Homophilic Dscam interactions control complex dendrite morphogenesis

Alternative splicing of the Drosophila gene Dscam results in up to 38,016 different receptor isoforms proposed to interact by isoform-specific homophilic binding. We report that Dscam controls cell-intrinsic aspects of dendrite guidance in all four classes of dendrite arborization (da) neurons. Loss of Dscam in single neurons causes a strong increase in self-crossing. Restriction of dendritic fields of neighboring class III neurons appeared intact in mutant neurons, suggesting that dendritic self-avoidance, but not heteroneuronal tiling, may depend on Dscam. Overexpression of the same Dscam isoforms in two da neurons with overlapping dendritic fields forced a spatial segregation of the two fields, supporting the model that dendritic branches of da neurons use isoform-specific homophilic interactions to ensure minimal overlap. Homophilic binding of the highly diverse extracellular domains of Dscam may therefore limit the use of the same "core" repulsion mechanism to cell-intrinsic interactions without interfering with heteroneuronal interactions.     

Patterning and organization of motor neuron dendrites in the Drosophila larva

Precise patterns of motor neuron connectivity depend on the proper establishment and positioning of the dendritic arbor. However, how different motor neurons orient their dendrites to selectively establish synaptic connectivity is not well understood. The Drosophila neuromuscular system provides a simple model to investigate the underlying organizational principles by which distinct subclasses of motor neurons orient their dendrites within the central neuropil. Here we used genetic mosaic techniques to characterize the diverse dendritic morphologies of individual motor neurons from five main nerve branches (ISN, ISNb, ISNd, SNa, and SNc) in the Drosophila larva. We found that motor neurons from different nerve branches project their dendrites to largely stereotyped mediolateral domains in the dorsal region of the neuropil providing full coverage of the receptive territory. Furthermore, dendrites from different motor neurons overlap extensively, regardless of subclass, suggesting that repulsive dendrite-dendrite interactions between motor neurons do not influence the mediolateral positioning of dendritic fields. The anatomical data in this study provide important information regarding how different subclasses of motor neurons organize their dendrites and establishes a foundation for the investigation of the mechanisms that control synaptic connectivity in the Drosophila motor circuit.     

Dendrite self-avoidance is controlled by Dscam

Dendrites distinguish between sister branches and those of other cells. Self-recognition can often lead to repulsion, a process termed "self-avoidance." Here we demonstrate that dendrite self-avoidance in Drosophila da sensory neurons requires cell-recognition molecules encoded by the Dscam locus. By alternative splicing, Dscam encodes a vast number of cell-surface proteins of the immunoglobulin superfamily. We demonstrate that interactions between identical Dscam isoforms on the cell surface underlie self-recognition, while the cytoplasmic tail converts this recognition to dendrite repulsion. Sister dendrites expressing the same isoforms engage in homophilic repulsion. By contrast, Dscam diversity ensures that inappropriate repulsive interactions between dendrites sharing the same receptive field do not occur. The selectivity of Dscam-mediated cell interactions is likely to be widely important in the developing fly nervous system, where processes of cells must distinguish between self and nonself during the construction of neural circuits.     

Drosophila sensory neurons require Dscam for dendritic self-avoidance and proper dendritic field organization

A neuron's dendrites typically do not cross one another. This intrinsic self-avoidance mechanism ensures unambiguous processing of sensory or synaptic inputs. Moreover, some neurons respect the territory of others of the same type, a phenomenon known as tiling. Different types of neurons, however, often have overlapping dendritic fields. We found that Down's syndrome Cell Adhesion Molecule (Dscam) is required for dendritic self-avoidance of all four classes of Drosophila dendritic arborization (da) neurons. However, neighboring mutant class IV da neurons still exhibited tiling, suggesting that self-avoidance and tiling differ in their recognition and repulsion mechanisms. Introducing 1 of the 38,016 Dscam isoforms to da neurons in Dscam mutants was sufficient to significantly restore self-avoidance. Remarkably, expression of a common Dscam isoform in da neurons of different classes prevented their dendrites from sharing the same territory, suggesting that coexistence of dendritic fields of different neuronal classes requires divergent expression of Dscam isoforms.     

Integrins regulate repulsion-mediated dendritic patterning of drosophila sensory neurons by restricting dendrites in a 2D space

Dendrites of the same neuron usually avoid each other. Some neurons also repel similar neurons through dendrite-dendrite interaction to tile the receptive field. Nonoverlapping coverage based on such contact-dependent repulsion requires dendrites to compete for limited space. Here we show that Drosophila class IV dendritic arborization (da) neurons, which tile the larval body wall, grow their dendrites mainly in a 2D space on the extracellular matrix (ECM) secreted by the epidermis. Removing neuronal integrins or blocking epidermal laminin production causes dendrites to grow into the epidermis, suggesting that integrin-laminin interaction attaches dendrites to the ECM. We further show that some of the previously identified tiling mutants fail to confine dendrites in a 2D plane. Expansion of these mutant dendrites in three dimensions results in overlap of dendritic fields. Moreover, overexpression of integrins in these mutant neurons effectively reduces dendritic crossing and restores tiling, revealing an additional mechanism for tiling.     

An autism-associated variant of Epac2 reveals a role for Ras/Epac2 signaling in controlling basal dendrite maintenance in mice

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity.     

Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion

BACKGROUND: Understanding how dendrites establish their territory is central to elucidating how neuronal circuits are built. Signaling between dendrites is thought to be important for defining their territories; however, the strategies by which different types of dendrites communicate are poorly understood. We have shown previously that two classes of Drosophila peripheral da sensory neurons, the class III and class IV neurons, provide complete and independent tiling of the body wall. By contrast, dendrites of class I and class II neurons do not completely tile the body wall, but they nevertheless occupy nonoverlapping territories. RESULTS: By developing reagents to permit high-resolution studies of dendritic tiling in living animals, we demonstrate that isoneuronal and heteroneuronal class IV dendrites engage in persistent repulsive interactions. In contrast to the extensive dendritic exclusion shown by class IV neurons, duplicated class III neurons showed repulsion only at their dendritic terminals. Supernumerary class I and class II neurons innervated completely overlapping regions of the body wall, and this finding suggests a lack of like-repels-like behavior. CONCLUSIONS: These data suggest that repulsive interactions operate between morphologically alike dendritic arbors in Drosophila. Further, Drosophila da sensory neurons appear to exhibit at least three different types of class-specific dendrite-dendrite interactions: persistent repulsion by all branches, repulsion only by terminal dendrites, and no repulsion.     

A novel forward genetic screen for identifying mutations affecting larval neuronal dendrite development in Drosophila melanogaster

Vertebrate and invertebrate dendrites are information-processing compartments that can be found on both central and peripheral neurons. Elucidating the molecular underpinnings of information processing in the nervous system ultimately requires an understanding of the genetic pathways that regulate dendrite formation and maintenance. Despite the importance of dendrite development, few forward genetic approaches have been used to analyze the latest stages of dendrite development, including the formation of F-actin-rich dendritic filopodia or dendritic spines. We developed a forward genetic screen utilizing transgenic Drosophila second instar larvae expressing an actin, green fluorescent protein (GFP) fusion protein (actin::GFP) in subsets of sensory neurons. Utilizing this fluorescent transgenic reporter, we conducted a forward genetic screen of >4000 mutagenized chromosomes bearing lethal mutations that affected multiple aspects of larval dendrite development. We isolated 13 mutations on the X and second chromosomes composing 11 complementation groups affecting dendrite outgrowth/branching, dendritic filopodia formation, or actin::GFP localization within dendrites in vivo. In a fortuitous observation, we observed that the structure of dendritic arborization (da) neuron dendritic filopodia changes in response to a changing environment.     

Cell type-specific dendritic polarity in the absence of spatially organized external cues

Pyramidal neurons of the hippocampus and cortex have polarized dendritic arbors, but little is known about the cellular mechanisms distinguishing apical and basal dendrites. We used morphometric analysis and time lapse imaging of cultured hippocampal neurons to show that glutamatergic neurons develop progressive dendritic asymmetry in the absence of polarized extrinsic cues. Thus, pyramidal neurons have a cellular program for polarized dendrite growth independent of tissue microenvironment.     

Differentiation of Apical and Basal Dendrites in Pyramidal Cells and Granule Cells in Dissociated Hippocampal Cultures

Hippocampal pyramidal cells and dentate granule cells develop morphologically distinct dendritic arbors, yet also share some common features. Both cell types form a long apical dendrite which extends from the apex of the cell soma, while short basal dendrites are developed only in pyramidal cells. Using quantitative morphometric analyses of mouse hippocampal cultures, we evaluated the differences in dendritic arborization patterns between pyramidal and granule cells. Furthermore, we observed and described the final apical dendrite determination during dendritic polarization by time-lapse imaging. Pyramidal and granule cells in culture exhibited similar dendritic patterns with a single principal dendrite and several minor dendrites so that the cell types were not readily distinguished by appearance. While basal dendrites in granule cells are normally degraded by adulthood in vivo, cultured granule cells retained their minor dendrites. Asymmetric growth of a single principal dendrite harboring the Golgi was observed in both cell types soon after the onset of dendritic growth. Time-lapse imaging revealed that up until the second week in culture, final principal dendrite designation was not stabilized, but was frequently replaced by other minor dendrites. Before dendritic polarity was stabilized, the Golgi moved dynamically within the soma and was repeatedly repositioned at newly emerging principal dendrites. Our results suggest that polarized growth of the apical dendrite is regulated by cell intrinsic programs, while regression of basal dendrites requires cue(s) from the extracellular environment in the dentate gyrus. The apical dendrite designation is determined from among multiple growing dendrites of young developing neurons.     

Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology

Neurons establish diverse dendritic morphologies during development, and a major challenge is to understand how these distinct developmental programs might relate to, and influence, neuronal function. Drosophila dendritic arborization (da) sensory neurons display class-specific dendritic morphology with extensive coverage of the body wall. To begin to build a basis for linking dendrite structure and function in this genetic system, we analyzed da neuron axon projections in embryonic and larval stages. We found that multiple parameters of axon morphology, including dorsoventral position, midline crossing and collateral branching, correlate with dendritic morphological class. We have identified a class-specific medial-lateral layering of axons in the central nervous system formed during embryonic development, which could allow different classes of da neurons to develop differential connectivity to second-order neurons. We have examined the effect of Robo family members on class-specific axon lamination, and have also taken a forward genetic approach to identify new genes involved in axon and dendrite development. For the latter, we screened the third chromosome at high resolution in vivo for mutations that affect class IV da neuron morphology. Several known loci, as well as putative novel mutations, were identified that contribute to sensory dendrite and/or axon patterning. This collection of mutants, together with anatomical data on dendrites and axons, should begin to permit studies of dendrite diversity in a combined developmental and functional context, and also provide a foundation for understanding shared and distinct mechanisms that control axon and dendrite morphology.     

The Proprotein Convertase KPC-1/Furin Controls Branching and Self-avoidance of Sensory Dendrites in Caenorhabditis elegans

Animals sample their environment through sensory neurons with often elaborately branched endings named dendritic arbors. In a genetic screen for genes involved in the development of the highly arborized somatosensory PVD neuron in C. elegans, we have identified mutations in kpc-1, which encodes the homolog of the proprotein convertase furin. We show that kpc-1/furin is necessary to promote the formation of higher order dendritic branches in PVD and to ensure self-avoidance of sister branches, but is likely not required during maintenance of dendritic arbors. A reporter for kpc-1/furin is expressed in neurons (including PVD) and kpc-1/furin can function cell-autonomously in PVD neurons to control patterning of dendritic arbors. Moreover, we show that kpc-1/furin also regulates the development of other neurons in all major neuronal classes in C. elegans, including aspects of branching and extension of neurites as well as cell positioning. Our data suggest that these developmental functions require proteolytic activity of KPC-1/furin. Recently, the skin-derived MNR-1/menorin and the neural cell adhesion molecule SAX-7/L1CAM have been shown to act as a tripartite complex with the leucine rich transmembrane receptor DMA-1 on PVD mechanosensory to orchestrate the patterning of dendritic branches. Genetic analyses show that kpc-1/furin functions in a pathway with MNR-1/menorin, SAX-7/L1CAM and DMA-1 to control dendritic branch formation and extension of PVD neurons. We propose that KPC-1/furin acts in concert with the ‘menorin’ pathway to control branching and growth of somatosensory dendrites in PVD.     

Sporoptosis: sowing the seeds of nuclear destruction

Previous models of neuronal dendrite arborization suggested that contact-dependent self-avoidance between dendrite branches prevents self-crossings within the arbor. Two papers in Neuron show how integrin-mediated adhesion to the extracellular matrix restricts dendrites to a two-dimensional space to optimize this mechanism (Han et al., 2012; Kim et al., 2012).     

Regulation of Dendritic Filopodial Interactions by ZO-1 and Implications for Dendrite Morphogenesis

Neuronal dendrites dynamically protrude many fine filopodia in the early stages of neuronal development and gradually establish complex structures. The importance of the dendritic filopodia in the formation of axo-dendritic connections is established, but their role in dendrite morphogenesis remains unknown. Using time-lapse imaging of cultured rat hippocampal neurons, we revealed here that many filopodia dynamically protruded from dendrites and transiently interacted with each other to form dendritic filopodia-filopodia contacts in the early stages of neuronal development. The MAGUK family member, Zonula Occludens-1 (ZO-1), which is known to be associated with the nectin and cadherin cell adhesion systems, was concentrated at these dendritic filopodia-filopodia contact sites and also at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology. Conversely, knockdown of ZO-1 decreased the formation of dendritic filopodia and their interactions, and induced abnormal dendrite morphology which was different from that induced by the overexpression of ZO-1. The components of the nectin and cadherin systems were co-localized with ZO-1 at the dendritic filopodia-filopodia contact sites, but not at the tips of free dendritic filopodia. Overexpression of ZO-1 increased the accumulation of these cell adhesive components at the dendritic filopodia-filopodia contact sites and stabilized their interactions, whereas knockdown of ZO-1 reduced their accumulation at the dendritic filopodia-filopodia contact sites. These results indicate that ZO-1 regulates dendritic filopodial dynamics, which is implicated in dendrite morphogenesis cooperatively with the nectin and cadherin systems in cultured neurons.     

Signaling mechanisms underlying reversible,activity-dependent dendrite formation

Neuronal activity and neurotrophins play a central role in the formation, maintenance, and plasticity of dendritic arbors. Here, we show that neuronal activity, mediated by electrical stimulation, KCl depolarization, or cholinergic receptor activation, promotes reversible dendrite formation in sympathetic neurons and that this effect is enhanced by NGF. Activity-dependent dendrite formation is accompanied by increased association of HMW MAP2 with microtubules and increased microtubule stability. Inhibition of either CaMKII or the MEK-ERK pathway, both of which phosphorylate MAP2, inhibits dendrite formation, but inhibition of both pathways simultaneously is required for dendrites to retract. These data indicate that neuronal activity signals via CamKII and the ERKs to regulate MAP2:microtubule interactions and hence reversible dendrite stability, and to provide a mechanism whereby activity and neurotrophins converge intracellularly to dynamically regulate dendritic morphology.     

Tiling of the Drosophila epidermis by multidendritic sensory neurons

Insect dendritic arborization (da) neurons provide an opportunity to examine how diverse dendrite morphologies and dendritic territories are established during development. We have examined the morphologies of Drosophila da neurons by using the MARCM (mosaic analysis with a repressible cell marker) system. We show that each of the 15 neurons per abdominal hemisegment spread dendrites to characteristic regions of the epidermis. We place these neurons into four distinct morphological classes distinguished primarily by their dendrite branching complexities. Some class assignments correlate with known proneural gene requirements as well as with central axonal projections. Our data indicate that cells within two morphological classes partition the body wall into distinct, non-overlapping territorial domains and thus are organized as separate tiled sensory systems. The dendritic domains of cells in different classes, by contrast, can overlap extensively. We have examined the cell-autonomous roles of starry night (stan) (also known as flamingo (fmi)) and sequoia (seq) in tiling. Neurons with these genes mutated generally terminate their dendritic fields at normal locations at the lateral margin and segment border, where they meet or approach the like dendrites of adjacent neurons. However, stan mutant neurons occasionally send sparsely branched processes beyond these territories that could potentially mix with adjacent like dendrites. Together, our data suggest that widespread tiling of the larval body wall involves interactions between growing dendritic processes and as yet unidentified signals that allow avoidance by like dendrites.     

Interaction between long-term potentiation and depression in CA1 synapses: temporal constrains, functional compartmentalization and protein synthesis

Information arriving at a neuron via anatomically defined pathways undergoes spatial and temporal encoding. A proposed mechanism by which temporally and spatially segregated information is encoded at the cellular level is based on the interactive properties of synapses located within and across functional dendritic compartments. We examined cooperative and interfering interactions between long-term synaptic potentiation (LTP) and depression (LTD), two forms of synaptic plasticity thought to be key in the encoding of information in the brain. Two approaches were used in CA1 pyramidal neurons of the mouse hippocampus: (1) induction of LTP and LTD in two separate synaptic pathways within the same apical dendritic compartment and across the basal and apical dendritic compartments; (2) induction of LTP and LTD separated by various time intervals (0-90 min). Expression of LTP/LTD interactions was spatially and temporally regulated. While they were largely restricted within the same dendritic compartment (compartmentalized), the nature of the interaction (cooperation or interference) depended on the time interval between inductions. New protein synthesis was found to regulate the expression of the LTP/LTD interference. We speculate that mechanisms for compartmentalization and protein synthesis confer the spatial and temporal modulation by which neurons encode multiplex information in plastic synapses.