An extracellular interactome of immunoglobulin and LRR proteins reveals receptor-ligand networks.

Extracellular domains of cell surface receptors and ligands mediate cell-cell communication, adhesion, and initiation of signaling events, but most existing protein-protein "interactome" data sets lack information for extracellular interactions. We probed interactions between receptor extracellular domains, focusing on a set of 202 proteins composed of the Drosophila melanogaster immunoglobulin superfamily (IgSF), fibronectin type III (FnIII), and leucine-rich repeat (LRR) families, which are known to be important in neuronal and developmental functions. Out of 20,503 candidate protein pairs tested, we observed 106 interactions, 83 of which were previously unknown. We "deorphanized" the 20 member subfamily of defective-in-proboscis-response IgSF proteins, showing that they selectively interact with an 11 member subfamily of previously uncharacterized IgSF proteins. Both subfamilies interact with a single common "orphan" LRR protein. We also observed interactions between Hedgehog and EGFR pathway components. Several of these interactions could be visualized in live-dissected embryos, demonstrating that this approach can identify physiologically relevant receptor-ligand pairs.

Electroretinograms in Drosophila: a robust and genetically accessible electrophysiological system for the undergraduate laboratory.

Laboratory courses in neurophysiology fulfill a critical need for inquiry-based training in undergraduate programs in neuroscience and biology. These courses typically use classical electrophysiological preparations to explore the basic features of neuronal function. However, current neuroscience research also focuses on elucidating the molecular and genetic mechanisms of neuronal function, using model systems that include mutant and transgenic animals. To bridge laboratory training in neurophysiology with modern molecular genetics, we describe a teaching model based on electroretinography of the fruit fly Drosophila melanogaster, a long-established model system for basic neuroscience research. Drosophila are easily maintained, economical, and have hundreds of neurophysiologically relevant mutant strains and genetic tools readily available. The Drosophila electroretinogram (ERG) is a simple and accessible extracellular recording of a neural signal in the fly eye in response to flashes of light. The signal is multifaceted and the response is sensitive to stimulation parameters such as intensity, duration and wavelength, thus forming a rich source of analysis for students. Most importantly, different mutations affecting key components of intracellular signaling, synaptic transmission or neuronal function can affect the ERG waveform in characteristic ways. Recording wild type and mutant ERGs allows students to examine firsthand the connection between genetics, biochemical pathways, and electrophysiology. This neurophysiology laboratory course can facilitate and enhance an understanding of the cellular and molecular contributions to neurophysiological recordings.

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development.

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.

The heparan sulfate proteoglycans Dally-like and Syndecan have distinct functions in axon guidance and visual-system assembly in Drosophila.

Heparan sulfate proteoglycans (HSPGs), a class of glycosaminoglycan-modified proteins, control diverse patterning events via their regulation of growth-factor signaling and morphogen distribution. In C. elegans, zebrafish, and the mouse, heparan sulfate (HS) biosynthesis is required for normal axon guidance, and mutations affecting Syndecan (Sdc), a transmembrane HSPG, disrupt axon guidance in Drosophila embryos. Glypicans, a family of glycosylphosphatidylinositol (GPI)-linked HSPGs, are expressed on axons and growth cones in vertebrates, but their role in axon guidance has not been determined. We demonstrate here that the Drosophila glypican Dally-like protein (Dlp) is required for proper axon guidance and visual-system function. Mosaic studies revealed that Dlp is necessary in both the retina and the brain for different aspects of visual-system assembly. Sdc mutants also showed axon guidance and visual-system defects, some that overlap with dlp and others that are unique. dlp+ transgenes were able to rescue some sdc visual-system phenotypes, but sdc+ transgenes were ineffective in rescuing dlp abnormalities. Together, these findings suggest that in some contexts HS chains provide the biologically critical component, whereas in others the structure of the protein core is also essential.

Heparan sulfate proteoglycans and the emergence of neuronal connectivity.

With the identification of the molecular determinants of neuronal connectivity, our understanding of the extracellular information that controls axon guidance and synapse formation has evolved from single factors towards the complexity that neurons face in a living organism. As we move in this direction - ready to see the forest for the trees - attention is returning to one of the most ancient regulators of cell-cell interaction: the extracellular matrix. Among many matrix components that influence neuronal connectivity, recent studies of the heparan sulfate proteoglycans suggest that these ancient molecules function as versatile extracellular scaffolds that both sculpt the landscape of extracellular cues and modulate the way that neurons perceive the world around them.

Axonal heparan sulfate proteoglycans regulate the distribution and efficiency of the repellent slit during midline axon guidance.

The presentation of secreted axon guidance factors plays a major role in shaping central nervous system (CNS) connectivity. Recent work suggests that heparan sulfate (HS) regulates guidance factor activity; however, the in vivo axon guidance roles of its carrier proteins (heparan sulfate proteoglycans, or HSPGs) are largely unknown. Here we demonstrate through genetic analysis in vivo that the HSPG Syndecan (Sdc) is critical for the fidelity of Slit repellent signaling at the midline of the Drosophila CNS, consistent with the localization of Sdc to CNS axons. sdc mutants exhibit consistent defects in midline axon guidance, plus potent and specific genetic interactions supporting a model in which HSPGs improve the efficiency of Slit localization and/or signaling. To test this hypothesis, we show that Slit distribution is altered in sdc mutants and that Slit and its receptor bind to Sdc. However, when we compare the function of the transmembrane Sdc to a different class of HSPG that localizes to CNS axons (Dallylike), we find functional redundancy, suggesting that these proteoglycans act as spatially specific carriers of common HS structures that enable growth cones to interact with and perceive Slit as it diffuses away from its source at the CNS midline.

Heparan sulfate proteoglycan specificity during axon pathway formation in the Drosophila embryo.

Axon guidance is influenced by the presence of heparan sulfate (HS) proteoglycans (HSPGs) on the surface of axons and growth cones (Hu, [2001]: Nat Neurosci 4:695-701; Irie et al. [2002]: Development 129:61-70; Inatani et al. [2003]: Science 302:1044-1046; Johnson et al. [2004]: Curr Biol 14:499-504; Steigemann et al. [2004]: Curr Biol 14:225-230). Multiple HSPGs, including Syndecans, Glypicans and Perlecans, carry the same carbohydrate polymer backbones, raising the question of how these molecules display functional specificity during nervous system development. Here we use the Drosophila central nervous system (CNS) as a model to compare the impact of eliminating Syndecan (Sdc) and/or the Glypican Dally-like (Dlp). We show that Dlp and Sdc share a role in promoting accurate patterns of axon fasciculation in the lateral longitudinal neuropil; however, unlike mutations in sdc, which disrupt the ability of the secreted repellent Slit to prevent inappropriate passage of axons across the midline, mutations in dlp show neither midline defects nor genetic interactions with Slit and its Roundabout (Robo) receptors at the midline. Dlp mutants do show genetic interactions with Slit and Robo in lateral fascicle formation. In addition, simultaneous loss of Dlp and Sdc demonstrates an important role for Dlp in midline repulsion, reminiscent of the functional overlap between Robo receptors. A comparison of HSPG distribution reveals a pattern that leaves midline proximal axons with relatively little Dlp. Finally, the loss of Dlp alters Slit distribution distal but not proximal to the midline, suggesting that distinct yet overlapping pattern of HSPG expression provides a spatial system that regulates axon guidance decisions.

Systematic screening of Drosophila deficiency mutations for embryonic phenotypes and orphan receptor ligands.

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency "kits" that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of approximately 400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers approximately 50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification.

LAR, liprin alpha and the regulation of active zone morphogenesis.

Active zones are protein-rich regions of neurons that act as sites of synaptic vesicle fusion and neurotransmitter release at the pre-synaptic terminus. Although the discovery that the receptor protein tyrosine phosphatase LAR and its cytoplasmic binding partner liprin alpha are essential for proper active zone formation is nearly a decade old, the underlying mechanisms are still poorly understood. Recent studies have identified a number of binding partners for both LAR and liprin alpha, several of which play key roles in active zone assembly. These include nidogen, dallylike and syndecan--extracellular ligands for LAR that regulate synapse morphogenesis. In addition, liprin-alpha-interacting proteins such as ERC2, RIM and the MALS/Veli-Cask-Mint1 complex cooperate to form a dense molecular scaffold at the active zone that is crucial for proper synaptic function. These studies allow us to propose testable models of LAR and liprin alpha function, and provide insights into the fundamental molecular mechanisms of synapse formation and stabilization.

Receptor protein tyrosine phosphatases in nervous system development.

Receptor protein tyrosine phosphatases (RPTPs) are key regulators of neuronal morphogenesis in a variety of different vertebrate and invertebrate systems, yet the mechanisms by which these proteins regulate central nervous system development are poorly understood. In the past few years, studies have begun to outline possible models for RPTP function by demonstrating in vivo roles for RPTPs in axon outgrowth, guidance, and synaptogenesis. In addition, the crystal structures of several RPTPs have been solved, numerous downstream effectors of RPTP signaling have been identified, and a small number of RPTP ligands have been described. In this review, we focus on how RPTPs transduce signals from the extracellular environment to the cytoplasm, using a detailed comparative analysis of the different RPTP subfamilies. Focusing on the roles RPTPs play in the development of the central nervous system, we discuss how the elucidation of RPTP crystal structures, the biochemical analysis of phosphatase enzyme catalysis, and the characterization of complex signal transduction cascades downstream of RPTPs have generated testable models of RPTP structure and function.