Transmembrane/juxtamembrane domain-dependent Dscam distribution and function during mushroom body neuronal morphogenesis

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.     

Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Here we demonstrate that specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminated in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets was abnormal. Target specificity for other ORNs was not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA were detected in the developing antenna, and Dscam protein was localized to developing ORN axons. We propose a role for Dscam protein diversity in regulating ORN target specificity.     

The cytoskeletal and signaling mechanisms of axon collateral branching

During development, axons are guided to their appropriate targets by a variety of guidance factors. On arriving at their synaptic targets, or while en route, axons form branches. Branches generated de novo from the main axon are termed collateral branches. The generation of axon collateral branches allows individual neurons to make contacts with multiple neurons within a target and with multiple targets. In the adult nervous system, the formation of axon collateral branches is associated with injury and disease states and may contribute to normally occurring plasticity. Collateral branches are initiated by actin filament– based axonal protrusions that subsequently become invaded by microtubules, thereby allowing the branch to mature and continue extending. This article reviews the current knowledge of the cellular mechanisms of the formation of axon collateral branches. The major conclusions of this review are (1) the mechanisms of axon extension and branching are not identical; (2) active suppression of protrusive activity along the axon negatively regulates branching; (3) the earliest steps in the formation of axon branches involve focal activation of signaling pathways within axons, which in turn drive the formation of actin-based protrusions; and (4) regulation of the microtubule array by microtubule-associated and severing proteins underlies the development of branches. Linking the activation of signaling pathways to specific proteins that directly regulate the axonal cytoskeleton underlying the formation of collateral branches remains a frontier in the field.     

Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.     

The molecular diversity of Dscam is functionally required for neuronal wiring specificity in Drosophila

Alternative splicing of Dscam generates an enormous molecular diversity with maximally 38,016 different receptors. Whether this large diversity is required in vivo is currently unclear. We examined the role of Dscam in neuron-target recognition of single mechanosensory neurons, which connect with different target cells through multiple axonal branches. Analysis of Dscam null neurons demonstrated an essential role of Dscam for growth and directed extension of axon branches. Expression of randomly chosen single isoforms could not rescue connectivity but did restore basic axonal extension and rudimentary branching. Moreover, two Dscam alleles were generated that each reduced the maximally possible Dscam diversity to 22,176 isoforms. Reduction of Dscam diversity resulted in specific connectivity defects of mechanosensory neurons. Furthermore, the observed allele-specific phenotypes suggest functional differences among isoforms. Our findings provide evidence that a very large number of structurally unique receptor isoforms is required to ensure fidelity and precision of neuronal connectivity.     

Developmental regulation of axon branching in the vertebrate nervous system

During nervous system development, axons generate branches to connect with multiple synaptic targets. As with axon growth and guidance, axon branching is tightly controlled in order to establish functional neural circuits, yet the mechanisms that regulate this important process are less well understood. Here, we review recent advances in the study of several common branching processes in the vertebrate nervous system. By focusing on each step in these processes we illustrate how different types of branching are regulated by extracellular cues and neural activity, and highlight some common principles that underlie the establishment of complex neural circuits in vertebrate development.     

Common mechanisms underlying growth cone guidance and axon branching

During development, growth cones direct growing axons into appropriate targets. However, in some cortical pathways target innervation occurs through the development of collateral branches that extend interstitially from the axon shaft. How do such branches form? Direct observations of living cortical brain slices revealed that growth cones of callosal axons pause for many hours beneath their cortical targets prior to the development of interstitial branches. High resolution imaging of dissociated living cortical neurons for many hours revealed that the growth cone demarcates sites of future axon branching by lengthy pausing behaviors and enlargement of the growth cone. After a new growth cone forms and resumes forward advance, filopodial and lamellipodial remnants of the large paused growth cone are left behind on the axon shaft from which interstitial branches later emerge. To investigate how the cytoskeleton reorganizes at axon branch points, we fluorescently labeled microtubules in living cortical neurons and imaged the behaviors of microtubules during new growth from the axon shaft and the growth cone. In both regions microtubules reorganize into a more plastic form by splaying apart and fragmenting. These shorter microtubules then invade newly developing branches with anterograde and retrograde movements. Although axon branching of dissociated cortical neurons occurs in the absence of targets, application of a target-derived growth factor, FGF-2, greatly enhances branching. Taken together, these results demonstrate that growth cone pausing is closely related to axon branching and suggest that common mechanisms underlie directed axon growth from the terminal growth cone and the axon shaft.     

Stromal cell‐derived factor‐1 and hepatocyte growth factor guide axon projections to the extraocular muscles

Vertebrate eye movements depend on the co‐ordinated function of six extraocular muscles that are innervated by the oculomotor, trochlear, and abducens nerves. Here, we show that the diffusible factors, stromal cell‐derived factor‐1 (SDF‐1) and hepatocyte growth factor (HGF), guide the development of these axon projections. SDF‐1 is expressed in the mesenchyme around the oculomotor nerve exit point, and oculomotor axons fail to exit the neuroepithelium in mice mutant for the SDF‐1 receptor CXCR4. Both SDF‐1 and HGF are expressed in or around the ventral and dorsal oblique muscles, which are distal targets for the oculomotor and trochlear nerves, respectively, as well as in the muscles which are later targets for oculomotor axon branches. We find that in vitro SDF‐1 and HGF promote the growth of oculomotor and trochlear axons, whereas SDF‐1 also chemoattracts oculomotor axons. Oculomotor neurons show increased branching in the presence of SDF‐1 and HGF singly or together. HGF promotes the growth of trochlear axons more than that of oculomotor axons. Taken together, these data point to a role for both SDF‐1 and HGF in extraocular nerve projections and indicate that SDF‐1 functions specifically in the development of the oculomotor nerve, including oculomotor axon branch formation to secondary muscle targets. HGF shows some specificity in preferentially enhancing development of the trochlear nerve. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 549–564, 2010     

The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord

Sensory axonal projections into the spinal cord display a highly stereotyped pattern of T- or Y-shaped axon bifurcation at the dorsal root entry zone (DREZ). Here, we provide evidence that embryonic mice with an inactive receptor guanylyl cyclase Npr2 or deficient for cyclic guanosine monophosphate-dependent protein kinase I (cGKI) lack the bifurcation of sensory axons at the DREZ, i.e., the ingrowing axon either turns rostrally or caudally. This bifurcation error is maintained to mature stages. In contrast, interstitial branching of collaterals from primary stem axons remains unaffected, indicating that bifurcation and interstitial branching are processes regulated by a distinct molecular mechanism. At a functional level, the distorted axonal branching at the DREZ is accompanied by reduced synaptic input, as revealed by patch clamp recordings of neurons in the superficial layers of the spinal cord. Hence, our data demonstrate that Npr2 and cGKI are essential constituents of the signaling pathway underlying axonal bifurcation at the DREZ and neuronal connectivity in the dorsal spinal cord.     

The zebrafish down syndrome cell adhesion molecule is involved in cell movement during embryogenesis

The Down syndrome cell adhesion molecule (Dscam) is a protein overexpressed in the brains of Down syndrome patients and implicated in mental retardation. Dscam is involved in axon guidance and branching in Drosophila, but cellular roles in vertebrates have yet to be elucidated. To understand its role in vertebrate development, we cloned the zebrafish homolog of Dscam and showed that it shares high amino acid identity and structure with the mammalian homologs. Zebrafish dscam is highly expressed in developing neurons, similar to what has been described in Drosophila and mouse. When dscam expression is diminished by morpholino injection, embryos display few neurons and their axons do not enter stereotyped pathways. Zebrafish dscam is also present at early embryonic stages including blastulation and gastrulation. Its loss results in early morphogenetic defects. dscam knockdown results in impaired cell movement during epiboly as well as in subsequent stages. We propose that migrating cells utilize dscam to remodel the developing embryo.     

Drosophila Dscam is an axon guidance receptor exhibiting extraordinary molecular diversity

A Drosophila homolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulin superfamily member, was isolated by its affinity to Dock, an SH3/SH2 adaptor protein required for axon guidance. Dscam binds directly to both Dock's SH2 and SH3 domains. Genetic studies revealed that Dscam, Dock and Pak, a serine/threonine kinase, act together to direct pathfinding of Bolwig's nerve, containing a subclass of sensory axons, to an intermediate target in the embryo. Dscam also is required for the formation of axon pathways in the embryonic central nervous system. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conserved architecture containing variable Ig and transmembrane domains. Alternative splicing can potentially generate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificity of neuronal connectivity.     

Dscam guides embryonic axons by Netrin-dependent and -independent functions

Developing axons are attracted to the CNS midline by Netrin proteins and other as yet unidentified signals. Netrin signals are transduced in part by Frazzled (Fra)/DCC receptors. Genetic analysis in Drosophila indicates that additional unidentified receptors are needed to mediate the attractive response to Netrin. Analysis of Bolwig's nerve reveals that Netrin mutants have a similar phenotype to Down Syndrome Cell Adhesion Molecule (Dscam) mutants. Netrin and Dscam mutants display dose sensitive interactions, suggesting that Dscam could act as a Netrin receptor. We show using cell overlay assays that Netrin binds to fly and vertebrate Dscam, and that Dscam binds Netrin with the same affinity as DCC. At the CNS midline, we find that Dscam and its paralog Dscam3 act redundantly to promote midline crossing. Simultaneous genetic knockout of the two Dscam genes and the Netrin receptor fra produces a midline crossing defect that is stronger than the removal of Netrin proteins, suggesting that Dscam proteins also function in a pathway parallel to Netrins. Additionally, overexpression of Dscam in axons that do not normally cross the midline is able to induce ectopic midline crossing, consistent with an attractive receptor function. Our results support the model that Dscam proteins function as attractive receptors for Netrin and also act in parallel to Frazzled/DCC. Furthermore, the results suggest that Dscam proteins have the ability to respond to multiple ligands and act as receptors for an unidentified midline attractive cue. These functions in axon guidance have implications for the pathogenesis of Down Syndrome.     

Wnt/PCP proteins regulate stereotyped axon branch extension in Drosophila

Branching morphology is a hallmark feature of axons and dendrites and is essential for neuronal connectivity. To understand how this develops, I analyzed the stereotyped pattern of Drosophila mushroom body (MB) neurons, which have single axons branches that extend dorsally and medially. I found that components of the Wnt/Planar Cell Polarity (PCP) pathway control MB axon branching. frizzled mutant animals showed a predominant loss of dorsal branch extension, whereas strabismus (also known as Van Gogh) mutants preferentially lost medial branches. Further results suggest that Frizzled and Strabismus act independently. Nonetheless, branching fates are determined by complex Wnt/PCP interactions, including interactions with Dishevelled and Prickle that function in a context-dependent manner. Branching decisions are MB-autonomous but non-cell-autonomous as mutant and non-mutant neurons regulate these decisions collectively. I found that Wnt/PCP components do not need to be asymmetrically localized to distinct branches to execute branching functions. However, Prickle axonal localization depends on Frizzled and Strabismus.     

Regulation of axon arborization pattern in the developing chick ciliary ganglion: Possible involvement of caspase 3

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase‐3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase‐3 activity using an in ovo electroporation method. The axon arborization pattern was 3‐dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6–8, there appeared to be a dynamic balance in the axon arborization pattern between the “targeting” mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the “pathfinding” mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.     

Beyond the cytoskeleton: The emerging role of organelles and membrane remodeling in the regulation of axon collateral branches

The generation of axon collateral branches is a fundamental aspect of the development of the nervous system and the response of axons to injury. Although much has been discovered about the signaling pathways and cytoskeletal dynamics underlying branching, additional aspects of the cell biology of axon branching have received less attention. This review summarizes recent advances in our understanding of key factors involved in axon branching. This article focuses on how cytoskeletal mechanisms, intracellular organelles, such as mitochondria and the endoplasmic reticulum, and membrane remodeling (exocytosis and endocytosis) contribute to branch initiation and formation. Together this growing literature provides valuable insight as well as a platform for continued investigation into how multiple aspects of axonal cell biology are spatially and temporally orchestrated to give rise to axon branches. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1293–1307, 2016     

Biochemical purification of a mammalian slit protein as a positive regulator of sensory axon elongation and branching

Many neurons in both vertebrates and invertebrates innervate multiple targets by sprouting secondary axon collaterals (or branches) from a primary axon shaft. To begin to identify molecular regulators of axon branch initiation or extension, we studied the growth of single sensory axons in an in vitro collagen assay system and identified an activity in extracts of embryonic spinal cord and of postnatal and adult brain that promotes the elongation and formation of extensive branches by these axons. Biochemical purification of the activity from calf brain extracts led to the identification of an amino-terminal fragment of Slit2 as the main active component and to the discovery of a distinct activity that potentiates its effects. These results indicate that Slit proteins may function as positive regulators of axon collateral formation during the establishment or remodeling of neural circuits.     

Computational modeling of retinotopic map development to define contributions of EphA-ephrinA gradients, axon-axon interactions, and patterned activity

The topographic projection of retinal ganglion cell (RGC) axons to mouse superior colliculus (SC) or chick optic tectum (OT) is formed in three phases: RGC axons overshoot their termination zone (TZ); they exhibit interstitial branching along the axon that is topographically biased for the correct location of their future TZ; and branches arborize preferentially at the TZ and the initial exuberant projection refines through axon and branch elimination to generate a precise retinotopic map. We present a computational model of map development that demonstrates that the countergradients of EphAs and ephrinAs in retina and the OT/SC and bidirectional repellent signaling between RGC axons and OT/SC cells are sufficient to direct an initial topographic bias in RGC axon branching. Our model also suggests that a proposed repellent action of EphAs/ephrinAs present on RGC branches and arbors added to that of EphAs/ephrinAs expressed by OT/SC cells is required to progressively restrict branching and arborization to topographically correct locations and eliminate axon overshoot. Simulations show that this molecular framework alone can develop considerable topographic order and refinement, including axon elimination, a feature not programmed into the model. Generating a refined map with a condensed TZ as in vivo requires an additional parameter that enhances branch formation along an RGC axon near sites that it has a higher branch density, and resembles an assumed role for patterned neural activity. The same computational model generates the phenotypes reported in ephrinA deficient mice and Isl2-EphA3 knockin mice. This modeling suggests that gradients of counter-repellents can establish a substantial degree of topographic order in the OT/SC, and that repellents present on RGC axon branches and arbors make a substantial contribution to map refinement. However, competitive interactions between RGC axons that enhance the probability of continued local branching are required to generate precise retinotopy.     

AMP‐activated protein kinase mediates activity‐dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 557–573, 2014     

Septin-driven coordination of actin and microtubule remodeling regulates the collateral branching of axons

Axon branching is fundamental to the development of the peripheral and central nervous system. Branches that sprout from the axon shaft are termed collateral or interstitial branches. Collateral branching of axons requires the formation of filopodia from actin microfilaments (F-actin) and their engorgement with microtubules (MTs) that splay from the axon shaft. The mechanisms that drive and coordinate the remodeling of actin and MTs during branch morphogenesis are poorly understood. Septins comprise a family of GTP-binding proteins that oligomerize into higher-order structures, which associate with membranes and the actin and microtubule cytoskeleton. Here, we show that collateral branching of axons requires SEPT6 and SEPT7, two interacting septins. In the axons of sensory neurons, both SEPT6 and SEPT7 accumulate at incipient sites of filopodia formation. We show that SEPT6 localizes to axonal patches of F-actin and increases the recruitment of cortactin, a regulator of Arp2/3-mediated actin polymerization, triggering the emergence of filopodia. Conversely, SEPT7 promotes the entry of axonal MTs into filopodia, enabling the formation of collateral branches. Surprisingly, septins provide a novel mechanism for the collateral branching of axons by coordinating the remodeling of the actin and microtubule cytoskeleton.