Slit and Receptor Tyrosine Phosphatase 69D Confer Spatial Specificity to Axon Branching via Dscam1.

Axonal branching contributes substantially to neuronal circuit complexity. Studies in Drosophila have shown that loss of Dscam1 receptor diversity can fully block axon branching in mechanosensory neurons. Here we report that cell-autonomous loss of the receptor tyrosine phosphatase 69D (RPTP69D) and loss of midline-localized Slit inhibit formation of specific axon collaterals through modulation of Dscam1 activity. Genetic and biochemical data support a model in which direct binding of Slit to Dscam1 enhances the interaction of Dscam1 with RPTP69D, stimulating Dscam1 dephosphorylation. Single-growth-cone imaging reveals that Slit/RPTP69D are not required for general branch initiation but instead promote the extension of specific axon collaterals. Hence, although regulation of intrinsic Dscam1-Dscam1 isoform interactions is essential for formation of all mechanosensory-axon branches, the local ligand-induced alterations of Dscam1 phosphorylation in distinct growth-cone compartments enable the spatial specificity of axon collateral formation.

Nuclear import of the DSCAM-cytoplasmic domain drives signaling capable of inhibiting synapse formation.

DSCAM and DSCAML1 are immunoglobulin and cell adhesion-type receptors serving important neurodevelopmental functions including control of axon growth, branching, neurite self-avoidance, and neuronal cell death. The signal transduction mechanisms or effectors of DSCAM receptors, however, remain poorly characterized. We used a human ORFeome library to perform a high-throughput screen in mammalian cells and identified novel cytoplasmic signaling effector candidates including the Down syndrome kinase Dyrk1a, STAT3, USP21, and SH2D2A. Unexpectedly, we also found that the intracellular domains (ICDs) of DSCAM and DSCAML1 specifically and directly interact with IPO5, a nuclear import protein of the importin beta family, via a conserved nuclear localization signal. The DSCAM ICD is released by γ-secretase-dependent cleavage, and both the DSCAM and DSCAML1 ICDs efficiently translocate to the nucleus. Furthermore, RNA sequencing confirms that expression of the DSCAM as well as the DSCAML1 ICDs alone can profoundly alter the expression of genes associated with neuronal differentiation and apoptosis, as well as synapse formation and function. Gain-of-function experiments using primary cortical neurons show that increasing the levels of either the DSCAM or the DSCAML1 ICD leads to an impairment of neurite growth. Strikingly, increased expression of either full-length DSCAM or the DSCAM ICD, but not the DSCAML1 ICD, significantly decreases synapse numbers in primary hippocampal neurons. Taken together, we identified a novel membrane-to-nucleus signaling mechanism by which DSCAM receptors can alter the expression of regulators of neuronal differentiation and synapse formation and function. Considering that chromosomal duplications lead to increased DSCAM expression in trisomy 21, our findings may help uncover novel mechanisms contributing to intellectual disability in Down syndrome.

Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy.

Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers.

Ultra-deep profiling of alternatively spliced Drosophila Dscam isoforms by circularization-assisted multi-segment sequencing.

The Drosophila melanogaster gene Dscam (Down syndrome cell adhesion molecule) can generate thousands of different ectodomains via mutual exclusive splicing of three large exon clusters. The isoform diversity plays a profound role in both neuronal wiring and pathogen recognition. However, the isoform expression pattern at the global level remained unexplored. Here, we developed a novel method that allows for direct quantification of the alternatively spliced exon combinations from over hundreds of millions of Dscam transcripts in one sequencing run. With unprecedented sequencing depth, we detected a total of 18,496 isoforms, out of 19,008 theoretically possible combinations. Importantly, we demonstrated that alternative splicing between different clusters is independent. Moreover, the isoforms were expressed across a broad dynamic range, with significant bias in cell/tissue and developmental stage-specific patterns. Hitherto underappreciated, such bias can dramatically reduce the ability of neurons to display unique surface receptor codes. Therefore, the seemingly excessive diversity encoded in the Dscam locus might nevertheless be essential for a robust self and non-self discrimination in neurons.

Axonal wiring in neural development: Target-independent mechanisms help to establish precision and complexity.

The connectivity patterns of many neural circuits are highly ordered and often impressively complex. The intricate order and complexity of neuronal wiring remain not only a challenge for questions related to circuit functions but also for our understanding of how they develop with such an apparent precision. The chemotropic guidance of the growing axon by target-derived cues represents a central paradigm for how neurons get connected with the correct target cells. However, many studies reveal a remarkable variety of important target-independent wiring mechanisms. These mechanisms include axonal sorting, axonal tiling, growth cone polarization, as well as cell-intrinsic mechanisms underlying growth cone sprouting, and neurite branching. Our review focuses on target independent wiring mechanisms and in particular on recent progress emerging from studies on three different sensory systems: olfactory, visual, and somatosensory. We discuss molecular mechanisms that operate during axon-axon interactions or constitute axon-intrinsic functions and outline how they complement the well-known target-dependent wiring mechanisms.

Dscam and DSCAM: complex genes in simple animals, complex animals yet simple genes.

Cadherins and the immunoglobulin (Ig) proteins give rise to a multitude of surface receptors, which function as diverse cell adhesion molecules (CAMs) or signal-transducing receptors. These functions are often interdependent, and rely on a high degree of specificity in homophilic binding as well as heterophilic interactions. The Drosophila receptor Dscam is an exceptional example of homophilic binding specificity involved in a number of important biological processes, such as neural wiring and innate immunity. Combinatorial use of alternatively spliced Ig-domains enables the generation of an estimated 18,000 isoform-specific homophilic receptor pairs. Although isoform diversity of Dscam is unique to arthropods, recent genetic analysis of vertebrate DSCAM (Down Syndrome Cell Adhesion Molecule) genes has revealed an intriguing conservation of molecular functions underlying neural wiring. This review covers the multiple functions of Dscam across different species highlighting its remarkable versatility as well as its conserved basic functions in neural development. We discuss how an unprecedented expansion of complex alternative splicing has been uniquely employed by arthropods to generate diverse surface receptors, important for cell-cell communication, molecular self-recognition in neurons, and innate immune defenses. We end with a speculative hypothesis reconciling the striking differences in Dscam and DSCAM gene structures with their conserved functions in molecular recognition underlying neural circuit formation.

VEGF modulates NMDA receptors activity in cerebellar granule cells through Src-family kinases before synapse formation.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).

Structural basis of Dscam isoform specificity.

The Dscam gene gives rise to thousands of diverse cell surface receptors thought to provide homophilic and heterophilic recognition specificity for neuronal wiring and immune responses. Mutually exclusive splicing allows for the generation of sequence variability in three immunoglobulin ecto-domains, D2, D3 and D7. We report X-ray structures of the amino-terminal four immunoglobulin domains (D1-D4) of two distinct Dscam isoforms. The structures reveal a horseshoe configuration, with variable residues of D2 and D3 constituting two independent surface epitopes on either side of the receptor. Both isoforms engage in homo-dimerization coupling variable domain D2 with D2, and D3 with D3. These interactions involve symmetric, antiparallel pairing of identical peptide segments from epitope I that are unique to each isoform. Structure-guided mutagenesis and swapping of peptide segments confirm that epitope I, but not epitope II, confers homophilic binding specificity of full-length Dscam receptors. Phylogenetic analysis shows strong selection of matching peptide sequences only for epitope I. We propose that peptide complementarity of variable residues in epitope I of Dscam is essential for homophilic binding specificity.

A New Technical Approach for Cross-species Examination of Neuronal Wiring and Adult Neuron-glia Functions.

Advances in single cell sequencing have enabled the identification of a large number of genes, expressed in many different cell types, and across a variety of model organisms. In particular, the nervous system harbors an immense number of interacting cell types, which are poorly characterized. Future loss- and gain-of-function experiments will be essential in determining how novel genes play critical roles in diverse cellular, as well as evolutionarily adapted, contexts. However, functional analysis across species is often hampered by technical limitations, in non-genetic animal systems. Here, we describe a new single plasmid system, misPiggy. The system is based around the hyperactive piggyBac transposon system, which combines stable genomic integration of transgenes (for long-term expression) with large cargo capacity. Taking full advantage of these characteristics, we engineered novel expression modules into misPiggy that allow for cell-type specific loss- and gain-of-gene function. These modules work widely across species from frog to ferret. As a proof of principle, we present a loss-of-function analysis of the neuronal receptor Deleted in Colorectal Cancer (DCC) in retinal ganglion cells (RGCs) of Xenopus tropicalis tadpoles. Single axon tracings of mosaic knock-out cells reveal a specific cell-intrinsic requirement of DCC, specifically in axonal arborization within the frog tectum, rather than retina-to-brain axon guidance. Furthermore, we report additional technical advances that enable temporal control of knock-down or gain-of-function analysis. We applied this to visualize and manipulate labeled neurons, astrocytes and other glial cells in the central nervous system (CNS) of mouse, rat and ferret. We propose that misPiggy will be a valuable tool for rapid, flexible and cost-effective screening of gene function across a variety of animal models.

Molecular diversity of Dscam: recognition of molecular identity in neuronal wiring.

Our understanding of how the enormously complex task of interconnecting millions of nerve cells is accomplished remains rudimentary. What molecular mechanisms control its exquisite specificity? Can we pinpoint single molecular interactions that might help to explain some of the specificity requirements that underlie neuronal wiring? A series of recent studies on the molecular diversity of the Drosophila melanogaster cell-surface receptor Down syndrome cell-adhesion molecule (Dscam) provide one exceptional example of a novel mechanistic model of neuronal-wiring specificity, progressing from structural studies of single protein-protein interactions to biochemical analysis in vitro and to an understanding of complex neuronal differentiation at the single-cell or tissue levels.

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.

Axonal branch patterning and neuronal shape diversity: roles in developmental circuit assembly: Axonal branch patterning and neuronal shape diversity in developmental circuit assembly.

Recent progress in human genetics and single cell sequencing rapidly expands the list of molecular factors that offer important new contributions to our understanding of brain wiring. Yet many new molecular factors are being discovered that have never been studied in the context of neuronal circuit development. This is clearly asking for increased efforts to better understand the developmental mechanisms of circuit assembly [1]. Moreover, recent studies characterizing the developmental causes of some psychiatric diseases show impressive progress in reaching cellular resolution in their analysis. They provide concrete support emphasizing the importance of axonal branching and synapse formation as a hotspot for potential defects. Inspired by these new studies we will discuss progress but also challenges in understanding how neurite branching and neuronal shape diversity itself impacts on specificity of neuronal circuit assembly. We discuss the idea that neuronal shape acquisition itself is a key specificity factor in neuronal circuit assembly.

Axon morphogenesis and maintenance require an evolutionary conserved safeguard function of Wnk kinases antagonizing Sarm and Axed.

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.

Subtle Roles of Down Syndrome Cell Adhesion Molecules in Embryonic Forebrain Development and Neuronal Migration.

Down Syndrome (DS) Cell Adhesion Molecules (DSCAMs) are transmembrane proteins of the immunoglobulin superfamily. Human DSCAM is located within the DS critical region of chromosome 21 (duplicated in Down Syndrome patients), and mutations or copy-number variations of this gene have also been associated to Fragile X syndrome, intellectual disability, autism, and bipolar disorder. The DSCAM paralogue DSCAM-like 1 (DSCAML1) maps to chromosome 11q23, implicated in the development of Jacobsen and Tourette syndromes. Additionally, a spontaneous mouse DSCAM deletion leads to motor coordination defects and seizures. Previous research has revealed roles for DSCAMs in several neurodevelopmental processes, including synaptogenesis, dendritic self-avoidance, cell sorting, axon growth and branching. However, their functions in embryonic mammalian forebrain development have yet to be completely elucidated. In this study, we revealed highly dynamic spatiotemporal patterns of Dscam and Dscaml1 expression in definite cortical layers of the embryonic mouse brain, as well as in structures and ganglionic eminence-derived neural populations within the embryonic subpallium. However, an in-depth histological analysis of cortical development, ventral forebrain morphogenesis, cortical interneuron migration, and cortical-subcortical connectivity formation processes in Dscam and Dscaml1 knockout mice (Dscam del17 and Dscaml1 GT ) at several embryonic stages indicated that constitutive loss of Dscam and Dscaml1 does not affect these developmental events in a significant manner. Given that several Dscam- and Dscaml1-linked neurodevelopmental disorders are associated to chromosomal region duplication events, we furthermore sought to examine the neurodevelopmental effects of Dscam and Dscaml1 gain of function (GOF). In vitro, ex vivo, and in vivo GOF negatively impacted neural migration processes important to cortical development, and affected the morphology of maturing neurons. Overall, these findings contribute to existing knowledge on the molecular etiology of human neurodevelopmental disorders by elucidating how dosage variations of genes encoding adhesive cues can disrupt cell-cell or cell-environment interactions crucial for neuronal migration.

Branch-restricted localization of phosphatase Prl-1 specifies axonal synaptogenesis domains.

Central nervous system (CNS) circuit development requires subcellular control of synapse formation and patterning of synapse abundance. We identified the Drosophila membrane-anchored phosphatase of regenerating liver (Prl-1) as an axon-intrinsic factor that promotes synapse formation in a spatially restricted fashion. The loss of Prl-1 in mechanosensory neurons reduced the number of CNS presynapses localized on a single axon collateral and organized as a terminal arbor. Flies lacking all Prl-1 protein had locomotor defects. The overexpression of Prl-1 induced ectopic synapses. In mechanosensory neurons, Prl-1 modulates the insulin receptor (InR) signaling pathway within a single contralateral axon compartment, thereby affecting the number of synapses. The axon branch-specific localization and function of Prl-1 depend on untranslated regions of the prl-1 messenger RNA (mRNA). Therefore, compartmentalized restriction of Prl-1 serves as a specificity factor for the subcellular control of axonal synaptogenesis.

Downstream of guidance receptors: entering the baroque period of axon guidance signaling.

The Roundabout ("Robo") family of transmembrane proteins are the receptors and mediators of the repellent axon guidance signal Slit. However, the molecular mechanisms by which Robo signaling leads to growth cone or neuron repulsion are still poorly understood. A study by Fan et al. in this issue of Neuron expands the repertoire of Robo pathway components and stimulates a new look at axon guidance signaling in general.

Generation of recognition diversity in the nervous system.

For decades, it has been suggested that complex neural wiring might be specified by extensive diversity in receptor isoforms. Dscam is a cell surface protein with 38,016 potential alternatively spliced isoforms in the fly nervous system. Remarkable binding studies now show that Dscam isoform diversity indeed results in an unprecedented level of recognition diversity, showing isoform-specific homophilic binding. In vivo studies have begun to suggest models for use of Dscam diversity in neuron-target recognition, axon fasciculation, and neuron self-recognition.

ABL to Integrate Opposing Cellular Growth Signals.

Growth factor signaling has long been known to stimulate cellular growth and motility. That it might also directly promote repulsive signaling, however, is a surprising finding reported by Yoon et al. (2017) in this issue of Developmental Cell.