Proteolytically released Lasso/teneurin-2 induces axonal attraction by interacting with latrophilin-1 on axonal growth cones

A presynaptic adhesion G-protein-coupled receptor, latrophilin-1, and a postsynaptic transmembrane protein, Lasso/teneurin-2, are implicated in trans-synaptic interaction that contributes to synapse formation. Surprisingly, during neuronal development, a substantial proportion of Lasso is released into the intercellular space by regulated proteolysis, potentially precluding its function in synaptogenesis. We found that released Lasso binds to cell-surface latrophilin-1 on axonal growth cones. Using microfluidic devices to create stable gradients of soluble Lasso, we show that it induces axonal attraction, without increasing neurite outgrowth. Using latrophilin-1 knockout in mice, we demonstrate that latrophilin-1 is required for this effect. After binding latrophilin-1, Lasso causes downstream signaling, which leads to an increase in cytosolic calcium and enhanced exocytosis, processes that are known to mediate growth cone steering. These findings reveal a novel mechanism of axonal pathfinding, whereby latrophilin-1 and Lasso mediate both short-range interaction that supports synaptogenesis, and long-range signaling that induces axonal attraction.


Introduction
Correct wiring of the nervous system critically depends on both long-range diffusible cues and shortrange contact-mediated factors which can be attractive or repulsive (Chen and Cheng, 2009). However, the relatively small repertoire of key molecules known to be involved in axon guidance or trans-synaptic adhesion cannot fully explain the complexity and specificity of synaptic connections. Indeed, new interacting partners and signal-modulating ligands are now being found for many well-established guidance factors (Karaulanov et al., 2009;Leyva-Díaz et al., 2014;Sö llner and Wright, 2009). Furthermore, our novel findings demonstrate that at least one receptor pair can both mediate cell contacts and, unexpectedly, also act as a long-range signaling factor and its receptor.
The second member of this receptor pair, Lasso, is a representative of teneurins (TENs), large single-pass transmembrane proteins (Baumgartner et al., 1994;Levine et al., 1994). Lasso is the splice variant of TEN2 (TEN2-SS) ( Figure 1A) that specifically binds LPHN1 in cell adhesion experiments (Li et al., 2018). Given also that only Lasso is isolated by affinity chromatography on LPHN1 (Silva et al., 2011), we will refer here to TEN2 that is able to bind LPHN1 as Lasso. All TENs possess a large C-terminal extracellular domain (ECD) containing a series of epidermal growth factor (EGF)like repeats and other repeat domains ( Figure 1A). Inter-chain disulfide bridges mediate TEN homodimerization ( Figure 1B, left) (Feng et al., 2002;Vysokov et al., 2016). Similar to Notch, during the intracellular processing of TENs, their ECDs are constitutively cleaved by furin at site 1 ( Figure 1A,B, eLife digest The brain is a complex mesh of interconnected neurons, with each cell making tens, hundreds, or even thousands of connections. These links can stretch over long distances, and establishing them correctly during development is essential. Developing neurons send out long and thin structures, called axons, to reach distant cells. To guide these growing axons, neurons release molecules that work as traffic signals: some attract axons whilst others repel them, helping the burgeoning structures to twist and turn along their travel paths.
When an axon reaches its target cell, the two cells join to each other by forming a structure called a synapse. To make the connection, surface proteins on the axon latch onto matching proteins on the target cell, zipping up the synapse. There are many different types of synapses in the brain, but we only know a few of the surface molecules involved in their creation -not enough to explain synaptic variety.
Two of these surface proteins are latrophilin-1, which is produced by the growing axon, and Lasso, which sits on the membrane of the target cell. The two proteins interact strongly, anchoring the axon to the target cell and allowing the synapse to form. However, a previous recent discovery by Vysokov et al. has revealed that an enzyme can also cut Lasso from the membrane of the target cell. The 'free' protein can still interact with latrophilin-1, but as it is shed by the target cell, it can no longer serve as an anchor for the synapse. Could it be that free Lasso acts as a traffic signal instead?
Here, Vysokov et al. tried to answer this by growing neurons from a part of the brain called the hippocampus in a special labyrinth dish. When free Lasso was gradually introduced in the culture through microscopic channels, it interacted with latrophilin-1 on the surface of the axons. This triggered internal changes that led the axons to add more membrane where they had sensed Lasso, making them grow towards the source of the signal.
The results demonstrate that a target cell can both carry and release Lasso, using this duplicitous protein to help attract growing axons as well as anchor them. The work by Vysokov et al. contributes to our knowledge of how neurons normally connect, which could shed light on how this process can go wrong. This may be relevant to understand conditions such as schizophrenia and ADHD, where patients' brains often show incorrect wiring.  Tucker and Chiquet-Ehrismann, 2006;Vysokov et al., 2016). However, the cleaved ECD remains tightly tethered to the cell surface due to its strong interaction with the transmembrane fragment ( Figure 1B, middle) (Vysokov et al., 2016).
However, our surprising finding (Vysokov et al., 2016) that Lasso/TEN2 is partially released from the cell surface by regulated proteolysis (at site 3; Figure 1B, right) was inconsistent with a solely cell-surface function of Lasso. On the other hand, we found that the released Lasso fragment retained its ability to bind cell-surface LPHN1 with high affinity and induce intracellular signaling (Silva et al., 2011;Vysokov et al., 2016). Thus, it was possible that the released, soluble ECD of Lasso/TEN2 could act as a diffusible (attractive or repulsive) factor and mediate some of the TEN2 functions in neurite pathfinding described above. Therefore, we hypothesized that the binding of soluble Lasso to LPHN1 on distant neurites could trigger important changes in their growth.
Here, we test this hypothesis using cultured hippocampal neurons. First, we show that developing neurons release a substantial proportion of Lasso ECD into the medium, while LPHN1 is concentrated on the leading edge of axonal growth cones. We then use microfluidic chambers to culture. Rat hippocampal neurons were cultured for 3, 7 and 14 days, and proportionate amounts of the conditioned media and cell lysates were separated by SDS-PAGE. A Western blot (representative of three independent experiments, which all gave similar results) was stained for Lasso, LPHN1, neurofilament-H (NF-H), and actinin. The doublet bands corresponding to splice variants of full-size Lasso (FS) and the fragment of ECD (Frag.) cleaved at site 1 are indicated by arrowheads. (D) Quantification of Western blots (as in C), using Lasso C-terminus staining data. (E) Axonal growth cones (white arrowheads) do not express Lasso/teneurin-2. Neurons in a 9 DIV hippocampal culture were permeabilized and stained for the axonal protein Tau (green) and Lasso (TN2C, red) (representative image from n = 5 experiments). (F) A detailed study of growth cones. Hippocampal neurons were transfected with a vector encoding GFP, then, after 14 DIV, stained for LPHN1 (PAL1 and Alexa 647-conjugated secondary antibody, magenta), and axonal growth cones were visualized by GFP fluorescence (green). (G, H) Correlation of LPHN1 polarization within a growth cone with its recent travel trajectory. G left, a fluorescent image of a growth cone stained for LPHN1 (magenta). G right, the same image in false color (contour based on GFP staining), demonstrating LPHN1 polarization on the right side. H left, the contours of 13 roughly symmetrical growth cones and their preceding axons were aligned to locate the stronger LPHN1 staining on the right. Note, that all axons approach growth cones from the right low quadrant. H right, the proportion of right-and left-turning growth cones plotted with Jeffreys 99.73% confidence intervals for a binomial parameter; ***, p<0.001; n = 13. (I). LPHN1 is found within filopodia and lamellipodia on the leading edge (left, arrowheads), but not on the trailing edge (right) of a growth cone. Green, GFP fluorescence; magenta, PAL1 staining for LPHN1. DOI: https://doi.org/10.7554/eLife.37935.003 The following source data and figure supplements are available for figure 1: Source data 1. Source data for  demonstrate that a spatio-temporal gradient of soluble Lasso attracts neuronal axons, but not dendrites, and that this process involves LPHN1 that is present on axonal growth cones. Using model cells expressing functional LPHN1, and mouse neuromuscular preparations, we also show that LPHN1 activation by soluble Lasso causes intracellular Ca 2+ signaling, which leads to increased exocytosis. This suggests a plausible cellular mechanism causing axons to turn in the direction of a gradient of soluble Lasso. Moreover, the LPHN1-Lasso pair illustrates a novel principle of chemical guidance whereby cell-surface receptors engage not only in short-range interactions, but also in long-range signaling, which can further contribute to the formation of complex neuronal networks.

Neurons partially cleave and release lasso
We previously showed in model cell lines and in adult brain that Lasso is cleaved at several sites (sites 1, 2, three in Figure 1A,B) and is released into the extracellular environment in a regulated manner (Vysokov et al., 2016). To test whether Lasso undergoes the same processing and release during neuronal development, we followed Lasso expression at different stages of neuron maturation in hippocampal cell cultures (Kaech and Banker, 2006). Soon after plating, embryonic (E18) rat hippocampal neurons produced Lasso, which was detectable at 3 days in vitro (DIV) ( Figure 1C,D). A large proportion of Lasso (~90%) was constitutively cleaved at site 1 during neuronal development in vitro ( Figure 1-figure supplement 1A). Increasing amounts of cleaved fragment also appeared in the medium at 7 and 14 DIV ( Figure 1D and Figure 1-figure supplement 1A, green), indicating a slow cleavage at site 3. Thus, Lasso is fully cleaved at site 1 and partially released by regulated cleavage at site 3 not only in transfected immortalized cells, but also in developing neurons and in the postnatal rat brain (Vysokov et al., 2016).
We also examined the neuronal structures that could release soluble Lasso ECD. We found that large amounts of Lasso were present on dendrites and dendritic growth cones (Figure 1-figure supplement 1B), while it was practically absent from axons and axonal growth cones ( Figure 1E). Since about 80% of Lasso was not normally released ( Figure 1D, Figure 1-figure supplement 1A), these data suggested that the compartments rich in Lasso, that is dendrites and dendritic growth cones, were the main source of the soluble Lasso fragment.

LPHN1 is expressed on growth cones of developing neurons
As early as 3 DIV, the developing neurons also expressed LPHN1, the high-affinity receptor for soluble Lasso ECD, and the amounts of LPHN1 continued to increase through all time points ( Figure 1B), in parallel with the increasing amounts of soluble Lasso (Figure 1-figure supplement 1A). This correlation between the soluble Lasso and cell-surface LPHN1 further supported the idea of their likely interaction during neuronal development.
Interestingly, in developing hippocampal neurons, LPHN1 was found concentrated in axons and especially in axonal growth cones, where it co-localized with synapsin ( Figure 1-figure supplement 1C, D, arrowheads). LPHN1 was also enriched in axonal varicosities, which were identified as en passant synapses by immunostaining for PSD-95 ( Figure 1-figure supplement 1D, asterisks).
We then studied the expression of LPHN1 in growth cones in more detail by transfecting hippocampal neurons with GFP, which greatly simplified the identification and tracking of axons and axonal growth cones. All GFP-labeled axonal growth cones showed a clear enrichment of endogenous LPHN1 ( Figure 1F,G,I). Conversely, when LPHN1 expression was knocked down by shRNA (delivered together with GFP in the same bicistronic vector), it clearly disappeared from the growth cones of transfected neurons, while the growth cones of non-transfected cells were not affected ( Figure 1figure supplement 1E, arrow and arrowhead, respectively).
We also discovered that endogenous LPHN1 expression within axonal growth cones was polarized in relation to the cone's symmetry axis, such that one side of each growth cone contained on average 1.88 ± 0.22 fold more LPHN1 than the other ( Figure 1G,H). To assess whether this LPHN1 enrichment correlated with the direction of axonal growth, we traced the growth trajectories of a number of symmetrical growth cones and compared these with the distribution of LPHN1. This analysis clearly demonstrated that LPHN1 polarization within the growth cones very strongly positively correlated with the direction of their turning ( Figure 1G,H). Moreover, in non-symmetrical growth cones, which had clearly started turning prior to fixation, LPHN1 expression had a bimodal distribution, being enriched not only near the 'neck' of a turning cone, but also close to its leading edge ( Figure 1-figure supplement 1F, G). Such leading-edge enrichment also extended into fine growth cone protrusions. Thus, filopodia and lamellipodia located on the leading edge of a growth cone ( Figure 1I, left, arrowheads) showed a much higher amount of LPHN1 than the processes on the trailing edge of the growth cone ( Figure 1I, right).
We concluded that LPHN1 expression within growth cones correlated positively with the global directionality of growth and with the fine structures that underpin the growth cone's extension.
Soluble Lasso binds to cell-surface LPHN1 Next, we tested the interaction between soluble Lasso and cell-surface LPHN1. For these tests we expressed a shorter, constitutively secreted construct, Lasso-D (Figure 2A, right) in HEK293A cells and affinity-purified it ( Figure 2B). 100 nM Lasso-D was incubated with neuroblastoma cells stably expressing (i) LPHN1, (ii) a chimeric construct LPH-82 containing ECD from EMR-2 used as a negative control, (iii) Lasso-A, or (iv) Lasso-FS (Figure 2A, left). As expected, Lasso-D did not interact with LPH-82 ( Figure 2C, panel 4). The lack of Lasso-D binding to Lasso-A and released fragment of Lasso-A binding to Lasso-FS ( Figure 2D, panels 2, 3; Figure 2-figure supplement 1B) was somewhat surprising, since homophilic interactions between membrane-bound and soluble TENs were reported previously (Bagutti et al., 2003;Beckmann et al., 2013;Hong et al., 2012;Rubin et al., 2002;Boucard et al., 2014), but this could be due to a relatively low affinity of Lasso-Lasso interaction and relatively long washes employed in our protocol. On the other hand, and consistent with previous reports of high affinity between LPH1 and Lasso (Silva et al., 2011;Boucard et al., 2014), Lasso-D and the released fragment of Lasso-A bound strongly to cells expressing LPHN1 ( Figure 2C, panels 2, three and To verify that the soluble ECD of Lasso, when proteolytically released from the cell-surface as depicted in Figure 2A (Lasso-A), could diffuse between individual cells and bind LPHN1 on distant cells, we co-cultured neuroblastoma cells stably expressing Lasso-A with cells stably expressing LPHN1. When co-cultured at high density, these cells formed clusters, held together by LPHN1/Lasso-A intercellular adhesion complexes ( Figure 2E, panel 1). In more sparsely plated co-cultures, the Lasso-A fragment was released into the medium, where it diffused and bound to cells expressing LPHN1, but not to the wild type (WT) neuroblastoma cells ( Figure 2E, panel 2, and Figure 2-figure supplement 1C). Interestingly, after binding Lasso, the LPHN1 staining appeared to concentrate in large patches, a pattern very different from LPHN1 distribution in control conditions ( Figure 2C, panel 1) (see also below). These experiments suggest that (i) when Lasso is released into the medium as a result of its regulated cleavage, it retains its affinity for LPHN1 and (ii) on reaching distant LPHN1-expressing cells by diffusion, Lasso causes LPHN1 redistribution on the cell surface.
We then asked whether the soluble Lasso ECD could similarly bind to LPHN1 in neurons and, more specifically, on axonal growth cones. To control for the specificity of Lasso binding to LPHN1, this experiment was carried out on cultured hippocampal neurons from LPHN1 WT (Adgrl1 +/+ ) and LPHN1 KO (Adgrl1 -/-) newborn mice (P0). Also, to unequivocally distinguish between the soluble and cell-surface Lasso, we used exogenous Lasso-D, which was detected using anti-FLAG antibody. As expected, in WT mouse neurons, LPHN1 was found mostly in axonal growth cones (arrowheads) and varicosities (asterisks) (

MAIDs as a tool to study axonal responses to chemoattractant gradients
Based on the data above, we hypothesized that the interaction of released Lasso ECD with LPHN1 on axonal growth cones could represent one of the mechanisms that underlie the previously formulated, but so far unexplained, role of TENs in axonal pathfinding and brain patterning (Antinucci et al., 2013;Hor et al., 2015;Leamey et al., 2007;Young et al., 2013). To study this effect, we developed a new method of long-term exposure of hippocampal axons to stable gradients of Lasso using 'microfluidic axon isolation devices' (MAIDs) ( Figure 3A). The advantage of this method over conventional ligand-puffing was that the MAIDs enabled exposure of axons to long-term stable gradients of Lasso, which was critical for our assay. The device used here had two compartments, each consisting of two cylindrical wells connected by a 'corridor'; a 150 mm-thick wall that separated the two corridors had multiple parallel microchannels (2-3 mm tall and 10 mm wide) connecting the two compartments ( Figure 3A, middle). When neurons are plated in one of the compartments (designated as the Somal Compartment), their neurites grow in all directions, but only the axons (identified by NF-H staining) readily penetrate the microchannels and cross into the empty, Axonal Compartment ( Figure 3A, right; 3B, C). While there is a large number of dendrites in the Somal Compartment (identified by microtubule-associated protein 2, MAP-2, staining), only a few of them enter the Axonal Compartment and then terminate close to the wall ( Figure 3B,C).
From the previously described physical characteristic of microfluidic chambers (Zicha et al., 1991), we predicted that a concentration gradient across the microchannels in our devices could be established over time. This was modelled by adding TRITC-conjugated BSA to one compartment and visualizing the dye in the microchannels ( Figure 3D). We found that a gradient was formed within the first 24 hr and remained stable over several days ( Figure 3D,E).
To test the functionality of the MAIDs for studying axonal guidance, we employed brain-derived neurotrophic factor (BDNF) known to act as an axonal chemoattractant (Li et al., 2005). Rat hippocampal neurons were plated into the Somal Compartment, and at 3 DIV, when axons normally start entering microchannels, BDNF was added to the Axonal Compartment (PBS was added to control cultures) ( Figure 3F). After a further 5 DIV, we observed a 2.2-fold higher number of axons crossing into the Axonal Compartment in the presence of BDNF compared with the control ( Figure 3G,H). This effect was statistically significant ( Figure 3H). This proof-of-concept experiment confirmed that MAIDs could be used to study the long-term effects of chemoattractant gradients on axonal migration.

A gradient of soluble lasso induces axonal attraction
We then used this methodology to study the reaction of LPHN1-expressing neuronal growth cones to a gradient of soluble released Lasso. Lasso-D was added to the Axonal Compartment ( Figure 4A), and the integrity of Lasso during the experiment was verified by Western blotting ( Figure 4B). Quantification of axons in Axonal Compartments by NF-H immunofluorescence ( Figure 4C,D) revealed a statistically significant 1.5-fold increase in axonal growth induced by Lasso-D. Thus, soluble Lasso-D clearly functioned as an attractant of axonal elongation and/or steering.
Since LPHN1 is present on axonal growth cones (  Lasso-A or Lasso-FS (D) or mutant LPH-82 (C, panel 4) are able to interact with Lasso-D or Lasso-A. E, panel 1. Short-term, high-density incubation of cells expressing LPHN1 and membrane-anchored Lasso-A allows these proteins to form inter-cellular contacts. E, panel 2. After a 48 hr co-culture, a sufficient amount of Lasso-A is released into the medium, diffuses away from Lasso-A expressing cells (arrowhead) and can be detected interacting with distant LPHN1-expressing cells (arrow). Images are representative of n = 6-7 independent experiments. DOI: https://doi.org/10.7554/eLife.37935.007 The following source data and figure supplements are available for figure 2: total amounts of neurites and cells in both compartments were quantified using the lipophilic membrane tracer DiO (see Materials and methods for details). The results clearly demonstrated that the neurites from LPHN1-expressing (WT) hippocampal neurons crossed into the Lasso D-containing Axonal Compartment 5.5-fold more readily than the neurites from neurons lacking this receptor ( Figure 4E, left). Importantly, this effect was not due to a lower viability of LPHN1 KO neurons, because there was no difference between the KO and WT cells within the Somal Compartment ( Figure 4E, right).  We also studied the behavior of axons in response to a spatio-temporal Lasso gradient in the corridor of the Axonal Compartment, by exposing axons to an increasing concentration of the attractant during the whole growth process. In order to achieve a stable increase in protein concentration over time, we seeded HEK293A cells stably expressing soluble Lasso-D (untransfected HEK293A cells were used in control) into the wells of the Axonal Compartment ( Figure 5A). The presence of secreted Lasso-D within the Axonal Compartments was verified at the end of each experiment ( Figure 5B), and the distribution of axons was quantified by NF-H immunofluorescence ( Figure 5C, D). In this experiment, we observed not only a significantly greater number of axons being attracted, but also axons growing deeper into the corridors of the Axonal Compartments ( Figure 5D). On the other hand, quantification of MAP-2 immunofluorescence demonstrated that released Lasso-D did not attract dendrites; in fact, there was a slight repulsive effect ( Figure 5E). Taken together, these experiments indicate that a gradient of the soluble Lasso fragment specifically induces axonal attraction.
Soluble Lasso fragment also induced strong axonal fasciculation (e.g. Figures 4C and 5C). This effect was quantified by measuring the width of axonal bundles at 100 mm from the separating wall, where axons grew mostly away from the wall rather than along it. Based on the average width of a single axon (1 mm), an average bundle contained 2-3 axons in control conditions, but more than five axons in the presence of 1.5 nM Lasso-D ( Figure 5F). Thus, Lasso fragment can induce axonal fasciculation in a concentration-dependent manner.
In order to rule out the possibility that the observed effects of the released Lasso fragment were due to a general positive trophic effect (e.g. an increase in axonal elongation speed), Lasso-D was added directly to cultures of hippocampal neurons. To visualize axons, neurons were transfected with GFP prior to plating and allowed to grow for 4 DIV, after which the longest neurites of GFPpositive neurons were traced and measured. We did not detect any increase in the length of neurites when neurons were exposed to Lasso-D ( Figure 5G,H).
Taken together, these data demonstrate unequivocally that a gradient of the soluble fragment of Lasso acts as an axonal attraction cue without affecting their overall growth.

The mechanism of axonal attraction by lasso
To determine the downstream effects of the interaction between soluble Lasso ECD and LPHN1, we used neuroblastoma cells stably expressing LPHN1. It was reported previously that the signaling machinery downstream of LPHN1 in these cells is similar to that in neurons (Silva et al., 2009;Volynski et al., 2004). When the LPHN1-expressing neuroblastoma cells are stimulated by the known LPHN1 ligand and potent secretagogue LTX N4C , the N-terminal and C-terminal fragments (NTF and CTF) of LPHN1 undergo rearrangement (as illustrated in Figure 6A, middle). In turn, this induces intracellular Ca 2+ signaling which involves the activation of Ga q and phospholipase C (PLC), and release of inositol 1,4,5-trisphosphate (IP 3 ) (Silva et al., 2009;Volynski et al., 2004).
These observations suggested that Lasso might also affect the distribution of NTF and CTF of LPHN1 in the plasma membrane. Indeed, we noticed that soluble Lasso-D or Lasso-A caused the NTF to aggregate into patches on the surface ( Figure 2C  with and without Lasso-D (3-way ANOVA: ***, p<0.001; F 1,144 = 12.92). Right, average integrated immunofluorescence at 0-500 mm from the wall, with and without Lasso-D (t-test: *, p=0.027; n = 7). (E) Knockout of LPHN1 blocks axonal attraction by soluble Lasso. Hippocampal neurons from Adgrl1 -/-(LPHN1 KO) and Adgrl1 +/+ (LPHN1 WT) mice were cultured in MAIDs and exposed to Lasso-D gradient. The amount of cellular material in each compartment was quantified by DiO labeling at 8 DIV. E. Left, LPHN1 KO cultures sent significantly fewer neurites to Lasso-containing Axonal Compartments compared to WT cultures (t-test: ***, p<0.001, n = 3). Right, there was no difference in the number of cells, dendrites and axons in the Somal Compartments between the two types of cultures (t-test: N.S., p=0.4, n = 3). DOI: https://doi.org/10.7554/eLife.37935.013 The following source data and figure supplement are available for figure 4: Source data 1. Source data for we applied Lasso-D to LPHN1-expressing cells and followed the fate of both NTF and CTF. We observed a dramatic rearrangement of both LPHN1 fragments in the membrane, leading to the formation of large molecular aggregates also containing Lasso ( Figure 6C). Similar clustering of both LPHN1 fragments was also induced by LTX N4C , a strong LPHN1 agonist ( Figure 6D). On the other hand, an antibody recognizing the V5 epitope at the N-terminus of NTF only caused NTF clustering, but did not affect the distribution of CTF ( Figure 6A, right; Figure 6E). Thus, soluble Lasso ECD, which causes the association of the LPHN1 fragments, might be a functional agonist of LPHN1, similar to LTX N4C . By analogy, this also indicated that the soluble Lasso fragment could induce signal transduction via the CTF of LPHN1 coupled to a G-protein.
The effect of LTX N4C can be assessed by monitoring cytosolic Ca 2+ (Silva et al., 2011;Volynski et al., 2004). We therefore investigated whether the soluble Lasso ECD could induce similar effects. LPH1-expressing neuroblastoma cells were stimulated with saturating concentrations of Lasso-D, LTX N4C (positive control) or buffer (negative control), while cytosolic calcium levels were monitored using an intracellular Ca 2+ -sensing dye, Fluo-4 (see Figure 7-figure supplement 1A for the scheme of experiment). Similar to LTX N4C , in the absence of extracellular Ca 2+ , Lasso-D did not cause any Ca 2+ signals in LPHN1-expressing NB2a cells ( Figure 7A). However, when extracellular Ca 2+ was added to the cells, the rise in intracellular Ca 2+ signal was significantly higher in the presence of the ECD of Lasso, compared to negative control ( Figure 7A). Thus, Lasso-D is able to cause intracellular Ca 2+ signaling in LPHN1-expressing cells.
One of the features of LTX N4C -induced effects (such as Ca 2+ signaling and neurotransmitter release) is that they develop with a delay of~20 min, which has been attributed to the time taken by the toxin to assemble the LPHN1 fragments together and cause its maximal activation (Volynski et al., 2004). We predicted, therefore, that the rearrangement of the NTF and CTF induced by soluble Lasso ( Figure 6C) should prepare the signaling machinery for stimulation by the toxin. To test this idea, we first treated the LPHN1-expressing cells with Lasso-D and then with LTX N4C (Figure 7-figure supplement 1B). When Lasso-D was applied in the presence of 2 mM Ca 2+ , it induced relatively short-lived intracellular Ca 2+ signaling ( Figure 7B, right, prior to the blue arrowhead). However, when LTX N4C was then added, it triggered Ca 2+ signaling after a shorter delay (~14 min), instead of the usual~23 min ( Figure 7C). This additivity of effects is consistent with soluble Lasso inducing intracellular Ca 2+ signaling via the same molecular mechanism as LTX N4C .
Another well-known effect of LTX N4C is the burst-like release of neurotransmitters, linked to the elevated levels of cytosolic Ca 2+ Volynski et al., 2003). As Lasso-D likewise increased intracellular Ca 2+ concentration, it might also trigger such transmitter exocytosis. To test this hypothesis, we applied a previously characterized (Silva et al., 2011), soluble, short C-terminal Lasso construct (Lasso-G, Figure 1A) to mouse neuromuscular preparations and recorded the spontaneous miniature end plate potentials (MEPPs), which correspond to individual exocytotic events. We found that incubation with Lasso-G significantly increased MEPPs frequency from 1.61 ± 0.27 Hz in control to 3.83 ± 0.79 Hz in the presence of Lasso-G ( Figure 7D,E). However, this was much less than the effect of LTX N4C , which triggered massive secretion of neurotransmitter reaching 29.5 ± 4.1 Left, profiles of axons in Axonal Compartments, identified by NF-H immunofluorescence, exposing a difference between control and Lasso-secreting cells (3-way ANOVA: **, p=0.006; n = 7, F 1,84 = 7.89). Right, average integrated axonal fluorescence at 0-500 mm from the wall, with control or Lasso-secreting cells (t-test: *, p=0.045; n = 7). (E) Left, profiles of dendrites in Axonal Compartments, identified by MAP-2 immunofluorescence, with control or Lasso-secreting cells (3-way ANOVA: non-significant, p=0.23; F 1,84 = 1.46). Right, average integrated dendritic fluorescence at 0-500 mm from the wall, with control or Lasso-secreting cells (t-test: non-significant, p=0.54; n = 7). (F) Soluble released Lasso-D induces axonal fasciculation. The width of all NF-H-positive axonal bundles was measured at 100 mm from the separating wall. The degree of fasciculation correlates with Lasso concentration (Pearson's correlation: R 2 = 0.43, p=0.041). (G) Soluble Lasso has no effect on axon length in cultured hippocampal cells. Left. Representative images of GFP-positive neurons immunostained for GAP-43 (red); after treatment with control medium (left) or with Lasso-D (right). Right. Quantification of the total neurite length in GFP-expressing neurons after the treatment (t-test: non-significant, p>0.05, n = 30 cells without Lasso-D and 61 cells with Lasso-D from three independent cultures). DOI: https://doi.org/10.7554/eLife.37935.016 The following source data is available for figure 5: Source data 1. Source data for Figure 5, Panels D-G. DOI: https://doi.org/10.7554/eLife.37935.017 Hz ( Figure 7F). To ascertain that both these effects were mediated by LPHN1, we used neuromuscular preparations from LPHN1 KO mice. Interestingly, unstimulated LPHN1 KO motor neurons showed an increased MEPPs frequency compared to synapses from WT animals (3.33 ± 0.79 Hz in KO synapses). However, neither Lasso-G, nor LTX N4C had any effect on exocytosis in preparations lacking LPHN1 ( Figure 7E,F; 3.4 ± 0.68 Hz with Lasso-G and 3.8 ± 1.4 Hz with LTX N4C ). In all the recordings, the mean amplitudes of MEPPs under any condition did not differ significantly (Figure 7-figure  supplement 1C), which indicated a purely presynaptic effect of the two LPHN1 agonists and of LPHN1 ablation. These results show that the soluble Lasso fragment can increase exocytosis at nerve terminals, and confirm the importance of LPHN1 in the observed effects of LTX and the ECD of Lasso.
From the results reported here, we hypothesize that the soluble Lasso fragment, released by developing neurons, interacts with LPHN1 on axonal growth cones and nerve terminals. It then induces clustering of LPHN1 fragments and activation of downstream signaling, causing an increase in cytosolic Ca 2+ and subsequent exocytosis. The latter two processes are known to be key regulators of axonal attraction (Tojima et al., 2011). Thus, the ability of soluble Lasso to activate these processes on axonal growth cones could underpin the mechanisms by which it attracts axons.

Discussion
This study provides evidence that Lasso (a splice variant of TEN2 lacking a 7-residue insert in the bpropeller domain, TEN2-SS) functions specifically as an attractant for axons expressing LPHN1, and proposes a molecular mechanism for this effect. By using microfluidic devices to create long-term gradients of soluble proteins (Figure 3), we demonstrate that a gradient of soluble ECD of Lasso can act as an attractant for axons from hippocampal neurons (Figures 4 and 5A-E). Importantly, growing hippocampal neurons in a medium containing a uniform concentration of Lasso had no effect on the length of their axons ( Figure 5G). This shows that Lasso plays an instructive role in the directionality, rather than the amount, of axonal growth. This is consistent with the effect of other axon attractants acting via similar mechanisms. For example, short-term exposure of axonal growth cones to gradients of BDNF stimulates IP 3 -induced Ca 2+ release (IICR) that causes axonal attraction without an overall effect on neurite extension (Li et al., 2005).
One interesting observation from this project was the fasciculation of neurites in response to soluble Lasso/TEN2 ( Figure 5C,F). Fasciculation of axons is one of the major mechanisms of axonal navigation, for example in limb development (Bastiani et al., 1986). While axonal fasciculation has not been previously linked to a soluble ECD of TEN, neurite bundling was actually observed in hippocampal cultures in response to TEN1 C-terminal peptide (TCAP-1) (Al Chawaf et al., 2007). Furthermore, knockdown of TEN1 in C. elegans resulted in de-fasciculation of the axons in the ventral nerve cord (Drabikowski et al., 2005). Potential mechanisms of axonal bundling include actin reorganization induced by an LPHN1-mediated rise in cytosolic Ca 2+ , other unknown interactions with cell adhesion molecules, or it could also be due to the divalent Lasso/TEN2 fragment crosslinking adjacent axons, thus promoting their parallel elongation.
The soluble Lasso/TEN2 fragment could potentially have two membrane-anchored receptors: (i) TEN2 itself, as a homophilic ligand (Bagutti et al., 2003;Rubin et al., 2002), or (ii) LPHN1, as a heterophilic ligand (Boucard et al., 2014;Silva et al., 2011). However, we have not observed TEN2 expression in growth cones of hippocampal axons ( Figure 1E), but found it to be abundant on In addition, homophilic interaction of Lasso/TEN2 actually has been reported to inhibit neurite outgrowth in neuroblastoma cells (Beckmann et al., 2013), while we saw an opposite effect (Figures 4 and 5). Thus, the potential Lasso/TEN2 homophilic interaction could not explain the observed axonal attraction. On the other hand, we found strong expression of LPHN1 on the axonal growth cones of cultured hippocampal neurons ( Figure 1E-I, Figure 1-figure supplement 1C-F) (Silva et al., 2011). Importantly, the released soluble ECD of Lasso strongly bound to LPHN1 that was expressed on neuroblastoma cells or neuronal growth cones (Figure 2, Figure 2-figure supplements 1-2). Furthermore, we found that deletion of LPHN1 precluded axonal attraction by Lasso (Figure 4), while it had no effect on neuronal cell bodies and dendrites in the Somal Compartment. These data strongly implicate LPHN1 in mediating Lasso-induced axon attraction.
Our studies also reveal the likely mechanism that underlies the Lasso/LPHN1-induced axonal attraction. LPHN1 is a G-protein-coupled receptor (GPCR) that physically and functionally links to Ga q/11 (Rahman et al., 1999). Activation of LPHN1 by its non-pore-forming agonist, LTX N4C , leads to aggregation of the NTF and CTF of LPHN1 (Silva et al., 2009;Volynski et al., 2004). This results in assembly of a functional GPCR, with subsequent activation of the downstream signaling cascade, which includes Ga q/11 , phospholipase C, production of IP 3 and IP 3 -receptor-mediated release of Ca 2+ from intracellular stores Lajus et al., 2006;Volynski et al., 2004), thus inducing IICR. IICR is also regulated and enhanced by increased cAMP levels (Tojima et al., 2011), and we previously demonstrated that activation of LPHN1 expressed in COS7 cells induces an increase in cAMP production (Lelianova et al., 1997). In line with this, the recent study by Li et al. (2018) confirmed the ability of LPHN1 to regulate cAMP signaling. In that work (Li et al., 2018), the cAMP signaling interference system was based on HEK293 cells expressing exogenous b 2 adrenoceptor (b2AR). Activation of b2AR by its agonist led to an increase in cAMP production, while a large excess of coexpressed LPHN1 interfered with b2AR signaling. This clearly suggests that LPHN1 uses the same cAMP signaling machinery as b2AR, and that when LPHN1 is not stimulated, it can titrate components of this machinery, decreasing their availability to b2AR.
In agreement with the role of Lasso as a functional LPHN1 agonist, the binding of the released Lasso fragment to LPHN1 similarly causes the re-association of LPHN1 fragments ( Figure 6) and Ca 2+ signaling ( Figure 7A-C). A rise in cytosolic Ca 2+ concentration, in turn, can increase the rate of exocytosis, and we indeed observed enhanced acetylcholine release in mouse neuromuscular junctions in response to soluble Lasso ( Figure 7D-F). This response to Lasso was clearly mediated by LPHN1, as it was not detected in neuromuscular preparations from LPHN1 KO mice ( Figure 7D-F).
On the other hand, the effect of soluble Lasso on vesicular exocytosis was much weaker -and probably more physiological -than the massive effect of LTX N4C .
In addition to Ca 2+ regulation, Lasso binding to LPHN1 can induce cAMP signaling. Indirect evidence for this is provided by the cAMP signaling interference experiments mentioned above (Li et al., 2018). When LPHN1 co-expressed with b2AR was stimulated for 24 hr with Lasso/TEN2 (expressed on the same or opposite cells), this strongly decreased cAMP levels induced by b2AR activation. The most likely reason could be that, following an initial Lasso-induced LPHN1 activation, which normally subsides within 30 min ( Figure 7B), the continued LPHN1 stimulation led to massive heterologous receptor desensitization (Kelly et al., 2008) and inhibition of b2AR-mediated effect.
Intriguingly, the effects of soluble Lasso resemble the well-known mechanism that underpins axonal attraction and consists of IP 3 receptor-mediated local release of Ca 2+ from intracellular stores, coupled with an increase in cAMP levels, that leads to increased exocytosis at the advancing edge of a growth cone (Akiyama et al., 2009;Qu et al., 2002;Tojima et al., 2011;Tojima and Kamiguchi, 2015). Thus, when a gradient of soluble Lasso ECD approaches one side of an axonal growth cone, it may cause local activation of LPHN1 and its downstream signaling, ultimately leading to IICR. Local IICR in growth cones can induce an increase in vesicular exocytosis (as observed in our experiments with Lasso-G, Figure 7) and the remodeling of actin filaments (Tojima et al., 2011). The resulting augmented membrane delivery and actin-driven extension of filopodia at the edge facing a Lasso gradient would support the growth cone's advance in this direction. Thus, based on all our data, we propose this chain of events (summarized in Figure 8) as a likely mechanism for axonal attraction by soluble Lasso observed in this study.
While TEN2 has been implicated in axon guidance in the visual pathway (Young et al., 2013), here we report that it can also trigger axonal steering in developing hippocampal neurons, which is consistent with the strong expression of both Lasso/TEN2 and LPHN1 in the hippocampus (Davletov et al., 1998;Otaki and Firestein, 1999). Furthermore, both proteins are expressed throughout the CNS, suggesting that this mechanism of soluble Lasso/LPHN1-mediated axonal attraction may apply widely across the brain, especially in such areas as the cortex, cerebellum, thalamus and spinal cord.
Interestingly, the splice variant of TEN2 (TEN2+SS), which contains the 7-amino acid insert in the b-propeller domain and cannot mediate cell adhesion via LPHN1 (Li et al., 2018), might attract dendrites instead of axons, in contrast to Lasso (TEN2-SS). Thus, in an artificial synapse formation experiment (Li et al., 2018), HEK293 cells expressing TEN2+SS were seen covered by neurites from cocultured hippocampal neurons that contained GABA A receptors. However, these processes did not show a proportionate accumulation of PSD-95 and thus probably represented en passant dendrites that were attracted to TEN2+SS cells, but unable to form mature inhibitory synapses with them. This could be a mechanism by which TEN2+SS could provide a substrate for the growth of dendrites searching for their ultimate target/s. Although the relative abundance of Lasso and TEN2+SS in the brain is unknown, these data suggest that various TEN isoforms could participate in distinct interactions, possibly with opposite results.
High expression of LPHN1 and Lasso/TEN2 throughout the CNS, combined with their fundamental role in axon guidance, is consistent with lethal phenotypes observed in simpler organisms (Langenhan et al., 2009;Mosca et al., 2012). In knockout mice, however, the phenotype is less severe (Tobaben et al., 2002;Young et al., 2013) (Ushkaryov, to be published elsewhere) suggesting that LPHN1 deletion is not completely penetrant, likely due to a compensatory effect of multiple LPHN and TEN homologs expressed in the mammalian brain. Indeed, LPHN1 can also weakly interact with TEN4 (Boucard et al., 2014), and LPHN3 can interact with TEN1 (O'Sullivan et al., 2014). Moreover, LPHN and TEN isoform expression patterns overlap (Oohashi et al., 1999;Sugita et al., 1998;Zhou et al., 2003). This predisposition to compensation further raises the possibility that the mechanism of axonal guidance involving the interaction of soluble TEN2 with LPHN1, described in this study, may occur between different members of the LPHN and TEN families. These observations provide evidence of further diversity of interactions and local specificity of developmental pathways for more accurate and plastic patterning of neural networks within the mammalian CNS. Figure 8. A proposed scheme of the mechanism of axonal attraction by released Lasso ECD. When Lasso binds the NTF of LPHN1, it causes its re-association with the CTF. This activates Ga q/11 and triggers the PLC signaling cascade. Downstream of this cascade, the local IP 3 -induced calcium release (IICR) from intracellular stores stimulates exocytosis and may also stimulate reorganization of actin through Ca 2+ /calmodulin-dependent protein kinase II (CaMKII), thus mediating axonal attraction. The dashed line represents LPHN1-mediated activation of neuronal adhesion molecules via an unknown mechanism that may lead to axonal fasciculation observed in the presence of soluble Lasso ( Figure 5C, F

Cell lines
The following cell lines were used: human embryonic kidney cells (HEK293A, purchased from ECCC; RRID:CVCL_6910); mouse neuroblastoma cells (NB2a, a kind gift from Dr. C. Isaac, Imperial College London; originally from ATCC and subsequently authenticated by ATCC using their proprietary methods.; RRID:CVCL_0470). Both cultures are mycoplasma-free, based on a mycoplasma test kit PlasmoTest (Invivogen).
A LPHN1 KO mouse (strain AG148-2, Adgrl1 -/-) was generated on the 129SvJ genetic background. Briefly (details to be published elsewhere), the LPHN1 gene was isolated from a BAC clone containing a 36-kbp fragment of mouse genomic DNA. This was used to design a transfer vector for homologous recombination, containing a 13-kbp gene fragment of the LPHN1 gene, in which the intron between exons 1 and 2 was replaced with a neomycin gene/promoter cassette flanked by two loxP sequences. This insert disrupted the open reading frame in the mRNA transcribed from the resulting mutated LPHN1 gene. The transfer vector, carrying also a negative selection marker (diphtheria toxin A-chain), was used to generate stably transfected 129Sv/J ES cell lines and chimeric mice, using standard transgenic techniques. Mice transmitting the inactivated LPHN1 gene through the germline were selected, inbred, back-crossed onto C57BL/6J background, and maintained at Charles River UK. LPHN1 gene disruption was confirmed by Southern blotting, PCR amplification using multiple primer pairs and Western blotting. The genotype of all animals used for breeding and tissue extraction was determined by PCR. All procedures (breeding and Schedule 1) were approved by the University of Kent Animal Welfare Committee and performed in accordance with Home Office regulations and the European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes. E18 hippocampi were obtained from rats (BrainBits UK, Rhp). P0 hippocampi were prepared from P0 mice (strains: C57BL/6J, Adgrl1 +/+ , LPHN1 WT, or AG148/2, Adgrl1 -/-, LPHN1 KO). Flexor digitorum brevis muscle preparations were isolated from P21 male mice (C57BL/6J or AG148/2).

Generation of stable cell lines
Human embryonic kidney (HEK293A) and rat neuroblastoma (NB2a) cell lines were cultured using standard techniques in DMEM with 10% heat-inactivated fetal bovine serum (FBS, PAA Laboratories), at 5% CO 2 and 37˚C. Stable cell lines were generated using the Escort III transfection reagent and Geneticin selection (Thermo Fisher Scientific). The positive cells were further enriched by fluorescence-assisted cell sorting (FACSCalibur, BD Biosciences). All NB2a cell cultures contain proliferating, spindle-like cells and differentiated, neuron-like cells. We have not observed any difference in Lasso or LPHN1 expression between these two types of cell in stably transfected NB2a cultures.

Protein purification
For increased expression of Lasso or LPH constructs, the complete medium was replaced with a serum-free DMEM (for HEK23A cells) or Neurobasal-A containing supplements (for NB2a cells). Lasso-D was purified by immunoaffinity chromatography. Briefly, serum-free medium conditioned by HEK293A cells expressing Lasso-D was filtered through 0.2 mm filters and incubated with anti-FLAG M2 affinity gel overnight at 4˚C. Lasso-D was then eluted with 20 mM triethylamine, neutralized with 1 M HEPES, dialyzed against PBS, sterile-filtered for use in cell culture and concentrated on sterile 30 kDa MWCO filtration units (Vivaspin, GE Lifesciences). Medium above non-transfected cells was processed in the same manner and used as a negative control. Amount and purity of concentrated Lasso-D were assessed by SDS-PAGE and Coomassie staining. Activity was confirmed by measuring its binding to cell-surface or soluble LPHN1 constructs (Silva et al., 2011).

Primary neuronal cultures
Hippocampal cultures were prepared from Sprague-Dawley E18 rat hippocampi (BrainBits UK), according to the supplier's instructions, or dissected from P0 AG148/2 mouse pups (Adgrl1 -/-, LPH1 KO) under sterile conditions. Hippocampi were digested with 2 mg/ml papain in Ca 2+ -free Hibernate-A medium and dissociated in Hibernate-A medium with B27 supplement using fire-polished Pasteur pipettes. Cells were seeded in Neurobasal-A/B27 medium on poly-D-lysine-coated 13 mm coverslips at 5 Â 10 4 cells/coverslip and maintained at 5% CO 2 and 37˚C. The medium was partially replaced at least once a week.

Electroporation of neurons
Primary hippocampal neurons were transfected using Amaxa Rat Neuron Nucleofector Kit (Lonza) as described by the manufacturer. Briefly, dissociated cells were resuspended in Rat Neuron Neucleofector Solution with Supplement, then mixed with 3 mg of pcDNA6-GFP and electroporated in Nucleofector using the G-013 program. The transfected cells were resuspended in 500 ml of a recovery medium, containing a 1:3 mixture of Hibernate-A/B27 and Ca-free Hibernate-A (BrainBits UK), and incubated at 37˚C for 15 min. Cells were plated at a higher concentration to compensate for cell death. Next day, 0.8 nM Lasso-D was added to the medium (PBS was added to control medium). At 4 DIV, the cultured hippocampal cells were fixed with 4% paraformaldehyde (PFA), stained and visualized as described below in Image Analysis.

Cultures in MAIDs
To investigate axonal responses to chemoattractant gradients, MAIDs ( Figure 5) with 150 mm separation walls (Xona Microfluidics LLC) were prepared in accordance with the manufacturer's guidelines (Harris et al., 2007a;Harris et al., 2007b). Briefly, MAIDs were sterilized with ethanol, washed with sterile water and dried. To facilitate firm attachment of MAIDs, 22 Â 22 mm coverslips (VWR International) were sonicated in water and ethanol, autoclaved, dried, then coated with 1 mg/ml poly-Dlysine overnight, washed, and dried overnight before the assembly.
For neuronal cell culture in MAIDs, E18 rat hippocampi were dissociated as above. Neurons (1.5 Â 10 5 /10 ml) were added to Somal Compartments and allowed to settle for 30 min. MAIDs were then filled with Neurobasal-A/B27. After 3 DIV, the medium in Axonal Compartments was carefully replaced with medium containing soluble Lasso-D or with control medium. Alternatively, HEK293A cells stably expressing Lasso-D (or untransfected) were plated in the wells of Axonal Compartment. At 8 DIV, the cells were fixed and processed as described below.

Protein diffusion in MAIDs
For diffusion modeling experiments, MAIDs were assembled as above and filled with PBS; then 0.1 mg/ml BSA-TRITC (Thermo Fisher Scientific) in PBS was added to Axonal Compartments without changing liquid level in any compartment (to avoid creating a hydrostatic pressure in the microchannels). BSA-TRITC diffusion in MAIDs was monitored by time-lapse fluorescent imaging of all compartments for 5 days under an Axiovert fluorescent microscope (Carl Zeiss) equipped with a temperature-and humidity-controlling enclosure, and a Canon G5 camera. Fluorescence intensity profiles across the microchannels at multiple time points were generated in ImageJ (NIMH, Bethesda; RRID:SCR_002285, RRID:SCR_003070) and normalized to the fluorescence profile of 100 ng/ml BSA-TRITC forced into the microchannels and both compartments.

Immunocytochemistry
Cells on coverslips or inside MAIDs were fixed for 10 min with 4% PFA (for staining requiring SDS treatment to aid epitope retrieval, the fixative also included 0.1% glutaraldehyde). Cells were permeabilized with 0.1% Triton X-100 (or 1% SDS for PAL1 and dmAb staining), washed, then blocked for 1 hr with 10% goat serum in PBS and incubated with primary antibodies in blocking solution (dilutions used were: PAL1, 3 ng/ml; dmAb, 1:300; anti-NF-H, anti-myc mAb, and anti-GFP, 1:1,000; anti-V5, 1:2,000) for 1 hr at room temperature (or overnight at 4˚C with PAL1 and dmAb). The coverslips or MAIDs were then washed three times and incubated for 1 hr with secondary antibodies in blocking solution, followed by three washes. Coverslips were mounted using FluorSave mounting medium (Calbiochem), while neurons in MAIDs were imaged within 4 hr after the washes.

Receptor patching
NB2a cells stably expressing LPH-42 were grown on poly-D-lysine-coated coverslips in DMEM, 10% fetal calf serum (PAA Laboratories) to 30-50% confluency and to test receptor clumping incubated at 0˚C for 20 min in PBS with one of the three potential LPHN1 ligands: (1) 20 nM Lasso-D, (2) 2 nM Alexa Fluor 647-labeled LTX N4C (Volynski et al., 2004), or (3) rabbit anti-NTF antibodies (RL1), followed by a 20 min incubation with Alexa Fluor 546-conjugated goat anti-rabbit IgG. In control, only the fluorescent secondary antibody was added for the last 20 min. The cells were then fixed for 10 min with 4% PFA in PBS, blocked with 10% goat serum in PBS, and subsequent procedures were designed to reveal the distribution of the three components of each assay (NTF, CTF, and ligand). First, in all experiments, the V5 epitope on LPHN1 NTF was detected with a rabbit anti-V5 antibody (1 hr in blocking solution), followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG and fixation. Subsequent staining depended on the ligand used: (1) Lasso-D was stained using a mouse anti-FLAG mAb and Alexa Fluor 546-conjugated goat anti-mouse IgG. For LPHN1 CTF detection, the cells were then permeabilized with 0.1% Triton X-100, incubated with a chicken anti-myc antibody, fixed, blocked, and stained with Alexa Fluor 647-conjugated anti-chicken antibody.
(2) With LTX N4Cinduced patching, the cells were permeabilized, incubated with a mouse anti-myc mAb, fixed, blocked, and stained with an Alexa Fluor 546-conjugated anti-mouse IgG.
(3) With RL1-induced patching (and in controls), the cells were permeabilized, incubated with the chicken anti-myc antibody, fixed, blocked, and stained with Alexa Fluor 647-conjugated anti-chicken antibody. The primary antibodies were used at 1:1000 dilution; the secondary antibodies, 1:2000; the cells were washed three times with PBS after each stage. At the end, the cells were briefly fixed, blocked, washed, and mounted using FluorSave reagent (Calbiochem, Cat. No. 345789).

Image acquisition
Images of axons in MAIDs were acquired on an Axiovert 200M microscope (Carl Zeiss) using LD Plan-Neofluar 20x objective and Volocity-controlled camera, filters, shutter, and stage. Images were taken with a 5% overlap to facilitate stitching (Perkin-Elmer; RRID:SCR_002668). Blank images were subtracted to correct for optical artifacts. The images were stitched automatically and 'despeckled', using a 3 Â 3 median filter (ImageJ). To correct for large illumination artifacts, background was subtracted in ImageJ using the 'Subtract background' plug-in, with a 100 mm window and the sliding paraboloid algorithm. Images of immunostained cells and neurons on coverslips (other than for neurite tracing) were acquired using an upright laser-scanning confocal microscope (LSM-510, Zeiss; RRID:SCR_014344) equipped with 40x or 100x oil-immersion objectives; 488, 543, and 633 nm lasers; and 505-530, 560-615, and >650 nm emission filters. Images for neurite tracing were acquired using Axio Observer.Z1 microscope (Zeiss) equipped with Hamamatsu ORCA-Flash 4 sCMOS camera, EC Plan-Neofluar 40x objective, Colibri 2 LED illumination and appropriate filters.

Image analysis
To correlate the polarity of LPH1 expression and growth cone turning, GFP images of growth cones and preceding axons were traced using CorelTRACE X3 (Corel, Canada). The obtained contour images were aligned along their median line, with all axons starting at the same point. The images were then flipped so that the higher LPHN1 staining was located in the right half of each growth cone. The trajectory of respective axons was then assessed: correlation was considered positive if the axon approached its cone from the right quadrant. To plot Jeffreys confidence intervals (CI) for a binomial distribution the standard formula was used: CI = p + z*sqrt(p*(1 p)/n), where z = 3 for confidence level CI = 0.9973.
For profiling of neurite growth within MAID Axonal Compartments, regions of interest encompassing the depth of the compartments, were selected, avoiding artefacts (e.g. antibody aggregates or HEK cell bodies). The average fluorescence was determined as a function of distance (see Figure 5A) from the separation wall and binned over 100 mm intervals. Background fluorescence in the areas beyond 1200 mm from the wall (that contained no axons) was subtracted from all other fluorescence values, and the results were used for statistical analysis as described below.
For axon fasciculation measurements in MAIDs, the width of each axon/bundle was determined in pixels at 100 mm from the separation wall and converted to mm.
Neurite tracing of GFP-positive neurons was performed in ImageJ (Schindelin et al., 2012) using default settings in Simple Neurite Tracer plug-in (Longair et al., 2011). The longest neurite for each cell was used as a single independent measurement (data obtained from three independent cultures).
Analysis of the co-localization of the NTF, CTF, and respective ligands in the plasma membrane was carried out using a method previously developed and tested (Silva et al., 2011). Here, the confocal images were obtained near the middle of each cell (optical plane, Z = 0.5 mm). For consistency, the recorded images were assigned false colors according to the detected protein, irrespective of the actual fluorescence wavelength used for detection. The fluorescence profiles for each protein along the cell's perimeter were collected using ImageJ. Pearson's correlation coefficient r was then calculated for the pairs of resulting profiles obtained from 4 to 7 independent experiments.
In the representative images that were used in the Figures, the contrast and brightness were enhanced in the same manner as in respective control images.

Fluorometry
For experiments with LPHN1 KO and WT/HET cultures in MAIDs, the membranes of cell bodies and axons were labeled using 5 mM DiO (Vybrant DiO, Life Technologies) in Neurobasal-A, containing B-27 supplement and 0.005% Pluronic F-127 (Sigma-Aldrich), which had been passed through a 0.2 mm filter. After 30 min incubation, the excess dye was carefully washed two times, and the cell bodies (Somal Compartments) and axons (Axonal Compartments) were solubilized in 1% Triton X-100 in PBS. The undiluted axonal and 10-fold diluted somal fractions were analyzed in microtiter plates using a Fluoroskan Ascent Fluorometer (485 nm excitation, 505 nm emission filters) (Thermo Fisher Scientific). In some experiments, 2 mL samples of lysates were individually measured using a Nano-Drop ND-3300 Fluorospectrometer (Thermo Fisher Scientific) with the following settings: 470 nm Blue LED excitation, 500-700 nm emission spectrum, quantified at 504 nm. The levels of fluorescence were proportional to the amount of axons/cells bodies present in respective compartments.

Quantification and statistical analysis
The data shown are the means ± SEM, unless otherwise stated. A Lilliefors test was applied to all data sets to assess normality in data distribution. Statistical significance was then determined using two-tailed heteroscedastic t-test, with Bonferroni correction in cases of multiple pair-wise comparisons. For non-normally distributed data, a Mann-Whitney test was applied. The axonal fluorescence curves obtained from image analysis in MAIDs were compared using n-way ANOVA algorithm (MAT-LAB; RRID:SCR_001622), where n reflected the number of factors involved in an assay (treatment type, distance from the separation wall and batch number) (Vysokov, 2018). To test for correlation in axonal fasciculation measurements, a Pearson correlation coefficient (R 2 ) and the p values (to test the correlation hypothesis) were calculated using MATLAB. Jeffreys confidence intervals were used to assess statistical significance of correlation between LPH1 enrichment and growth cone turning direction. Differences were considered significant if p<0.05. The specific p and n values are indicated in corresponding figure legends or the following notation is used to denote statistical significance: NS (non-significant), p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001. The investigators were blinded to the identity of samples during data collection and analysis in all experiments involving LPHN1 KO.