant targeting of β1-integrin to focal adhesions in endothelial cells. The current findings of biochemical interaction and colocalization of Ret with the α/β-integrins mys and mew in C4da neuron dendrites argue in favor of direct receptor interaction and subsequent activation of a common signaling pathway (Soba, 2015).

    Integrins and RTKs like Ret do share some of the same intracellular signaling components. These comprise, among others, the MAPK (mitogen-activated protein kinase) pathway, Pi3-Kinase (Pi3K), and Rho family GTPases including Rac1. Previous studies provide evidence for Ret-integrin-Rac1 interplay in vitro showing that Ret can enhance integrin mediated adhesion and induce Rac1 dependent lamellipodia formation in cell culture models. In primary chick motor neurons, Rac1 is involved in neurite outgrowth on the integrin substrates laminin and fibronectin. Interestingly, Rac1 has previously been shown to regulate dendrite branching in C4da neurons, however a role in dendrite adhesion in vivo has not been described before. This study shows that Rac1 is required for dendrite-ECM adhesion similarly to what has been described for integrins, and Ret and integrin dependent adhesion was genetically linked with Rac1 function. In Drosophila, MAPK, Src and PI3K can be activated by constitutively active Ret overexpression in the compound eye. Moreover, novel inhibitors of Ret signaling targeting Raf, Src, and S6-Kinase (S6K) prevent lethality induced by Ret over-activation in a Drosophila multiple endocrine neoplasia (MEN2) model. Interestingly, S6K has been shown to be involved in dendrite growth but not tiling in C4da neurons. It remains to be shown if these pathways play a direct role in Ret function in dendrite adhesion and growth (Soba, 2015).

    Notwithstanding important commonalities, Ret function in C4da neurons cannot be fully explained by crosstalk with integrins and rac1. Reduced dendritic field coverage, likely due to the observed increase in dendrite turnover, is only evident in Ret but not in integrin or rac1 mutant C4da neurons. Moreover, increasing integrin expression in a Ret mutant background did not rescue dendrite coverage defects, albeit it prevented dendrite crossing. These findings indicate that Ret has additional functions in dendritic branch growth and stability that require as yet unknown extracellular and intracellular mediators. This is also supported by the aberrant F-actin localization in neurons lacking Ret. In this study, Ret dependent intracellular effectors are likely important for F-actin assembly to support directed dendrite growth and stabilization, and their localization and activity might be deregulated in the absence of Ret (Soba, 2015).

    Drosophila Ret is a highly conserved molecule, its cognate vertebrate ligand GDNF, however is not (Airaksinen, 2006). In addition, Drosophila Ret can neither bind GDNF nor transduce GDNF signaling, although it has been shown to contain a functional tyrosine kinase domain. In mammals, the GFRα co-receptors are essential components of GDNF/Ret signaling. A Drosophila GFR-like homolog (dGFRL) has recently been characterized and was found to function and interact with the NCAM homolog FasII. Therefore, it appears that Ret's functional interaction partners in dendrite development differ significantly from the previously described co-factors in other systems. It is interesting to speculate that a yet undiscovered Ret ligand is involved in Ret mediated dendrite growth and branch stabilization, which might have implications for mammalian Ret function as well: due to its role in the maintenance of dopaminergic neurons and motor axon growth in mouse, adhesion related signaling via integrins could well be important during these processes. Moreover, the formation of a dorsal root ganglia derived mechanosensory neurons and their afferent and efferent fiber growth and innervation depends on Ret expression. It will be interesting to investigate the functional interplay of Ret and integrins in central and peripheral target innervation and neurite maintenance in these systems, given the interdependent function of Ret and integrins in sensory dendrite growth as shown in this study (Soba, 2015).

    In summary, this study describes a novel role for the Ret receptor in dendrite branch growth and stability in Drosophila C4da neurons. This role involves cell-autonomous effects of Ret on ECM adhesion, and F-actin localization in these neurons. Moreover, dendritic adhesion defects attributable to Ret have been linked to integrin and rac1 function featuring a novel and possibly conserved mode of action for Ret in dendrite development (Soba, 2015).

    Structural homeostasis: Compensatory adjustments of dendritic arbor geometry in response to variations of synaptic input

    As the nervous system develops, there is an inherent variability in the connections formed between differentiating neurons. Despite this variability, neural circuits form that are functional and remarkably robust. One way in which neurons deal with variability in their inputs is through compensatory, homeostatic changes in their electrical properties. This study shows that neurons also make compensatory adjustments to their structure. The development of dendrites on an identified central neuron (aCC) was studied in the late Drosophila embryo at the stage when it receives its first connections and first becomes electrically active. At the same time, the distribution of presynaptic sites on the developing postsynaptic arbor was charted. Genetic manipulations of the presynaptic partners demonstrate that the postsynaptic dendritic arbor adjusts its growth to compensate for changes in the activity and density of synaptic sites. Blocking the synthesis or evoked release of presynaptic neurotransmitter results in greater dendritic extension. Conversely, an increase in the density of presynaptic release sites induces a reduction in the extent of the dendritic arbor. These growth adjustments occur locally in the arbor and are the result of the promotion or inhibition of growth of neurites in the proximity of presynaptic sites. Evidence is provided that suggest a role for the postsynaptic activity state of protein kinase A in mediating this structural adjustment, which modifies dendritic growth in response to synaptic activity. These findings suggest that the dendritic arbor, at least during early stages of connectivity, behaves as a homeostatic device that adjusts its size and geometry to the level and the distribution of input received. The growing arbor thus counterbalances naturally occurring variations in synaptic density and activity so as to ensure that an appropriate level of input is achieved (Tripodi, 2008).

    Since cholinergic neurons provide the only known excitatory input to Drosophila motor neurons in the embryo, the effect of the lack of neurotransmitter (synthesis) in the cholinergic neurons was examined on the development of the aCC dendritic arbor. Acetyl choline is not synthesized in animals mutant for choline acetyl transferase (Chal13), which are therefore immobile and unable to hatch. The development of aCC dendritic arborisations was examined in animals homozygous for the null mutation Chal13. In Cha mutant embryos, it was found that the development of the aCC dendritic arbor proceeds normally until 16 h AEL. However, in the interval between 16 to 18 h AEL, Cha mutants, unlike controls, fail to reduce the rate of dendritic growth. As a result, at 18 h AEL, the extent of the aCC dendritic arbor is increased by about 26% in Cha mutants as compared to controls. It is concluded that in the window of development, when in normal embryos acetyl choline-dependent excitation of aCC first begins and the rate of dendritic growth declines, the absence of neurotransmitter in presynaptic neurons allows postsynaptic growth to continue linearly at an undiminished constant rate. These findings suggest that the aCC dendritic arbor reacts to the loss of synaptic input by increasing in size. A consequence of this increase in overall dendritic length is that it allows the dendritic arbor to explore a larger portion of the neuropile than in normal animals. In fact, it was observed that in Cha mutants, the dendritic arbor of aCC extends into regions of the neuropile that are not normally invaded in control conditions (Tripodi, 2008).

    Growth adjustments by the aCC arbor appear to operate sequentially at two levels. In the first instance, the dendritic arbor determines the presence or absence of presynaptic partners. This event is independent of presynaptic activity. It is also a local event that appears to affect primarily neurites receiving presynaptic sites, which act as a local stop-growing signal. The second step depends on the activity of the synapse. In normal conditions, presynaptic sites are able to inhibit growth, both in synaptic neurites as well as neighbouring nonsynaptic sister neurites. Interestingly, the dendritic arbor does not measure the efficacy of synapses (since there is no significant change in arbor size in Ace mutants as compared to wild type), but simply determines whether a synapse is active or not. However, the possibility of there being a compensatory response to reduced efficacy cannot be excluded. This phase of activity-dependent inhibition of dendritic growth is mediated by postsynaptic activation of PKA (Tripodi, 2008).

    The morphology of dendrites is likely to be an important determinant of the connectivity state of a nervous system. It is not by chance that even in complex nervous systems, one of the most distinctive feature of different classes of neurons is probably the morphology of their dendritic arbors. Indeed, the anatomical discrimination of different classes of neurons, based on their dendritic morphology, has often anticipated and predicted molecular, electrophysiological, and computational differences (Tripodi, 2008).

    Therefore, an appreciation of the logic governing the assembly of a nervous system must include an understanding of how dendritic morphology is acquired. Although many of the distinctive features that differentiate the dendritic morphologies of different classes of neurons are likely to be cell-autonomously and genetically determined, the effect of partner-derived cues, as shown in this study, can have a substantial impact in modulating these features. This investigation was initiated by analysing the effect of presynaptic transmission in shaping the morphology of the postsynaptic dendritic arbor. Previous studies on this issue have not reached a clear agreement on the role of activity in regulating dendritic growth. Even though in some instances it has been reported that activity has no effect on regulating arbor growth, in the majority of studies, activity emerges clearly as an essential modulator of dendritic remodelling. The main issue has been whether the incoming presynaptic input acts as a trophic factor that promotes arbor growth or whether it delivers a stop-growing signal. Unfortunately, with a few notable exceptions, many of these studies were carried out in different animals, in different neural populations, at different developmental stages, and by using different experimental approaches (genetic manipulation or pharmacological treatments), thus making it difficult to find a common theme in the results. One major source of variation that could explain the differences in the results of previous studies is likely to be the developmental stage at which manipulations were applied. For instance, a single class of tectal neurons in Xenopus tadpoles appears to respond in opposite ways to presynaptic input at different developmental stages. In immature neurons, presynaptic input acts as a growth-promoting signal, whereas in mature neurons, it acts as a stop-growing or stabilization signal. In this study, no evidence was found for a growth-promoting effect of synaptic activity. Instead, it was shown that synaptic input inhibits dendritic growth starting from the earliest stages at which neurotransmission occurs (16-18 h AEL). Altering activity before the onset of evoked synaptic transmission causes no dendritic phenotype (14-16 h AEL). It appears that developing Drosophila embryonic motor neurons behave like mature tectal neurons in Xenopus (Tripodi, 2008).

    The difference in the effect obtained at different stages in Xenopus tectal neurons nicely correlates with a change in their molecular characteristics. At later stages, when synaptic input acts as a stop-growing signal, the tectal neurons in Xenopus have acquired the ability to respond to local calcium increases via the activation of a calcium-dependent protein kinase CamKII. In Drosophila, aCC appears be sensitive to the activation state of a different protein kinase, PKA (which is also regulated, albeit indirectly, by intracellular calcium levels) throughout the interval of development that was studied (Tripodi, 2008).

    A great deal of attention has been given to global changes in postsynaptic activity and how these changes might regulate global dendritic growth. However, although this is an extremely interesting question, it is difficult to imagine how global variations in the state of activation of the whole dendritic arbor or the soma might contribute to fine-tuning the morphology of dendritic arbors. Far more compelling would be a system that was able to calculate and respond to local changes in activity levels. It is well known that calcium levels can be altered locally at the synaptic site following synaptic input. It is also known that changes in dendritic levels of calcium can induce dramatic changes in dendritic morphology. Nevertheless, there have been few investigations of how dendritic morphology might be altered locally by synaptic activity (Tripodi, 2008).

    Because the system used allows the study a single identified postsynaptic neuron whose presynaptic input can be altered, this study begins to address the issue of local versus global changes of dendritic morphology induced by synaptic activity. Analyzing the branching pattern of the dendritic arbor with respect to the position of its synaptic inputs highlights some interesting and unexpected features. By simply looking in wild-type animals, it is clear that neurites bearing presynaptic sites branch less than nonsynaptic neurites of the same arbor. Through experimental manipulation, this study has shown that this local inhibition of the growth and branching of synaptic neurites appears to be mediated by contact between pre- and postsynaptic partners and does not require evoked transmission. The lack of this contact-dependent inhibition of dendritic growth is already apparent when the terminals normally presynaptic to aCC are mistargeted before they can form functional synapses at 16 h AEL. Moreover, this effect cannot be attributed to an interference with transmission, since Cha mutants of the same stage (16 h AEL) do not show this dendritic overgrowth phenotype. Loss of neurotransmitter release, on the other hand, can also induce an overgrowth of the postsynaptic dendritic arbor, though only after synapses would have been active for 2 h in normal conditions (i.e., 18 h AEL). It was found that this neurotransmitter-dependent overgrowth is due to increased extension of nonsynaptic segments that are immediately adjacent to neurites receiving presynaptic sites. Thus, neurotransmitter release at the synaptic sites acts on the nonsynaptic sister neurites to inhibit their extension (Tripodi, 2008).

    It is concluded that the geometry of the dendritic tree is regulated by two partner-dependent mechanisms. First, contact with presynaptic terminals locally inhibits dendritic growth and branching in an activity-independent fashion. A second inhibitory effect requires evoked presynaptic neurotransmitter release. It extends from these sites to the immediate proximity, affecting nonsynaptic sister branches. This second 'neighbourhood effect' could be mediated by local increases in dendritic calcium levels (Tripodi, 2008).

    Neurotransmission-dependent variations in intracellular calcium levels are an attractive mechanism that might implement activity-dependent local rearrangement of the dendritic arbor geometry. Therefore the role of the protein kinase PKA in dendritic remodelling was investigated, since its activity is regulated directly or indirectly by calcium levels. PKA is activated by increases in the intracellular levels of cAMP. It has been shown that levels of cAMP are finely regulated by intracellular levels of calcium, which in turn respond to levels of presynaptic neurotransmitter release (Tripodi, 2008).

    PKA signalling in postsynaptic cells has been shown to mediate homeostatic responses. For instance, at the neuromuscular junction, postsynaptic PKA signalling modulates quantal size (Davis, 1998), while centrally, it mediates homeostatic change in the electrical excitability of aCC neurons following alterations to presynaptic input (Baines, 2003). This study has shown that PKA activity is also a potent modulator of dendritic morphology. PKA signalling is probably downstream of presynaptic transmission mediating the inhibition of dendritic growth. Overexpression of a constitutively active form of PKA (PKAact) is able to rescue the dendritic overgrowth phenotype that ensues in the absence of presynaptic transmission in Cha mutants. However, overexpression of PKAact in aCC in control animals has no measurable effect on dendritic development, suggesting that PKA signalling operates to saturation under normal levels of synaptic input. Although neurotransmission is clearly one signal that regulates dendritic development through downstream PKA signalling, it is not necessarily the only one (Tripodi, 2008).

    The term homeostasis has classically been used to refer to compensatory variations in the electrical properties of neurons that tend to counterbalance changes in their synaptic input. This paper proposes that neurons might combine the homeostatic regulation of electrical properties with compensatory structural adjustments of their dendritic geometry. It is argued that variations in the morphology of aCC dendritic arbors represent such compensatory adjustment of dendritic growth and branching (Tripodi, 2008).

    These experimental observations indicate that eliminating synaptic input induces a compensatory dendritic overgrowth in the postsynaptic neuron, whereas an increase in the density of active synapses induces the opposite effect. Interestingly, increasing the overall level of neurotransmitter released at presynaptic terminals without altering the density of presynaptic sites on the arbor does not induce compensatory adjustments of the postsynaptic arborisation. This suggests that the compensatory changes in dendritic arbor morphology that were observed act to compensate for variations in the density of active synaptic release sites rather than variations in the global state of dendritic or neuronal depolarization. This is in agreement with observations that changes in dendritic morphology in response to changes in presynaptic input or synaptic density appear to be implemented locally rather than across the entire arbor. This structural homeostasis, therefore, seems to work on a local scale, allowing particular regions of the dendritic arbor to compensate for variation in their inputs while leaving other regions of the arbor substantially unchanged. This could be an effective mechanism for neurons that use distinct regions of their dendritic arbor to independently compute different inputs (Tripodi, 2008).

    It will be interesting to understand whether electrical homeostasis and what is proposed to be a structural homeostasis operate in concert to shape the postsynaptic response, or whether one or the other is preferentially deployed depending on the circumstances. At the moment, it is not possible to answer this question, and more experiments are required (Tripodi, 2008).

    Nonetheless, one can begin to envisage how PKA signalling in the context of electrical homeostasis might be integrated with the structural homeostasis that was shown in this study. As shown by Baines (2003), postsynaptic overexpression of a constitutively active form of PKA induces a change in the electrical properties of aCC, resulting in a decrease in excitability. Overexpression of a PKA inhibitor, on the other hand, does not modulate the excitability of aCC. This study has described a perfect mirror image of this situation, namely that the expression of a PKA inhibitor induces a dendritic overgrowth phenotype, while expression of a constitutively active form of PKA does not. Bringing these two lines of observations together suggests the following model: following a decrease in presynaptic input (and PKA signalling), the postsynaptic neuron expands its receptive field so as to increase the number of presynaptic sites that it contacts. In these circumstances, an increase in postsynaptic excitability would not be required. On the other hand, following an increase in presynaptic input, the postsynaptic neuron decreases its excitability (via increased PKA signalling) without the need to reduce its receptive field (Tripodi, 2008).

    The view is favoured that electrical and structural homeostatic mechanisms might indeed be integrated by neurons. This would enable the cells to implement compensatory changes that resulted in adjustments to their electrical characteristics and dendritic geometry, so as to ensure that in an inherently variable environment an adequate pattern and level of connectivity and excitability is achieved (Tripodi, 2008).

    Growing dendrites and axons differ in their reliance on the secretory pathway

    Little is known about how the distinct architectures of dendrites and axons are established. From a genetic screen, dendritic arbor reduction (dar) mutants were isolated with reduced dendritic arbors but normal axons of Drosophila neurons. dar2, dar3, and dar6 genes were identified as the homologs of Sec23, Sar1, and Rab1 of the secretory pathway. In both Drosophila and rodent neurons, defects in Sar1 expression preferentially affected dendritic growth, revealing evolutionarily conserved difference between dendritic and axonal development in the sensitivity to limiting membrane supply from the secretory pathway. Whereas limiting ER to Golgi transport resulted in decreased membrane supply from soma to dendrites, membrane supply to axons remained sustained. It was also shown that dendritic growth is contributed by Golgi outposts (see Horton, 2003), which are found predominantly in dendrites. The distinct dependence between dendritic and axonal growth on the secretory pathway helps to establish different morphology of dendrites and axons (Ye, 2007).

    This study has demonstrated that growing dendrites and axons display different sensitivity to changes in the activity of the secretory pathway. These findings add to the accumulating evidence that the secretory pathway is involved in cell polarity and provide one of the first evidence of the importance of the satellite secretory pathway in dendrite development (Ye, 2007).

    The secretory pathway is important for cell polarity. For example, during the cellularization of Drosophila embryos, membrane growth is tightly controlled in a polarized fashion. New membrane produced through the secretory pathway is predominantly added to the apical side early in polarization and to the lateral side at later stages. Other examples of targeted membrane transport during cell polarization include the apical-basolateral polarization of Madin-Darby canine kidney epithelial cells, cell cycle of the budding yeast, and cell migration. Thus, membrane trafficking is tightly controlled in order to supply membrane to specific subcellular compartments of polarized cells (Ye, 2007).

    The establishment of dendritic and axonal arbors is a major compartmentalization process of neurons. Before dendrites and axons are formed, selective delivery of post-Golgi vesicles to one neurite precedes the specification of that neurite as the axon. After axon specification, large amount of plasma membrane is added to these two compartments with distinct architecture. Whether the secretory pathway is involved in the differential growth of dendrites and axons is an important but poorly understood question. Horton (2005) found that blockade of post-Golgi trafficking by expressing a dominant-negative protein kinase D1 (PKD-KD) causes a preferential reduction in dendritic growth. Although this result may appear similar to the current results from manipulation of Sar1 function, there is in fact an important difference. PKD is required for cargo trafficking to the basolateral but not the apical membrane in epithelial cells. Since the targeting of dendritic proteins shares some of the mechanisms of the basolateral protein transport in epithelial cells, PKD-KD may only affect dendrite-specific cargo transport. This study assessed how membrane resource is allocated between growing dendrites and axons when global membrane production is limited. It is possible that the efficiency of dendrite-and axon-specific transport, including those mediated by PKD, changes differently in response to global restriction of membrane resource (Ye, 2007).

    The plasma membrane of cells can be inserted via exocytosis and internalized through endocytosis. The expansion of plasma membrane in growing dendrites and axons is achieved through the interplay between these two antagonistic processes. Before dendrites are formed in cultured hippocampal neurons, membrane is selectively added to the growth cone of growing axons as well as minor neurites that will later become dendrites. In more mature neurons, there is insertion of new membrane to the axonal growth cone but not to the dendritic growth cone. This raises the possibility that plasma membrane is added to either multiple sites along the dendritic surface or throughout the dendritic surface. It is conceivable that dendritic Golgi outposts play a role in such membrane addition given their requirement for dendritic growth as shown in this study (Ye, 2007).

    It has been reported that plasma membrane of cultured hippocampal neurons is internalized throughout the dendrites but only in the presynaptic terminals in axons. It was also found that essentially all internalized structures, from both dendritic and axonal surface, move in the retrograde direction to the soma. The rate of membrane removal from dendritic and axonal surfaces were compared and it was found that endocytosis is more prominent in dendrites than in axons. The plasma membrane of dendrites was endocytosed 8.27 times faster than that of axons at 3 days in vitro. Thus, the demand for membrane supply to dendrites is likely greater than what appears based on the length of dendrites (Ye, 2007).

    The difference in the ways that membrane is supplied to growing dendrites and axons is poorly understood. Notwithstanding much progress made on dendritic traffic of the temperature sensitive mutant of the vesicular stomatitis virus glycoprotein (VSVG) fused to GFP (Horton, 2003; Horton, 2005), the preferential targeting of VSVG to dendrites precludes its application for comparing dendritic and axonal membrane dynamics. This work compared the membrane supply from soma to dendrites and axons using the FRAP analysis. Fluorescence recovery was sampled in the dendritic and axonal segment 5 microm away from the soma as an indicator of the amount of the membrane passing though this segment. The sampling approach was taken because even with moderate magnification (20X) only a fraction of the arbors can be included in the imaging field, precluding the monitoring of fluorescence recovery along the entire dendritic and axonal arbor. This FRAP analysis revealed that, when the secretory pathway function is limiting, membrane supply from soma to growing dendrites is preferentially affected (Ye, 2007).

    Notably, the secretory pathway with reduced activity could still provide sufficient membrane to support axonal growth at least during the period described in this study. It is possible that there are mechanisms involving post-Golgi carriers to allocate different amount of membrane to dendrites and axons. Alternatively, endocytosed dendritic membrane can also support axonal growth via the transcytosis pathway. Lastly, trafficking that bypasses COPII-based ER to Golgi transport, although not widely observed, might also contribute to axonal growth (Ye, 2007).

    The finding that Golgi outposts are present in dendrites has generated great interests in the function of these structures. Two main functions have been proposed for Golgi outposts. One hypothesis is that they are involved in local protein translation (Steward, 2003). This is supported by the presence of protein synthesis machinery in the dendrites and findings of local translation of membrane proteins in dendrites suggest that Golgi outposts, together with somatic Golgi complex, participate in forming the apical dendrites of pyramidal neurons (Ye, 2007).

    In this study, the dynamics of Golgi outposts were monitored in intact neurons of live Drosophila larvae, and it was observed that the direction of outpost movement correlates with dendrite branch dynamics. This suggests that Golgi outposts are probably involved in both the extension and retraction of a dendritic branch. Indeed, laser damaging of the outposts in a branch reduced branch dynamics, making the dendrite excessively stable, which will lead to retarded growth during the expansion of dendritic arbors. It should be noted that, in the laser damaging experiments, the nature and extent of damages to Golgi outposts are unclear. It is indeed technically challenging to manipulate organelle function in a small, localized subcellular region. The laser damaging experiment is considered one of the first attempts of such manipulations. Although it can be concluded that Golgi outposts are the most sensitive locations for affecting dendritic branch dynamics among the various target locations, alternative approaches are needed to test the function of Golgi outposts. The present study complemented the laser damaging experiment with genetic manipulations to redistribute Golgi outposts. It was found that dendritic branching patterns changed with the redistribution of Golgi outposts by LvaDN and Lva-RNAi. High level of LvaDN expression led to the strongest defect in Golgi outpost distribution and dendritic branching pattern. Although it is difficult to rule out the contribution of non-specific effects of LvaDN, there is very good correlation between the distribution of Golgi outposts and that of dendritic branches in neurons with varying extent of disruption of Lva function. These findings underscore the importance of Golgi outposts in dendritic growth (Ye, 2007).

    It remains unclear how Golgi outposts are produced, transported to dendrites, and largely excluded from axons. The results on Lva suggest that they are probably produced in the soma and transported to dendrites. The presence of Golgi outposts in axons of LvaDN-expressing neurons suggest that they might be actively transported out of axons under normal condition. The absence of Golgi outposts and exuberant branches in the proximal axons of neurons expressing Lva-RNAi is likely due to partial interference of Lva function as similar phenotype was seen in neurons expressing low level of LvaDN. The increase in the size of Golgi outposts and dendritic branches in neurons expressing high level of LvaDN possibly reflect the requirement of Lva-dynactin interaction for the budding of Golgi outposts, a phenomenon that was observed in this study(Ye, 2007).

    Although it is difficult to rule out the presence of outposts that are below the detection sensitivity in this study, axonal Golgi outposts, if any, must be dramatically smaller and/or fewer than dendritic outposts. Therefore, the requirement of Golgi outposts for dendritic growth provides a mechanism to differentially control dendrite and axon growth (Ye, 2007).

    The secretory pathway is possibly under regulation to control dendritic growth The distinct dependence of dendrites and axons on the secretory pathway, as well as the involvement of Golgi outposts for local dendritic dynamics, raise the possibility that the secretory pathway may be regulated to influence the elaboration of dendrites. Such regulation might involve both genetic programs for specifying intrinsic differences in dendritic patterning of different neurons and activity-dependent modifications of dendritic arbors (Ye, 2007).

    Several molecules are known to be specifically involved in dendritic but not axonal growth. Some of these molecules function in a neural activity-dependent fashion, such as the calcium/calmodulin-dependent protein kinase IIα (CaMKIIα), the transcription factor NeuroD, and the Ca2+-induced transcriptional activator CREST, while others are neural activity-independent such as bone morphogenetic protein 7 (BMP-7) and Dasm1. It will be important to find out whether the secretory pathway contributes to their regulation of dendritic growth (Ye, 2007).

    In summary, this study has demonstrated that dendritic and axonal growth exhibit different sensitivity to changes in membrane supply from the secretory pathway. These findings raise a number of questions regarding membrane trafficking in dendrite and axon development. Answers to these questions will provide cell biological basis for understanding how the tremendous diversity of neuron morphology is achieved during development and how the changes in morphology happen in pathological conditions (Ye, 2007).

    Nak regulates localization of clathrin sites in higher-order dendrites to promote local dendrite growth

    During development, dendrites arborize in a field several hundred folds of their soma size, a process regulated by intrinsic transcription program and cell adhesion molecule (CAM)-mediated interaction. However, underlying cellular machineries that govern distal higher-order dendrite extension remain largely unknown. This study shows that Numb-associated kinase (Nak), a clathrin adaptor-associated kinase, promotes higher-order dendrite growth through endocytosis. In nak mutants, both the number and length of higher-order dendrites are reduced; these characters phenocopied by disruptions of clathrin-mediated endocytosis. Nak interacts genetically with components of the endocytic pathway, colocalizes with clathrin puncta and is required for dendritic localization of clathrin puncta. More importantly, these Nak-containing clathrin structures preferentially localize to branching points and dendritic tips that are undergoing active growth. Evidence is presented that the Drosophila L1-CAM homolog Neuroglian is a relevant cargo of Nak-dependent internalization, suggesting that localized clathrin-mediated endocytosis of CAMs facilitates the extension of nearby higher-order dendrites (Yang, 2011; see video abstract).

    Postmitotic neurons elaborate highly branched, tree-like dendrites that display distinct patterns in accordance with their input reception and integration. Therefore, regulation of dendrite arborization during development is crucial for neuronal function and physiology. Dendrite morphogenesis proceeds in two main phases: lower-order dendrites first pioneer and delineate the receptive field and then higher-order dendrites branch out to fill in gaps between existing ones (Jan, 2010). This process is exemplified by the morphogenesis of Drosophila dendritic arborization (da) neurons, which have a roughly fixed pattern of lower-order dendrites in early larval stages. Higher-order dendrites then branch out to reach the order of more than six, covering the entire epidermal area. These distinct phases of dendrite arborization are manifested by the difference in underlying cytoskeletal composition. While lower-order dendrites are structurally supported by rigid microtubules, higher-order dendrites contain actin and loosely packed microtubules. It is thought that the structural flexibility of higher-order dendrites allows dynamic behaviors like extension, retraction, turning and stalling to explore unfilled areas (Yang, 2011).

    The da neurons are classified into four types (I-IV) according to branching pattern and complexity of dendrites. The most complex class IV da neurons have a unique pattern, in which few branches are sent out from proximal dendrites, while dendrites grow extensively in distal regions. Polarized growth of higher-order dendrites requires specialized cellular machineries. For instance, disruption of the ER-to-Golgi transport in class IV ddaC neurons preferentially shreds higher-order dendrites, suggesting that the secretory pathway is needed to sustain membrane addition during dendrite formation. Golgi outposts, hallmark of the satellite secretory pathway in dendrites, move anterogradely and retrogradely during extension and retraction of terminal dendrites, respectively. Arborization in the distal field demands active transport systems mediated by microtubule-based motors, as mutations in dynein light intermediate chain (dlic) or kinesin heavy chain (khc) fail to elaborate branches in the distal region of class IV ddaC neurons. The transport of Rab5-positive endosomes allows branching of distal dendrites, suggesting that the endocytic pathway also has a role in dendrite morphogenesis (Yang, 2011).

    The growth of higher-order dendrites seems to require elevated level of endocytosis. Endocytosis is more active in dendrites than in axons in cultured hippocampal neurons. Dynamic assembly and disassembly of clathrin-positive structures, indicative of active endocytosis, are seen at dendritic shafts and tips of young hippocampal neurons. These clathrin-positive structures become stabilized in mature neurons. Endocytosis is known to regulate the polarized distribution of the cell adhesion molecule NgCAM in hippocampal neurons, which is first transported to the somatodendritic membrane and then transcytosed to the axonal surface. Endocytosis is also important for transporting NMDAR to synaptic sites during their formation in dendrites of young cortical neurons. The NMDAR packets transported along microtubules are intermittently exposed to the membrane surface by cycles of exocytosis and endocytosis, at sites coinciding with the clathrin 'hotspots'. Endocytosis can regulate the activities of transmembrane receptors whose signaling activity is important to dendrite growth and maintenance. For instance, the neurotrophin-Trk receptor-mediated signaling that depends on endocytosis could be importantfor dendrite morphogenesis. However, how endocytosis regulates dendrite morphogenesis is not yet clear (Yang, 2011).

    Clathrin-mediated endocytosis (CME) is the major route for selectively internalizing extracellular molecules and transmembrane proteins from the plasma membrane. Transmembrane cargos destined for internalization are recruited into clathrin-coated pits through interaction with appropriate clathrin adaptors. One such accessory factor is adaptor protein 2 (AP2), a heterotetrameric complex consisting of a, b, m and s subunits. AP2-dependent cargo recruitment can be regulated by reversible protein phosphorylation by actin-related kinase (Ark) family serine/threonine kinases. In yeast, Ark family genes are known to influence endocytosis by phosphorylating Pan1, an Eps15 homolog, to regulate actin dynamics. Mammals contain two Ark family genes, cyclin G-associated kinase (GAK) and adaptor-associated kinase 1 (AAK1) and both have been implicated in vesicular transport. GAK, best known for its role in the disassembly of clathrin coats from clathrin-coated vesicles, has multiple functions during clathrin cycle. AAK1 has been shown to bind the a subunit of AP2, phosphorylate the cargo-binding m2 subunit and promote receptor-mediated transferrin uptake. AAK1 also participates in transferrin receptor recycling from the early/sorting endosome in a kinase activity-dependent manner (Yang, 2011).

    Numb-associated kinase (Nak), the Drosophila Ark family member, contains the conserved Ark kinase domain and several motifs (DPF, DLL and NPF) mediating interactions with endocytic proteins. To study the function of Nak in development, nak deletion mutants and RNAi lines were generated and it was shown that depletion of nak activity in da neurons disrupts higher-order dendrite development. This function of Nak in dendritic morphogenesis is likely mediated through CME, as Nak exhibits specific genetic interactions with components of CME, colocalizes with clathrin in dendritic puncta and is required for the presence of clathrin puncta in distal higher-order dendrites. More importantly, live-imaging analysis shows that the presence of these clathrin/Nak puncta at basal branching sites correlates with extension of terminal branches. In addition, evidence is presented that the localization of Neuroglian (Nrg) in higher-order dendrites requires Nak, implying that regional internalization of a cell adhesion molecule is crucial for dendrite morphogenesis (Yang, 2011).

    This study has shown that disruption of nak during dendrite arborization of da neurons significantly reduces both number and length of dendritic branches. Multiple classes of da neurons were analyzed for the lack of Nak activity, which suggests that its general role in higher-order dendrite morphogenesis. The function of Nak in dendrite arborization is required cell autonomously, as dendritic defects in nak mutants could be rescued by neuron-specific expression of wild-type Nak (Yang, 2011).

    Several lines of evidence suggest a functional link between Nak and AP2, the endocytosis-specific clathrin adaptor, in dendrite morphogenesis. First, coimmunoprecipitation results show that Nak predominantly associates with AP2. Second, Nak colocalizea well with GFP-Clc and alpha-adaptin but not with AP1 and AP3 in S2 cells. Third, neuron-specific depletion of AP2 mimics the dendritic defect in nak mutants and reduction of AP2 gene dose enhances nak-induced dendritic defect. These genetic interactions are specific, as mutations in components of AP1 (AP47SAE-10) and AP3 (garnet1) showed no enhancement of nak-associated dendritic phenotypes (Yang, 2011).

    Mutations in Nak DPF motifs that are known to interact with alpha-adaptin (DPF-to-AAA), reducing interaction with AP2, render Nak incapable of rescuing the dendritic defects. As AP2 acts to recruit clathrin to endocytic sites, this functional link between Nak and AP2 implies that the dendritic defect in nak mutants is caused by the disruption of Clathrin-mediated endocytosis (CME). Consistent with this notion, mutations in Chc also interact genetically with nak in dendrite morphogenesis and Nak and clathrin are colocalized in dendrites. Thus, it is suggested that Nak functions through CME to promote dendrite development. Being an Ark family kinase implicated in CME, Nak might function similarly to AAK1 that is known to regulate the activities of clathrin adaptor proteins via phosphorylation in cultured mammalian cells). Consistently, it was shown that Nak kinase activity is indispensable for its ability to rescue dendritic defects. Disrupting dynamin activity in shits1-expressing neurons exhibited stronger defects than nak mutants. In addition to endocytosis, dynamin is known to act in the secretory pathway. Given the known role of the secretory pathway in dendrite morphogenesis, it is possible that only endocytic aspect is disrupted in nak mutants, but both secretory and endocytic aspects are affected in shi mutants (Yang, 2011).

    Clathrin- and Nak-positive structures in da neurons are preferentially localized to the branching points of higher-order dendrites. Unlike Rab4, Rab5 and Rab11 that are mobile in dendrites, these clathrin/Nak puncta are stationary. Importantly, it was possible to correlate the localization of these stationary clathrin/Nak puncta with motility of local terminal dendrites. The clathrin puncta in higher-order dendrites probably represent sites where populations of clathrin-coated vesicles actively participate in endocytosis. Consistent with this, these clathrin-positive structures are enriched with PI4,5P2, which is known to assemble endocytic factors functioning in the nucleation of clathrin-coated pits. The proximity and tight association between localized endocytic machinery and polarized growth have been described in several systems, including the extension of root hair tips, the budding of yeast cells and the navigation of axonal growth cones. Thus, while the mechanism remains to be determined, the requirement of CME in cellular growth appears conserved (Yang, 2011).

    How does regionalized endocytosis contribute to dendrite branching? It is proposed that region-specific internalization and recycling of the cell adhesion molecule Nrg is a mechanism for generating local Nrg concentration optimized for higher-order dendrite morphogenesis. In the advance of mammalian axonal growth cones, adherent L1 can provide the tracking force for growth cone extension. As the growth cone advances, L1 is endocytosed in the central region to release unnecessary adhesion and recycled back to the peripheral region. Similarly, continuously recycling of Nrg along the dendritic membrane may help its delivery to growing dendrites that potentially function in promoting dendrite extension or stabilizing newly formed dendrites. Excessive Nrg in higher-order dendrites as in da neurons overexpressing Nrg may inhibit dendrite arborization by generating superfluous adhesion. Thus, Nak-mediated endocytosis could alleviate this inhibition by internalizing Nrg from the cell surface, allowing dendrite elongation (Yang, 2011).

    Arborization of higher-order dendrites in Drosophila da neurons requires branching out new dendrites and elongation of existing ones, which requires two other cellular machineries. First, transporting the branch-promoting Rab5-positive organelles to distal dendrites by the microtubule-based dynein transport system is essential for branching activity. In the absence of Rab5 activity, dendritic branching is largely eliminated and lacking the dynein transport activity limits branching activity to proximal dendrites. Second, the satellite secretory pathway contributes to dendrite growth by mobilizing Golgi outposts to protruding dendrites. Similar to Rab proteins, the Golgi outposts labeled by ManII-GFP were only partially colocalized with YFP-Nak and their dendritic distribution is independent of Nak activity. Also, in lva-RNAi larvae in which the transport of Golgi outposts is disrupted, YFP-Nak puncta were localized normally to distal dendrites. These findings suggest that localization of Golgi outposts in dendrites is not dependent on Nak activity and localization of YFP-Nak is not dependent on transport of Golgi outposts. It is envisioned that arborization of dendrites is achieved by transporting the branch-promoting factors like Rab5 distally via the dynein transport system. Following the initiation of new branches, dendrite extension requires growth-promoting activity provided by the anterograde Golgi outposts and localized clathrin puncta to promote local growth. To actively distribute clathrin puncta in distal dendrites that are far away from the soma, Nak can participate in the condensation of efficient endocytosis into the punctate structures in higher-order dendrites. It is possible that both stationary Nak/clathrin puncta and secretory Golgi outposts are spatially and temporally coupled to promote extension of dendrites, thus coordinating several events like adhesion to the extracellular matrix, membrane addition/extraction, cargo transport, and cytoskeletal reorganization, eventually building up the sensory tree in the target field (Yang, 2011).

    Endocytic pathways downregulate the L1-type cell adhesion molecule Neuroglian to promote dendrite pruning in Drosophila

    Pruning of unnecessary axons and/or dendrites is crucial for maturation of the nervous system. However, little is known about cell adhesion molecules (CAMs) that control neuronal pruning. In Drosophila, dendritic arborization neurons, ddaCs, selectively prune their larval dendrites. This study reports that Rab5/ESCRT-mediated endocytic pathways are critical for dendrite pruning. Loss of Rab5 or ESCRT function leads to robust accumulation of the L1-type CAM Neuroglian (Nrg) on enlarged endosomes in ddaC neurons. Nrg is localized on endosomes in wild-type ddaC neurons and downregulated prior to dendrite pruning. Overexpression of Nrg alone is sufficient to inhibit dendrite pruning, whereas removal of Nrg causes precocious dendrite pruning. Epistasis experiments indicate that Rab5 and ESCRT restrain the inhibitory role of Nrg during dendrite pruning. Thus, this study demonstrates the cell-surface molecule that controls dendrite pruning and defines an important mechanism whereby sensory neurons, via endolysosomal pathway, downregulate the cell-surface molecule to trigger dendrite pruning (Zhang, 2014).

    Endocytic pathways profoundly regulate turnover and homeostasis of various cell-surface adhesion proteins and guidance receptors in the developing nervous systems. Perturbation of endocytic pathways often leads to a variety of neurodegenerative diseases, such as frontotemporal dementia, amyotrophic lateral sclerosis, Alzheimer's disease, lysosomal storage diseases, and Niemann-Pick disease. In Drosophila, the endolysosomal pathway is activated in neighboring glia to engulf degenerating axon/dendrite fragments for their subsequent breakdown during pruning, suggesting a non-cell-autonomous role. This study reports that Rab5 and the ESCRT complexes, two key endocytic regulators, cell autonomously promote dendrite pruning in ddaC neurons. Consistent with these findings, the endocytic pathways also play a cell-autonomous role in axon pruning of MB γ neurons. How do Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning in ddaC neurons at the cellular level? This study has identified a cell-surface adhesion protein, namely the L1-type CAM Nrg, as a target of Rab5/ESCRT-dependent endocytic pathways (Zhang, 2014).

    Drosophila Nrg and the mammalian L1-type CAMs regulate axonal growth and guidanc, synaptic stability and function, and axon/dendrite morphogenesis. Mutations in the human L1 CAM gene have been reported to cause a broad spectrum of neuronal disorders. This study has identified the Drosophila L1-type CAM Nrg as the key cell-surface molecule that inhibits dendrite pruning in ddaC neurons. The extracellular domains of the L1-type CAMs can regulate cell-cell adhesion via homophilic and/or heterophilic interactions, whereas their intracellular domains can link the proteins with F-actin/spectrins to stabilize the cytoskeletal architecture. In C. elegans, a ligand-receptor complex of cell adhesion molecules containing the nematode Nrg homolog controls dendrite-substrate adhesion to stabilize and pattern dendritic arbors in certain sensory neurons. In Drosophila, Nrg-mediated cell adhesion plays an essential role in stabilizing synapse growth and maintenance at the larval neuromuscular junction. Likewise, Nrg may also mediate adhesion of the dendrites to their adjacent epidermis to stabilize the dendritic architecture in ddaC sensory neurons, whereas downregulation of Nrg may reduce dendritic adhesion/stability and result in disassembly of dendrites. Consistent with the potential adhesive role, structure-function analysis indicates that the ECD of Nrg is important for its function in stabilizing dendrite and/or inhibiting dendrite pruning in ddaC neurons. The fact that overexpression of the ICD-deleted Nrg protein partially rescued the nrg14 mutant phenotype suggests that, in addition to the adhesion function of the ECD, the ICD of Nrg may recruit cytoskeletal components to stabilize dendritic branches in ddaC neurons. The model is therefore favored that the adhesive role of Nrg is a potential mechanism for inhibiting pruning in ddaC sensory neurons (Zhang, 2014).

    Another major class of CAMs, integrins, were shown to regulate dendrite-substrate interactions and anchor ddaC dendritic arbors to the extracellular matrix. However, unlike Nrg, integrins do not accumulate on enlarged endosomes in Rab5 or ESCRT ddaC neurons, implying that integrins are not regulated by Rab5/ESCRT-dependent endocytic pathways in ddaC neurons. Moreover, other cell-surface molecules Robo and N-Cad, albeit regulated by the endolysosomal pathway in motor neurons, photoreceptors, or sensory neurons, are dispensable for normal progression of dendrite pruning in ddaC neurons. Thus, this study highlights an important role of the L1-type CAM Nrg in inhibiting dendrite pruning of ddaC sensory neurons (Zhang, 2014).

    Interestingly, loss of nrg function causes precocious dendrite pruning without affecting the axonal integrity and connectivity in ddaC neurons, underscoring a specific requirement of Nrg in stabilizing the dendrites, but not the axons. Downregulation of Nrg may reduce dendritic adhesive properties of ddaC sensory neurons and thereby make the dendritic architecture more susceptible to pruning. It is conceivable that Nrg-independent mechanisms may be utilized to protect the axonal structure from the pruning machinery in ddaC neurons. Moreover, both nrg loss of function and gain of function did not affect axon pruning in MB γ neurons (data not shown), further supporting the conclusion that Nrg plays a specific role in dendrite pruning in ddaC sensory neurons. Future studies may elucidate whether and how Nrg mediates its dendritic adhesive properties to inhibit dendrite pruning (Zhang, 2014).

    In summary, this study shows that Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning of ddaC neurons by downregulating the Drosophila L1-type CAM Nrg during early metamorphosis. This study also demonstrated the role of the cell-surface adhesion protein Nrg in inhibiting dendrite pruning in ddaC sensory neurons. Thus, this study opens the door for further studies of the functions of cell-surface molecules in the regulation of dendritic adhesion during neuronal remodeling (Zhang, 2014).

    Synaptic control of secretory trafficking in dendrites

    Localized signaling in neuronal dendrites requires tight spatial control of membrane composition. Upon initial synthesis, nascent secretory cargo in dendrites exits the endoplasmic reticulum (ER) from local zones of ER complexity that are spatially coupled to post-ER compartments. Although newly synthesized membrane proteins can be processed locally, the mechanisms that control the spatial range of secretory cargo transport in dendritic segments are unknown. This study, carried out in mammalian neuronal cell cultures, monitored the dynamics of nascent membrane proteins in dendritic post-ER compartments under regimes of low or increased neuronal activity. In response to activity blockade, post-ER carriers are highly mobile and are transported over long distances. Conversely, increasing synaptic activity dramatically restricts the spatial scale of post-ER trafficking along dendrites. This activity-induced confinement of secretory cargo requires site-specific phosphorylation of the kinesin motor Kif17 (see Drosophila KIF17) by Ca2+/calmodulin-dependent protein kinases (CaMK) (see for example Drosophila CaMKII). Thus, the length scales of early secretory trafficking in dendrites are tuned by activity-dependent regulation of microtubule-dependent transport (Hunus, 2014. PubMed ID: 24931613).

    Regulation of dendrite growth and maintenance by exocytosis

    Dendrites lengthen by several orders of magnitude during neuronal development, but how membrane is allocated in dendrites to facilitate this growth remains unclear. This study reports that Ras opposite (Rop), the Drosophila ortholog of the key exocytosis regulator Munc18-1, is an essential factor mediating dendrite growth. Neurons with depleted Rop function exhibit reduced terminal dendrite outgrowth followed by primary dendrite degeneration, suggestive of differential requirements for exocytosis in the growth and maintenance of different dendritic compartments. Rop promotes dendrite growth together with the exocyst, an octameric protein complex involved in tethering vesicles to the plasma membrane, with Rop-exocyst complexes and exocytosis predominating in primary dendrites over terminal dendrites. By contrast, membrane-associated proteins readily diffuse from primary dendrites into terminals, but not in the reverse direction, suggesting that diffusion, rather than targeted exocytosis, supplies membranous material for terminal dendritic growth, revealing key differences in the distribution of materials to these expanding dendritic compartments (Peng, 2015).

    Sec71 functions as a GEF for the small GTPase Arf1 to govern dendrite pruning of Drosophila sensory neurons

    Pruning, whereby neurons eliminate their exuberant neurites, is central for the maturation of the nervous system. In Drosophila, sensory neurons, ddaCs, selectively prune their larval dendrites without affecting their axons during metamorphosis. However, it is unknown whether the secretory pathway plays a role in dendrite pruning. This study shows that the small GTPase Arf1, an important regulator of secretory pathway, is specifically required for dendrite pruning of ddaC/D/E sensory neurons but dispensable for apoptosis of ddaF neurons. Analyses of the GTP and GDP-locked forms of Arf1 indicate that the cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. Sec71 was identified as a guanine nucleotide exchange factor for Arf1 that preferentially interacts with its GDP-bound form. Like Arf1, Sec71 is also important for dendrite pruning, but not apoptosis, of sensory neurons. Arf1 and Sec71 are interdependent for their localizations on Golgi. Finally, Sec71/Arf1-mediated trafficking process is a prerequisite for Rab5-dependent endocytosis to facilitate endocytosis and degradation of the cell adhesion molecule Neuroglian (Wang, 2017).

    In the developing nervous systems, neurons often extend excessive neurites and form superfluous connections at early stages. Subsequent removal of those exuberant or inappropriate neurites without causing the death of parental neurons, a process known as pruning, is crucial for the refinement of neural circuits at late developmental stages. Neuronal pruning is a conserved process widely occurring in both vertebrates and invertebrates. In vertebrates, many neurons in the neocortex, neuromuscular system and hippocampal dendate gyrus prune their unwanted neurites to control the proper wiring of the nervous systems. In invertebrates, such as Drosophila, the nervous systems undergo drastic remodelling during metamorphosis, a transition stage from a larva to an adult fly. In the central nervous system (CNS), mushroom body (MB) γ neurons prune their dorsal and medial axon branches as well as entire dendrites. In the peripheral nervous system (PNS), some dorsal dendritic arborization (da) neurons, ddaC, ddaD and ddaE, selectively eliminate their larval dendrites without affecting their axons, whereas ddaF neurons are apoptotic during early metamorphosis. The pruning event involves both local degeneration and retraction, resembling neurodegeneration associated with brain injury and neurodegenerative diseases. Thus, a complete understanding of cellular and molecular mechanisms of developmental pruning would shed some light on pathological neurodegeneration following neurological diseases and injury (Wang, 2017).

    In Drosophila, ddaC sensory neurons have emerged as an attractive model system to elucidate the molecular and cellular mechanisms of dendrite-specific pruning during early metamorphosis. In response to the steroid-molting hormone 20-hydroxyecdysone (ecdysone) at the late larval stage, ddaC neurons sever the proximal region of their dendrites and subsequently undergo rapid fragmentation of the severed dendrites as well as dendritic clearance via phagocytosis. It has been well documented that the Ecdysone Receptor and its co-receptor Ultraspiracle are required to activate the expression of several downstream targets to initiate dendrite pruning. Endocytic pathways have been identified that are critical for dendrite pruning. Rab5/Avalanche and ESCRT complexes, the components of the endocytic pathways, are required for downregulation of the L1-type cell adhesion molecule (L1-CAM) Neuroglian (Nrg). Nrg is drastically redistributed to endosomes and its protein levels are strongly downregulated prior to pruning, suggesting that massive Nrg endocytosis promotes dendrite pruning. It is conceivable that Nrg endocytosis might be triggered by secreted ligands/signals through the secretory pathway. However, it is completely unknown whether the secretory pathway, an opposite route of the endocytic pathway, also plays a role in dendrite pruning of ddaC neurons (Wang, 2017).

    The primary sites of the secretory pathway consist of the endoplasmic reticulum (ER), the Golgi apparatus and the trans-Golgi network in eukaryotic cells. Newly synthesized membrane proteins and lipids exit from the ER, pass through the Golgi complexes and are delivered to the plasma membrane via the post-Golgi exocytosis. In developing neurons, the continuous addition of membrane proteins and lipids via the secretory pathway plays a key role in the outgrowth and elongation of dendrites and axons. Disruption of the ER-to-Golgi transport leads to the inhibition of dendritic or axonal growth in Drosophila sensory neurons and rodent hippocampal neurons. In an attempt to isolate novel players of dendrite pruning, a large-scale RNA interference (RNAi) screen was carried out, and ADP-ribosylation factor 1(Arf1), also known as ADP-ribosylation factor at 79F, was identified as an important player for dendrite pruning in ddaC sensory neurons. Arf1 is a small GTPase and belongs to the Class I Arf family. It has been reported that Arf1 can recruit COPI coat proteins on cis-Golgi and clathrin adaptor proteins, such as AP-1, AP-3, and GGAs, on trans-Golgi in a GTP- dependent manner, and thereby facilitate vesicle formation and trafficking. Studies from yeast and mammals indicate that Arf1 is activated by two conserved families of guanine nucleotide exchange factors (GEFs), including the Gea1/GBF1 family on cis-Golgi and Sec7/BIG family on trans-Golgi. Arf1 cycles between GDP- and GTP-bound forms, and both the GTP- and GDP-locked forms can interfere with its functions and disrupt secretory trafficking. In mammalian hippocampal neurons, overexpression of the GTP-locked form of Arf1 (Arf1Q71L), which abolishes Arf1 activity, inhibits dendrite growth. The mammalian Arf1GEF, BIG2, is required for vesicle trafficking and mutations in human BIG2 gene lead to autosomal recessive periventricular heterotopia with microcephaly (ARPHM), a brain disorder characterized by defective neural proliferation and migration. Thus, various studies have documented that the secretory pathway plays a critical role in neurite growth and extension in developing neurons. However, very little is known about its role in regulating neurite pruning, a developmental degenerative process (Wang, 2017).

    This study reports the identification of Arf1 as an important player of dendrite pruning in ddaC sensory neurons. The cycling of Arf1 between GDP-bound and GTP-bound forms is essential for dendrite pruning. Sec71 was identified as a GEF for Arf1 in Drosophila. Sec71, like Arf1, is cell-autonomously required for dendrite pruning of ddaC/D/E sensory neurons but not for apoptosis of ddaF neurons during early metamorphosis. Arf1 and Sec71 co-localize on Golgi apparatus and regulate secretory vesicle biogenesis in ddaC neurons. Furthermore, it was shown that Sec71/Arf1-dependent secretory pathway acts upstream of Rab5-dependent endocytosis and facilitates the internalization and downregulation of the cell adhesion molecule Nrg prior to dendrite pruning. Thus, this study demonstrates a novel and important role of Arf1/Sec71-mediated secretory pathway in promoting developmental pruning via the regulation of Nrg endocytosis (Wang, 2017).

    The small G protein Arf1 regulates vesicular trafficking in eukaryotes and is activated on cis-Golgi by the Gea1/GBF1 family of Arf1GEF or on trans-Golgi by the Sec7/BIG1 family . It has been reported that Drosophila Arf1 regulates hematopoietic niche maintenance, blood cell precursor differentiation in vivo, planar cell polarity, and lamellipodium formation in S2 cells. Arf1 and other Golgi proteins were reported to exhibit upregulation of their transcripts in axon pruning of MB γ neurons during the larval-pupal transition. This study reports an important role of Arf1 in regulating dendrite pruning of ddaC sensory neurons. Arf1 puncta overlap with the trans- Golgi marker GalT and partially with the cis-Golgi marker GM130, suggesting that Arf1 is primarily localized on trans-Golgi in ddaC sensory neurons. A specific GEF for Arf1 has not been identified in Drosophila. In vivo and in vitro data provide compelling evidence that Sec71 is a GEF for Arf1 in Drosophila. First, Sec71 was co-localized with Arf1 on Golgi and their localizations were inter-dependent in ddaC neurons. Second, Sec71 preferentially bound to the GDP-bound form of Arf1 instead of GTP-bound form. Third, in the GEF assays Sec71 accelerated the release of GDP from Arf1. Fourth, both Arf1 and Sec71 are required for dendrite pruning, as loss of Sec71 or Arf1 led to comparable pruning defects in ddaC sensory neurons. Finally, the expression of Arf1 fully restored WP dendrite morphology and rescued the pruning defects in Sec71 RNAi ddaC neurons. Thus, Sec71 is specifically required for the GDP-to-GTP exchange of Arf1. Structure-function analyses indicate that DCB domain of Sec71 is important for its targeting on Golgi. In contrast, another small GTPase Arl1 was reported to bind to the N-terminal region of Sec71 (DCB and HUS1 domains) and recruit Sec71 on trans-Golgi apparatus in Drosophila S2 cells, and mammalian Arl1 is required for the recruitment of BIG1/2 (mammalian homologues of Sec71) on trans-Golgi (Wang, 2017).

    Secretory pathway plays a novel and important role in governing neurite pruning Extensive studies have attempted to understand roles of post-Golgi trafficking in outgrowth and elaboration of dendrites in growing neurons. Post-Golgi trafficking is polarized toward apical dendrites of rodent hippocampal neurons and selectively regulates the growth of dendrites. The dynamics of the Golgi outposts, mediated by the Golgin Lava Lamp, dynein-dynactin complex and Leucine- rich repeat kinase (Lrrk), is important for dendrite growth in Drosophila class IV da neurons. A small GTPase Rab10, which mediates post-Golgi vesicle trafficking, regulates dendrite growth and branching of multi-dendritic sensory neurons in both C. elegans and Drosophila (Wang, 2017).

    This study provides compelling evidence to demonstrate that post-Golgi trafficking plays a crucial role in proper dendrite pruning in sensory neurons. First, the key small GTPase Arf1 was identified that is important for post-Golgi trafficking regulates secretory vesicle biogenesis and dendrite pruning in sensory neurons during metamorphosis. Second, a Sec7-domain-containing protein Sec71 acts as a specific GEF for Arf1 and co- localizes with Arf1. Like Arf1, Sec71 is also an essential factor for regulating dendrite pruning. Third, given that both Arf1 and Sec71 also regulate dendrite growth and arborization in ddaC neurons, a critical role of Arf1 and Sec71 in dendrite pruning was further controlled using the Gene-Switch system. Pulse induction of Arf1T31N or Sec71DN at the middle third instar larval stage when the complete larval dendrite arbors form in ddaC neurons consistently caused much more severe dendrite pruning defects. These results highlight that the secretory pathway play separable roles in two distinct processes, namely dendrite growth and dendrite pruning. Arf1 was reported to regulate post-Golgi secretion by recruiting its downstream effectors, including the clathrin adaptors AP-1 and AP-3, and GGA. Post-Golgi trafficking pathways include the transport from Golgi to plasma membrane (exocyst complex-mediated), from Golgi to early/sorting endosomes (AP-1-meditated), from Golgi to late endosomes (Golgi-localized Gamma-ear containing Arf-binding protein or GGA-mediated) as well as from Golgi to lysosomes (AP-3-mediated). It is conceivable that at least one of these post-Golgi trafficking routes is involved in dendrite pruning of sensory neurons (Wang, 2017).

    It has been reported previously that Rab5/ESCRT-dependent endocytic pathways facilitate dendrite pruning by downregulating the L1-CAM Nrg in ddaC neurons during metamorphosis. In MB γ neurons, the JNK pathway promotes axon pruning by downregulating another adhesion molecule Fasciclin II. These studies suggest a general mechanism whereby cell adhesion molecules are internalized and downregulated to destabilize dendrites and/or axons during neurite pruning. However, the mechanism that triggers Nrg endocytosis is poorly understood. This study demonstrated that Arf1/Sec71-mediated secretory pathway promotes endocytosis and downregulates Nrg prior to dendrite pruning. First, while Nrg levels were strongly reduced prior to dendrite pruning, loss of Arf1 or Sec71 led to elevated levels of Nrg protein in dendrites, axons and soma, comparable to Rab5 mutant neurons. Second, Nrg was no longer redistributed on FYVE-positive endosomes in Arf1 or Sec71 mutant ddaC neurons, suggesting a blockage of Nrg endocytosis. Third, while Rab5 mutant neurons exhibited robust ubiquinated protein aggregates and enlarged endosomes, further removal of either Arf1 or Sec71 suppressed these rab5 mutant phenotypes, suggesting that the secretory pathway acts upstream of Rab5 to positively regulates endocytosis. Finally, knockdown of Nrg significantly suppressed the dendrite pruning defect in Arf1 or Sec71 mutant neurons, supporting the notion that the secretory pathway promotes Nrg endocytosis and downregulation. Thus, the secretory pathway not only secretes the cell adhesion molecules to the dendrite surface and stabilize dendrites, but also unexpectedly promotes the internalization and turnover of the adhesion molecules. It is conceivable that in response to ecdysone pulse, the secretory pathway might be required to specifically secrete an as-yet-unidentified ligand to trigger massive endocytosis of the L1-CAM Nrg and thereby leads to degeneration of larval dendrites. Further studies may continue to elucidate what ligand or secreted protein promotes Nrg endocytosis (Wang, 2017).

    Golgi outpost synthesis impaired by toxic polyglutamine proteins contributes to dendritic pathology in neurons

    Dendrite aberration is a common feature of neurodegenerative diseases caused by protein toxicity, but the underlying mechanisms remain largely elusive. This study shows that nuclear polyglutamine (polyQ) toxicity resulted in defective terminal dendrite elongation accompanied by a loss of Golgi outposts (GOPs) and a decreased supply of plasma membrane (PM) in Drosophila class IV dendritic arborization (da) (C4 da) neurons. mRNA sequencing revealed that genes downregulated by polyQ proteins included many secretory pathway-related genes, including COPII genes regulating GOP synthesis. Transcription factor enrichment analysis identified CREB3L1/CrebA, which regulates COPII gene expression. CrebA overexpression in C4 da neurons restores the dysregulation of COPII genes, GOP synthesis, and PM supply. Chromatin immunoprecipitation (ChIP)-PCR revealed that CrebA expression is regulated by CREB-binding protein (CBP), which is sequestered by polyQ proteins. Furthermore, co-overexpression of CrebA and Rac1 synergistically restores the polyQ-induced dendrite pathology. Collectively, these results suggest that GOPs impaired by polyQ proteins contribute to dendrite pathology through the CBP-CrebA-COPII pathway (Chung, 2017).

    Temporal coherency between receptor expression, neural activity and AP-1-dependent transcription regulates Drosophila motoneuron dendrite development

    Neural activity has profound effects on the development of dendritic structure. Mechanisms that link neural activity to nuclear gene expression include activity-regulated factors, such as CREB, Crest (Ca2+-responsive transactivator, a syntaxin-related nuclear protein that interacts with CREB-binding protein and is expressed in the developing brain) or Mef2, as well as activity-regulated immediate-early genes, such as fos and jun. This study investigates the role of the transcriptional regulator AP-1, a Fos-Jun heterodimer, in activity-dependent dendritic structure development. Genetic manipulation, imaging and quantitative dendritic architecture analysis were combined in a Drosophila single neuron model, the individually identified motoneuron MN5. First, Dalpha7 nicotinic acetylcholine receptors (nAChRs) and AP-1 are required for normal MN5 dendritic growth. Second, AP-1 functions downstream of activity during MN5 dendritic growth. Third, using a newly engineered AP-1 reporter it was demonstrated that AP-1 transcriptional activity is downstream of Dalpha7 nAChRs and Calcium/calmodulin-dependent protein kinase II (CaMKII) signaling. Fourth, AP-1 can have opposite effects on dendritic development, depending on the timing of activation. Enhancing excitability or AP-1 activity after MN5 cholinergic synapses and primary dendrites have formed causes dendritic branching, whereas premature AP-1 expression or induced activity prior to excitatory synapse formation disrupts dendritic growth. Finally, AP-1 transcriptional activity and dendritic growth are affected by MN5 firing only during development but not in the adult. These results highlight the importance of timing in the growth and plasticity of neuronal dendrites by defining a developmental period of activity-dependent AP-1 induction that is temporally locked to cholinergic synapse formation and dendritic refinement, thus significantly refining prior models derived from chronic expression studies (Vonhoff, 2013).

    By combining genetic and neuroanatomical tools with imaging in a single-cell model, the adult MN5 in Drosophila, this study demonstrates that: (1) AP-1 is transcriptionally active during all stages of postembryonic motoneuron dendritic growth, (2) AP-1 and excitatory cholinergic inputs are required for normal dendrite growth in MN5, (3) AP-1 transcriptional activity is enhanced via a CaMKII-dependent mechanism by increased neural activity during pupal development but not in the adult, and (4) both activity and AP-1 can promote or inhibit dendritic branching, depending on the developmental stage. AP-1 is required for normal MN5 dendrite growth downstream of activity and CaMKII (Vonhoff, 2013).

    Although AP-1 has been thought to regulate dendrite development in an activity-dependent manner via global changes in gene expression, probably in a calcium-dependent manner as described for CREB or Crest, direct evidence for this hypothesis was sparse (Vonhoff, 2013).

    This study demonstrated that excitatory cholinergic input to MN5 and AP-1 transcriptional activity were required for normal dendrite growth of MN5 during pupal life. MN5 total dendritic length and branch numbers were significantly reduced (~50%) by inhibition of AP-1 [by Jbz (a dominant-negative form of Jun) expression] and in Dα nAChR mutants. Conversely, overexpression of AP-1 or increased MN5 excitability as induced by potassium channel knockdown (by EKI) increased dendritic branching (Duch, 2008). Clearly, AP-1 acted downstream of activity as inhibition of AP-1 by Jbz completely attenuated EKI (electrical knock-in) mediated dendritic growth and branching (Vonhoff, 2013).

    A new AP-1 reporter was employed to measure activity-induced AP-1 transcriptional activity by imaging, and to gain insight into the pathway that might connect MN5 activity to AP-1-dependent transcription. Although the detection threshold of this reporter might be too low to detect small changes in AP-1 activity, sensitivity was sufficient to reliably report increased AP-1 activity following overexpression of fos and jun, inhibition of AP-1 transcriptional activity by Jbz expression, and changes in AP-1 activity as induced by various manipulations of cellular signaling. Therefore, the reporter was deemed suitable for testing changes in AP-1 transcriptional activity in MN5 (Vonhoff, 2013).

    Targeted expression of TrpA1 channels in MN5 allowed the induction of firing in vivo by temperature shifts during selected developmental periods. Activation of MN5 during pupal life for 36 hours (P9 to adult) or longer (P5 to adult) caused significant increases in AP-1-induced nuclear GFP fluorescence. By contrast, in adults neither similar nor longer durations of TrpA1 activation resulted in any detectable increase in AP-1 reporter-mediated nuclear GFP fluorescence in MN5. Similarly, live imaging in semi-intact adult preparations did not reveal any detectable AP-1 activity upon acute TrpA1 activation for various durations. This indicated that activity-dependent AP-1 activation was restricted to pupal life. However, whether AP-1 activation in the adult MN5 occurred upon patterned activity was not tested. Spaced stimuli that reflect endogenous activity patterns are required for insect motoneuron axonal and dendritic development and can regulate mammalian neuron dendritic morphology. However, during flight, MN5 fires tonically at frequencies between 5 and 20 Hz, a pattern that is well reflected by temperature-controlled TrpA1 channel activation. Therefore, adult flight behavior is unlikely to induce AP-1 activity, which is involved in dendrite and synapse development (Freeman, 2010). This is consistent with the assumption that dendritic structure is fairly stable in the adult (Vonhoff, 2013).

    cAMP and Jun N-terminal kinase (Jnk) have been implicated as potential links between activity and AP-1 activation. Cell culture studies on Drosophila larval motoneurons and giant neurons demonstrate a role of calcium. This study showed that Dα7 nAChRs, which are highly permeable to calcium, were required for normal MN5 dendritic growth. Combining genetic manipulation of Dα7 nAChRs, AP-1 and CaMKII with imaging of AP-1 reporter activity revealed that CaMKII was required downstream of Dα7 nAChRs to cause AP-1-dependent transcription. These data show that activity-dependent calcium influx through nAChRs might activate AP-1 during pupal life via a CaMKII-dependent mechanism in vivo. Activity and AP-1 can promote or inhibit dendritic growth during pupal life, depending on timing (Vonhoff, 2013).

    In larval motoneurons, AP-1 is required for dendritic overgrowth as induced by artificially increased activity (Hartwig, 2008). In MN5, AP-1 is required downstream of nAChRs and CaMKII for normal dendritic growth. By contrast, premature expression of AP-1 in MN5 inhibited dendritic growth. These data were consistent with the hypothesis that timing is the crucial factor. First, P103.3 and D42 both caused similar overgrowth but exhibited fairly different expression patterns. Second, C380-GAL4 and Dα7 nAChR-GAL4 both inhibited MN5 dendrite growth but expressed in largely different sets of neurons. Therefore, the common factor of C380 and Dα7 nAChR on the one hand and D42 and P103.3 on the other hand was timing. Third, shifting the timing of C380-GAL4-driven AP-1 expression to later stages prevented dendritic defects. Fourth, imposed activity prior to P5 by TrpA1 activation also inhibited dendritic branching. Dendritic defects as induced by imposed premature activity were rescued by inhibition of AP-1 via Jbz expression in MN5 (Vonhoff, 2013).

    MN5 early dendritic growth starts at early pupal stage 5 (P5), and expression of Dα7 nAChRs begins 2.5 hours later, at mid stage P5. Similarly, Xenopus optic tectal and turtle cortical neurons receive glutamatergic and GABAergic inputs as soon as the first dendrites are formed. In vertebrates, early synaptic inputs and neurotransmitters play essential roles in dendrite development. The current data are consistent with the hypothesis that the endogenous expression of nAChRs caused increased activity throughout the developing motor networks, which, in turn, upregulated AP-1-dependent transcription and dendritic growth via a CaMKII-dependent mechanism. During zebrafish spinal cord development, activity is required for strengthening functional central pattern generator (CPG) connectivity. As dendrites are the seats of input synapses to motoneurons, an activity-dependent component in motoneuron dendritic growth that follows early synaptogenesis might function to refine dendrite shape during the integration into the developing CPG (Vonhoff, 2013).

    Dendritic growth gated by a steroid hormone receptor underlies increases in activity in the developing Drosophila locomotor system

    As animals grow, their nervous systems also increase in size. How growth in the central nervous system is regulated and its functional consequences are incompletely understood. These questions were explored using the larval Drosophila locomotor system as a model. In the periphery, at neuromuscular junctions, motoneurons are known to enlarge their presynaptic axon terminals in size and strength, thereby compensating for reductions in muscle excitability that are associated with increases in muscle size. This work examined how motoneurons change in the central nervous system during periods of animal growth. I was found that within the central nervous system motoneurons also enlarge their postsynaptic dendritic arbors, by the net addition of branches, and that these scale with overall animal size. This dendritic growth is gated on a cell-by-cell basis by a specific isoform of the steroid hormone receptor ecdysone receptor-B2, for which functions have thus far remained elusive. The dendritic growth is accompanied by synaptic strengthening and results in increased neuronal activity. Electrical properties of these neurons, however, are independent of ecdysone receptor-B2 regulation. It is proposed that these structural dendritic changes in the central nervous system, which regulate neuronal activity, constitute an additional part of the adaptive response of the locomotor system to increases in body and muscle size as the animal grows (Zwart, 2013).

    The implications of these observations are twofold: First, neurons can deploy structural changes in their dendritic trees as a central mechanism with which to regulate and adjust levels of neuronal activity; second, in terms of connectivity, the size of the postsynaptic dendritic arbor seems to be decisive in determining the number of connections that neurons form among available presynaptic terminals (Zwart, 2013).

    Increases in body or organ size are normally accompanied by matching changes in innervation, required to maintain appropriate neuronal control. One of the best-studied examples is the neuromuscular junction where increases in muscle size lead to biophysical changes in muscle physiology, which are compensated for by a matching enlargement of the neuromuscular junction. The neural and cellular mechanisms that regulate these homeostatic adjustments have been studied in detail. This study has identified a potential additional, central mechanism associated with adjustment to growth in the neuromuscular system: In the central nervous system, motoneurons also enlarge their postsynaptic dendritic arbors as animals increase in body size; this leads to increased synaptic drive and thus prolonged periods of bursting activity (Zwart, 2013).

    Morphometric quantifications of dendritic arbors during larval stages, show that motoneuron dendritic trees increase their overall dendritic length proportionately to body size. The growth of these arbors occurs by addition of new dendritic segments that 'fill in' existing territory, as well as at the perimeter of the tree, thus widening its reach. Such scaling growth of dendritic arbors in relation to body size is a widespread phenomenon and has previously been observed in different types of nerve cells, including Purkinje, pyramidal, olfactory mitral cells and sympathetic ganglionic neurons (Zwart, 2013).

    An interesting discovery of this study is that the growth that normally occurs during larval stages is regulated cell-autonomously. Specifically, this study established that the B2 isoform of the ecdysone steroid hormone receptor is required cell-autonomously in motoneurons for normal dendritic growth. Expression of a dominant-negative form of EcRB2, UAS-EcR-B2W650A, in single cells prevents the characteristic increase in motoneuron dendritic arbor size during the second larval instar stage and seems to arrest neural arbors structurally at a young larval stage, despite being embedded in an otherwise normally developing ganglion. Electrical excitability of aCC and RP2 motoneurons, however, are not affected by expression of EcR-B2W650A. It was found EcR-B2 is the only isoform expressed in the larval nerve cord during early larval stages, in agreement with and complementary to previously published data. Although different functions have been ascribed to the other two EcR isoforms, A and B1, the role of the B2 isoform had until now remained unknown. This study has uncovered an important role for the EcR-B2 isoform in nervous system development, namely to permit growth in larval stages. The role of EcR-B2 is intrepeted as being a permissive factor for dendritic growth for two reasons. First, the pattern of dendritic growth during larval stages does not follow ecdysteroid titers but may be exponential. Second, precocious or overexpression of the wild-type form of EcR-B2 does not cause abnormal dendritic growth. Because the nervous system is one of the most metabolically expensive tissues, it is conceivable that gating the decision through EcR-B2 on whether or not neurons grow may provide a strategy to synchronize neural growth with the growth of the animal as a whole, as it progresses from one developmental stage to the next. Indeed, production of ecdysteroids in the Drosophila larva is under nutritional control (Zwart, 2013).

    It is likely that other signaling pathways determine the extent to which neurons grow. For example, in rat superior cervical ganglion cells dendritic growth correlates with peripheral target size and NGF has been implicated. In Drosophila, three neurotrophic factors have been identified, expressed in subsets of body-wall muscles at embryonic stages, although none has thus far been reported to be expressed in the dorsal musculature, whose innervating aCC and RP2 motoneurons were analyzed in this study. Other muscle and associated glia-derived retrograde regulators of neuromuscular junctions include the TGF-β homologs Glass bottom boat and Maverick and the Wnt family member Wingless. It is conceivable that these could regulate the growth of motoneuron dendritic arbors in synchrony with that of presynaptic axon terminals. Indeed, it was fiynd that, in addition to its role in regulating dendritic growth, EcR-B2 is also required for normal neuromuscular junction growth, suggesting that EcR-B2 itself regulates the development of both pre- and postsynaptic compartments (Zwart, 2013).

    As the Drosophila larva grows around 100-fold in surface area with matching increases in body wall muscle size, appropriate levels of muscle depolarization have to be maintained. The larval body-wall muscles are virtually isopotential, and as their input resistance goes down with increasing muscle size, presynaptic output at the neuromuscular junction increases in a compensatory fashion: By adjusting both terminal size and synaptic strength the amplitude of postsynaptic responses in the muscle is maintained. Similarly, at the growing vertebrate neuromuscular junction, motor endplates expand as muscles enlarge, enhancing neurotransmitter release. The findings of this study show that in addition to the well-characterized regulation of neuromuscular junction strength, in the central nervous system, motoneurons also adjust the size of their dendritic terminals. Previous studies have demonstrated that during the initial assembly of the locomotor network in the embryo neurons deploy their dendritic arbors as structural homeostatic devices, adjusting their extent to compensate for naturally occurring variations in the density of synaptic partner terminals. This study has shown that in subsequent larval stages, when animals grow rapidly, extension and elaboration of dendritic arbors leads to greater numbers of presynaptic inputs and thus increased synaptic drive. Specifically, with progression from the second to the third larval instar the duration of action potential bursts approximately doubles. Longer action potential burst periods might increase and potentially prolong muscle contractions; they could also enhance facilitation. Furthermore, the time course of excitatory junctional potentials (EJPs) at the Drosophila neuromuscular junction changes as muscles enlarge: Time constants describing both the rise and fall of EJPs increase, which, taking into account the bursting input the muscle receives, will result in a larger envelope of depolarization as the animals grows. It is therefore conceivable that the increased number of action potentials fired per burst that were observed in this study affects the strength of the neuromuscular synapse by enhancing both the process of facilitation and the envelope of depolarization (Zwart, 2013).

    Having identified EcR-B2 as a regulator of dendritic growth of motoneurons, this study has investigated how dendritic growth relates to synaptic drive in this system. During normal development, motoneurons increase their dendritic arbor proportionally to animal body size. A biophysical consequence of increased neuronal size is increased capacitance and decreased input resistance, both of which reduce the cell’s intrinsic excitability. Indeed, this study found that motoneurons in third instar larvae (48 h ALH) have larger dendritic arbors and are less excitable than smaller cells of younger, second instar (24 h ALH) animals. It was also found that increases in dendritic arbor size are accompanied by increases in the frequency of spontaneous mEPSPs, suggesting that the dendritic growth during larval development facilitates the addition of synapses. It was asked how these changes in synaptic input, from the second to the third larval instar, might lead to extended bursting periods. Most likely, this requires the addition of synapses from new premotor partner neurons. Interestingly, at 48 h ALH EcRB2W650A– expressing motoneurons, despite being located within an otherwise unmanipulated nervous system with its third instar complexity and density of presynaptic release sites, are indistinguishable from younger neurons in a younger ganglion, in terms of dendritic arbor size, distribution of dendritic branches in the neuropil, number of synaptic sites on these, and activity patterns. This suggests that the size and geography of these dendritic arbors is decisive in determining their connectivity. Because increases were found in the duration of action potential bursts over developmental time, it is likely that as they grow aCC and RP2 motoneuron arbors establish new presynaptic contacts from additional interneurons, some of which may be in adjacent segments. These could include segmental homologs of those with whom they already form connections more proximally at earlier stages. Such a scenario could extend the duration of the synaptic drive, as was observe: As each wave of activity passes through the nerve cord during locomotion cycles, synapses in adjacent segments would have different timings that when combined on one dendritic arbor could lead to prolonged periods of action potential bursts. Indeed, tentative evidence has shown that larval Drosophila motoneurons change their connectivity qualitatively, in that neurons begin to show inhibitory responses during larval development (Zwart, 2013).

    Comparable strategies have been documented in other systems. For instance, the substantia nigra compacta dopaminergic neurons change their dendritic architecture to alter the number and identity of synapses they receive and thereby also their functional properties within the network. In the case of the aCC and RP2 motoneurons in Drosophila that are studied here in this work, the identity of their excitatory presynaptic partners has not yet been characterized beyond being cholinergic and so at this point cannot be resolved conclusively (Zwart, 2013).

    The electrical excitability of aCC/RP2 is not affected by expression of EcRB2W650A. The excitability of a cell is determined by the input resistance, the sum of all leak currents at rest, and the voltage-sensitive conductances that generate the action potential. The input resistance of a cell is normally inversely proportional to its size. Consistent with this notion, this study found that during normal development the electrical excitability of these neurons decreases during the second larval instar stage, as cells increase in size. Moreover, no changes were recorded in the relationship between membrane potential and action potential firing, suggesting there is no net change in the voltage-sensitive conductances that generate the action potential, and this aspect is not affected by EcR-B2W650A expression. However, the inverse relationship between cell size and excitability no longer holds for cells expressing EcR-B2W650A: At 48 h ALH, the intrinsic excitability of these comparatively small cells, which are reduced both in dendritic arbor and soma size, is similar to that of their age-matched, larger, control counterparts. These findings are compatible with at least two scenarios. First, it is possible that the location of the action potential initiation zone, a key determinant of excitability of these neurons, changes from the second to the third larval instar stage. For example, the positioning of the action potential initiation zone relative to the proximal primary neurite, which integrates dendritic currents, may change as nerve cords enlarge, and this could be independent of EcR-B2 signaling in individual motoneurons. However, in agreement with an earlier study, this study found that the intrinsic excitability of aCC and RP2 motoneurons strongly correlates with the amplitude of synaptic input (78). In this model, EcR-B2W650A–expressing neurons would undergo homeostatic adjustment to remain within the normal range of neuronal activity. Given that no changes were detected in the voltage-sensitive conductances that generate the action potential, but measured a reduction in membrane resistance over developmental time, leak channels could be involved (Zwart, 2013).

    Dendritic refinement of an identified neuron in the Drosophila CNS is regulated by neuronal activity and Wnt signaling

    The dendrites of neurons undergo dramatic reorganization in response to developmental and other cues, such as stress and hormones. Although their morphogenesis is an active area of research, there are few neuron preparations that allow the mechanistic study of how dendritic fields are established in central neurons. Dendritic refinement is a key final step of neuronal circuit formation and is closely linked to emergence of function. This is a study of a central serotonergic neuron in the Drosophila brain, the dendrites of which undergo a dramatic morphological change during metamorphosis. Using tools to manipulate gene expression in this neuron, the refinement of dendrites during pupal life was examined. This study shows that the final pattern emerges after an initial growth phase, in which the dendrites function as 'detectors', sensing inputs received by the cell. Consistent with this, reducing excitability of the cell through hyperpolarization by expression of K(ir)2.1 results in increased dendritic length. Sensory input, possibly acting through NMDA receptors, is necessary for dendritic refinement. These results indicate that activity triggers Wnt signaling, which plays a 'pro-retraction' role in sculpting the dendritic field: in the absence of sensory input, dendritic arbors do not retract, a phenotype that can be rescued by activating Wnt signaling. These findings integrate sensory activity, NMDA receptors and Wingless/Wnt5 signaling pathways to advance understanding of how dendritic refinement is established. The maturation of sensory function is shown to interact with broadly distributed signaling molecules, resulting in their localized action in the refinement of dendritic arbors (Singh, 2010).

    This study focuses on a specific phase during the metamorphosis of the dendrites of a central serotonergic neuron, in which excess growth is removed by a process that has been termed refinement. Genetic analyses using loss-of-function mutants and RNAi-mediated knockdown of specific genes has led to a postulated a link between neuronal activity, synaptic input and Wnt signaling in this process. The sparse dendrites innervating the adult antennal lobe, present on the wide-field serotonergic neurons (CSDn) during the larval stage, are removed early in pupation by pruning, followed by a period of exuberant growth. The arrival of sensory neurons at the antennal lobe correlates well with when growth of the CSDn dendrites ceases and removal of the excess branches occurs. The CSDn must be active for the refinement process to occur, as refinement fails when neuronal activity is inhibited or when the sensory neurons are absent. Phenotypes observed in the latter case can be rescued by ectopic activation of the neuron using the temperature-sensitive dTrp-A1 channel. It is suggested that activity within the CSDn, possibly together with activity in presynaptic neurons, acts to provide the correlated activity required to trigger activation of NMDARs. Knockdown of NMDARs affects the refinement process, although identifying its specific action requires further study. A possible consequence of the activity-dependent process is activation of the Wg pathway, as the phenotype observed in aristalless mutants can also be rescued by ectopic expression of Dishevelled (Dsh) in the CSDn. It seems unlikely that activity within the CSDn leads to the release of Wnt ligands, but rather that dendrites respond locally to Wnt ligands in the region of a dendrite that is receiving input. Although other interpretations of the data are possible, a hypothesis is favored whereby specific synapses are stabilized as a result of correlated neuronal activity, and that excess dendritic branches are removed by Wnt signaling (Singh, 2010).

    Perturbations in neuronal activity can be compensated by changes at multiple levels, including alterations in the expression of ion channels and in synaptic strength. Tripodi (2008) provides evidence for structural homeostasis whereby alterations in afferent input during development can be compensated by changes in dendritic geometry. This suggests that dendritic arbors serve as sensors for input levels, thus allowing the self-organization of circuits that is necessary for robust behavioral outputs (Tripodi, 2008). The current studies in the CSDn support these observations: reduced activation of the cell by targeted expression of Kir2.1 results in a greatly enlarged dendritic field in the adult. This phenotype can be explained by a mechanism in which the absence of electrical activity results in a failure of the signaling mechanisms that stop growth of the arbors and that remove additional branches. Reduced excitability could also drive the homeostatic mechanisms towards making more arbors and to suppress the refinement program (Singh, 2010).

    Dendritic growth and refinement are closely associated with input activity and synapse formation during development. Activity-dependent development of circuits is thought to utilize mechanisms similar to those involved in Hebbian learning and plasticity. NMDARs are ideal candidates for detecting correlated pre- and postsynaptic activity, which is crucial in the Hebbian model of learning and plasticity. Strengthening of synapses, as in this study, leads to the stabilization and extension of dendrites, whereas weakening of synapses leads to the destabilization and elimination of dendritic branches (Espinosa, 2009; Cline, 2008; Constantine-Paton, 1998). During vertebrate hippocampal development, NMDAR activation has been shown to limit synapse number and reduce dendritic complexity. The stabilization of a particular synapse or arbor possibly attenuates the formation of new branches or synapses, thus limiting further dendritic growth. In such a scenario, knocking down NMDAR levels would be expected to result in increased dendritic complexity, as indeed has been observed in this study. The mechanism by which 'appropriately connected' synapses are strengthened, whereas suboptimal contacts are eliminated, needs to be studied in thus system. In other systems, Ca2+, which is released upon NMDAR activation, impinges on various intracellular effectors that regulate dendritic morphogenesis. In addition, selective stabilization/destabilization of dendritic arbors could be affected by the local release of growth factors in response to activity (Singh, 2010).

    This study shows that activity-dependent activation of the Wnt pathway facilitates retraction of dendritic arbors. Arbors that receive appropriate input are somehow protected and stabilized. These experiments suggest that Wnt-dependent refinement functions through a non-nuclear pathway and could act by impinging directly on cytoskeletal dynamics (Schlessinger, 2009; Salinas, 2008). Disruption of the microtubule cytoskeleton is a key feature of dendritic pruning in Drosophila during metamorphosis. GSK3β (Shaggy in Drosophila) an intracellular inhibitor of the Wnt pathway, has been shown to act as a sensor of inputs for neuronal activity (Chiang, 2009) and a potent regulator of microtubule dynamics in axons. In the Drosophila embryonic CNS, the Src family of tyrosine kinases (SFKs) is required for Wnt5/Drl-mediated signaling. Interestingly, SFKs seem to act as a crucial point of convergence for multiple signaling pathways that enhance NMDAR activity and hence are thought to act as molecular hubs for the control of NMDARs. It is tempting to envisage a scenario in which there is cross-talk between Wnt5/Drl signaling-mediated activation of SFKs and NMDAR signaling during refinement (Singh, 2010).

    In summary, this study shows that the dendritic refinement of a central modulatory serotonergic neuron is regulated by electrical activity, NMDAR and Wnt signaling. Similar mechanisms have been implicated in dendritic growth and refinement of excitatory neurons in vertebrates. This study provides a model neuron preparation in which the dendritic growth and refinement of a modulatory neuron can be analyzed genetically. It was demonstrated that the dendrites of CSDn receive input from sensory neurons from the arista, supporting previous suggestions that mechanosensory input could alter sensitivity to odorant stimuli. In both Drosophila (Dacks, 2009) and the mammalian olfactory bulb (Petzold, 2009), serotonin gates the odor-evoked sensory response. CSDn sends projections to higher brain centers and multiglomerular projections to the contralateral antennal lobe and hence it is likely to influence the overall properties of the olfactory circuit. This study suggests that the structural and resulting functional properties of this neuron emerge from an interaction between partner neurons, together with input from intrinsic and extrinsic cues (Singh, 2010).

    Intra-neuronal competition for synaptic partners conserves the amount of dendritic building material

    Brain development requires correct targeting of multiple thousand synaptic terminals onto staggeringly complex dendritic arbors. The mechanisms by which input synapse numbers are matched to dendrite size, and by which synaptic inputs from different transmitter systems are correctly partitioned onto a postsynaptic arbor, are incompletely understood. By combining quantitative neuroanatomy with targeted genetic manipulation of synaptic input to an identified Drosophila neuron, this study shows that synaptic inputs of two different transmitter classes locally direct dendrite growth in a competitive manner. During development, the relative amounts of GABAergic and cholinergic synaptic drive shift dendrites between different input domains of one postsynaptic neuron without affecting total arbor size. Therefore, synaptic input locally directs dendrite growth, but intra-neuronal dendrite redistributions limit morphological variability, a phenomenon also described for cortical neurons. Mechanistically, this requires local dendritic Ca2+ influx through Dα7 nAChRs or through low-voltage-activated channels following GABAA receptor-mediated depolarizations (Ryglewski, 2017).

    Mitochondrial dysfunction induces dendritic loss via eIF2α phosphorylation

    Mitochondria are key contributors to the etiology of diseases associated with neuromuscular defects or neurodegeneration. How changes in cellular metabolism specifically impact neuronal intracellular processes and cause neuropathological events is still unclear. This study dissects the molecular mechanism by which mitochondrial dysfunction induced by Prel aberrant function mediates selective dendritic loss in Drosophila melanogaster class IV dendritic arborization neurons. Using in vivo ATP imaging, it was found that neuronal cellular ATP levels during development are not correlated with the progression of dendritic loss. By searching for mitochondrial stress signaling pathways that induce dendritic loss it was found that mitochondrial dysfunction is associated with increased eIF2α phosphorylation, which is sufficient to induce dendritic pathology in class IV arborization neurons. It was also observed that eIF2α phosphorylation mediates dendritic loss when mitochondrial dysfunction results from other genetic perturbations. Furthermore, mitochondrial dysfunction induces translation repression in class IV neurons in an eIF2α phosphorylation-dependent manner, suggesting that differential translation attenuation among neuron subtypes is a determinant of preferential vulnerability (Tsuyama, 2017).

    In vivo dendrite regeneration after injury is different from dendrite development

    Neurons receive information along dendrites and send signals along axons to synaptic contacts. The factors that control axon regeneration have been examined in many systems, but dendrite regeneration has been largely unexplored. This study reports that, in intact Drosophila larvae, a discrete injury that removes all dendrites induces robust dendritic growth that recreates many features of uninjured dendrites, including the number of dendrite branches that regenerate and responsiveness to sensory stimuli. However, the growth and patterning of injury-induced dendrites is significantly different from uninjured dendrites. Regenerated arbors cover much less territory than uninjured neurons, fail to avoid crossing over other branches from the same neuron, respond less strongly to mechanical stimuli, and are pruned precociously. Finally, silencing the electrical activity of the neurons specifically blocks injury-induced, but not developmental, dendrite growth. By elucidating the essential features of dendrites grown in response to acute injury, this work builds a framework for exploring dendrite regeneration in physiological and pathological conditions (Thompson-Peer, 2016).

    Nutrient-dependent increased dendritic arborization of somatosensory neurons

    Suboptimal nutrition imposes developmental constraints on infant animals, which marshal adaptive responses to eventually become mature adults. Such responses are mounted at multiple levels from systemic to cellular. Little is known about how growth of postmitotic and morphologically complex cells, such as neurons, is controlled by nutritional status. This question was addressed using Class I and Class IV dendritic arborization neurons in Drosophila larvae. Class IV neurons have been shown to sense nociceptive thermal, mechanical and light stimuli, whereas Class I neurons are proprioceptors. Larvae were reared on diets with different protein and carbohydrate content throughout larval stages, and how morphologies of Class I or Class IV neurons were affected was examined. Dendritic arbors of Class IV neurons became more complex when larvae were reared on a low-yeast diet, which contains lower amounts of amino acids and other ingredients, compared to a high-yeast diet. In contrast, such low-yeast-dependent hyperarborization was not seen in Class I neurons. The physiological and metabolic implications of the hyperarborization phenotype are discussed in relation to a recent hypothesis that Class IV neurons sense protein-deficient stress and to this characterization of how the dietary yeast contents impacted larval metabolism (Watanabe, 2016).

    Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila.

    A major gap in understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, this study identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1 (Pickpocket), another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception (Gorczyca, 2014).

    This study has identified Ppk26, an DEG/ENaC channel subunit that is coexpressed with Ppk1 in class IV da neurons. Consistent with the model that Ppk26 and Ppk1 may be subunits of the same channel, it was found that Ppk1 and Ppk26 colocalize in class IV da neurons, they form a complex in heterologous expression systems, and they show nonadditive and nonredundant mutant phenotypes in vivo. Ppk26 protein was found in somatic, dendritic, and axonal compartments, and plasma membrane insertion was observed in terminal dendrites. Ppk1 and Ppk26 were reciprocally required for normal trafficking and/or insertion to the plasma membrane, further supporting the notion that these two channel subunits interact in vivo. This study shows that, as is the case for MEC Degenerin channels, mutations at the Degenerin position of Ppk26 lead to loss of class IV da neuron integrity. It was also found that Ppk26 function plays essential roles in normal larval locomotion, particularly in turning behavior (Gorczyca, 2014).

    Overexpression of EGFP-tagged MEC channels in C. elegans has been reported to result in a punctate localization, leading to suggestions that each of these puncta represents a mechanosensory apparatus. In light of the speculation that contact of dendrites with subcuticular epidermis is part of the apparatus that senses mechanical stimuli, it is intriguing that both Ppk1 and Ppk26 were found on the surface of distal and higher-order dendrites, consistent with channel function in this compartment. Mutants in Ppk1 and Ppk26 showed defects in the frequency of turning of freely crawling larvae. Moreover, loss of function of either or both Ppk26 and Ppk1 had the same effect on larval locomotion. These findings support the notion that Ppk26 and Ppk1 may act in the same pathway—perhaps in the same channel complex—in mechanosensation that is important for proper locomotion (Gorczyca, 2014).

    Class IV da neurons are known to express the DEG/ENaC channel subunit Ppk1 (Adams, 1998), an observation that was confirmed in this study. Given that DEG/ENaC channels are trimeric ion channels that typically require the assembly of different subunits for proper function, the specific coexpression of Ppk26 in class IV da neurons raises the possibility that Ppk1 and Ppk26 may correspond to different subunits of the same mechanosensory channel in vivo. This is supported by the findings of biochemical interactions between Ppk1 and Ppk26, their null mutant phenotypes that indicate nonredundancy, and by the reciprocal requirements of Ppk1 and Ppk26 for normal trafficking or insertion to the plasma membrane . The mechanism for this mutual dependence is currently unknown (Gorczyca, 2014).

    Notably, inspection of the developmental expression profile of ppk1 RNA and ppk26 RNA revealed a similar time course, with the absolute levels of RNA for ppk26 twice as high as those of RNA for ppk1 throughout development. Although it is uncertain that this quantitative difference in RNA levels reflects a similar quantitative difference in the levels of Ppk1 and Ppk26 subunits, it is tempting to speculate that two Ppk26 subunits and one Ppk1 subunit may be assembled to form a surface-expressed trimeric channel (Gorczyca, 2014).

    Studies in many systems have suggested that mutations in the Degenerin domain of DEG/ENaC lead to loss of cell integrity and perhaps degeneration; however, this behavior has not yet been observed in Drosophila Pickpocket family members. The Degenerin position is localized in the wrist region, close to the mouth of the pore. Thus, it has been suggested that Degenerin mutations change the properties of the channel, increasing open time probability and perhaps shifting its ion selectivity from Na+ to Ca2+. Given the tight regulation of Ca2+ within cells and its involvement in critical cellular processes, this increase in Ca2+ permeability may lead to loss of cellular homeostasis, in a process that has been dubbed excitotoxicity. Consistent with these findings, it was also observed that when a Degenerin mutation was introduced into Ppk26 that was overexpressed in class IV da neurons, it resulted in a marked reduction in dendritic arbor size. This suggests that the Ppk26-Deg mutation leads to toxicity in the sensory neurons, as has been observed in DEG/ENaC channels in C. elegans, but not in Ppk1. Whereas it is likely that the function of the pore structure of these channels is evolutionarily conserved, it is unclear why Ppk26-Deg has a more potent effect than Ppk1-Deg. One possibility is that the Ppk26-Deg residue makes a larger contribution to pore structure than Ppk1-Deg, because of its intrinsic structure or because of a potential 2:1 stoichiometry in vivo (Gorczyca, 2014).

    Larval locomotion is likely regulated by sensory input provided by sensory neurons in the body wall, which may in turn modulate the motoneurons innervating the body wall. The C. elegans mechanosensitive TRPN channel TRP-4 acts in the DVA neuron to coordinate bending behavior and body posture through positive and negative modulation. It seems likely that class IV da neurons likewise provide some information for sensory modulation of locomotion through a mechanosensory mechanism, but future work will need to determine if this is indeed the case (Gorczyca, 2014).

    Proprioception is a mechanosensory process involving sensory neurons that transduce the mechanical information related to body position or characteristics of the environment for the generation of appropriate behavioral output, such as the turning locomotor behavior that is essential for foraging larvae. This study has identified a member of the DEG/ENaC family of proteins, Ppk26, which acts together with Ppk1 likely as subunits of a channel important for mechanosensation. The results suggest that perhaps a major site of mechanosensory transduction is located in class IV da neuron dendritic processes. The behavioral phenotypes of larvae with Ppk1 and Ppk26 knockdown in class IV da neurons, as well as the respective null mutants or double mutants, suggest that a deficit in these channels interferes with the ability of the animal to execute proper turning behavior, raising the possibility that the two subunits could be involved in proprioceptively sensing the deformation of the cuticle. Whether class IV neurons function as proprioceptors still needs to be directly demonstrated, and future experiments will be needed to address the relationship between class IV neural activity and body position (Gorczyca, 2014).

    Numerous studies have implicated the class IV da neurons in both thermal and mechanical nociception behavior. This study found that whereas Ppk1 and Ppk26 are important for mechanical nociception behavior, they are dispensable for thermal nociception behavior. While Ppk1 and Ppk26 channels indeed moonlight during two processes in the same neuron, namely, mechanical nociception and proprioception, these channels must be playing a specific role as they are not involved in thermal response by the same neuron (Gorczyca, 2014).

    Whereas the class I da neurons and bd neurons are implicated in proprioception for the regulation of sequential contractions use the TRPN channel NompC as the sensor, class IV da neurons rely on the DEG/ENaC channel likely composed of Ppk26 and Ppk1 for the regulation of turning behavior as well as mechanical nociception, perhaps through sensing a mechanical signal at the cuticle. Interestingly, this study found that Piezo, a bona fide mechanotransducing ion channel involved in class IV da neuron mechanotransduction and required for mechanical nociception, does not appear to be involved in turning behavior, suggesting that different combinations of ion channels may serve different mechanosensory functions in the same neuron (Gorczyca, 2014).

    Dendrites are dispensable for basic motoneuron function but essential for fine tuning of behavior

    Dendrites are highly complex 3D structures that define neuronal morphology and connectivity and are the predominant sites for synaptic input. Defects in dendritic structure are highly consistent correlates of brain diseases. However, the precise consequences of dendritic structure defects for neuronal function and behavioral performance remain unknown. This study probed dendritic function by using genetic tools to selectively abolish dendrites in identified Drosophila wing motoneurons without affecting other neuronal properties. These motoneuron dendrites were unexpectedly found to be dispensable for synaptic targeting, qualitatively normal neuronal activity patterns during behavior, and basic behavioral performance. However, significant performance deficits in sophisticated motor behaviors, such as flight altitude control and switching between discrete courtship song elements, scale with the degree of dendritic defect. These observations provide the first direct evidence that complex dendrite architecture is critically required for fine-tuning and adaptability within robust, evolutionarily constrained behavioral programs that are vital for mating success and survival. It is speculated that the observed scaling of performance deficits with the degree of structural defect is consistent with gradual increases in intellectual disability during continuously advancing structural deficiencies in progressive neurological disorders (Ryglewski, 2014).

    A bidirectional circuit switch reroutes pheromone signals in male and female brains

    The Drosophila sex pheromone cVA elicits different behaviors in males and females. First- and second-order olfactory neurons show identical pheromone responses, suggesting that sex genes differentially wire circuits deeper in the brain. Using in vivo whole-cell electrophysiology, this study has shown that two clusters of third-order olfactory neurons have dimorphic pheromone responses. One cluster responds in females; the other responds in males. These clusters are present in both sexes and share a common input pathway, but sex-specific wiring reroutes pheromone information. Regulating dendritic position, the Fruitless transcription factor both connects the male-responsive cluster and disconnects the female-responsive cluster from pheromone input. Selective masculinization of third-order neurons transforms their morphology and pheromone responses, demonstrating that circuits can be functionally rewired by the cell-autonomous action of a switch gene. This bidirectional switch, analogous to an electrical changeover switch, provides a simple circuit logic to activate different behaviors in males and females (Kohl, 2013).

    This study reveals principles of neural circuit organization and development that are of general significance. First, it was shown that two populations of neurons, present in both sexes, show reciprocal, sex-specific responses to the same stimulus. Second, it was demonstrated that these responses result from differential wiring of a common input to different outputs. Together, these results define an elegant principle of neural circuit organization: a developmental circuit switch directly analogous to an electrical changeover (or single pole, double throw, SPDT) switch that efficiently reroutes a common input signal to one of two possible outputs. This model appears directly applicable to sex-specific processing of mouse pheromones, including ESP1 and Darcin (Haga, 2010; Stowers, 2010), but not to Caenorhabditis elegans ascarosides, where recent data suggest wiring differences may not be required. The electrical changeover switch is the prototype for a wide-range of electrical switches in which concerted changes involving three or more contacts reroute signals; it is very likely that neural circuits, including those involved in pheromone processing, contain more complex switches or assemblies of multiple switches that elaborate on the basic mechanism that are described in this study. Indeed, over 700 sites of dimorphic neuronal overlap have been identified that may form such switches in other sensory pathways, multimodal interneurons, or motor circuits across the fly brain (Cachero, 2010). Third, sex-specific placement of target neuron dendrites were identified as the primary cellular basis of the switch that is described in this study. This contrasts with earlier studies of this circuit that proposed that axonal dimorphism or neurons present only in one sex were the key dimorphic element. Regarding axonal dimorphism, Datta (2008) hypothesized that a male-specific extension of DA1 PN axon terminals is the basis of differential wiring in this system, and Ruta (2010) subsequently proposed that this extension synapses with the dendrites of aSP-f LHNs in males. The large shifts in dendritic position that were observed in aSP-f and aSP-g neurons mean the male-specific extension of DA1 PNs cannot be sufficient for rewiring. Is it necessary? In mosaic masculinization experiments, aSP-f and aSP-h neurons adopt male morphology and pheromone responses in a brain in which other neurons (including DA1 PNs) are female. Therefore, the male-specific ventral extension is either not necessary for differential wiring or is a secondary consequence of changes in the dendrites of post-synaptic LHNs. Of course, this extension may increase contact between DA1 PNs and aSP-f and aSP-h LHNs, strengthening responses of those LHNs in males. All three mechanisms (dendritic and axonal dimorphisms, dimorphic cell numbers) are likely relevant to different degrees in different circuits (Kohl, 2013).

    Fourth, having defined this bi-directional switch, it was demonstrated that its male form is specified by the fruitless gene. This transcription factor has a dual function, coordinating the disconnection of one group of target neurons and the connection of the other. Fifth, it was shown that masculinization of third-order neurons alone is sufficient for functional rewiring. Although previous studies have demonstrated a cell-autonomous effect of fruitless on neuronal morphology, this study now demonstrates a difference in functional connectivity. This is surprising because many would predict that connectivity changes would depend on coordinate regulation of genes in synaptic partner neurons. Such simplicity has evolutionary implications: it may allow variation in circuit structure and ultimately in behavior, through evolution of cis-regulatory elements, as previously shown for somatic characters, such as wing spots (Kohl, 2013).

    Sixth, studies of pheromone processing in general and cVA processing in particular have emphasized a labeled line processing model. However, the current data indicate that both narrowly (aSP-f) and broadly tuned (aSP-h) cVA-responsive neurons coexist in males. Likewise in females, aSP-g neurons respond to cVA and general odors, such as vinegar, but only cVA responses depend on the Or67d receptor. It will be very interesting to determine the circuit origin and behavioral significance of this integration of odor channels. For example, it seems reasonable to speculate that coincidence of cVA and food odors could interact in a supralinear way to promote female courtship or egg laying. This parallels the convergence in the lateral horn of a labeled line responsive to non-cVA fly odors (Or47b/VA1lm neurons) and one responsive to a specific food odorant, phenylacetic acid, that acts as a male aphrodisiac (Kohl, 2013).

    This study naturally raises additional questions. The action of fruitless within fewer than 5% of the neurons in the fly brain can specify behavior, and this study now shows that it can reroute pheromone signals within those neurons. But what is the behavioral relevance of this particular bidirectional switch? Testing this will require the development of sensitive behavioral assays of cVA processing and a reliable genetic approach to control this switch without affecting the many other dimorphic elements in sensory and motor circuits. Indeed, it remains to be seen whether flipping a single switch in sensory processing is sufficient to engage motor behavior typical of the opposite sex without masculinizing downstream circuitry. It is noted that it is possible to force the production of courtship song by activating fruitless-positive neurons in headless females, but this was almost never successful in intact females (Kohl, 2013).

    Another open question concerns the functional significance of female aSP-f and male aSP-g neurons, which do not respond to cVA or other tested odors. Do they receive input at all? One possibility, based on in silico analysis of the brain-wide 3D maps is that they receive gustatory input, perhaps from contact pheromones, although further work is necessary to test this hypothesis. Finally, which genes does fruitless regulate in order to differentially wire the switch? Clonal transformation experiments strongly support the earlier proposal that male and female aSP-f/g/h clusters are generated by neuroblasts common to both sexes but that those neurons develop in a sex-specific manner. Therefore, cell-surface molecules required for dendritic guidance are plausible targets. It will be intriguing to see if the same fru-dependent factor(s) direct(s) male aSP-f and female aSP-g dendrites to the ventral lateral horn and, more generally, whether fruitless acts on conserved downstream targets across all the dimorphic neurons in the fly brain (Kohl, 2013).

    The stum gene is essential for mechanical sensing in proprioceptive neurons

    Animal locomotion depends on proprioceptive feedback, which is generated by mechanosensory neurons. A genetic screen for impaired walking was performed in Drosophila, and a gene, stumble (stum), was isolated. The Stum protein has orthologs in animals ranging from nematodes to mammals and is predicted to contain two transmembrane domains. Expression of the mouse orthologs of stum in mutant flies rescued their phenotype, which demonstrates functional conservation. Dendrites of stum-expressing neurons in legs were stretched by both flexion and extension of corresponding joints. Joint angles that induced dendritic stretching also elicited elevation of cellular Ca(2+) levels-not seen in stum mutants. Thus, this study has identified an evolutionarily conserved gene, stum, which is required for transduction of mechanical stimuli in a specific subpopulation of Drosophila proprioceptive neurons that sense joint angles (Desai, 2014).

    Animal locomotion is achieved by coordination of motor activity according to proprioceptive mechanosensory inputs. In Drosophila, mechanosensation is mediated either by ciliated or multidendritic receptor neurons. Multidendritic neurons can respond to direct application of mechanical force to their membranes. It is less clear, however, how multidendritic mechanosensory neurons can be tuned to one mechanical modality, such as joint angle, and disregard other mechanical stimuli that may originate from external impacts or changes in the shape of muscles during contraction. In order to identify genes involved in proprioceptive sensation, uncoordination was screened for in a collection of ethyl methanesulfonate-mutagenized Drosophila lines. Lines that exhibited walking impairments were selected, and the phenotype severity was quantified by measuring climbing speed. Three lines, 204, 922, and 4487, were identified that showed lack of coordination in homozygous flies and did not complement one another, which suggested that they represent alleles of the same gene. Two of the lines, 204 and 4487, showed severe uncoordination, and the phenotype in 922 was mild. Deficiency mapping pointed to a gene, CG30263, predicted to encode a large protein with 1870 or 1959 amino acids (depending on the splice variant). Because the mutants had a walking impairment phenotype, this gene was named stumble (stum). The stum ortholog in humans is C1orf95, and it was categorized as a member of the SPEC3 family (UNIPROT, INTERPRO), with unknown function. The stum gene was sequenced in the three mutant lines, and it was found that stum204, stum4487, and stum922 had stop codons at amino acid positions 171, 202, and 1081, respectively (Desai, 2014).

    Transgenic flies that express stum cDNA in neurons were generated and it was found that the stum phenotype was partially rescued, which indicated that the mutations in stum were underlying the uncoordination phenotype. It is noted that the mouse ortholog of stum (National Center for Biotechnology Information, the U.S. National Institutes of Health, reference sequence: NP_001074696.1), which is only 141 amino acids long and shares 33% sequence identity with Drosophila stum, was also able to substantially rescue the uncoordination phenotype. Therefore, the function of stum appears to be conserved between distant animal species. Moreover, the rescue of the phenotype with such a short form of stum suggests that the C-terminal region of the fly protein constitutes the functional core (Desai, 2014).

    Proprioceptive defects in adult flies have been attributed to malfunction of type I (ciliated) mechanoreceptor neurons. To test whether such defects also underlie the phenotype in the stum mutant, electrophysiological recordings were performed of mechanical responses from the ciliated mechanoreceptor neuron of the anterior notopleural bristle. Type I neurons of stum mutants were indistinguishable from controls. Therefore, unlike known proprioception mutants, the phenotype in stum mutants does not arise from a general defect in type I mechanoreceptor neurons. To identify which cells give rise to the phenotype, the genomic regulatory region of stum was used to drive a CD8 fused to green fluorescent protein (CD8GFP) reporter [stum-Gal4 driving the upstream activation sequence (UAS)–CD8GFP]. It was found that stum expression in the legs was localized to three labeled neurons: one at the femur-tibia joint, the second at the tibia-tarsus joint, and the third spanning the second tarsal segment. The cell bodies of these stum-expressing neurons were located near the distal end of each leg segment, and their dendrites terminated at the corresponding joints (Desai, 2014).

    To study whether there are stum-expressing cells within the ventral nerve cord (VNC), it was examined in flies that express CD8GFP using stum-Gal4. The only fluorescent signal in the VNC originated from the axons of the leg neurons. The axons terminated within the neuropil that corresponds to each particular leg, branching into a bowl shape. This pattern is typical of neurons that take part in proprioception, such as the hair plate neurons. Therefore, the stum-expressing cells in the Drosophila body have characteristics of proprioceptive neurons that sense a property of specific joints (Desai, 2014).

    Confocal imaging was performed of the dendritic region of stum-expressing neurons, and it was found that, close to its tip, the dendrite branches toward the lateral aspect of the joint, and this side branch terminates at a short distance from the cuticle. The tips of dendrite were not associated with a cuticular structure or a scolopale. These structural features are typical features of type II (multidendritic) neurons but are incompatible with type I mechanoreceptor neurons that terminate with a ciliary structure. Thus, stum uncoordination mutations affect type II mechanoreceptor neurons. Furthermore, the entire dendritic terminal of these neurons was located in a region that is devoid of musculature, which suggests that they do not sense the mechanical properties of muscles. Taken together, these data suggest that stum-expressing neurons sense a mechanical property of the joint (Desai, 2014).

    To test whether stum-positive neurons encode joint angles, high-resolution imaging of the tibia-tarsus joint area was performed at different angles. At each angle of the joint, the total length of the sensory dendrite and its side branch was measured. It was found that the total dendrite length had a minimum typically at 130o to 170o, and it increased when the joint was shifted to either more obtuse or more acute angles. These morphological changes indicate that these neurons are mechanically affected by the position of the joint. Because the tip of the side branch is stationary, it is likely that the change in total length results from the coupling of the tip of the main dendrite to the motion of the distal joint segment. The position of the dendrite and the susceptibility of its morphology to joint angle suggest that the role of stum-positive neurons is to sense and encode the angle of the joint (Desai, 2014).

    Ca2+ fluorescence was measured while forcing the tibia-tarsus joint to different angles, and the Ca2+ fluorescence in stum-expressing neurons was found to correlate with the angle of the joint. As in the morphological changes, the responses correlated with joint angle in a U-shaped manner, where both acute and obtuse angles induced increasing Ca2+ elevations. These results indicate that the stum-positive neurons encode proprioceptive information about the angle of joints. It is noteworthy that a similar U-shaped encoding of joint angles has also been described in receptor neurons of mammalian joints, which suggests that sensing the deviations from a neutral joint range is universally critical for motor function (Desai, 2014).

    The walking impairment in the stum mutant fly suggests that the gene is necessary for generating the proper proprioceptive responses in stum-expressing neurons. To test whether the stum mutations affect coupling between joint angles and dendritic stretching, the stretching was quantitated in mutant flies that have stum-expressing neurons labeled with CD8GFP. In the stum mutant, the dendritic stretching in response to joint angles was comparable to that of control flies. Thus, stum is not required for the mechanical coupling between joint angles and stretching of the sensory dendrites (Desai, 2014).

    Although the dendritic stretching was not significantly affected in the stum mutant, the Ca2+ responses to both acute and obtuse joint angles were abolished in the mutant. Therefore, it is concluded that stum is essential for transducing dendrite stretching into cellular responses (Desai, 2014).

    The morphology of stum-expressing neurons were examined, and it was found that, although axons and cell bodies of stum mutants were indistinguishable from controls, the sensory dendrite in mutants exhibited abnormalities. Most notably, in some of the stum-expressing neurons the tip of the dendrite was overgrown and extended into the distal segment of the joint. Thus, the absence of stum leads to a morphological defect, possibly because of the lack of mechanical responsiveness. The occasional morphological differences may account for the slight difference in the stretching profile between control and mutant dendrites. The fraction of neurons demonstrating the abnormal morphology in the mutant increased from the day of eclosion to the following day. As the addition of morphological changes takes place after eclosion, it is possible that stum-dependent activity is essential for late shape determination that can take place in adult multidendritic neurons (Desai, 2014).

    Transgenic flies were generated that express a GFP fused with the N terminus of Stum under UAS regulation (UAS-GFP-Stum). The Stum fusion protein was found to be specifically localized to the distal part of the sensory dendrite, although it did not accumulate substantially in any other part of the cell. The fluorescent signal started at the region of bifurcation and extended to both distal tips of the dendrite. This specific localization suggests that Stum functions in the part of the dendrite that senses stretching (Desai, 2014).

    Taken together, stum expression in mechanosensory neurons, Stum localization to the sensory dendrite, and the abolition of responses to stretching in the stum mutant suggest that stum has an essential role in mediating mechanical sensing in receptor neurons. Because the Stum protein in most species is very small and because Drosophila stum is expressed in limited populations of receptor neurons, it is proposed that stum is not the mechanically activated channel. Rather, stum may serve as an accessory module that is essential for the proper localization or function of the transduction channels (Desai, 2014).

    The stretch-receptor neurons that express stum present an elegant engineering solution for generating specificity to the modality of mechanical stimulus. The distal part of their dendrite bifurcates into two branches whose tips are anchored to parts of the joint that shift their relative positions. Sensing the stretching only between the two dendritic tips may tune the nerve responses to joint motions and filter out the effect of irrelevant mechanical impacts. This specificity enables the sensory neuron to relay reliable proprioceptive information to the central nervous system (Desai, 2014).

    The RNA-binding protein Caper is required for sensory neuron development in Drosophila melanogaster

    Alternative splicing mediated by RNA-binding proteins (RBPs) is emerging as a fundamental mechanism for the regulation of gene expression. Alternative splicing has been shown to be a widespread phenomenon that facilitates the diversification of gene products in a tissue specific manner. Although defects in alternative splicing are rooted in many neurological disorders, only a small fraction of splicing factors have been investigated in detail. This study finds that the splicing factor Caper is required for the development of multiple different mechanosensory neuron subtypes at multiple life stages in Drosophila melanogaster. Disruption of Caper function causes defects in dendrite morphogenesis of larval dendrite arborization neurons, neuronal positioning of embryonic proprioceptors, as well as the development and maintenance of adult mechanosensory bristles. Additionally, Caper dysfunction was found to result in aberrant locomotor behavior in adult flies. Transcriptome-wide analyses further support a role for Caper in alternative isoform regulation of genes that function in neurogenesis. This results provide the first evidence for a fundamental and broad requirement for the highly conserved splicing factor Caper in the development and maintenance of the nervous system and provide a framework for future studies on the detailed mechanism of Caper mediated RNA regulation (Olesnicky, 2017).

    Neuronal processing of noxious thermal stimuli mediated by dendritic Ca influx in somatosensory neurons

    Adequate responses to noxious stimuli causing tissue damages are essential for organismal survival. Class IV neurons in Drosophila larvae are polymodal nociceptors responsible for thermal, mechanical, and light sensation. Importantly, activation of Class IV provoked distinct avoidance behaviors, depending on the inputs. Noxious thermal stimuli, but not blue light stimulation, was shown to cause a unique pattern of Class IV, which were composed of pauses after high frequency spike trains and a large Ca2+ rise in the dendrite (the Ca2+ transient). Both of these responses depended on two TRPA channels and the L-type voltage-gated calcium channel (L-VGCC), showing that the thermosensation provokes Ca2+ influx. The precipitous fluctuation of firing rate in Class IV neurons enhanced the robust heat avoidance. It is hypothesized that the Ca2+ influx can be a key signal encoding a specific modality (Terada, 2016).

    Enclosure of dendrites by epidermal cells restricts branching and permits coordinated development of spatially overlapping sensory neurons

    Spatial arrangement of different neuron types within a territory is essential to neuronal development and function. How development of different neuron types is coordinated for spatial coexistence is poorly understood. In Drosophila, dendrites of four classes of dendritic arborization (C1-C4da) neurons innervate overlapping receptive fields within the larval epidermis. These dendrites are intermittently enclosed by epidermal cells, with different classes exhibiting varying degrees of enclosure. The role of enclosure in neuronal development and its underlying mechanism remain unknown. This study shows that the membrane-associated protein Coracle acts in C4da neurons and epidermal cells to locally restrict dendrite branching and outgrowth by promoting enclosure. Loss of C4da neuron enclosure results in excessive branching and growth of C4da neuron dendrites and retraction of C1da neuron dendrites due to local inhibitory interactions between neurons. It is proposed that enclosure of dendrites by epidermal cells is a developmental mechanism for coordinated innervation of shared receptive fields (Tenenbaum, 2017).


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    date revised: 15 October 2016

    Genes involved in organ development

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