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.

The brain is the most complex organ in the body, with trillions of specific synapses whose formation depends on the precise targeting of axons and dendrites during nervous system development. Axons are guided to their appropriate targets by a number of conserved guidance cues and their receptors, which enable neurons to form specific connections and establish functional neural circuits. The axon guidance receptors that mediate axonal guidance and targeting are tightly regulated to achieve a controlled balance between attractive and repulsive signaling, and disruption of this process results in a number of movement disorders and other neurological deficits (Bosley et al., 2005; Depienne et al., 2011; Jen et al., 2004). Specifically, the Roundabout (Robo) family of repulsive axon guidance receptors has been implicated in many neurodevelopmental disorders like autism spectrum disorder, dyslexia, horizontal gaze palsy, and others (Anitha et al., 2008; Hannula-Jouppi et al., 2005; Jen et al., 2004; Suda et al., 2011). Elucidating the mechanisms by which these guidance receptors function is crucial for understanding the formation of neural circuits both during development and in disease pathogenesis.

The Drosophila midline is analogous to the vertebrate spinal cord and serves as an intermediate target for commissural axons that cross from one side of the body to the other (Klämbt et al., 1991; Seeger et al., 1993). The Drosophila ventral nerve cord has a ladder-like structure consisting of 13 repeated segments, each containing an anterior commissure and a posterior commissure into which commissural neurons extend their axons to cross the midline. Midline glial cells secrete a number of guidance cues that act on their cognate receptors present on axon growth cones to induce attraction toward or repulsion away from the midline. Slit is secreted by midline glia and acts as a repulsive ligand for the Robo family of receptors (Brose et al., 1999; Kidd et al., 1999; Kidd et al., 1998). There are three Robo receptors in Drosophila and four in vertebrates. The Robo receptors are transmembrane proteins with an ectodomain consisting of five immunoglobulin-like domains and three fibronectin repeats, and an intracellular domain containing short, highly conserved cytoplasmic (CC) motifs (Bashaw et al., 2000; Kidd et al., 1998). Robo1 induces repulsion in growth cones of navigating axons primarily by modulating the actin cytoskeletal network. Previous work has identified some downstream effectors for Robo1 including Ena, an uncapping protein for actin filaments (Bashaw et al., 2000), and Son of Sevenless (SOS), a GEF for Rac1 (Yang and Bashaw, 2006). However, downstream signaling of Robo1 is not completely understood, especially in relation to effectors that directly link Robo1 to the actin cytoskeleton and the nature of cytoskeletal changes orchestrated by Robo1. While it seems intuitive for repulsive signaling to induce depolymerization of the actin network, a recent study reports that dorsal root ganglion axons first extend actin-rich filopodia toward a source of Slit before retracting away from it (McConnell et al., 2016). This challenges the prevailing notion that repulsive signaling primarily relies on actin depolymerization and suggests that the actin rearrangements occurring downstream of Robo1 are more nuanced and complex than previously thought. Indeed, several of the well-known downstream effectors of Robo1 signaling, namely Ena and Rac1, are documented enhancers of actin polymerization (Barzik et al., 2005; Ridley et al., 1992).

The Scar or WAVE regulatory complex (WRC) is a heteropentameric complex consisting of five different proteins: Scar/WAVE, CYFIP/Sra1, Kette/Nap1, HSPC300/Brick1, and Abi (Eden et al., 2002). Scar or WAVE contains a VCA (verprolin homology, cofilin homology, acidic) region and serves as a nucleation-promoting factor for Arp2/3, thereby driving branched actin polymerization. While mammals have multiple orthologs of these proteins, Drosophila has single homologs of all five members of the complex, making it a simpler, more tractable model system for studying the WRC. The WRC has been previously implicated in axon guidance and targeting in Drosophila and Caenorhabditis elegans (Shakir et al., 2008; Stephan et al., 2011; Xu and Quinn, 2012); however, if and how it is recruited and activated downstream of guidance receptors is not known. Recent work identified a unique binding site for the WRC known as the WRC-interacting receptor sequence (WIRS) motif (Chen et al., 2014a). The WIRS motif is a short six amino acid peptide sequence characterized by a bulky hydrophobic residue at position 1 and a threonine or a serine at position 3, followed by a phenylalanine at position 4. The WIRS motif is present in a number of transmembrane proteins including Robo1 (Chen et al., 2014a). Robo1 has a WIRS motif between its CC0 and CC1 domains that is conserved across species, including humans. Previously, the WIRS motif has been shown to be important for recruitment of the WRC by neuroligins and SYG-1 in synapse formation (Chia et al., 2014; Xing et al., 2018) and for neogenin function in maintaining the stability of adherens junctions (Lee et al., 2016). To our knowledge, this study is the first to demonstrate a role for the WIRS-WRC interaction in axon guidance.

Here, we show that the WRC is required for Slit-Robo1 repulsive signaling at the Drosophila midline. We present evidence that Robo1 interacts with the WRC partially via its WIRS motif and that this interaction is enhanced in the presence of Slit. We show that the WIRS motif in Robo1 is important for its ability to induce ectopic repulsion in vivo. Using rescue assays, we show that Robo1 also requires its WIRS motif to mediate repulsion in ipsilateral axons in vivo. In addition, using CRISPR-Cas9-mediated mutagenesis, we show that the WIRS motif is important for endogenous Robo1 function as mutating the endogenous WIRS motif results in the complete loss of Robo1 repulsion at the midline. Finally, we use mouse dorsal spinal cord explants and growth cone collapse assays in mouse commissural neurons, together with gain-of-function experiments in chick embryos, to demonstrate that the WIRS motif is also important for vertebrate Robo1 repulsive signaling. We propose a model in which Slit binding induces recruitment of the WRC to the WIRS motif of Robo1 where it functions in Robo1-mediated repulsion at the midline.

In this article, we have documented a conserved role for the WRC in Slit-mediated Robo1 repulsive signaling. Using the developing Drosophila embryonic CNS, we demonstrate a series of dose-dependent genetic interactions between components of the WRC and Slit-Robo1 signaling, which show that the WRC functions in vivo to regulate Robo1 repulsive signaling at the midline. Biochemical experiments in cultured cells show that Robo1 can bind to the WRC partially via its WIRS motif and that Slit stimulation can induce recruitment of the WRC to Robo1. Further, we present several lines of evidence to demonstrate that the WIRS motif is important for Robo1 function in vivo. First, mutating the WIRS motif results in a significantly weaker gain-of-function phenotype when Robo1 is misexpressed in commissural axons. Second, the Robo1 variant with mutations in its WIRS motif fails to rescue the robo1 mutant phenotype as effectively as wild-type Robo1. Finally, mutating the WIRS motif in the endogenous robo1 locus using the CRISPR-Cas9 system results in embryos with severe ectopic crossing defects that phenocopy robo1 mutants. These data demonstrate a severe decline in Robo1 function upon disruption of the WRC binding site. Together, our observations support the model that Slit stimulation results in recruitment of the WRC to the WIRS motif in Robo1, which is vital to Robo1-mediated repulsive signaling at the midline (Figure 9). Further, using genetic and biochemical approaches, we show that the Arp2/3 complex functions in the Slit-Robo signaling pathway and undergoes a WIRS-dependent recruitment to Robo1 in response to Slit. We propose that downstream of Robo1 the WRC functions to recruit the Arp2/3 complex to initiate localized cytoskeletal remodeling. We also present several lines of evidence that support an evolutionarily conserved role for the WIRS motif in vertebrate Robo1 signaling. First, we show that Robo1∆WIRS is less effective at mediating repulsion in response to Slit in explants from the mouse dorsal spinal cord. In addition, Robo1∆WIRS is less responsive to the collapsing activity of Slit in dissociated spinal commissural axons. Finally, we show that mutating the WIRS motif in human Robo1 results in a reduced ability to induce ectopic repulsion in embryonic chicken commissural axons as compared to wild-type human Robo1. These data highlight a vital conserved role for the WIRS motif in Robo1 function.

Figure 9

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In this study, we used a series of complementary approaches to evaluate the importance of the WIRS motif for Robo1 repulsive signaling. While it is generally assumed that the high expression levels resulting from the Gal4/UAS system are unlikely to reflect normal spatial and temporal regulation, it remains unclear to what extent this might confound comparisons between different mutant variants of a given protein. Our results with Robo1 indicate that Gal4-UAS/directed expression significantly hinders the detection of critical structural elements of the receptor. For example, when using a pan-neuronal driver to reintroduce Robo1 into the robo1 mutant embryos, we observe only very modest differences between the wild-type and WIRS mutant forms of overexpressed Robo1. In contrast, we see much more severe phenotypes when the WIRS motif is disrupted under conditions that more closely match the endogenous robo1 levels using the robo1 genomic rescue constructs or when mutating the WIRS motif in the endogenous robo1 locus using the CRISPR-Cas9 system. These direct comparisons between different assays to measure protein function suggest that rescue experiments using the Gal4/UAS system must be interpreted with caution. This also suggests that our results comparing the gain-of-function effects of Robo1 and Robo1∆WIRS in vertebrate systems are likely to underestimate the significance of the WIRS motif and recruitment of the WRC for Robo1 repulsion.

Here, we have shown that the WRC is an important component of the Slit-Robo1 repulsive pathway at the midline. But what happens once the WRC is recruited to Robo1? The VCA region of Scar/Wave is sequestered within the complex until activation, which triggers a conformational change releasing the VCA domain (Chen et al., 2010; Ismail et al., 2009). Rac1 is an important activator of the WRC and has been previously found to be activated downstream of Robo1 in both Drosophila and mouse (Fan et al., 2003; Wong et al., 2001). The genetic interaction between sos and cyfip suggests that these proteins function cooperatively to regulate midline repulsion. We propose a model where the WRC is recruited to the WIRS motif in response to Slit where it is activated by increased local Rac1 signaling. Active WRC can then promote Arp2/3-meditated actin assembly. Such a direct interaction with the WRC via the WIRS motif would allow for localized WRC activity at desired subdomains to achieve tighter spatiotemporal control and support directional actin changes. At first glance, the initiation of actin polymerization downstream of Robo1 might seem paradoxical; however, many repulsive guidance cues recruit downstream effectors that enhance actin polymerization. One recent study demonstrates that dorsal root ganglion neurons initially extend filopodia toward a source of Slit before retracting (McConnell et al., 2016). The McConnell study highlights the nuanced and complex actin rearrangements that occur downstream of guidance cues, which potentially contribute to sensing of the environment for improved resolution of a guidance gradient. The WRC might play an important role in the generation of these Slit-induced filopodia by initiating the formation of branched actin filaments that are subsequently rebundled to form filopodia as suggested by the convergent elongation model that supports a role for Arp2/3 in filopodia formation in neurons (Yang and Svitkina, 2011). Ena/VASP proteins, downstream effectors of Robo1, are important for these Slit-induced filopodial extensions (McConnell et al., 2016) and on account of their actin bundling activity are perfectly poised to orchestrate this actin reorganization in order to drive filopodia formation. The WRC has also been shown to be important for receptor endocytosis (Basquin et al., 2015; Xu et al., 2016), and another possible outcome of initiating localized actin polymerization is the endocytosis and recycling of Robo1. Indeed, previous work has demonstrated that endocytosis of Drosophila Robo1 upon Slit stimulation is essential for Robo1 repulsive signaling (Chance and Bashaw, 2015) and that vertebrate Robo1 also undergoes endocytosis and recycling following Slit stimulation (Kinoshita-Kawada et al., 2019), suggesting a conservation of this regulatory mechanism. However, our data suggests that mutating the WIRS motif has no detectable effect on the surface localization of Robo1 either in Drosophila or in cultured mouse commissural neurons. Future studies are needed to decipher the exact nature and function of the local actin remodeling induced by the Rac-WRC-Arp2/3 complex signaling axis downstream of Robo1. Additionally, other known downstream effectors of Robo1 like Ena and Abl have also been shown to influence WRC activity (Chen et al., 2014b; Leng et al., 2005). It would thus be interesting to dissect how this inter-regulation between these different components of Robo1 signaling contributes to fine-tuning of WRC activity to generate a specific output for Slit-Robo1 repulsion.

Previously, the WIRS motif has been shown to be important in Neuroligins and Syg-1 for proper synapse formation (Chia et al., 2014; Xing et al., 2018) as well as in Neogenin for the maintenance of adherens junctions (Lee et al., 2016). In these contexts, it is apparent that the WRC reinforces the F-actin network at these membrane junctions. However, it is unclear if the WIRS-WRC interactions are subject to regulation by the respective ligands or if the WRC performs more of a scaffolding function. In the context of axon guidance, our work demonstrates a ligand-dependent recruitment of the WRC to the WIRS domain of Robo1 suggestive of both spatial and temporal specificity of WRC activation. To our knowledge, this study is the first to demonstrate that the WRC can be recruited to a guidance receptor in response to a ligand.

In addition to functioning as part of the actin-regulating complex, WRC members can have functions independent of the complex as well. For example, CYFIP proteins can interact with fragile X mental retardation protein (FMRP) to regulate mRNA localization and protein translation (Abekhoukh and Bardoni, 2014; Schenck et al., 2003; Schenck et al., 2001) and Abi can interact with WASP and Diaphanous to regulate F-actin (Bogdan et al., 2005; Ryu et al., 2009). While we cannot entirely rule out a role for WRC-independent functions of these proteins in Robo1 signaling, several lines of evidence point to the involvement of the WRC as a whole downstream of Robo1. First, two separate WRC members, cyfip and hscp300, show genetic interactions with the Slit-Robo pathway. Second, the physical interaction between Robo1 and HSPC300 is dependent on the WIRS motif, which requires a binding interface formed by CYFIP and Abi that comes together only in the fully assembled WRC (Chen et al., 2014a). Additionally, the strong midline crossing phenotypes we see upon manipulating the WIRS motif suggests that it is indeed this interaction with the fully assembled WRC that is important for Robo1 signaling in vivo. Finally, we tested whether the Drosophila homolog of FMRP, dfmr1, genetically interacts with the Slit-Robo1 pathway. In contrast to cyfip and hspc300, completely removing dfmr1 has no effect on the slit, robo transheterozygous phenotype (Figure 6—figure supplement 1J). This data further supports a WRC-dependent function for cyfip in Slit-Robo signaling and suggests that CYFIP/dFMR interactions are not important for Robo repulsion.

Drosophila Robo1 has numerous functions in development outside of its role in midline repulsion, and the robo1ΔWIRS CRISPR mutants generated here also provide an opportunity to discern which developmental functions of Robo1 require the WRC in future studies. Robo1 regulates the migration of chordotonal sensory neurons (Kraut and Zinn, 2004) and mesodermal migration for muscle patterning (Kramer et al., 2001). Embryos lacking robo1 show defects in heart lumen formation (Qian et al., 2005) and tracheal migration (Englund et al., 2002). In mammals, Robo1 also plays important roles outside of the nervous system, including the formation of blood vessels (Rama et al., 2015) and organs like the heart (Mommersteeg et al., 2013) and the mammary glands (Macias et al., 2011), and it can also regulate stem cell proliferation (Ballard et al., 2015). Finally, the Slit-Robo pathway has been shown to regulate tumor angiogenesis along with tumor cell migration and metastasis (Tong et al., 2019). Mis-regulation of Slit-Robo signaling has been implicated in multiple types of tumorigenesis, making it a promising target for cancer treatments (Koohini et al., 2019). Such therapeutic avenues require a comprehensive understanding of Slit-Robo signaling in specific cancers, highlighting the importance of investigating the WRC as a downstream effector of Robo in disease contexts as well.

In addition to Robo1, other Robo receptors also contain WIRS motifs in their cytoplasmic domains. Drosophila Robo2 plays a minor role in midline repulsion and, together with Robo3, also regulates lateral positioning of the longitudinal fascicles (Evans and Bashaw, 2010; Rajagopalan et al., 2000). As Robo2 and Robo3 do not contain CC2 and CC3 domains, to which most of the known Robo1 effectors bind, very little is known about their downstream signaling. Vertebrate Robo3 can induce repulsive signaling in response to a recently identified ligand, NELL2 (Jaworski et al., 2015) Vertebrate Robo3 also contains a WIRS motif in its cytoplasmic domain, raising the possibility that these Robo receptors could share a common cellular mechanism for repulsion, despite responding to distinct ligands. Additionally, attractive axon guidance receptors like Fra and its vertebrate ortholog DCC also contain WIRS motifs in their cytoplasmic domains. Unsurprisingly, many core actin-modifying proteins that act downstream of repulsive cues like Ena/VASP and Abl kinase also function in attractive signaling (Forsthoefel et al., 2005; Gitai et al., 2003). We can speculate that the WRC might also function in both repulsion and attraction by regulating different actin-based processes like membrane trafficking versus growth cone advancement. Alternatively, as other studies have shown the importance of an initial growth cone extension toward repulsive cues, it is likely that tight spatiotemporal activation along with regulation by other effector molecules can result in fine-tuning of WRC activity to contribute to distinct cytoskeletal outputs downstream of different guidance receptors.

All data generated or analysed during this study are included in the manuscript and supporting files. Confocal stacks were collected using a spinning disk confocal system (Perkin Elmer) built on a Nikon Ti-U inverted microscope with Volocity imaging software. Images were processed using NIH ImageJ and Adobe Photoshop software. All statistics and graphs were generated using GraphPad Prism 9.

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Author details

1. Karina Chaudhari

   Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3533-3027

2. Madhavi Gorla

   Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Chao Chang

   1. Institut de recherches cliniques de Montréal (IRCM), Montréal, Canada
   2. Department of Anatomy and Cell Biology and Division of Experimental Medicine, McGill University, Montréal, Canada

   Contribution

   Investigation, Methodology

   Competing interests

   No competing interests declared

4. Artur Kania

   1. Institut de recherches cliniques de Montréal (IRCM), Montréal, Canada
   2. Department of Anatomy and Cell Biology and Division of Experimental Medicine, McGill University, Montréal, Canada

   Contribution

   Supervision, Funding acquisition, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5209-2520

5. Greg J Bashaw

   Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   gbashaw@pennmedicine.upenn.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6146-0962

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