Commissureless acts as a substrate adapter in a conserved Nedd4 E3 ubiquitin ligase pathway to promote axon growth across the midline.

In both vertebrates and invertebrates, commissural neurons prevent premature responsiveness to the midline repellant Slit by downregulating surface levels of its receptor Roundabout1 (Robo1). In Drosophila, Commissureless (Comm) plays a critical role in this process; however, there is conflicting data on the underlying molecular mechanism. Here, we demonstrate that the conserved PY motifs in the cytoplasmic domain of Comm are required allow the ubiquitination and lysosomal degradation of Robo1. Disruption of these motifs prevents Comm from localizing to Lamp1 positive late endosomes and to promote axon growth across the midline in vivo. In addition, we conclusively demonstrate a role for Nedd4 in midline crossing. Genetic analysis shows that nedd4 mutations result in midline crossing defects in the Drosophila embryonic nerve cord, which can be rescued by introduction of exogenous Nedd4. Biochemical evidence shows that Nedd4 incorporates into a three-member complex with Comm and Robo in a PY motif-dependent manner. Finally, we present genetic evidence that Nedd4 acts with Comm in the embryonic nerve cord to downregulate Robo1 levels. Taken together, these findings demonstrate that Comm promotes midline crossing in the nerve cord by facilitating Robo ubiquitination by Nedd4, ultimately leading to its degradation.

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

DrosoPHILA: A Partnership between Scientists and Teachers That Begins in the Lab and Continues into City Schools.

Here, we describe the development, structure, and effectiveness of an outreach program, DrosoPHILA, that leverages the tools of our fly neurodevelopmental research program at the University of Pennsylvania to reinforce the biology curriculum in local public schools. DrosoPHILA was developed and is sustained by a continued collaboration between members of the Bashaw lab, experienced outreach educators, and teachers in the School District of Philadelphia. Since the program's inception, we have collaborated with 18 teachers and over 2400 students. Student outcome data indicates significant positive attitude shifts around science identity and grade-appropriate knowledge gains.

Intracellular Trafficking Mechanisms that Regulate Repulsive Axon Guidance.

Friedrich Bonhoeffer made seminal contributions to the study of axon guidance in the developing nervous system. His discoveries of key cellular and molecular mechanisms that dictate wiring specificity laid the foundation for countless investigators who have followed in his footsteps. Perhaps his most significant contribution was the cloning and characterization of members of the conserved ephrin family of repulsive axon guidance cues. In this review, we highlight the major contributions that Bonhoeffer and his colleagues made to the field of axon guidance, and discuss ongoing investigations into the diverse array of mechanisms that ensure that axon repulsion is precisely regulated to allow for accurate pathfinding. Specifically, we focus our discussion on the post-translational regulation of two major families of repulsive axon guidance factors: ephrin ligands and their Eph receptors, and slit ligands and their Roundabout (Robo) receptors. We will give special emphasis to the ways in which regulated endocytic trafficking events allow navigating axons to adjust their responses to repellant signals and how these trafficking events are intimately related to receptor signaling. By highlighting parallels and differences between the regulation of these two important repulsive axon guidance pathways, we hope to identify key outstanding questions for future investigation.

Tace/ADAM17 is a bi-directional regulator of axon guidance that coordinates distinct Frazzled and Dcc receptor signaling outputs.

Frazzled (Fra) and deleted in colorectal cancer (Dcc) are homologous receptors that promote axon attraction in response to netrin. In Drosophila, Fra also acts independently of netrin by releasing an intracellular domain (ICD) that activates gene transcription. How neurons coordinate these pathways to make accurate guidance decisions is unclear. Here we show that the ADAM metalloprotease Tace cleaves Fra, and this instructs the switch between the two pathways. Genetic manipulations that either increase or decrease Tace levels disrupt midline crossing of commissural axons. These conflicting phenotypes reflect Tace's function as a bi-directional regulator of axon guidance, a function conserved in its vertebrate homolog ADAM17: while Tace induces the formation of the Fra ICD to activate transcription, excessive Tace cleavage of Fra and Dcc suppresses the response to netrin. We propose that Tace and ADAM17 are key regulators of midline axon guidance by establishing the balance between netrin-dependent and netrin-independent signaling.

A Nedd4 E3 Ubiquitin Ligase Pathway Inhibits Robo1 Repulsion and Promotes Commissural Axon Guidance across the Midline.

Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. In insects and mammals, negative regulation of the surface expression of Roundabout (Robo) receptors prevents premature response to Slit. We previously identified two mammalian Nedd4 interacting proteins, Ndfip1 and Ndfip2, that act analogously to Drosophila Commissureless (Comm) to recruit mammalian Robo1 to late endosomes. However, whether Nedd4 E3 ubiquitin ligases are required for Ndfip-mediated Robo1 regulation and midline axon crossing in vivo is not known. Here, we show using in vitro biochemical techniques and genetic analysis using embryonic mice of either sex that Nedd4-1 and Nedd4-2 are specifically required for Robo1 regulation and spinal commissural axon guidance. Biochemical data indicate that Robo1, Ndfip and Nedd4 form a ternary protein complex that depends on the presence of Ndfip, and these interactions are required for Robo1 endosomal sorting, ubiquitylation and degradation. Nedd4-1 and Nedd4-2 are expressed in commissural neurons in the developing spinal cord, and conditional deletion of Nedd4-1 or Nedd4-2 results in dose-dependent defects in midline crossing. We propose that Nedd4 E3 Ubiquitin ligases and their adaptor proteins Ndfip1 and Ndfip2 constitute a vital intracellular trafficking pathway required to downregulate Robo1 and promote midline crossing of commissural axons.SIGNIFICANCE STATEMENT During the development of the nervous system, many neurons extend their axons across the midline to establish circuits that are important for sensory, motor and cognitive functions. In order to cross the midline, axon responses to midline-derived cues must be precisely regulated. Here, we characterize an important intracellular trafficking pathway that regulates the membrane expression of the conserved Roundabout (Robo) axon guidance receptor- the receptor for the midline repellant Slit. We show that Nedd4 E3 Ubiquitin ligases and their Ndfip adapter proteins inhibit premature responses to Slit by promoting Robo degradation in precrossing commissural neurons in the developing spinal cord.

Diverse roles for axon guidance pathways in adult tissue architecture and function.

Classical axon guidance ligands and their neuronal receptors were first identified due to their fundamental roles in regulating connectivity in the developing nervous system. Since their initial discovery, it has become clear that these signaling molecules play important roles in the development of a broad array of tissue and organ systems across phylogeny. In addition to these diverse developmental roles, there is a growing appreciation that guidance signaling pathways have important functions in adult organisms, including the regulation of tissue integrity and homeostasis. These roles in adult organisms include both tissue-intrinsic activities of guidance molecules, as well as systemic effects on tissue maintenance and function mediated by the nervous and vascular systems. While many of these adult functions depend on mechanisms that mirror developmental activities, such as regulating adhesion and cell motility, there are also examples of adult roles that may reflect signaling activities that are distinct from known developmental mechanisms, including the contributions of guidance signaling pathways to lineage commitment in the intestinal epithelium and bone remodeling in vertebrates. In this review, we highlight studies of guidance receptors and their ligands in adult tissues outside of the nervous system, focusing on in vivo experimental contexts. Together, these studies lay the groundwork for future investigation into the conserved and tissue-specific mechanisms of guidance receptor signaling in adult tissues.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

Robo recruitment of the Wave regulatory complex plays an essential and conserved role in midline repulsion.

The Roundabout (Robo) guidance receptor family induces axon repulsion in response to its ligand Slit by inducing local cytoskeletal changes; however, the link to the cytoskeleton and the nature of these cytoskeletal changes are poorly understood. Here, we show that the heteropentameric Scar/Wave Regulatory Complex (WRC), which drives Arp2/3-induced branched actin polymerization, is a direct effector of Robo signaling. Biochemical evidence shows that Slit triggers WRC recruitment to the Robo receptor's WRC-interacting receptor sequence (WIRS) motif. In Drosophila embryos, mutants of the WRC enhance Robo1-dependent midline crossing defects. Additionally, mutating Robo1's WIRS motif significantly reduces receptor activity in rescue assays in vivo, and CRISPR-Cas9 mutagenesis shows that the WIRS motif is essential for endogenous Robo1 function. Finally, axon guidance assays in mouse dorsal spinal commissural axons and gain-of-function experiments in chick embryos demonstrate that the WIRS motif is also required for Robo1 repulsion in mammals. Together, our data support an essential conserved role for the WIRS-WRC interaction in Robo1-mediated axon repulsion.

The brain is the most complex organ in the body. It contains billions of nerve cells, also known as neurons, with trillions of precise and specific connections, but how do these neurons know where to go and which connections to make as the brain grows? Neurons contain a small set of proteins known as guidance receptors. These receptors respond to external signals that can be attractive or repulsive. They instruct neurons to turn towards, or away from, the source of a signal. During embryonic development, neurons use these signals as guideposts to find their way to their destination. One such guidance receptor-signal pair consists of a receptor called Roundabout, also known as Robo, and its cue, Slit. Robo, which is located on the neuron's surface, responds to the presence of Slit in the environment, by initiating a set of signalling events that instruct neurons to turn away. Neurons make the turn by rearranging their internal scaffolding, a network of proteins called the actin cytoskeleton. How Robo triggers this rearrangement is unclear. One possibility relies on a group of proteins called the WAVE regulatory complex, or the WRC for short. Researchers have already linked the WRC to nerve cell guidance, showing that it can trigger the growth of new filaments in the actin cytoskeleton. Proteins can activate the WRC by binding to it using a set of amino acids called a WRC-interacting receptor sequence, or WIRS for short, which Robo has. Chaudhari et al. used fruit flies to find out how Robo and the WRC interact. The experiments revealed that when Slit binds to Robo on the outside of a nerve cell, the WRC binds to Robo via its WIRS sequence on the inside of the cell. This attracts proteins inside the cell involved in rearranging the actin cytoskeleton. Disrupting this interaction by mutating either WRC or WIRS leads to severe errors in pathfinding, because when the WRC cannot connect to Robo, neurons cannot find their way. Experiments in mouse and chicken embryos showed that vertebrates use the WIRS sequence too, indicating that evolution has conserved this method of passing signals from Robo to the cytoskeleton. The fact that Slit and Robo work in the same way across fruit flies and vertebrates has implications for future medical research. Further work could explain how the brain and nervous system develop, and what happens when development goes wrong, but Slit and Robo control more than just nerve cell pathfinding. Research has linked disruptions in both proteins to many types of cancer, so a better understanding of how Robo interacts with the WRC could lead to new developments in different fields.

Conserved and divergent aspects of Robo receptor signaling and regulation between Drosophila Robo1 and C. elegans SAX-3.

The evolutionarily conserved Roundabout (Robo) family of axon guidance receptors control midline crossing of axons in response to the midline repellant ligand Slit in bilaterian animals including insects, nematodes, and vertebrates. Despite this strong evolutionary conservation, it is unclear whether the signaling mechanism(s) downstream of Robo receptors are similarly conserved. To directly compare midline repulsive signaling in Robo family members from different species, here we use a transgenic approach to express the Robo family receptor SAX-3 from the nematode Caenorhabditis elegans in neurons of the fruit fly, Drosophila melanogaster. We examine SAX-3's ability to repel Drosophila axons from the Slit-expressing midline in gain of function assays, and test SAX-3's ability to substitute for Drosophila Robo1 during fly embryonic development in genetic rescue experiments. We show that C. elegans SAX-3 is properly translated and localized to neuronal axons when expressed in the Drosophila embryonic CNS, and that SAX-3 can signal midline repulsion in Drosophila embryonic neurons, although not as efficiently as Drosophila Robo1. Using a series of Robo1/SAX-3 chimeras, we show that the SAX-3 cytoplasmic domain can signal midline repulsion to the same extent as Robo1 when combined with the Robo1 ectodomain. We show that SAX-3 is not subject to endosomal sorting by the negative regulator Commissureless (Comm) in Drosophila neurons in vivo, and that peri-membrane and ectodomain sequences are both required for Comm sorting of Drosophila Robo1.

New insights into the molecular mechanisms of axon guidance receptor regulation and signaling.

As the nervous system develops, newly differentiated neurons need to extend their axons toward their synaptic targets to form functional neural circuits. During this highly dynamic process of axon pathfinding, guidance receptors expressed at the tips of motile axons interact with soluble guidance cues or membrane tethered molecules present in the environment to be either attracted toward or repelled away from the source of these cues. As competing cues are often present at the same location and during the same developmental period, guidance receptors need to be both spatially and temporally regulated in order for the navigating axons to make appropriate guidance decisions. This regulation is exerted by a diverse array of molecular mechanisms that have come into focus over the past several decades and these mechanisms ensure that the correct complement of surface receptors is present on the growth cone, a fan-shaped expansion at the tip of the axon. This dynamic, highly motile structure is defined by a lamellipodial network lining the periphery of the growth cone interspersed with finger-like filopodial projections that serve to explore the surrounding environment. Once axon guidance receptors are deployed at the right place and time at the growth cone surface, they respond to their respective ligands by initiating a complex set of signaling events that serve to rearrange the growth cone membrane and the actin and microtubule cytoskeleton to affect axon growth and guidance. In this review, we highlight recent advances that shed light on the rich complexity of mechanisms that regulate axon guidance receptor distribution, activation and downstream signaling.

Molecular mechanisms regulating axon responsiveness at the midline.

During embryonic development in bilaterally symmetric organisms, correct midline crossing is important for the proper formation of functional neural circuits. The aberrant development of neural circuits can result in multiple neurodevelopmental disorders, including horizontal gaze palsy, congenital mirror movement disorder, and autism spectrum disorder. Thus, understanding the molecular mechanisms that regulate proper axon guidance at the midline can provide insights into the pathology of neurological disorders. The signaling mechanisms that regulate midline crossing have been extensively studied in the Drosophila ventral nerve cord and the mouse embryonic spinal cord. In this review, we discuss these axon guidance mechanisms, highlighting the most recent advances in the understanding of how commissural axons switch their responsiveness from attractants to repellents during midline crossing.

Molecular identification of a BAR domain-containing coat complex for endosomal recycling of transmembrane proteins.

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.

Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord.

Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. This switch prevents axons from re-entering the midline. In insects and mammals, negative regulation of Roundabout (Robo) receptors prevents premature response to the midline repellant Slit. In Drosophila, the endosomal protein Commissureless (Comm) prevents Robo1 surface expression before midline crossing by diverting Robo1 into late endosomes. Notably, Comm is not conserved in vertebrates. We identified two Nedd-4-interacting proteins, Ndfip1 and Ndfip2, that act analogously to Comm to localize Robo1 to endosomes. Ndfip proteins recruit Nedd4 E3 ubiquitin ligases to promote Robo1 ubiquitylation and degradation. Ndfip proteins are expressed in commissural axons in the developing spinal cord and removal of Ndfip proteins results in increased Robo1 expression and reduced midline crossing. Our results define a conserved Robo1 intracellular sorting mechanism between flies and mammals to avoid premature responsiveness to Slit.

Brain Tumor promotes axon growth across the midline through interactions with the microtubule stabilizing protein Apc2.

Commissural axons must cross the midline to establish reciprocal connections between the two sides of the body. This process is highly conserved between invertebrates and vertebrates and depends on guidance cues and their receptors to instruct axon trajectories. The DCC family receptor Frazzled (Fra) signals chemoattraction and promotes midline crossing in response to its ligand Netrin. However, in Netrin or fra mutants, the loss of crossing is incomplete, suggesting the existence of additional pathways. Here, we identify Brain Tumor (Brat), a tripartite motif protein, as a new regulator of midline crossing in the Drosophila CNS. Genetic analysis indicates that Brat acts independently of the Netrin/Fra pathway. In addition, we show that through its B-Box domains, Brat acts cell autonomously to regulate the expression and localization of Adenomatous polyposis coli-2 (Apc2), a key component of the Wnt canonical signaling pathway, to promote axon growth across the midline. Genetic evidence indicates that the role of Brat and Apc2 to promote axon growth across the midline is independent of Wnt and Beta-catenin-mediated transcriptional regulation. Instead, we propose that Brat promotes midline crossing through directing the localization or stability of Apc2 at the plus ends of microtubules in navigating commissural axons. These findings define a new mechanism in the coordination of axon growth and guidance at the midline.

Axon guidance pathways and the control of gene expression.

Axons need to be properly guided to their targets to form synaptic connections, and this requires interactions between highly conserved extracellular and transmembrane ligands and their cell surface receptors. The majority of studies on axon guidance signaling pathways have focused on the role of these pathways in rearranging the local cytoskeleton and plasma membrane in growth cones and axons. However, a smaller body of work has demonstrated that axon guidance signaling pathways also control gene expression via local translation and transcription. Recent studies on axon guidance ligands and receptors have begun to uncover the requirements for these alternative mechanisms in processes required for neural circuit formation: axon guidance, synaptogenesis, and cell migration. Understanding the mechanisms by which axon guidance signaling regulates local translation and transcription will create a more complete picture of neural circuit formation, and they may be applied more broadly to other tissues where axon guidance ligands and receptors are required for morphogenesis. Developmental Dynamics 247:571-580, 2018. © 2017 Wiley Periodicals, Inc.

Islet Coordinately Regulates Motor Axon Guidance and Dendrite Targeting through the Frazzled/DCC Receptor.

Motor neuron axon targeting in the periphery is correlated with the positions of motor neuron inputs in the CNS, but how these processes are coordinated to form a myotopic map remains poorly understood. We show that the LIM homeodomain factor Islet (Isl) controls targeting of both axons and dendrites in Drosophila motor neurons through regulation of the Frazzled (Fra)/DCC receptor. Isl is required for fra expression in ventrally projecting motor neurons, and isl and fra mutants have similar axon guidance defects. Single-cell labeling indicates that isl and fra are also required for dendrite targeting in a subset of motor neurons. Finally, overexpression of Fra rescues axon and dendrite targeting defects in isl mutants. These results indicate that Fra acts downstream of Isl in both the periphery and the CNS, demonstrating how a single regulatory relationship is used in multiple cellular compartments to coordinate neural circuit wiring.

Sema-1a Reverse Signaling Promotes Midline Crossing in Response to Secreted Semaphorins.

Commissural axons must cross the midline to form functional midline circuits. In the invertebrate nerve cord and vertebrate spinal cord, midline crossing is mediated in part by Netrin-dependent chemoattraction. Loss of crossing, however, is incomplete in mutants for Netrin or its receptor Frazzled/DCC, suggesting the existence of additional pathways. We identified the transmembrane Semaphorin, Sema-1a, as an important regulator of midline crossing in the Drosophila CNS. We show that in response to the secreted Semaphorins Sema-2a and Sema-2b, Sema-1a functions as a receptor to promote crossing independently of Netrin. In contrast to other examples of reverse signaling where Sema1a triggers repulsion through activation of Rho in response to Plexin binding, in commissural neurons Sema-1a acts independently of Plexins to inhibit Rho to promote attraction to the midline. These findings suggest that Sema-1a reverse signaling can elicit distinct axonal responses depending on differential engagement of distinct ligands and signaling effectors.

Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.