Explore the vast range of titles available.

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.

Eph receptor tyrosine kinases participate in a variety of normal and pathogenic processes during development and throughout adulthood. This versatility is likely facilitated by the ability of Eph receptors to signal through diverse cellular signalling pathways: primarily by controlling cytoskeletal dynamics, but also by regulating cellular growth, proliferation, and survival. Despite many proteins linked to these signalling pathways interacting with Eph receptors, the specific mechanisms behind such links and their coordination remain to be elucidated. In a proteomics screen for novel EPHB2 multi-effector proteins, we identified human MYC binding protein 2 (MYCBP2 or PAM or Phr1). MYCBP2 is a large signalling hub involved in diverse processes such as neuronal connectivity, synaptic growth, cell division, neuronal survival, and protein ubiquitination. Our biochemical experiments demonstrate that the formation of a complex containing EPHB2 and MYCBP2 is facilitated by FBXO45, a protein known to select substrates for MYCBP2 ubiquitin ligase activity. Formation of the MYCBP2-EPHB2 complex does not require EPHB2 tyrosine kinase activity and is destabilised by binding of ephrin-B ligands, suggesting that the MYCBP2-EPHB2 association is a prelude to EPHB2 signalling. Paradoxically, the loss of MYCBP2 results in increased ubiquitination of EPHB2 and a decrease of its protein levels suggesting that MYCBP2 stabilises EPHB2. Commensurate with this effect, our cellular experiments reveal that MYCBP2 is essential for efficient EPHB2 signalling responses in cell lines and primary neurons. Finally, our genetic studies in Caenorhabditis elegans provide in vivo evidence that the ephrin receptor VAB-1 displays genetic interactions with known MYCBP2 binding proteins. Together, our results align with the similarity of neurodevelopmental phenotypes caused by MYCBP2 and EPHB2 loss of function, and couple EPHB2 to a signalling effector that controls diverse cellular functions.

A recently developed Phox2a::Cre mouse line has been shown to capture anterolateral system (ALS) projection neurons. Here, we used this line to test whether Phox2a-positive cells represent a distinct subpopulation among lamina I ALS neurons. We show that virtually all lamina I Phox2a cells can be retrogradely labelled from injections targeted on the lateral parabrachial area (LPb), and that most of those in the cervical cord also belong to the spinothalamic tract. Phox2a cells accounted for ~ 50–60% of the lamina I cells retrogradely labelled from LPb or thalamus. Phox2a was preferentially associated with smaller ALS neurons, and with those showing relatively weak neurokinin 1 receptor expression. The Phox2a cells were also less likely to project to the ipsilateral LPb. Although most Phox2a cells phosphorylated extracellular signal-regulated kinases following noxious heat stimulation, ~ 20% did not, and these were significantly smaller than the activated cells. This suggests that those ALS neurons that respond selectively to skin cooling, which have small cell bodies, may be included among the Phox2a population. Previous studies have defined neurochemical populations among the ALS cells, based on expression of Tac1 or Gpr83. However, we found that the proportions of Phox2a cells that expressed these genes were similar to the proportions reported for all lamina I ALS neurons, suggesting that Phox2a is not differentially expressed among cells belonging to these populations. Finally, we used a mouse line that resulted in membrane labelling of the Phox2a cells and showed that they all possess dendritic spines, although at a relatively low density. However, the distribution of the postsynaptic protein Homer revealed that dendritic spines accounted for a minority of the excitatory synapses on these cells. Our results confirm that Phox2a-positive cells in lamina I are ALS neurons, but show that the Phox2a::Cre line preferentially captures specific types of ALS cells.

Peripheral and intraspinal feedback is required to shape and update the output of spinal networks that execute motor behavior. We report that lumbar dI2 spinal interneurons in chicks receive synaptic input from afferents and premotor neurons. These interneurons innervate contralateral premotor networks in the lumbar and brachial spinal cord, and their ascending projections innervate the cerebellum. These findings suggest that dI2 neurons function as interneurons in local lumbar circuits, are involved in lumbo-brachial coupling, and that part of them deliver peripheral and intraspinal feedback to the cerebellum. Silencing of dI2 neurons leads to destabilized stepping in posthatching day 8 hatchlings, with occasional collapses, variable step profiles, and a wide-base walking gait, suggesting that dI2 neurons may contribute to the stabilization of the bipedal gait.

  Overexpression experiments argue that only Isl1-Lhx8 hexamer is able to activate cholinergic features in the forebrain, while Isl1-Lhx3 can only induce motor-neuron-specific genes such as Hb9 and Isl2 in the spinal cord, arguing that the cellular context is critical for the function of the Isl1-LhxX hexamers. [...]another important general question is that of the maintenance of neuronal identity, including NtI.

The development of the chronic neuropathic pain state often originates at the level of peripheral sensory neurons, whose abnormal function elicits central sensitization and maladaptive plasticity in the nociceptive circuits of the spinal dorsal horn. These changes eventually reach supraspinal areas bringing about cognitive and affective co-morbidities of chronic pain such as anxiety and depression. This transmission presumably relies on the function of spinal projection neurons at the origin of the anterolateral system (AS). However, the identity of these neurons and the extent of their functional contribution remain unknown. Here, we asked these questions in the context of the mouse AS neurons that require the transcription factor Phox2a for their normal target connectivity and function in transmitting acute nociceptive information to the brain. To this end, we examined the effects of a spinal cord-specific loss of Phox2a (Phox2acKO) on the development of central sensitization evoked by the spared nerve injury (SNI) model of chronic pain. We found that SNI-treated Phox2acKO mice developed normal reflexive spinal responses such as mechanical allodynia evidenced by a decreased withdrawal threshold to von Frey filament stimulation and dynamic brush. On the other hand, Phox2acKO attenuated the development of cold but not mechanical hyperalgesia, in behavioral paradigms that require the relay of nociceptive information to the brain. Furthermore, Phox2acKO attenuated anxio-depressive-like behaviors evoked by SNI, measured by performance in the open field test and tail suspension test. Thus, Phox2a AS neurons play a critical role in the generation and maintenance of chronic neuropathic pain.

Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cellsurface cell-adhesion receptors. We previously showed that the mammalian orthologh of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protien coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion.

Myoblast fusion is tightly regulated during development and regeneration of muscle fibers. BAI3 is a receptor that orchestrates myoblast fusion via Elmo/Dock1 signaling, but the mechanisms regulating its activity remain elusive. Here we report that mice lacking BAI3 display small muscle fibers and inefficient muscle regeneration after cardiotoxin-induced injury. We describe two proteins that repress or activate BAI3 in muscle progenitors. We find that the secreted C1q-like1–4 proteins repress fusion by specifically interacting with BAI3. Using a proteomic approach, we identify Stabilin-2 as a protein that interacts with BAI3 and stimulates its fusion promoting activity. We demonstrate that Stabilin-2 activates the GPCR activity of BAI3. The resulting activated heterotrimeric G-proteins contribute to the initial recruitment of Elmo proteins to the membrane, which are then stabilized on BAI3 through a direct interaction. Collectively, our results demonstrate that the activity of BAI3 is spatiotemporally regulated by C1qL4 and Stabilin-2 during myoblast fusion. Myoblast fusion is an essential step in muscle growth and regeneration, and is regulated by the G-protein coupled receptor (GPCR) BAI3. Here Hamoud et al. show that the GPCR activity of BAI3 is spatiotemporally regulated during myoblast fusion, and identify C1qL4 and Stabilin-2 as, respectively, negative and positive regulators of its activity.

During neural circuit assembly, axonal growth cones are exposed to multiple guidance signals at trajectory choice points. While axonal responses to individual guidance cues have been extensively studied, less is known about responses to combination of signals and underlying molecular mechanisms. Here, we studied the convergence of signals directing trajectory selection of spinal motor axons entering the limb. We first demonstrate that Netrin-1 attracts and repels distinct motor axon populations, according to their expression of Netrin receptors. Quantitative in vitro assays demonstrate that motor axons synergistically integrate both attractive or repulsive Netrin-1 signals together with repulsive ephrin signals. Our investigations of the mechanism of ephrin-B2 and Netrin-1 integration demonstrate that the Netrin receptor Unc5c and the ephrin receptor EphB2 can form a complex in a ligand-dependent manner and that Netrin–ephrin synergistic growth cones responses involve the potentiation of Src family kinase signaling, a common effector of both pathways. The ability of animals to walk and perform skilled movements depends on particular groups of muscles contracting in a coordinated manner. Muscles are activated by nerve cells called motor neurons found in the spinal cord. The connections between the motor neurons and muscles are established in the developing embryo. Each motor neuron produces a long projection called an axon whose growth is guided towards the target muscle by signal proteins. The motor neurons are exposed to many such signal proteins at the same time and it is not clear how they integrate all this information so that their axons target the correct muscles. Poliak, Morales et al. used a variety of genetic and biochemical approaches to study the formation of motor neuron and muscle connections in the limbs of mice and chicks. The experiments show that a signal protein called Netrin-1 is produced in the limbs of developing embryos and attracts the axons of some types of motor neurons and repels others. This is due to the motor neurons producing different types of receptor proteins to detect Netrin-1. Further experiments show that individual axons can combine information from attractive or repulsive Netrin-1 signals together with repulsive signals from another family of proteins called ephrins in a 'synergistic' manner. That is, the combined effect of both cues is stronger than their individual effects added together. This synergy involves ligand-dependent interactions between the Netrin-1 and ephrin receptor proteins, and the activation of a common enzyme. Poliak, Morales et al.’s findings reveal a new role for Netrin-1 in guiding the development of motor neurons in the limb. Future work will focus on further understanding the mechanism of synergy between Netrin-1 and ephrins. Netrin-1 and ephrins are also involved in the formation of blood vessels and many other developmental processes, so understanding how they work together would have a wide-reaching impact on research into human health and disease.

Key Points Eph Tyr kinases and ephrins can simultaneously act as receptors and ligands, allowing unidirectional or bidirectional signalling, which in turn can lead to parallel and antiparallel signalling modes between two neighbouring cells (in trans configuration). Ephs and ephrins can also interact on the surface of the same cell (in cis ), and this attenuates Eph signalling, possibly by inhibiting the formation of Eph clusters. Eph-triggered cellular responses lead to cytoskeletal rearrangements, such as the collapse of the cytoskeleton, by controlling the balance between small GTPase activation and inactivation. Eph forward signalling often results in cell repulsion, whereas ephrin reverse signalling elicits either cell repulsion or adhesion. The formation of topographic maps, in which the positions of projection neurons relative to one another are maintained in the relative positions of the innervated targets, is mediated by ephrin–Eph signalling. Ephrin–Eph signalling regulates cell migration in neurons, allowing the cells to reach their final destination, guides axons along intermediate choice points to reach their final targets, and mediates synapse formation and plasticity. In blood and lymphatic vasculature, ephrin–Eph signalling establishes borders between different compartments and helps the remodelling process by regulating the effects of vascular growth factors. Ephrin–Eph signalling plays complex parts in several adult stem cell niches and can be both inhibitory and beneficial for central and peripheral nervous system regeneration, respectively. Ephrin–Eph signalling also has complex roles in cancer by controlling tissue architecture and cellular motility. These proteins are therefore important targets in therapies against various human diseases. Ephrin ligands and Eph receptor Tyr kinases are transmembrane proteins that elicit short-distance cell–cell signalling when they interact. As both Eph kinases and ephrins exist in various isoforms and function as receptors or ligands, this signalling evokes versatile responses, which regulate a plethora of morphogenetic and homeostatic processes. Eph receptor Tyr kinases and their membrane-tethered ligands, the ephrins, elicit short-distance cell–cell signalling and thus regulate many developmental processes at the interface between pattern formation and morphogenesis, including cell sorting and positioning, and the formation of segmented structures and ordered neural maps. Their roles extend into adulthood, when ephrin–Eph signalling regulates neuronal plasticity, homeostatic events and disease processes. Recently, new insights have been gained into the mechanisms of ephrin–Eph signalling in different cell types, and into the physiological importance of ephrin–Eph in different organs and in disease, raising questions for future research directions.