Aurora Pignata

Hugo Ducuing

Val Erie Castellani
email: valerie.castellani@univ-lyon1.fr

Thibault Gardette

Servane Tauszig-Delamasure

Valérie Castellani

Commissural axon navigation: Control of midline crossing in the vertebrate spinal cord by the semaphorin 3B signaling

Keywords: axon guidance, commissural neurons, midline, semaphorins, spinal cord development Axon guidance Commissural axon Midline crossing Guidance signaling Repulsion Attraction Growth cone

The mechanisms governing the navigation of commissural axons during embryonic development have been extensively investigated in the past years, often using the drosophila ventral nerve cord and the spinal cord as model systems. Similarities but also specificities in the general strategies, the molecular signals as well as in the regulatory pathways controlling the response of commissural axons to the guidance cues have been found between species. Whether the semaphorin signaling contributes to midline crossing in the fly nervous system remains unknown, while in contrast, it does play a prominent contribution in vertebrates. In this review we discuss the functions of the semaphorins during commissural axon guidance in the developing spinal cord, focusing on the family member semaphorin 3B (Sema3B) in the context of midline crossing in the spinal cord.

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Accurate perception of guidance cues is crucial for axonal pathfinding. During their initial navigation in the spinal cord, commissural axons are kept insensitive to midline repellents.

Through yet unclear mechanisms acting during midline crossing in the floor plate, they switch on responsiveness to various repulsive signals, that establish a permanent midline barrier and propel the axons for exit. Whether these gains of response are coupled to occur in synchrony or rather are independently activated through signaling-specific programs is fully unknown. We set-up a paradigm for live imaging and super resolution analysis of guidance receptor dynamics during commissural growth cone navigation in chick and mouse embryos. We uncovered a remarkable program of delivery and allocation of receptors at the growth cone surface, generating receptor-specific spatial and temporal profiles. This reveals a mechanism whereby commissural growth cones can discriminate coincident repulsive signals that they functionalize at different time points of their navigation.

In a broad range of biological contexts, cells are exposed to a complex array of environmental cues from which they receive specific instructions. This is well exemplified by the model of axon responses to guidance cues during the formation of neuronal circuits. Axons navigate highly diverse environments to reach their targets. Unique trajectories emerge from the perception by axon tips, the growth cones, of combinations of extracellular cues exposed in choice points along the paths. A typical case is provided by commissural neurons, which must project their axons across the midline to build with contralateral target cells circuits integrating left and right neuronal activities [START_REF] Evans | Axon guidance at the midline: of mice and flies[END_REF][START_REF] Pignata | Commissural axon navigation: control of midline crossing in the vertebrate spinal cord by the semaphorin 3B signaling[END_REF]Stoeckli, 2018).

Midline crossing of commissural axons in the floor plate (FP) of the developing spinal cord has been extensively worked out to explore axon guidance mechanisms, especially those regulating growth cone sensitivity to guidance cues. Various repulsive forces provided by proteins of the Slit and Semaphorin families are needed to prevent midline re-crossing and expel the axons away towards their next step. They are also thought to control the lateral position relative to the FP that commissural axons take to navigate their rostrally-oriented longitudinal path after the crossing [START_REF] Long | Conserved roles for Slit and Robo proteins in midline commissural axon guidance[END_REF][START_REF] Jaworski | Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance[END_REF]. Semaphorin3B (Sema3B) acting via Neuropilin2 (Nrp2)-PlxnA1 receptor complex, N-terminal and C-terminal Slit fragments resulting from Slit processing acting respectively via Roundabout (Robo)1/2 and PlexinA1 (PlxnA1) receptors are guidance cues all found expressed in the FP and playing instructive roles during commissural axon navigation [START_REF] Zou | Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord[END_REF][START_REF] Long | Conserved roles for Slit and Robo proteins in midline commissural axon guidance[END_REF][START_REF] Jaworski | Collaborative and specialized functions of Robo1 and Robo2 in spinal commissural axon guidance[END_REF][START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF][START_REF] Delloye-Bourgeois | PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments[END_REF].

Manipulations of Semaphorin and Slit repulsive signaling in mouse and chicken embryo models brought the view that the sensitivity of commissural axons to midline repellents must be silenced in a first step, prior to the crossing, and switched on in a second step to allow repulsive forces to set a midline barrier and expel the growth cones away. Consistently, manipulations presumably inducing premature sensitization or preventing it resulted in failure of FP crossing, with axons arrested before or within the FP, turning back or longitudinally before reaching the contralateral side [START_REF] Chen | Alternative splicing of the Robo3 axon guidance receptor governs the midline switch from attraction to repulsion[END_REF][START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]. Noteworthy, the FP navigation is not a synchronous process. For example, it extends over several days in the mouse embryo, from the first axon wave of earliest-born commissural neurons at E9.5 to the latest one at E12.5 [START_REF] Wilson | A molecular program for contralateral trajectory: Rig-1 control by LIM homeodomain transcription factors[END_REF][START_REF] Pignata | Commissural axon navigation: control of midline crossing in the vertebrate spinal cord by the semaphorin 3B signaling[END_REF]. The repellents are expressed in the FP over the entire period of commissural navigation [START_REF] Pignata | Commissural axon navigation: control of midline crossing in the vertebrate spinal cord by the semaphorin 3B signaling[END_REF]. Thus, independently from the ligand expression profiles, the switch towards sensitivity upon crossing has to be set at individual growth cone level. Whether the sensitization of commissural axons to the various repellents they encounter in the FP occurs in synchrony, or rather presents signaling-specific features is unknown. Also, whether repulsive guidance receptors distribute homogeneously or present spatial specificities at the growth cone surface is undetermined. Insights are still scarce essentially due to the deficit of experimental paradigms giving access to molecular events in single living commissural axons navigating in their native context.

We postulated that functional engagement of midline repellents could arise from peculiar dynamics of guidance receptors at the surface of navigating growth cones. To address this hypothesis, we investigated the cell surface dynamics of four receptors mediating repulsion by midline cues, Nrp2, PlxnA1, Robo1 and Robo2, in chick and mouse embryo models. Surprisingly, we found striking differences between Robo1 and Robo2 temporal patterns, which excludes Robo2 as a mediator of Slit repulsion during FP crossing but places it as a major player of the lateral funiculus navigation. Our study also revealed exquisite specificities of PlxnA1 and Robo1 dynamics. Both receptors are not only sorted at different timing of FP navigation but also are distributed in distinct domains of the growth cones. This spatial and temporal compartmentalization is achieved at post-translational and post-intra-axonal trafficking levels, specifically at the step of membrane delivery in the growth cones. Analysis of the dynamics of PlxnA1-Robo1 chimeric receptors demonstrated that the intracellular domain of PlxnA1 but not that of Robo1, is sufficient for coding the receptor-specific temporal pattern. Finally, FRAP analysis in growth cones navigating the FP further confirmed dynamics specificities of these two receptors. Our study thus revealed remarkably unique spatial and temporal sequences of cell surface sorting of guidance receptors during the navigation of spinal commissural axons. This mechanism enables the growth cones to discriminate in time and space coincident guidance signals and provides a basis for these cues to exert non-redundant and concerted actions.

Development of an experimental paradigm to visualize cell surface receptor dynamics in navigating commissural axons

We setup time lapse imaging to monitor the cell surface dynamics of Semaphorin and Slit receptors in commissural axons navigating the FP in native spinal cords of chicken embryos. Nrp2, PlxnA1, Robo1 and Robo2 receptors were fused to the pH-sensitive GFP, pHLuorin (pHLuo), whose fluorescence at neutral pH enables to report membrane protein pools and cloned in vectors with ires-mb-tomato (Fig. 1a) (Jacob et al., 2005;[START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]Delloye-Bourgeois et al., 2014). The pH-dependency of receptor fluorescence was verified by in vitro cell-line transfections (Supplementary Fig. 1a) (Delloye-Bourgeois et al., 2014). The vectors were then transferred to spinal cord commissural neurons using in ovo neural tube electroporation (Fig. 1b). Then, isolated spinal cords were dorsally opened and imaged over several hours for mapping the receptor cell surface sorting reported by pHLuo fluorescence.

The FP entry and exit limits were delineated using DIC channel or based on the observation of some typical features of axon trajectory, such as the presence of wrinkles when axons enter the FP and the rostral turning when axons exit the FP (Supplementary Fig. 1b).

PlxnA1 and Robo1 are specifically and successively sorted to the growth cone surface during FP navigation

Using our setup, we analyzed individual growth cone trajectories from time-lapse sequences.

We plotted the position of the growth cones that turned on the pHLuo fluorescence to build cartographies of receptor cell surface sorting positions along the navigation. First, we observed that Nrp2 is exposed at the commissural growth cone surface from the pre-crossing stage and remains over entire FP crossing (Fig. 1c,f,h,i; Supplementary Movie 1,2). In contrast, we found that the membrane sorting of both PlxnA1 and Robo1 specifically occurs during FP navigation. Interestingly, the timing of their sorting significantly differed. PlxnA1 was addressed to the surface when commissural growth cones navigate the first half of the FP, thus from the FP entry point to the midline (Fig. 1d,g,h,i; Supplementary Movie 3,4), while Robo1 was sorted during the navigation of the second FP half, from the midline to the FP exit point (Fig. 1e,g,h,i; Supplementary Movie 5,6). Next, we assessed whether these temporal patterns are profiled by control of receptor cell surface sorting or rather by control of protein availability within the axon. Spinal cord open books were fixed with paraformaldehyde (PFA) at neutral pH to detect the total pHLuo receptor pool. While in live axons pHLuo signal was found restricted to the growth cones during FP crossing, we observed in contrast for both receptors that the total pools had much broader distribution than the surface ones. In 90% of the cases for PlxnA1 and 75% for Robo1, the precrossing axon segment immediately adjacent to the FP entry of growth cones that were found to navigate within the FP, contained pHLuo-receptors (Fig. 2a,b). We also measured pHLuo + pre-crossing segment length in the fixed samples and found significant difference between PlxnA1 and Robo1, the latter having more restricted length and punctate pattern than the first one (Fig. 2c). These observations are consistent with previous works that reported in cultured commissural neurons Robo1 intra-axonal vesicular trafficking [START_REF] Philipp | RabGDI controls axonal midline crossing by regulating Robo1 surface expression[END_REF] and PlxnA1 processed within axons to prevent its membrane expression [START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]. Thus, PlxnA1 and Robo1 are available within commissural axons and their cell surface sorting is spatially and temporally controlled in a receptor-specific manner.

We found in previous work that medium conditioned by cultured isolated FP tissues (FP cm ) could trigger PlxnA1 cell surface expression [START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]. Such medium was also reported to induce in commissural growth cones Robo3.2, the Robo3 isoform expressed in postcrossing axons [START_REF] Colak | Regulation of axon guidance by compartmentalized nonsense-mediated mRNA decay[END_REF], providing the evidence that local signals emanating from the FP are implicated in synchronizing the sorting of these receptors with midline crossing.

How is triggered the sorting of Robo1 is yet unknown. We thus examined whether it could also be under local FP control. We treated dorsal spinal cord explants electroporated with pHLuo-Robo1-ires-mb-tomato with FP cm and ctrl medium and recorded Robo1 dynamics by measuring pHLuo fluorescence in the growth cones at T0 and T1, 20 min later (Fig. 2d). We observed a significant increase of pHLuo fluorescence at T1 compared to T0 for the FP cm but not the control condition (Fig. 2e,f), thus indicating that FP cells release cues triggering Robo1 at the growth cone surface.

Next, we assessed whether disturbing the temporal pattern of receptor sorting impacts on growth cone behaviors. Open-books were electroporated with high concentration of vectors (3µg/µl and 4µg/µl) to overcome the internal control of PlxnA1 and Robo1 surface sorting in commissural neurons and create premature surface expressions. We monitored individual growth cones and found that commissural growth cones having premature cell surface receptor exposure failed to cross the FP, rather turning or stalling before or within the FP (Supplementary Fig. 2; Supplementary Movie 7,8). These findings indicated that pHLuoreceptors are functional and confirmed that the temporal pattern of receptor sorting is critical for proper FP navigation.

Next, we asked whether the gain of PlxnA1 and Robo1 at the surface could be correlated with acquisition of novel behavioral properties of the navigating growth cones. To address this question, we analyzed in time-lapse movies growth cone trajectories at time-points preceding and succeeding the pHLuo flashes, by measuring the deviation angles of growth cone direction from the trajectory baseline (Fig. 2g). Interestingly for Robo1, we observed that acquisition of surface receptor was coincident with a significant increase of exploratory behavior along the rostro-caudal axis as if the growth cones were starting sensing cues that will direct their longitudinal turning at the FP exit. In contrast, we found no difference of exploration for PlxnA1 (Fig. 2g,h,i).

Robo2 is sorted not in the FP but in the post-crossing lateral funiculus

Next, we investigated the dynamics of Robo2. In sharp contrast with Robo1, we found that Robo2 was absent from the surface of commissural growth cones navigating the FP and turning longitudinally at medial position in the ventral funiculus (VF). Instead, we observed that it was specifically sorted in post-crossing axons that chose to navigate in the lateral funiculus and turned longitudinally to the FP (LF) (Fig. 3a,b,c; Supplementary Movie 9,10). To assess if Robo2 cell surface sorting correlates with this change of trajectory, we measured the angle formed by a vector aligned along the axon tip and the FP axis at the two time points framing Robo2 pHLuo flash. Interestingly, we found that the angle was significantly more pronounced at post-flash time points compared with pre-flash ones, supporting that Robo2 sorting contributes to directional growth cone changes along the longitudinal axis (Fig. 3d,e). Thus interestingly, signaling by Robo1 and Robo2 appear to have similar outcome on growth cone behaviors, that they control at two different time points of commissural navigation.

The temporal sequence of receptor sorting is conserved in the mouse

Next, we studied whether the temporal control of receptor sorting during FP navigation uncovered in the chicken embryo is conserved in the mouse and whether it also instructs growth cone guidance choices. We electroporated pHLuo-PlxnA1 and pHLuo-Robo1 constructs in the developing spinal cord of E12 wild-type mouse embryos. We plotted the position of fluorescent growth cones in living open-books at fixed time point, 48 hours post-electroporation, when many FP crossing are ongoing, as depicted by the distribution of mb-tomato + growth cones (Fig. 4c,d). In pHLuo-PlxnA1 electroporated spinal cords, growth cones exposing the pHLuo were distributed almost homogenously in all FP and post-crossing compartments (Fig. 4a,b,c) whereas in the pHLuo-Robo1 electroporated littermates, most of the growth cones exposing Robo1 were situated between the midline and FP exit ( Fig4. a,d,e). Therefore, the spatial and temporal cell surface pattern of PlxnA1 and Robo1 observed in chick spinal cord is conserved in mice (Fig. 4e).

We then electroporated pHLuo-Robo1 construct in Robo1/2 -/-open books to determine whether reducing Robo1 dose by removal of the endogenous pool results in a modified Robo1 temporal pattern. We found that the profile of receptor sorting was identical to that observed after electroporation of Robo1/2 +/+ embryos (Fig. 4f,g,h). Thus, our experimental conditions of expression are likely to model the dynamics of endogenous receptor. This result also established that Robo1 sorting at the plasma membrane is Robo2 independent.

Next, we investigated whether the re-expression of pHLuo-Robo1 in Robo1/2 -/-mice could rescue the previously reported stalling phenotypes resulting from Robo1/2 deletion (Long et al., 2004;[START_REF] Delloye-Bourgeois | PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments[END_REF]. We analyzed the distribution of mb-tomato + growth cones over pre-to post-crossing steps, distinguishing growth cones that exposed Robo1 at their surface (mb-tomato + pHLuo + ) from those that did not (mb-tomato + pHLuo -) in Robo1/2 +/+ and Robo1/2 -/-embryos. We observed that Robo1/2 loss resulted in significantly shifted distribution of mb-tomato + pHLuo -towards the first FP half (Fig. 4i,j). Interestingly, the expression of Robo1 at the growth cone surface was sufficient to rescue the distribution observed in the WT condition, as observed by the matching of the distribution of mb-tomato + pHLuo + growth cones in Robo1/2 -/-and Robo1/2 +/+ embryos (Fig. 4k). Thus, re-expression of Robo1 coding sequence in commissural neurons and subsequent cell surface exposure at a time when the growth cone navigates the second half of the FP is sufficient to rescue proper navigation. Moreover, and consistent with its observed temporal sorting profile, Robo2 is dispensable for FP crossing and Robo1 surface exposure. Thus, this supports that this temporal profile properly reports the dynamics of endogenous Robo1 receptor. Lower panel: cumulative fractions of mb-tomato + pHLuo + and total mb-tomato + (P value is from the Kolmogorov-Smirnov (KS) test). (i) Representative images of mb-tomato + growth cones illustrating reduced number of growth cones (asterisks) on their way for FP exit in Robo1/2 -/- compared to Robo1/2 +/+ open-books. (j) Cumulative fractions reporting the distribution of mbtomato + pHLuo -growth cones in Robo1/2 +/+ and Robo1/2 -/-embryos, showing a significantly shifted distribution of tomato + pHLuo -growth cones towards the first FP half in Robo1/2 -/- embryos. (k) Cumulative fractions reporting similar distribution of pHLuo-Robo1 + growth cones in open-books of Robo1/ +/+ and Robo1/2 -/-embryos (P value is from the Kolmogorov-Smirnov (KS) test). Scale bars in a and f 20 µm, in i, 50 µm.

Introduction to the semaphorin family

The semaphorins form a large family of secreted and membrane-associated molecules present from virus to human. 32 By activating a range of holoreceptors and downtream intracellular cascades, the semaphorins regulate the molecular machinery controlling actin and microtubule dynamics, thus contributing to a variety of processes implicating cell movements, from cell migration and axon migration in the nervous system, organ morphogenesis, to immunology and tumor metastasis. 16,29,32 Many contributions of the semaphorins have been discovered in the developing and adult spinal cord, both in physiological and pathological contexts such as spinal cord injury, which exemplify the diversity of functional properties that these cues exert (O' Malley et al., 2014). In this review, we concentrate on the role of the secreted semaphorin 3B (Sema3B) during the formation of the spinal commissures.

Crossing the midline of the central nervous system: An obligatory step for all commissural axons

In bilateral organisms, multiple reciprocal neuronal projections interconnect the 2 halves of the central nervous system (CNS), forming a dense network of commissures that allow integration and coordination of left-right neuronal activities. 9,36 During embryonic and early postnatal developmental periods, commissural axons navigate through the central midline at all axial levels, crossing from one side of the CNS to the other one at specific time points and positions. 9 Commissural axons can be surrounded by or mixed with ipsilateral axons that are committed to build circuits between neurons of the same side, and that never cross the midline. For instance, in the visual circuits of organisms with binocular vision, ipsilateral and contralateral ganglion cell axons exit the retina and navigate together, segregating at the optic chiasm when the contralateral axon tract achieves midline crossing. 18 Similarly, tracts of ipsilateral and contralateral axons navigate in close proximity in the developing spinal cord 41 (Fig. 1A).

Specialized groups of local cells lying at the CNS midline are instrumental in segregating the ipsilateral and contralateral axon populations, allowing only the latter to cross the midline (Evans and Bashaw, 2009 9,36 ). In the developing spinal cord, midline crossing takes place ventrally through the floor plate (FP), a crucial patterning center composed of glial cells, which contribute to the specification of the neuronal lineages of the neural tube and adjacent territories by secreting the morphogen SHH. 17,24 The drosophila midline glia plays equivalent roles, secreting a TGF homolog to direct ventral cell fates of the CNS, the ectoderm and the mesoderm. 5 The FP also expressing guidance molecules at these early stages. It was recently discovered that semaphorin 3B (Sema3B) already has an instructive role prior to the stage of axon navigation. Sema3B is already detected at E9 and is secreted in the cerebrospinal fluid. Collected by receptors of neuroepithelial progenitors that undergo mitosis at the apical border lining the central canal, Sema3B triggers an intracellular signaling which regulates microtubule stability and promotes planar orientation of the cell divisions at the onset of the neuro-genesis. 2 Beyond this function, the FP source of Sema3B has been well characterized for its contribution to commissural axon guidance, a role that we will discuss in detail below.

Commissural axon navigation in the spinal cord: The prototypical dl1 tract

The dorsal interneuron lineages in the developing spinal cord are specified by transcriptional programs, according to their position, birthdate and pattern of connections. Various classes of commissural interneurons have been identified among which the commissural component of the dl1 population, born from a Math 1C progenitor pool that lies close to the roof plate and which also generates an ipsilateral component. 22 dl1 commissural neurons, specified by Lhx2/9 transcription factors, 44 elaborate a typical pattern of axonal projections, whose navigation has been widely investigated. The axons extend ventrally toward the FP, cross the midline, exit the FP and turn rostrally to ascend toward supraspinal levels, to convey proprioceptive information to the cerebellum. 1 The generation of transgenic mice expressing LacZ or GFP under the control of the Math1 promotor has allowed a precise spatial and temporal mapping of commissural dl1 axon navigation in the mouse embryo. Born from around E10, dl1 commissural neurons extend waves of axons toward the FP starting at this stage. Some of them already cross the midline as early as E10.5. It is widely accepted that most of them have crossed the midline by E12.5, and are already navigating distant longitudinal routes at E13 11,25,26,27,30,44 (Fig. 1B).

Repulsive signaling controlling midline crossing in the vertebrate spinal cord and invertebrate nerve cord

The FP and midline glia are sources of both attractive and repulsive cues for commissural axons. A temporal sequence has been proposed which orchestrates the different steps of commissural axon navigation by controlling axon responsiveness to the FP attractive and repulsive cues, thus preventing conflict of guidance information. First, the commissural growth cones perceive chemoattractants, including Netrin, which orient their trajectory toward the midline. Commissural growth cones then interact with local cells to navigate the FP/ midline glia. Next, upon crossing, they gain responsiveness to FP/midline glia-derived repulsive cues, which prevent them from turning back and re-crossing the midline, and also push them out of the FP toward the contralateral side. Finally, after FP exit, rostro-caudal gradients of guidance cues elicit a longitudinal turning of commissural axons, accompanied by a sorting of the axons into the ventral and lateral funiculi in which they navigate rostrally (Fig. 1C).

Such a temporal sequence of guidance programs relies on a tight control of growth cone sensitivity to the guidance cues. In particular, the sensitivity to the midline repellents must be first silenced before crossing to be triggered only after the crossing. The premature action of the repellents would prevent the axons from entering the FP.

The repulsive signaling found to regulate midline crossing in vertebrates and invertebrates present some degrees of conservation, although vertebrates evolved significant differences. 36 In drosophila, midline repulsion was shown to be ensured by SLIT, a gene encoding a protein which acts through binding to Roundabout (Robo) Robo 1 and Robo2 receptors on commissural axons. Similarly, in vertebrates, 3 SLIT genes (Slit1,2,3) were shown to act in synergy to control midline crossing, mediating their effects through Robo1 and Robo2 receptors. 6 The N-terminal (140 kDa) and C-terminal products (55-60 kDa) resulting from Slit protein cleavage were recently found to both contribute to the FP navigation in vertebrates. Until this recent work, Slit-Ns, which contain the binding sequence for Robo receptors were logically considered as the bioactive protein fragments. In contrast, the Slit-C fragments, for which no receptor was identified, were thought to be inactive. From initial analysis of mutant mouse models, deletion of Slit1-3 or Robo1/2 genes in mice were both known to disturb commissural navigation. However unexpectedly, phenotypic differences were noted between the 2 deletion contexts. Midline re-crossing was observed after Slit1-3 but not Robo1/2 deletion. 27,33 These studies raised the idea that Slits exhibit some Robo-independent functions that might be mediated by an as yet-unknown receptor. This receptor turned out to be PlexinA1, 15 a receptor shared by members of another prominent axon guidance family, the semaphorins. 32 Indeed, PlexinA1 deletion was found to induce the re-crossing phenotype, and also to confer the re-crossing phenotype to Robo1/2 mutants. Various combinations of PlexinA1 and Slit1/2/3 allelic deletions also confirmed that Slits and PlexinA1 both participate in preventing midline re-crossing during commissural axon guidance. Moreover, biochemical revealed that PlexinA1 binds Slit full-length and Slit-Cs, but not Slit-Ns, and mediates a repulsive action of the Slit-C fragments. 15 Slit processing also occurs in invertebrates. However, in transgenic rescue assays in drosophila, expression of a Slit mutant that resists proteolytic cleavage in the midline glia lineage was shown to rescue midline crossing defects resulting from general Slit loss equally as well as the wild type Slit, thus supporting that Slit processing is dispensable for midline navigation. 13 PlexinA1 was identified in previous work as the Plexin A member which associates with Neuropilin2 (Nrp2). Nrp2/PlexinA1 complex forms a functional commissural receptor for Sema3B, a semaphorin that was demonstrated to act as a FP repellent for post-crossing commissural axons in the mouse. 34,38 Mouse embryos lacking Nrp2 were shown to present FP crossing defects consistent with a role of semaphorin ligands as repellents for commissural axons after midline crossing. Among the class3 semaphorins, Sema3B expressed by FP cells, exhibits the expected profile for exerting this role. In contrast, Sema3F, another prominent Nrp2 ligand, instead of being expressed by the FP, localizes in a domain adjacent to the FP. 34,38 Consistently, Sema3B deletion was reported to result in alterations of FP crossing and these defects were phenocopied in PlexinA1 null mutant embryos. 34 Implication of semaphorins in the FP navigation of spinal commissural axons also came from studies in the chick model. Over-expression of a dominant negative PlexinA, abolishing the signaling by all PlexinAs, resulted in strong alterations of crossing and post-crossing axon trajectories. 15,34 Similarly, specific knock-down of individual PlexinAs such as PlexinA1, PlexinA2 and PlexinA4, all induced stalling at the FP exit and failure of rostral turning. 1 Altogether, these studies suggest that several repulsive signaling mechanisms operate during FP crossing in the mouse, such as Slit-N/Robo, Slit-C/PlexinA1 and Sema3B/PlexinA1-Nrp2 (Fig. 1D). How these signals are acting and which specific aspects of commissural growth cone behavior they control remains unclear. Mouse models allowing the investigation of the specific contributions of Slit-Cs and Slit-Ns still lack, in part because the nature of the protease(s) responsible for Slit cleavage remains unknown.

Silencing of the semaphorin signaling during FP navigation

Based on a variety of experimental set-ups, several studies provided evidence that the commissural axon responsiveness to Slits and Sema3B-derived FP repellents is silenced before the crossing. In early work, spinal cord open books were co-cultured with COS cell aggregates secreting Slits or Sema3B. 47 The behavior of commissural axons emerging from the explant border facing the cell aggregates was examined. In these spinal cord preparations, the endogenous FP was removed to mimic a precrossing context, so that commissural axons exiting the ventral side of the open-book had not experienced FP crossing in their native tissue before growing out of the explant. Under these conditions, similar growth toward control, Sema3B or Slit2-secreting cell aggregates was observed, indicating a lack of sensitivity of commissural axons to Sema3B and Slit2. These experiments were reproduced for Sema3B and the data were confirmed in more recent works 10,15,38 (Fig. 2A).

A second assay was used to demonstrate the lack of sensitivity to FP repellents at the pre-crossing stage which consisted in grafting FP and roof plate (RP) tissues as well as cell aggregates along the lateral side of an intact spinal cord. This assay challenges the ability of cues released by the ectopic tissue or the cell aggregate to re-route the trajectory of commissural axons toward or away from their natural path to the endogenous FP. 47 Such a model had been developed to show that the RP releases a repellent for pre-crossing commissural axons, whose action is to orient their initial trajectory toward the ventral side of the spinal cord. 3 Using this assay, commissural axons were found re-routed toward FP tissue, and Netrin1 expressing cell aggregates, reflecting that they are subjected to attractive cues. In contrast to ectopic RP, both Slit2 and Sema3B-secreting cell aggregates failed to deflect commissural axons away, confirming their lack of sensitivity to the FP repellents prior to crossing (Fig. 2B).

Finally, a last paradigm was employed in several studies, to assess the individual response of commissural axons to Sema3B. 15,34,43 Growth cone behavior to Sema3B application was examined in dissociated commissural neuron cultures collected from E11 to E13, which revealed their inability to undergo the collapse response, normally observed when growth cones perceive repulsive cues (Fig. 2C). Collapse assays were also conducted on dorsal spinal cords (lacking endogenous FP) collected from Atoh1-tauGFP and NeuroG2-tauGFP, transgenic mouse embryos, GFP identifying the dl1 and dl4 populations of commissural interneurons respectively. Both populations were found unresponsive to Sema3B. 43

Activation of the semaphorin signaling after midline crossing

Spinal cord open-books and commissural neuron cultures were used to establish that commissural axons acquire sensitivity to the FP repellents after the crossing.

First, the behavior of commissural axons emerging in front of Sema3B or Slit-secreting cell aggregate was examined in open-books in which the endogenous FP was left intact. In contrast to what was observed when the FP had been removed, the growth of commissural axons that experienced FP crossing was strongly prevented by Slit2 and Sema3B released from the cell aggregate 47 (Fig. 2D). Second in collapse assays, commissural growth cones insensitive to Sema3B at basal condition acquired a strong collapse response induced by application of FP conditioned medium (FP cm ). The sensitization was obtained in condition of co-application but also when FP cm was applied first and Sema3B treatment was applied after washing. This indicated that some FP signals prime commissural growth cones for Sema3B repulsion. 10,34 In collapse assays conducted on intact open-books from Atoh1-tauGFP and Neurog-tauGFP transgenic mouse embryos (having endogenous FP through which the axons navigate), dl1 but not dl4 commissural growth were reported to collapse in response to Sema3B. Thus, some cell-type specificities might exist in the responsiveness of post-crossing commissural axons to the FP repellents. 43 Mechanisms mediating pre-crossing silencing of repulsive signaling

The control of the sensitivity of commissural axons to midline repellents has been the topic of extensive investigations over the years, and pioneered by studies of midline crossing in the drosophila ventral nerve cord. This work uncovered key molecular mechanisms acting within commissural neurons to control guidance receptor trafficking and consequently responsiveness to midline repellents. Thereafter, in vertebrates, a panel of molecular mechanisms which regulate guidance receptors has been discovered, showing that, similar to the control of midline crossing in drosophila, strict control of receptor distribution and function is required for setting the temporal sequence of responsiveness to midline repellents. 36

Silencing by receptor degradation

In drosophila, the silencing of pre-crossing commissural axons to Slit repulsion was found to be achieved by active degradation of Robo receptors. The endosomal protein Comm plays a key role in preventing the presence of Robo at the growth cone surface, acting essentially but not exclusively by sorting the receptor to lysosomal degradation. After crossing, Comm is down-regulated, and as a consequence, Robos become available in commissural growth cones to transduce the Slit repulsive signal. 36 A key aspect of this regulatory pathway thus resides in the spatio-temporal control of Comm expression to restrict its action to the period of axon crossing. It was recently discovered that cleavage of Frazzled/DCC in commissural neurons, releases an intracellular fragment that acts as a transcriptional activator of Comm. 36

Silencing by receptor-receptor trans interactions

A second mechanism has been recently reported, which complements the Comm action by blocking the Robo receptors that start reaching the commissural growth cone surface before the crossing is completed. Such a situation is likely to occur, during crossing, after the onset of Comm down-regulation. This mechanism is mediated by trans interactions of this cell surface pool of Robo with Robo2 expressed by midline glial cells, which result in preventing Slit binding or activity. Such coupled mechanisms might ensure the robustness of the spatial and temporal control of Robo receptor availability during midline crossing 19 (Fig. 3A).

Notably, although the pre-crossing silencing of the midline repellents is conserved in vertebrates, the underlying mechanisms appears to differ sharply from those identified in the fly. First, the vertebrate genome was found to lack Comm. Second, active degradation of Robo receptors at the pre-crossing stage has not been reported yet. However, in the chick embryo, Robo1 was reported to distribute in intracellular vesicles, which might be the mechanism that maintains cell surface Robo at low levels before the crossing 39 (Fig. 3B). In addition, the silencing of Slit signaling was proposed to be achieved by Robo3, a divergent Robo family member having several binding partners including DCC and neural epidermal growth factor-like-like 2 (NELL2). 28,46 An isoform of Robo3 gene, Robo3.1 was reported to have a distribution restricted to the pre-crossing commissural axons. In vivo manipulations in chick and mouse embryos resulted in alterations of FP navigation, consistent with a function in blocking Robo/Slit activity before the crossing. 11 How the silencing of Slit-Robo signaling by Robo3.1 is achieved remains to be understood.

Silencing by prevention of receptor cell surface sorting

Although distinct from degradation, the sensitivity of spinal commissural axons to Sema3B also appears to be controlled through regulation of the Sema3B receptor. The signaling moiety of the complex, PlexinA1, was found to be a target of calpains, proteases known to process rather than degrade targets, capable of modulating both their functions and binding partner interactions. 8,45 Active calpain was shown to cleave PlexinA1, as well as other PlexinAs, generating 2 distinct PlexinA1 fragments. Both PlexinA1 integral and cleaved fragments could be detected by immunoblotting of lysates of dorsal spinal cord tissue. Treatment of fresh dorsal spinal cord tissue with calpain inhibitor prior to immunoblotting induced an increase of full-length PlexinA1 at the expense of the cleaved forms. 34 In the developing spinal cord, Nrp2 and PlexinA1 transcripts are both detected in commissural neurons at stages of pre-crossing navigation. Nevertheless, at protein levels, differences between the 2 receptor distributions were observed in embryonic immunolabelled sections. Using an anti-PlexinA1 antibody directed against an extracellular epitope and likely recognizing the integral protein, very low labeling was found in pre-crossing axon segments in stark contrast labeling was very strong on crossing and post-crossing axon segments. 34 Interestingly, Nrp2 was detected in both pre-crossing and post crossing commissural axon segments. Ex vivo, immunolabeling of PlexinA1 in DCC commissural axons emerging from spinal cord open-books with and without endogenous FP revealed that integral PlexinA1 labeling was only observed in commissural axons that experienced FP crossing. Finally, in vivo experiments consisting of expressing PlexinA1 fused to the pH sensitive GFP pHLuo, a selective reporter of the cell surface protein pool, in commissural neurons of the chick embryo showed that in a very large majority of the the green fluorescence was detected in commissural growth cones undergoing FP crossing. 34 The link between calpain-mediated PlexinA1 processing and commissural axon sensitivity to Sema3B was further investigated using in vitro and in vivo approaches. Inhibiting calpains was sufficient to confer a growth cone collapse response of commissural neurons to Sema3B. Administration of a pharmacological calpain inhibitor to pregnant mice resulted in strong alteration of FP crossing in the embryos. Commissural axons stalled at the FP entry, consistent with the acquisition of a premature sensitivity to Sema3B preventing them from entering the FP due to increased PlexinA1 cell surface levels. Similarly, overexpression of PlexinA1 leading to increased Plex-inA1 levels at the pre-crossing stage also resulted in stalling at the FP entry. 34 Thus, post-translational regulation of PlexinA1 levels appears to be a first mechanism to prevent pre-crossing commissural axons from responding to Sema3B (Fig. 3B).

Silencing by ligand-receptor cis and trans complex

A variety of additional mechanisms have been characterized to control the semaphorin signaling during midline crossing. First, macro-complexes of receptors were shown to orchestrate midline crossing in the optic chiasm of vertebrates. NrCAM, which participates in the semaphorin signaling, and PlexinA1 from midline glia cells were found to interact with NrCAM and PlexinA1 from retinal commissural axons, temporarily switching repulsive effects of Sema6B at the midline into attraction, to allow the crossing. 31 Second in the chick embryo, a recent study investigated the PlexinA/Semaphorin signaling during spinal commissural axon guidance. 1 PlexinAs were noted to have dynamic spatio-temporal expression patterns, with some members being expressed by commissural axons and FP cells such as PlexinA1 and PlexinA2, and others expressed only by commissural axons such as PlexinA4. Knock-down of individual Plexin A1,-A2, and -A4 in commissural neurons were all found to result in commissural axon stalling at the FP exit and failure of post-crossing rostral turning. Specific knockdown of FP-PlexinA2 also resulted in stalling, as did so the specific knock-down of Sema6B, which is endogenously expressed in commissural neurons. Thus, this identified a first signaling for crossing and post-crossing commissural axons arising from Sema6B acting as a commissural receptor for FP-PlexinA2, acting non-cell autonomously as a ligand. 1 In addition, PlexinA2 over-expression in pre-crossing commissural neurons strongly altered their ability to reach the FP, a phenotype that was interpreted as resulting from oversensitivity to ventral spinal cord/ FP repellents. The observation in cultured commissural neurons that Sema6B co-localized with PlexinA2 led the authors to propose a model whereby prior to the crossing, the Sema6B/PlexinA2 cis complex prevents commissural axons from sensing the FP repellents. The mechanism that silences the PlexinA4/Semaphorin signaling might be different. In cultured commissural neurons, while PlexinA2 was detected along axon shafts and growth cones together with Sema6B, PlexinA4 was reported to have in contrast a vesicular punctate pattern, and was excluded from the growth cones. 1 Overall, these mechanisms illustrate in various contexts that the silencing of semaphorin repellents is achieved through cell autonomous mechanisms desensitizing pre-crossing commissural axons, by controlling the cell surface sorting or the signaling activity of Plexi-nAs (Fig. 3B).

Silencing by ligand trapping

Recently, long-term (24h) application of Sema3B to dorsal spinal cord explant cultures was reported to result in reduced axon growth, with strong inhibition at high dose. 23 These data suggested that commissural axons at the pre-crossing stage might be able to perceive Sema3B. Indeed, since several previous studies failed to detect any Sema3B repulsion and collapse on pre-crossing commissural axons using a panoply of different paradigms 15,34,38,43,47 it could be that Sema3B exerts 2 distinct and independent effects on commissural axons, acting as a growth regulator at the pre-crossing stage and a repulsive cue at the post-crossing stage.

How the growth-inhibition effect is achieved and whether it is mediated by PlexinA1, as suggested by the authors, remains to be determined. It could be that the precrossing sensitivity of commissural axons to Sema3B reported by the authors is mediated by other PlexinAs, several being expressed by commissural growth cones 34 This would be consistent with previous findings that the repulsive post-crossing response is conferred by a dual mechanism which first prevents PlexinA1 to be available at the growth cone surface before the crossing and second triggers cell surface expression when commissural axons navigate the FP. It could also be that low levels of PlexinA1 present in precrossing commissural growth cones are sufficient for Sema3B to elicit a long-term growth response, but not to produce a repulsive effect. The implication of PlexinA1 was suggested by immunohistochemistry with home-made antibody directed against an C-terminal epitope of PlexinA1, which was observed to label pre-crossing commissural axons, 23 while a commercial antibody directed against the extracellular PlexinA1 domain only revealed substantial PlexinA1 levels in crossing and post-crossing axons. 10 Since PlexinA1 cleaved fragments were found present lysates from dorsal commissural tissue, 34 it could be that these Plex-inA1 forms are those recognized in the pre-crossing commissural axons. Whatever the case, PlexinA1 contribution could be addressed by blocking PlexinA1 in commissural explants to examine whether long term Sema3B exposure still elicits growth-inhibition.

Beyond, is this growth inhibition acting at the pre-crossing stage or is it silenced as is Sema3B repulsion? In their study, the authors reported that deletion of Nrp2 in the FP affects the commissure formation. Measures of the ventral part of the pre-crossing tract and the crossing tract in embryonic transverse sections showed reduced thickness in the mutants compared with wild-types, suggesting that FP-Nrp2 has a non-cell autonomous role. Whether this defect results from loss of fibers, growth delay or increased fasciculation remains to be determined. The reduction of commissure thickness was no longer observed when FP-Nrp2 deletion was combined with a general loss of PlexinA1. In the scenario proposed by the authors, Nrp2 would trap Sema3B in the FP, thus silencing Sema3B by making it inaccessible to commissural axons. FP-Nrp2 deletion would then result in Sema3B release, inducing pre-crossing growth inhibitory effect and subsequently reduction of the ventral commissure (Fig. 4A). Additional ablation of PlexinA1 would then induce pre-crossing commissural axons to loose their sensitivity to Sema3B, thus rescuing the normal size of the commissure.

According to this model, Sema3B-mediated growth inhibition would not play an instructive role at the precrossing stage and needs to be suppressed. The transition toward sensitivity to Sema3B would not be triggered by changing of Sema3B responsiveness between the pre-crossing and the post-crossing stages. Rather it would be achieved through unmasking of Sema3B after the crossing, proposed by the authors to result from downregulation of Nrp2 transcripts. Nevertheless, Nrp2 is detected in the FP over the entire period of FP navigation (from E10 to E13.5 on the least; 7,19,34 Hernandez-Enriques et al, 2015), during which asynchronous waves of commissural axons navigate the FP. Thus without any changing between pre-crossing and post-crossing stages, it is difficult to understand how this sole mechanism would be responsible for switching on Sema3B repulsion. Moreover, interpretations of the mouse phenotypes are particularly complex. Indeed, first, not only Nrp2 but also PlexinA1 is expressed by both commissural axons and FP glial cells. Second as shown in the chick embryo for PlexinA2, 1 FP-PlexinA1 could have non cell autonomous functions. Thus, a key experiment would be to test which of commissural-or FP-specific PlexinA1 deletion rescues the ventral commissure thickness in context of FP-Nrp2 deletion.

An alternative scenario can be proposed, which would fully mirror the mechanisms of Slit silencing by Robo/Robo2 interactions in the drosophila context. 19 Rather than trapping Sema3B, FP-Nrp2 (complexed or not with PlexinA1) could interact in trans with Nrp2 on commissural axons approaching and entering the FP. This receptor trapping would silence Sema3B responsiveness until the crossing is accomplished by preventing axonal Nrp2 from forming cis complexes with the PlexinA1 receptor pool accumulating at the cell surface (Fig. 4B). FP-Nrp2 ablation would prevent this effect, resulting in premature action of Sema3B. Loss of PlexinA1 would desensitize commissural axons to Sema3B, thus rescuing the commissure. In this scenario, both cell autonomous (axonal PlexinA1 processing) and non-cell autonomous (via FP-Nrp2 and possibility FP-PlexinA1) would act in synergy to accurately control the silencing of pre-crossing/crossing commissural axon responsiveness to Sema3B.

Finally, the outcome of Sema3B trapping by Nrp2 might not be to mask Sema3B but rather to control its spatial distribution, restricting the cue to the FP, where it could be active to slow down commissural axon growth. Previous work already established that cues released by the FP regulate the outgrowth of commissural axons. Such a property was reported for the Stem Cell Factor (SCF), which promotes the growth of post-crossing commissural axons. 21 Thus a balance of growth-promoting and growth-inhibitory effects could set a precise temporal pattern of FP navigation, adapting growth cone motility to the guidance decisions that have to be made.

Mechanisms controlling the transition from precrossing silencing to post-crossing sensitization to Slit and Sema3B repellents

The mechanisms controlling the switch of sensitivity to midline repellents after the crossing also appear to be highly diverse, depending on the signaling and the species (Fig. 5). In drosophila, down-regulation of COMM allows Robo to accumulate at the cell surface of commissural growth cones, resulting in the gain of sensitivity to Slit. 20 Studies conducted in the chick model revealed that Robo1 cell surface expression is up-regulated between the pre-crossing and post-crossing navigation, through transcriptional control of RabGDI, a key component of the exocytosis machinery. 39 Although different, these 2 mechanisms have in common that they control the temporal activity of Slit-Robo signaling at the midline by regulating guidance receptor cell surface levels. In the mouse, transition from Robo3.1 at the pre-crossing stage to Robo3.2 at the post-crossing stage was proposed to switch on the sensitivity to Slit repellents. 11 this transition is accomplished has been partially resolved by the findings that Robo3.2 mRNA is locally translated in crossing commissural axons, under the action of FP signals. 12 The release of Sema3B silencing has been investigated in the mouse model, with the goal to identify cues present in the FP conditioned medium which conferred a collapse response of commissural growth cones to Sema3B. Two FP cues were identified acting in synergy, the Ig SuperFamily Cell Adhesion Molecule NrCAM, probably released by ectodomain shedding, and the neurotrophic factor GDNF, which was found to provide the major contribution. 10 Double GDNF/NrCAM deletion in mice resulted in strong alteration of PlexinA1 levels in crossing/post-crossing commissural axons, with synergistic effects compared with the single knockouts. Reductions of PlexinA1 levels were also correlated with FP crossing defects. In co-cultures of In the mouse, upon the crossing, GDNF secreted by FP glial cells inhibits Calpain activity and allows PlexinA1 to reach the surface, to associate with Nrp2 and to mediate Sema3B repulsive response. Gain of cell surface PlexinA1 also switches on repulsion by Slit-C fragments. (c) In the chick, the pre-crossing PlexinA2/Sema6B cis complex is released, and replaced by a Sema6B/PlexinA2 trans interaction, releasing commissural PlexinA2 which become available for semaphorin repulsion. PlexinA4 might be sorted at the cell surface to also mediate semaphorin repulsion.

dorsal spinal cord explants with COS cell aggregates, commissural axons were to gain repulsion to Sema3B when the cell aggregate secreted both Sema3B and GDNF, compared to aggregate only secreting Sema3B. Both GDNF and NrCAM increased PlexinA1 levels in the growth cones of cultured commissural neurons. GDNF acting independently from RET via the NCAM receptor and GFRa1, both expressed by commissural axons, was able to inhibit calpain activity, switching off the mechanism ensuring Sema3B silencing. An additional FP cue, SHH, was reported to trigger gain of sensitivity to Sema3B, by down-regulating the activity of cAMP-dependent protein kinase A (PKA) in commissural growth cones. 38 Thus overall, these studies support that release from the pre-crossing Sema3B silencing is triggered by FP cues, through changes of the guidance machinery of commissural axons. 10,34 In the chick, the model proposed is that the PlexinA2/ Sema6B cis complex formed in pre-crossing commissural axons would be released, allowing FP-PlexinA2 to bind to Sema6B in trans, and commissural PlexinA2 to bind to FP semaphorin repellents. In addition, as is the case for PlexinA1 in the mouse, PlexinA4 might be sorted to the growth cones during FP crossing to allow them to sense the semaphorin repellents. 1 Thus in conclusion, given the diversity of possibilities by which the semaphorin signaling can be modulated, significant issues remain unclear. In particular, it will be important to better characterize the dynamics of Plexins, Neuropilin receptors and their semaphorin ligands as well as their cell-autonomous and non-cell autonomous functions. Addressing these questions is required to obtain a clear picture of how the silencing of midline repellents is achieved and released during spinal commissural axon navigation of the FP.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Introduction

Early theories of chemotropism and chemoaffinity by Ramon Y Cajal and Sperry provided the basis for more than a century of research on axon guidance mechanisms [1,2]. These theories pos- on growth cones [3]. The property of growth cone structures to retract after contact with some other axonal membrane surfaces was then discovered by Kapfhammer and Raper [4]. These observations echoed those of Verna, who wrote that dorsal root sensory neurons "interact differently with dermal than with epidermal cells. While nerve fibres readily extend over dermal cells, forming close membrane associations with some of them, they demonstrate a strong avoidance reaction with epidermal cells by changing their direction of extension" [5]. He postulated that molecules released by epidermal cells might deflect away nerve fibre growth trajectory, oppositely to those which were found to attract the axons, namely at that time, the neurotrophic factor NGF [6]. From these pioneer findings, repulsive forces have been demonstrated to play instrumental roles in a large range of developing neuronal circuits. As evidenced by numerous studies [7,8], repulsive forces can constrain axon navigation in various ways, channelling axonal bundles, deflecting away the growth cone trajectory, and creating sharp boundaries to delineate non-permissive territories (Fig. 1). As in a pinball, repulsive forces would act as launch pad, bumpers, and slingshots to propel and dynamically impact on the axon/ball trajectory. The development of commissural axons provides an appealing context to investigate how such repulsive forces can direct axon navigation. We review here the principal yet identified sources of repulsive cues, the nature of their influences and the molecular signals mediating their action during commissural axon navigation in the spinal cord.

Formation of commissural circuits

In Bilateria, commissural neurons form complex circuits that interconnect both sides of the central nervous system (CNS). They are essential for the correct processing and coordination of various sensory modalities, motor responses, and other brain functions [9]. These interneurons extend their axon across the midline at various axial levels of the CNS. For instance, the corpus callosum enables communication between the left and right cortical areas, the optic chiasm allows organisms with bilateral vision to correctly integrate visual cues, and spinal commissures ensure the correct coordination of various motor commands. These commissures are established during embryonic and early post-natal development in a highly specific spatial and temporal manner [10]. Defects in the correct wiring of commissural circuits have been observed in many neurodevelopmental disorders. However, if malformations of the corpus callosum have been well correlated with various human disorders, little is known of the consequences of spinal commissures defects. Indeed, patients having mutations in ROBO3 gene, affecting commissures of the hindbrain and the spinal cord, have no large sensorimotor deficit. Rather, they exhibit a very specific disease referred to as horizontal gaze palsy with progressive scoliosis (HGPPS) [11]. This suggests high degree of compensation of commissural defects with developmental origin.

Development of the dorsal commissural tract

The spinal commissural neurons are a heterogeneous population subdivided in several pools, differing in their location and timing of birth, each of them specified by various transcription factors [12,13]. Among them, dI1 interneurons settle early in the most dorsal part of the spinal cord, close to the Roof Plate (RP). They arise from a MATH1-positive pool of progenitors, that generates both ipsilateral and commissural lineages and are specified by LHX2/LHX9 transcription factors [14,15]. dI1 commissural (dI1c) neurons trajectory is highly stereotyped and has been extensively studied in the mouse, notably by using MATH1::LacZ and MATH1::GFP transgenic mice [16]. dI1c axons first extend ventrally, turning away from the RP and laying close to the pial surface (Fig. 2). At around mid-distance of the ventral border they break away from the lateral border to re-orient medially towards the central Floor Plate (FP) by running along the motoneuron domain. Such break of trajectory is also typical of chick commissural axons, apart from the pioneer ones which course with circumferential trajectory. In contrast, in xenopus and zebrafish embryos, the axons course by following the circumference of the tube until reaching the FP [17,18]. Next, commissural axons enter the FP, cross it and turn rostrally without ever crossing the midline again to connect their final targets. Commissural neurons arise around E9.5 in the mouse and navigate towards the FP from this stage. By E10.5, some of them have already crossed the midline and by E12.5, most of them have. By E13.5, they are navigating towards their final target following longitudinal routes [19][20][21].

Guidepost territories instructing commissural axon navigation in the spinal cord through repulsive action

Historically, the main intermediate target and crucial signaling hub for commissural axon navigation has been found to be the FP. Spinal commissural axon trajectory is highly stereotyped. dI1c axons (in black) arise from a dorsal territory. They extend ventrally, cross the midline through the floor plate (FP), and then turn rostrally without ever crossing the midline again to connect their final target. This trajectory is influenced by various guidepost cells along the way. First, axons are pushed away by chemorepulsive cues emanating from the roof plate (RP) (in blue) and follow the pial surface (in green). Meninges (in grey) and the DREZ/DRBZ (in red) keep the axons away from the CNS/PNS boundary through diffusible cues. At around mid-distance from the ventral side, axons turn towards the FP (in purple), then run along the motoneurons domain (in red). Axons never enter the motoneuron domain nor the ventricular zone (in light green), these territories channeling the commissural tract towards the FP. As they reach the FP, axons interact with the basal lamina (in green) and navigate through the FP glial cells radial processes. Upon crossing, they gain sensitivity to repulsive cues emanating from the FP, that they did not perceive before and thus exit the FP, accomplish a sharp turning in the rostral direction and navigate longitudinally in bundles, guided by various gradients of guidance cues, including repulsive ones.

FP cells as guidepost cells. The FP has been extensively studied and many of its molecular mechanisms have been unveiled. However, a variety of other cells within the spinal cord bring contributions to the navigation of commissural axons, including glial cells, neurons and progenitors. These different cell-types contribute together to sharply delineate the path of commissural neurons.

1.2.1. Kick off repulsive forces to orient commissural axon navigation 1.2.1.1. The roof plate. The RP is probably the second most studied group of guidepost cells after the FP. It is composed of glial cells that lay on the dorsal midline of the spinal cord. These cells come from progenitors that are induced in the most lateral regions of the neural folds [22]. This induction relies heavily on BMP signalling, mediated by the transcription factors LMX1A/B [23]. Upon neural tube closure, they are not easily distinguishable from other cells, in particular neural crest cells, but as they differentiate they start to express specific markers, notably BMPs and WNTs [22]. The RP is the first dorsal structure to differentiate and then impacts all other dorsal populations differentiation. Electro-microscopy studies revealed that, when differentiated, RP cells have two small processes extending radially towards the pial surface and the central canal [24,25]. The RP acts as a barrier that no axon can cross before E16.5, when a dorsal commissure is established [26]. Interestingly, the RP itself undergoes rather important morphological changes between E11.5 and E16.5, from an arch structure to a thin wall-like structure [25]. The RP is a crucial organizing centre of the different dorsal lineages. Notably through BMPs and WNTs, it specifies several classes of adjacent dorsal interneurons and regulates their proliferation, migration, and guidance [27].

Beyond this patterning function, RP cells provide the driving force which propels dl1c axons emerging from their soma away from the dorsal side. This effect was shown to be mediated by the morphogens BMPs, namely by GDF7:BMP7 heterodimers acting via the BMPRIB receptor [28,29] (Fig. 3). Not only their direction but their growth rate also appears regulated by BMPs [30]. An additional repellent protein was found produced by the RP, a secreted factor named Draxin. Draxin mutant mice display commissural axonal migration and fasciculation defects consistent with a repulsive mode of action [31]. Draxin shares UNC5, DCC, and Neogenin receptors with Netrin-1, a secreted molecule initially discovered to act as a chemoattractant [32,33]. Although Draxin has been shown to bind UNC5 and Neogenin in vitro [34], its repellent role in vivo was reported to be triggered via its binding to DCC [35] (Fig. 3). It can also be noted that the repulsive factor Slit2 is highly expressed at the RP at E13 [36]. Most dorsal commissural axons are already on their way in the contralateral side at this stage, whether this source contributes to the kick off of commissural axons is therefore questionable.

Repulsive forces to confine commissural axon navigation in the central nervous system

Commissural axons are destined to connect neurons of the central nervous system (CNS) and must be consequently strictly confined within the spinal cord. This is not true for all spinal cord axons, since on the contrary, those of the motoneurons project out of the CNS through the Motor Exit Point (MET). Moreover, in this case, only the axons exit the CNS, the neuronal soma being confined within the CNS. Conversely, sensory axons of the dorsal root ganglia penetrate the spinal cord via the Dorsal Root Entry Zone (DREZ), while their soma remain outside. In contrast, some non-neuronal cells enter the CNS, such as endothelial cells which infiltrate the CNS tissue to build the blood vessels. Thus, cells and neurites trafficking across the CNS/PNS frontier is strictly controlled, from the onset and throughout life.

1.2.2.1. Confinement by the meninges. Meninges are a protective multi-layered structure that envelops the brain and the spinal cord. They are mainly composed of fibroelastic cells and blood vessels. Meninges originate from somatic mesoderm that covers the neural tube shortly after neural tube closure, around E9 in the mouse embryo [37,38]. They act as barriers throughout life, controlling exchanges between the central nervous system and what lays outside. In the brain, meninges have been shown to initiate a morphogenic signaling cascade that regulates the development of a major dorsal commissure, the corpus callosum. The meninges inhibit callosal axon outgrowth through BMP7. WNT3, expressed by pioneer callosal axons, later counters this effect. WNT3 expression is regulated by another member of the BMP family, GDF5, expressed by adjacent Cajal-Retzius neurons, which in turn is regulated by a soluble inhibitor, DAN, expressed by the meninges [37]. In the spinal cord, in vivo studies lack to highlight the role of the meninges on commissural neuron development. However, a recent in vitro study showed that the meninges are able to produce secreted cues that can either attract or repulse different neuronal populations. Consistent with in vivo behaviours, these experiences showed that motoneurons and sensory neurons are attracted by meninges while ipsilateral and commissural neurons are repelled by them [38]. 1.2.2.2. Closing the CNS/PNS gate: confinement by the dorsal root entry zone (DREZ)/ dorsal root bifurcation zone (DRBZ). Commissural axons navigate at close proximity of the pia. Early studies established that their growth cones rarely, if ever, contact the basal lamina (Holley & Silver 1987, Yaginuma et al 1991). The basal lamina is punctuated by the DREZ and the MET, which ensure the circulation between the central Nervous System (CNS) and the surrounding tissues. The DREZ consists of a break in the lamina and a cluster of specialized cells arising from the neural crest, the boundary cap cells, which prevent both cell bodies and their axons from leaving the CNS, and gaps between the glial end-feet [39]. Around E11, the peripheral sensory neurons send axons towards the spinal cord through the DREZ and start to form the dorsal root bifurcation zone (DRBZ), where they project in an anterior-posterior direction within the tract [40]. Therefore, the DRBZ is in direct apposition to the DREZ. Some cues released from these gates to confine commissural axons have been identified. Netrin-1 is expressed at the border of the DRBZ between E11.5 and E12.5 [34]. In Netrin-1 mutant embryos, commissural axons invade the DREZ and DRBZ. The presence of ectopic axons can even be detected in the dorsal root ganglia (DRG) [34]. This study thus indicates that Netrin-1 participates in forming an inhibitory boundary at the border of the DRBZ and the DREZ [34]. The nature of the Netrin receptors mediating these effects has also been investigated. Ectopic axons were detected in the DRBZ of both DCC and UNC5C mutant embryos, while only in DCC mutant was their presence observed in the DREZ, suggesting a differential contribution of these receptors [34] (Fig. 4A). Other receptors could also potentially transduce a Netrin-1 signal. Down's syndrome Cell Adhesion Molecule (DSCAM), whose expression is high in the DREZ and in commissural axons, was indeed shown to modulate Netrin-1 attractive response with or without DCC [35] (Fig. 4A). In the chick which lacks DCC [41], a candidate could be Neogenin, a Netrin-1 receptor which is expressed in some commissural axons [42] (Fig. 4A). The mode of action of Netrin-1 remains unclear. A simple view would be that it acts as a repellent in this context. Moreover, it should be noted that Netrin-1 is not detected in the dorsolateral region of the spinal cord prior to E11.5 whereas ectopic axons are already observed at E10.5 in the DRBZ of Netrin-1 and DCC mutant embryos [34]. Thus, other Netrin sources might act to confine the axons at the early stages, whose release in the mutants result in their ectopic position in the DRBZ. Additional repulsive forces might also be involved in this confinement. Draxin, transducing repulsive signals via axonal DCC, is namely also expressed at the dorsal pial surface and in the DREZ [27] (Fig. 4A).

Furthermore, the role of extracellular matrix (ECM) components and glycoproteins in the confinement of axons along the pial surface might be essential. Laminin is present all along the pial surface in close contact with the commissural axons during their navigation [43]. Type IV Collagen and Heparan sulfate proteoglycans are also components of the basement membrane and their deposition is spatially and temporally controlled in coordination with morphogenesis [44]. Along this line, the glycoprotein Dystroglycan, an important scaffold for ECM proteins including Laminin, has been shown to interact with Slit and this interaction is detected all along the pial surface [43]. In the visual system, Laminin has been shown to modulate the attractive response of retinal axons to Netrin-1 by turning it into a repulsive signal [45]. Thus, co-expression of Netrin-1 and Laminin could, as well, contribute to set the pia as a repulsive barrier, explaining the observed lack of contacts between the basal lamina and commissural growth cones (Fig. 4A).

Repulsive forces to channel commissural path

Channelling of axon tracts can be achieved by dual lateral repulsive sources constraining their growth in between. Once reaching the half ventral half of the spinal cord towards the FP, commissural axons modify their initial circumferential trajectory and re-orient medially towards the FP, navigating at the border of the ventral motoneuron domain. A triad of three territories, the ventricular zone, the basal lamina, and the motoneuron domain, might thus act in synergy to channel the pre-crossing path of commissural axons in between these different territories.

1.2.3.1. The ventricular zone. The ventricular zone (VZ) is composed of the neuronal progenitors, laying the central canal. The different populations of progenitors are specified by a combinatorial code of transcriptional factors [12]. These factors are activated by opposing gradients of BMP/WNT and SHH emanating respectively from the RP and the FP [12]. Neural progenitors of the CNS have a bipolar morphology, extending two processes, one connecting the pial surface and the other connecting the central canal [46]. During the cell cycle, their nuclei oscillate between the apical and basal pole, a process referred to as the interkinetic nuclear migration. Postmitotic neurons born from neurogenic divisions detach their apical anchor and migrate laterally to establish themselves in the mantle. Strikingly, dI1c axons navigate at the VZ/mantle interface but never enter the VZ. The mechanisms underlying this navigation choice are unclear. The Netrin-1 attractant produced by the progenitors and transported via their basal process for lateral deposition at the basal lamina was recently proposed to direct commissural axon growth out of the VZ and close to the pia [47]. Interestingly, releasing the lateral deposition of this cue by specific deletion of Netrin-1 in progenitors induced some commissural axons, identified by their ROBO3 expression, to invade the VZ [47]. Nevertheless, some others, expressing the commissural marker TAG1, were still constrained out of the VZ [47]. A tempting model is that some repulsive forces also directly emanate from the VZ (Fig. 4B). Indeed, such a mechanism was reported to prevent cortical and thalamic axons from invading the proliferative regions of the developing brain [START_REF] Leyva-Díaz | In and out from the cortex: development of major forebrain connections[END_REF]. This would be consistent with reported expressions of transcripts encoding guidance molecules bearing repulsive activity, such as the Semaphorins and the Eph/Ephrins in mouse and chick spinal cord VZ [START_REF] Püschel | The sensory innervation of mouse spinal[END_REF][START_REF] Moret | Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding[END_REF][START_REF] Sanyas | Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins[END_REF][START_REF] Arbeille | Cerebrospinal fluid-derived Semaphorin3B orients neuroepithelial cell divisions in the apicobasal axis[END_REF][START_REF] Laussu | Eph/ephrin signaling controls progenitor identities in the ventral spinal cord[END_REF].

1.2.3.2. The motoneuron domain. Motoneurons arise at around E9.5 in the mouse embryo, from a pool of ventral progenitors. They are specified by a set of homeodomain transcription factors (notably HB9, LHX3, ISL2, and ISL3) [START_REF] Dasen | Hox Networks and the Origins of Motor Neuron Diversity[END_REF]. Post-mitotic motoneurons migrate out of the VZ in streams, and cluster at various medio-lateral levels of the mantle to form distinct and adjacent pools. It is noticeable that commissural axons break their circumferential path when they reach the emerging mass of motoneurons accumulating in the ventral horn. Nevertheless, whether this reflects an instructive role of this territory in the reorientation of commissural axons is unclear. Indeed, in mouse and chick embryos in which the FP has been genetically or experimentally ablated, commissural axons no longer break their circumferential trajectory, reaching the FP through a path that resembles that of xenopus and zebrafish embryos, all along the pial surface. However, ablating the FP and/or the notochord in these experimental contexts, simultaneously prevented the specification of motoneurons, which were thus also lacking [START_REF] Bovolenta | Perturbation of neuronal differentiation and axon guidance in the spinal cord of mouse embryos lacking a floor plate: analysis of Danforth's short-tail mutation[END_REF][START_REF] Yamada | Control of cell pattern in the developing nervous system: polarizing activity of the floor plate and notochord[END_REF]. Interestingly, motoneurons express various guidance molecules that could define this territory as non-permissive for commissural axons, such as the Slits and the Semaphorins (Fig. 4B). A recent study features NELL2, expressed mainly in the motor columns, as mediating such a repulsive action of the motoneuron domain [START_REF] Jaworski | Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2[END_REF]. In vitro, NELL2 could repel commissural axons and this effect was found exerted via ROBO3. An in vivo contribution of signaling is suggested by the analysis of NELL2 -/-ROBO3 -/-embryos, in which many commissural axons were observed to defasciculate and invade the motor columns [START_REF] Jaworski | Operational redundancy in axon guidance through the multifunctional receptor Robo3 and its ligand NELL2[END_REF].

The Slit/ROBO signaling pathway has been shown to be essential for the maintenance of boundaries, compartmentalizing the visual centres in the Drosophila brain [START_REF] Tayler | Compartmentalization of visual centers in the Drosophila brain requires Slit and Robo proteins[END_REF]. Slits are interesting candidates to consider in this channelling. Slit1/2 mRNAs are produced by both spinal progenitors and motoneurons [36]. The Semaphorin3F (SEMA3F) is also highly detected in the motoneuron domain and its repulsive action on spinal commissural axons has been evidenced in vitro, although it was reported to concern post-crossing rather than pre-crossing axons [START_REF] Zou | Squeezing axons out of the gray matter: a role for Slit and semaphorin proteins from midline and ventral spinal cord[END_REF]. Several other members of the Semaphorin family are indeed expressed both by motoneurons and progenitors. In the chick embryo, this is for example the case of SEMA3C and SEMA3A [START_REF] Moret | Semaphorin and neuropilin co-expression in motoneurons sets axon sensitivity to environmental semaphorin sources during motor axon pathfinding[END_REF][START_REF] Sanyas | Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins[END_REF]. Specific deletion of these candidates in progenitors and motoneurons would be highly informative to address their contribution to the channelling of commissural axons.

Travelling a repulsive field: navigation across the midline in the floor plate

Being a prototypical example of intermediate target for long distance connections, the FP has been, by far, the most studied group of guidepost cells. The FP is composed of glial cells that lay ventrally at the midline of the embryo. Though discrepancies exist regarding its exact developmental origin and the signaling pathways involved in its specification between species, the FP influences neuronal differentiation and axon pathfinding in the spinal cord of all vertebrates. In the mouse, SHH through GLI2 and then FOXA2 is a key factor in FP induction [START_REF] Strähle | Vertebrate floor-plate specification: variations on common themes[END_REF][START_REF] Placzek | The floor plate: multiple cells, multiple signals[END_REF][START_REF] Ribes | Distinct sonic hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube[END_REF]. FP cells display a morphology that resembles the one of RP cells. They possess 2 processes that extend radially, the shorter one towards the central canal and the longer one towards the basal lamina [24,[START_REF] Campbell | Expression of a lacZ transgene reveals floor plate cell morphology and macromolecular transfer to commissural axons[END_REF]. The FP is a unique group of guidepost cells for several reasons. First, up to now, it is the only one the commissural axons go through, instead of just passing by. Indeed, commissural axons enter the FP and then navigate through a meshing of FP cells basal processes, hugging the basal lamina [START_REF] Yaginuma | Pathfinding by growth cones of commissural interneurons in the chick embryo spinal cord: a light and electron microscopic study[END_REF]. FP cells and commissural axons are known to establish close contacts [START_REF] Okabe | Axonin-1, Nr-CAM, and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons[END_REF]. Interestingly, it is probably the territory in which the dI1c axons form the most compact bundle during their navigation. Second, the FP is not only a group of guidepost cells but also an intermediate and not a final target. Thus, intermediate target must combine the properties of a target (a territory that the axons select to grow within) and a non-target (a territory in which the axons do not stop). Studies of FP crossing helped resolving some aspects of this apparent paradox, by bringing to the scheme the notion of temporality. Indeed, temporal regulation of axon sensitivity to the guidance cues emanating from the intermediate target is the key to endow it first, with the properties of a target and second, with those of a non-target.

Signals conferring to the FP the properties of a target tissue are the cues that are perceived first by commissural axons on their way for midline crossing. These signals are thought to combine both short-range and long range attractive/promoting effects. Cell adhesion molecules play a crucial role at a short-range level. Commissural axons and FP glial cells engage in dynamic and complex cis and trans interactions via various Cell Adhesion Molecules of the IgSuperfamily such as L1/NgCam, NrCam, and TAG1 [66-69]. Long range attraction by Netrins, and additionally SHH and VEGF, have been thought to provide major signals guiding commissural axons towards the FP [33,[START_REF] Serafini | Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system[END_REF][START_REF] Charron | The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance[END_REF][START_REF] De Almodovar | VEGF mediates commissural axon chemoattraction through its receptor Flk1[END_REF]. This view has been recently revisited by studies demonstrating that Netrin-1 might indeed rather act much more locally and, as described in the above paragraphs, from spinal cord sources other than the FP, essentially neural progenitors [47,[START_REF] Dominici | Floor-plate-derived netrin-1 is dispensable for commissural axon guidance[END_REF]. Once the midline crossed, the FP must acquire the properties of non-target territory, to prevent axons to stall and terminate their navigation. This process, which has been the focus of recent reviews [START_REF] Pignata | Commissural axon navigation: control of midline crossing in the vertebrate spinal cord by the semaphorin 3B signaling[END_REF][START_REF] Neuhaus-Follini | Crossing the embryonic midline: molecular mechanisms regulating axon responsiveness at an intermediate target[END_REF], appeared from several works to be achieved, not by expressing novel molecules with repulsive activity in the FP, but rather by sensitizing commissural axons to repulsive cues yet present but that had been ignored until midline crossing. The temporality of the target to non-target switch is crucial. Prematurely releasing the sensitivity to the repellents would be disastrous, as it would block commissural axons entry in the FP and midline crossing. The mechanisms controlling the switch have started to be decrypted in the recent years and turn out to be highly complex. A broad panoply of transcriptional and post-translational mechanisms is indeed deployed to first silence the perception of the repellents and second to release this silencing and set the repulsive commissural response. In parallel, the properties that made initially the FP as a target -i.e. attractive cues-have to be shut-down. Shirasaki and collaborators demonstrated twenty years ago that this is indeed occurring. In ex vivo assays, grafting an ectopic FP close to commissural axons coursing towards their endogenous FP induced their re-orientation towards the ectopic FP. In contrast, exposing commissural axons which have already crossed the endogenous FP to ectopic FP had no effect [START_REF] Shirasaki | Change in chemoattractant responsiveness of developing axons at an intermediate target[END_REF]. It was subsequently proposed that Slit signaling blunts Netrin-1 attraction. cis interaction between ROBO and DCC was reported to silence Netrin-1 attractive signaling [START_REF] Stein | Hierarchical organization of guidance receptors: silencing of netrin attraction by slit through a Robo/DCC receptor complex[END_REF]. In parallel, SHH has been proposed to be sequestered by its FP specific receptor HHIP (Hedgehog Interacting Protein) to turn off its attractive function [START_REF] Bourikas | Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord[END_REF].

1.2.4.1. The molecular players mediating repulsive forces. Repulsion involves several couples of ligands/receptors (Fig. 5). The secreted Semaphorin 3B (SEMA3B) mediates repulsion at the midline by activating a complex formed of NEUROPILIN 2 (NP2) and PLEX-INA1 (PLEXA1) receptors [START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]. In addition, the membrane-bound SEMA6B is expressed by commissural axons when they cross the FP and was reported to interact with PLEXA2 expressed by FP glial cells [START_REF] Andermatt | Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance[END_REF]. Slit proteins are produced by FP cells and are submitted to proteolytic processing through yet unidentified protease(s). This cleavage releases N-terminal Slit fragment (SlitN), which binds to ROBO receptors, and C-terminal Slit (SlitC) which binds to PLEXA1 [START_REF] Delloye-Bourgeois | PlexinA1 is a new Slit receptor and mediates axon guidance function of Slit C-terminal fragments[END_REF]. B-class Ephrins can function as ligands or receptors, mediating forward or reverse signaling respectively [START_REF] Cowan | Ephrins in reverse, park and drive[END_REF]. In the FP, midline glial ephrin-B3 interacts with commissural Eph-B3 receptors [START_REF] Kadison | EphB receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord[END_REF]. Inhibitory effects of Nogo are carried out through the interaction to the Nogo receptor complex [START_REF] Domeniconi | Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth[END_REF][START_REF] Wang | p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp[END_REF]. Nogo is expressed by radial glia at the ventral midline of the spinal cord and Nogo receptors (NogoR) are detected in commissural axons extending through the FP [START_REF] Wang | Localization of an axon growth inhibitory molecule Nogo and its receptor in the spinal cord of mouse embryos[END_REF]. Recently, blocking NogoR was found to result in axon stalling at the FP and therefore in a reduction of the number of commissural fibers properly reaching the contralateral side of the spinal cord. Furthermore, it has been reported that the ligand interacting with NogoR is a truncated form of Nogo released by glial cells [START_REF] Wang | Nogo-B is the major form of Nogo at the floor plate and likely mediates crossing of commissural axons in the mouse spinal cord[END_REF].

1.2.4.2. Temporal regulation of commissural axon sensitivity to the repellents. Pioneer studies performed in Drosophila revealed that ROBO protein is degraded in pre-crossing commissural axons by the endosomal protein COMM [START_REF] Keleman | Comm sorts Robo to control axon guidance at the Drosophila midline[END_REF][START_REF] Keleman | Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo[END_REF]. Yet, ROBO mutant phenotype is rescued by a mutated version of ROBO that cannot be sorted by COMM. COMM thus probably regulates Slit/ROBO interaction through an additional, sorting-independent mechanism [START_REF] Gilestro | Redundant mechanisms for regulation of midline crossing in Drosophila[END_REF]. ROBO2 is also expressed by midline glial cells and has been shown to interact with ROBO1 in trans. This interaction would occur upon crossing, when ROBO1 receptors reach the cell surface consequently of COMM suppression and it would repress Slit repulsion until the crossing is completed [START_REF] Evans | Robo2 acts in trans to inhibit Slit-Robo1 repulsion in pre-crossing commissural axons[END_REF].

How are ROBO distribution and activity regulated in time are therefore key questions to further understand the navigation of the midline. COMM expression was shown to be controlled at least in part by Frazzled/DCC in pre-crossing [START_REF] Yang | A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance[END_REF], while it remains unknown how it is repressed after midline crossing. The nature of mechanisms silencing Slit repulsion before the crossing has been thought to totally differ in vertebrates since no COMM homolog was found in their genomes. Nevertheless, vesicular trafficking also appears as an important process in vertebrates. Indeed, vesicles containing ROBO1 were observed in commissural axons. Calsyn-tenin1 and RabGDI were found to regulate their trafficking and to allow the pulse exposure of ROBO1 at the growth cone surface [START_REF] Alther | Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point[END_REF][START_REF] Philipp | RabGDI controls axonal midline crossing by regulating Robo1 surface expression[END_REF]. Interestingly too, a recent study identified PRRG4, a protein which displays structural similarities with COMM, as capable of relocalizing ROBO1 at the cell surface in vitro [START_REF] Justice | The WAGR syndrome gene PRRG4 is a functional homologue of the commissureless axon guidance gene[END_REF]. Another reported regulator of Slit/ROBO signaling is an isoform of ROBO3, a divergent member of the ROBO family, ROBO3.1. Its distribution is restricted to the pre-crossing commissural axons and it is thought to facilitate midline crossing by antagonizing Slit/ROBO1/2 mediated repulsion [21]. The underlying mechanism is not yet known. It might not involve ROBO3 as a Slit receptor since ROBO3 was shown to have lost its affinity to bind Slit ligands with evolution, instead rather acting as a DCC co-receptor for NETRIN-1 [START_REF] Zelina | Signaling switch of the axon guidance receptor Robo3 during vertebrate evolution[END_REF]. Thus, progress as yet to be accomplished to better understand how the regulators of ROBO receptors are temporally controlled and their activity synchronized with midline crossing. Similarly, axon sensitivity to Semaphorin repulsive signaling is tightly controlled and PLEXA1 sorting at the axon surface in the FP is thought to be a key event switching on the sensitivity of commissural axons to SEMA3B. We indeed found that PLEXA1 is processed by Calpains in pre-crossing axons. This processing prevents PLEXINA1 cell surface sorting, and thus the association with NP2 co-receptor needed to transduce SEMA3B signaling [START_REF] Nawabi | A midline switch of receptor processing regulates commissural axon guidance in vertebrates[END_REF]. We also found that this silencing of SEMA3B responsiveness is released by local FP cues, GDNF and NrCAM, which trigger PLEXINA1 cell surface sorting by inhibiting Calpain activity [START_REF] Charoy | Gdnf activates midline repulsion by Semaphorin3B via NCAM during commissural axon guidance[END_REF]. In addition, SEMA6B and PLEXA2 were also shown to interact in cis at the growth cone surface and to silence the sensitivity of pre-crossing axons to midline-associated SEMA6B [START_REF] Andermatt | Semaphorin 6B acts as a receptor in post-crossing commissural axon guidance[END_REF]. Thus, as it is the case for the ROBO/Slit pathway, post-translational mechanisms appear to be instrumental in regulating the timing of activity of the Semaphorin repellents.

After FP crossing: a new pinball game starts

A second pinball game starts when FP repulsive signals propel commissural axons out of the FP. Concomitantly, a drastic change of direction is accomplished by the dl1c axons which turn in the rostral direction to navigate longitudinally to the FP. Two opposite chemotropic gradients control this guidance choice: a caudal high to rostral low repulsive one shaped by SHH, and a caudal low to rostral high attractive one shaped by WNT. Thus, as during the precrossing navigation game, repulsive forces play an important role in propelling the axons and imprinting the direction of their longitudinal growth. A temporal control of these forces is also needed. SHH has a reported pre-crossing attractive activity, shown to be mediated by SMO and one of its receptors, BOC (Brother of CDO) [START_REF] Charron | The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance[END_REF][START_REF] Okada | Boc is a receptor for sonic hedgehog in the guidance of commissural axons[END_REF][START_REF] Sloan | Integration of shallow gradients of Shh and netrin-1 guides commissural axons[END_REF]. Upon crossing, SHH attraction must be switched into repulsion, a process proposed in the chick to be achieved via the implication of another SHH receptor, HHIP (Hedgehog Interacting Protein), in addition to SMO in the mouse [START_REF] Bourikas | Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord[END_REF][START_REF] Yam | 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing[END_REF] (Fig. 6). The cytoplasmic adaptor protein, 14-3-3, increases in amount during the pre-crossing navigation to culminate at the post-crossing stage, during which it is required for SHH-dependent repulsion [START_REF] Yam | 14-3-3 proteins regulate a cell-intrinsic switch from sonic hedgehog-mediated commissural axon attraction to repulsion after midline crossing[END_REF]. In parallel, the sensitivity of the complementary rostro-caudal chemoattractive gradient of WNT is also switched on upon the crossing. A mechanism was recently reported implicating a molecular cascade during which SHH/SMO downregulates transcript levels of Shisa2 in commissural neurons. Down-regulation of Shisa2 allowed the WNT receptor Frizzled3 to be glycosylated and translocated to the surface of the commissural axon growth cones [START_REF] Onishi | Sonic hedgehog switches on Wnt/planar cell polarity signaling in commissural axon growth cones by reducing levels of Shisa2[END_REF].

Once the rostrocaudal direction is given, commissural axons segregate in several tracts. Reorganizations of post-crossing commissural axons during this step likely implicate selective fasciculation. In xenopus, live monitoring of commissural axons in the spinal cord revealed striking changes in the behaviors of the growth cones during crossing process. In fact, during pre-crossing navigation, growth cones were observed to avoid each others whereas in the post-crossing stage (after their longitudinal turning), they accomplished a series of fasciculation choices which suggest a process of bundle selection [START_REF] Moon | Adjacent pioneer commissural interneuron growth cones switch from contact avoidance to axon fasciculation after midline crossing[END_REF]. Several cell adhesion molecules are up-regulated in post-crossing commissural axons, such as the IgSFCAM L1 in the mouse [START_REF] Imondi | Mis-expression of L1 on pre-crossing spinal commissural axons disrupts pathfinding at the ventral midline[END_REF], which could contribute to this recognition process.

In Drosophila, commissural axons form three longitudinal tracts [START_REF] Evans | Axon guidance at the midline: of mice and flies[END_REF]. Their sorting and their position relative to the midline was shown to be controlled by a combination of ROBO receptors, differing between the tracts and thought to confer them different levels of sensitivity to midline Slit repellent [START_REF] Long | Conserved roles for Slit and Robo proteins in midline commissural axon guidance[END_REF]. A theoretical model predicts that a ROBO code based on quantitative differences of ROBO proteins could be on its own sufficient to generate different lateral tract position [START_REF] Goodhill | A theoretical model of axon guidance by the Robo code[END_REF].

In vertebrates, post-crossing commissural axons are sorted into two main tracts, navigating the ventral and lateral funiculi. The mechanisms underlying this sorting are still elusive. An implication of the Slit-ROBO signaling was reported from the analysis of Slit and ROBO null embryos, in which this sorting is defective [START_REF] Jaworski | Autocrine/juxtaparacrine regulation of axon fasciculation by Slit-Robo signaling[END_REF]. An interplay of Robo and N-cadherin was also found to contribute to the lateral sorting of post-crossing commissural axons [START_REF] Sakai | Axon sorting within the spinal cord marginal zone via Robo-mediated inhibition of N-cadherin controls spinocerebellar tract formation[END_REF]. The Ephrin signaling is also involved in the mediolateral positioning of the longitudinal tracts. Blocking EphB3/EphB signaling was reported to result in a lateral shift of commissural axons [START_REF] Kadison | EphB receptors and ephrin-B3 regulate axon guidance at the ventral midline of the embryonic mouse spinal cord[END_REF][START_REF] Imondi | Commissural axon pathfinding on the contralateral side of the floor plate: a role for B-class ephrins in specifying the dorsoventral position of longitudinally projecting commissural axons[END_REF].

Even though the topography of post-crossing axons differs from that of pre-crossing ones, the longitudinal navigation after midline crossing also appears constrained and channeled by the FP, the lateral basal lamina and the motoneuron domain. Whether the underlying mechanisms and signaling pathways are similar to those operating at the pre-crossing stage remains to be determined. After crossing the floor plate (FP), commissural axons turn rostrally in response to gradients of attractive and repulsive cues. Diffusible Shh from the FP glial cells interacts with the axonal receptor HHIP. In vertebrates, post-crossing commissural axons form two main tracts, the first turns in the ventral funiculus, while the second turns in the lateral funiculus. Slit contributes to this sorting through interaction with axonal ROBO1 and ROBO2. An interplay of ROBO and N-cadherin was also found to contribute to the lateral sorting of post-crossing commissural axons. Trans-interactions also occur between glial Ephrin-B3 (Eph-B3) and axonal Eph-B receptor to assign the medio-lateral position of post-crossing tracts.

Conclusion and perspectives

Important progress has been made over the years to identify components of the molecular machinery controlling axon pathfinding. The versatility of growth cone responsiveness to guidance cues endows the axon with multiple possibilities to interpret in a highly specific manner the topographic signals and the physical elements encountered in the environment during its navigation. Recent tissue-specific deletions of guidance molecules have revealed unexpected contributions of territories surrounding axonal pathways. This provides a better integrated picture, which would require to be further extended, on how axon guidance proceeds in an environment that is also subjected to continuous developmental constraints. In addition, a series of molecular mechanisms has been discovered, which brings to light the importance of spatial and temporal controls of growth cone sensitivity to guidance cues. Altogether, this raises the need of better understanding at protein levels how topographic cues are distributed and deposited in the tissues and how the activity of guidance receptors and downstream effectors in the growth cones is focused and fine-tuned.

In the context of commissural axon guidance, many types of repulsive forces which remain underestimated are likely to play key contributions in the robustness of commissural axon pathfinding. While it is rather straightforward to conceive how a repulsive cue repel away a growth cone, it is much less simple to conceive how commissural growth cone can navigate a field expressing repellents to which it progressively acquires sensitivity. Several theoretical models of axon guidance have been elaborated to explain the building of topographic maps. A recent one was based on the principle that growth cones undergo concentration-dependent alternated choices of repulsive and attractive response to guidance cues [START_REF] Naoki | Revisiting chemoaffinity theory: chemotactic implementation of topographic axonal projection[END_REF]. Interesting insights would come from such models of FP navigation that would integrate the different forces at play in time and space.

Fig. 1 :

 1 Fig. 1: PlxnA1 and Robo1 are successively sorted to the growth cone surface during FP navigation (a) Schematic drawings of pHLuo vector and pH-dependent pHLuo fluorescence activity in cells reporting cell surface protein pool. pHLuorin-receptor and mb-tomato coding sequences were cloned in pCAG vector, spaced by an ires. (b) In ovo electroporation procedure. 48h after electroporation, spinal cords were dissected and mounted as open books for time lapse microscopy. pHLuo fluorescence was monitored in 3 compartments of the open-books: the precrossing, where the cell bodies of commissural neurons are located, the FP, in which commissural axons cross the midline, and the post-crossing, in which commissural axons chose between ventral and lateral longitudinal trajectories. VF: ventral funiculus, LF: lateral funiculus. (c-e) Time-lapse sequences illustrating the spatial and temporal dynamics of pHLuo-Nrp2, pHLuo-PlxnA1 and pHLuo-Robo1 during FP navigation. The asterisks point growth cone positions before pHLuo flashes and the white arrowheads those of pHLuo flashes and subsequent growth cone positions. (f) Cartography of pHLuo-Nrp2 dynamics from movie analysis. Dashed lines indicate the overall trajectory of single growth cones and green spots the first pHLuo detection. Nrp2 is exposed at the growth cone surface since the onset of spinal cord navigation (Nrp2: N=5 embryos, 6 movies, 27 growth cones). (g) Cartography of pHLuo-PlxnA1 and pHLuo-Robo1 dynamics, plotting position of pHLuo flashes. The upper panel illustrates pHLuo-PlxnA1 sorting in the first FP half, the lower panel that of pHLuo-Robo1 in the second FP half (PlxnA1: N=5 embryos, 9 movies, 32 growth cones; Robo1: N= 9 embryos, 10 movies, 21 growth cones). (h) Cumulative fractions showing differential pHLuo-Nrp2, pHLuo-PlxnA1 and pHLuo-Robo1 dynamics during FP navigation (P value is from the Kolmogorov-Smirnov (KS) test). (i) Summary of the temporal sequence of pHLuo-Nrp2, pHLuo-PlxnA1 and pHLuo-Robo1 membrane sorting during FP navigation. Scale bars in c-e, 10 µm.

Fig. 2 :

 2 Fig. 2: Cell surface sorting of PlxnA1 and Robo1 is temporally controlled and coincident with changes of exploratory behavior for Robo1 (a) Microphotographs of open books illustrating pHLuo-PlxnA1 and pHLuo-Robo1 membrane pool (left panel) and total (intracellular + membrane) receptor pool (right panel). Arrowheads point discrete pHLuo + growth cones and axon segments. (b) Quantification of the % of growth cone population observed to navigate within the FP and containing pHLuo-receptor in the precrossing segment immediately adjacent to the FP entry. Histograms show much broader total than surface fluorescence in this segment (total PlxnA1: N=4 embryos, 443 growth cones; membrane PlxnA1: N=5 embryos, 106 growth cones; total Robo1: N=4 embryos, 184 growth cones; membrane Robo1: N=17 embryos, 166 growth cones. Error bars indicate mean ± SEM; * p <0.05; ** p <0.01; *** p <0.001. P values are from Mann-Whitney test). (c) Histograms of measured lengths of pre-crossing segment containing total pHLuo-PlxnA1 or pHLuo-Robo1 in growth cones observed to navigate the FP, showing a more restricted pHLuo-Robo1 pattern, compared to that of pHLuo-PlxnA1. (d) Electroporated dorsal explant cultures treated with Ctrl cm (left panel) or FP cm (right panel) showing pHLuo-Robo1 increase at the growth cone membrane after FP cm application. (e) Quantitative analysis showing the increase of pHLuo-Robo1 at the growth cone surface after 20 min (T1) of FP cm treatment. For each growth cone, pHLuo is normalized to mb-tomato signal (3 independent experiments; Ctrl: N=19 explants, 48 growth cones; FP cm : N=18 explants, 46 growth cones. Error bars indicate mean ± SEM; * p <0.05; ** p <0.01; *** p <0.001. P values are from paired Student t test). (f) Quantification of pHLuo-Robo1 signal variation between T0 and T1 in Ctrl cm and FP cm conditions, showing the increase of surface Robo1 after FP cm application (Error bars indicate mean ± SEM; * p <0.05; ** p <0.01; *** p <0.001. P values are from unpaired Student t test). (g) Photomicrographs of pHLuo-PlxnA1 (left panel) and pHLuo-Robo1 (right panel) sorting at the growth cone membrane. Arrowheads in pHLuo-Robo1 condition point the exploratory behavior of growth cones after pHLuo sorting. (h) Quantitative analysis of the total angle explored by growth cones from the time point just preceding the flash (T0) to 1,5 hours after the flash (T3) (PlxnA1: N= 3 embryos, 32 growth cones; Robo1: N=10 embryos, 21 growth cones. Error bars indicate mean ± SEM. P value is from the Kolmogorov-Smirnov (KS) test). (i) Graphic summary of the exploratory behavior of the growth cone following pHluo-Robo1 surface sorting. Scale bars in a and d, 10 µm; scale bars in g, 5 µm.

Fig. 3 :

 3 Fig. 3: Robo2 is not sorted in growth cones navigating the FP but during post-crossing pathfinding of the lateral funiculus (a) Time-lapse sequences of open-books illustrating pHLuo-Robo2 spatio-temporal dynamics in commissural growth cones. The asterisks point growth cone positions before pHLuo flashes and the white arrowheads those of pHLuo flashes and subsequent growth cone positions. (b) Cartography of pHLuo-Robo2 flashes. Dashed lines indicate the overall trajectory of individual growth cones, from imaging onset to the time point of flash occurrence (Robo2: N=4 embryos, 5 movies, 29 growth cones). (c) Cumulative fractions showing differential pHLuo-Robo1 and pHLuo-Robo2 dynamics during spinal cord navigation (P value is from the Kolmogorov-Smirnov (KS) test). (d) Representative images from time-lapse sequence illustrating a shift of growth cone orientation subsequent to pHLuo-Robo2 flash. (e) Schematic drawing and quantification of growth cone turning after pHLuo-Robo2 flashes (Robo2: N=3 embryos, 30 growth cones. Error bars indicate mean ± SEM; * p <0.05; ** p <0.01; *** p <0.001. P value is from Mann-Whitney test). Scale bars in a, 50 µm, in d, 10 µm.

Fig. 4 :

 4 Fig. 4: chick PlxnA1 and Robo1 temporal sequences are conserved in the mouse (a) Microphotographs of PlxnA1 +/+ and Robo1 +/+ open-books illustrating pHLuo-PlxnA1 + (left panel) and pHLuo-Robo1 + (right panel) growth cones pointed by white arrowheads. (b) Upper panel: cartography of pHLuo-PlxnA1 + growth cones (N=4 embryos, 60 growth cones); lower panel: cartography of pHLuo-Robo1 + growth cones (N=4 embryos, 54 growth cones). (c) Upper panel: distribution of mb-tomato + pHLuo + and mb-tomato + pHLuo -populations in PlxnA1 +/+ open books. Lower panel: cumulative fraction of mb-tomato + pHLuo + and total mb-tomato + (composed of mb-tomato + pHLuo + and mb-tomato + pHLuo -) populations showing that mbtomato + pHLuo + growth cones are detected from the onset of FP navigation (P value is from the Kolmogorov-Smirnov (KS) test). (d) Upper panel: distribution of mb-tomato + pHLuo + and mbtomato + pHLuo -populations in the spinal cord of Robo1/2 +/+ open books. Lower panel: cumulative fractions of mb-tomato + pHLuo + and total mb-tomato + (composed of mbtomato + pHLuo + and mb-tomato + pHLuo -) populations showing that whereas the total mbtomato + population distributes from the first FP half to the post-crossing compartment, mbtomato + pHLuo + growth cones are only detected from the second half of the FP (P value is from the Kolmogorov-Smirnov (KS) test). (e) Cumulative fraction of pHLuo-PlxnA1 + and pHLuo-Robo1 + growth cones, showing the differential timing of sorting of the receptors in the FP (P value is from the Kolmogorov-Smirnov (KS) test). (f) Microphotographs of Robo1/2 -/-openbooks illustrating pHLuo-Robo1 + growth cones pointed by white arrowheads. (g) Cartography of pHLuo-Robo1 + growth cones (N=3 embryos, 55 growth cones). (h) Upper panel: distribution of mb-tomato + pHLuo + and the mb-tomato + pHLuo -populations in Robo1/2 -/-open books.

Figure 1 .

 1 Figure 1. (A) Schematic representations of various commissural projections. In mammals, neocortical callosal axons (in red) turn medially and navigate toward the midline. They are guided by cues emanating from various sources: the midline zipper glia (in purple), the glial wedges (in light blue), the indusium griseum glia (in dark blue), and migrating neurons (in green). Ganglion cell axons exiting the retina connect both sides of the brain, forming ipsilateral (in red) and contralateral (in blue) tracts, the latter crossing the midline at the optic chiasm (LE: left eye; RE: right eye; LT: left thalamus; RT: right thalamus). In the Drosophila ventral nerve cord, ipsilateral (in red) and contralateral (in blue) neurons extend their axons medially toward the midline, but ipsilateral axons turn before crossing while contralateral ones turn after the crossing. In vertebrates, spinal commissural axons navigate first ventrally toward the floor plate (FP) lying at the ventral edge of the central canal (cc), cross the midline and then turn rostrally. (B) Temporal sequence of dl1 commissural interneuron generation and axon navigation. The first spinal commissural neurons are born from a dorsal territory around E9.5. Some of them already extend their axons across the midline at E10.5. By E12.5, most of them have completed midline crossing and all navigate post-crossing longitudinal routes by E.13.5. (C) Temporal sequence of guidance signaling controlling the navigation of the midline. As they navigate toward the FP, the sensitivity to midline repellents is silenced in spinal commissural growth cones. During FP crossing, commissural growth cones gain responsiveness to these FP repulsive cues, which prevent them from re-crossing and drive them out of the FP. At the exit, they follow rostro-caudal gradients of guidance cues, turning rostrally in the ventral (VF) or lateral (LF) funiculus. (D) Representation of the principal signaling implicated in the midline repulsion. Slit/Robo in the Drosophila, Robo1-2/SlitN, PlexA1/SlitC, and Sema3B/Nrp2/PlexA1 in the mouse.

Figure 2 .

 2 Figure 2. Experimental paradigms to assess commissural axon sensitivity to FP repellents. (A) Spinal cord open-book co-cultured with control (ctrl) COS cells or COS cells secreting Sema3B or Slit2. In the absence of endogenous FP, comparable axonal growth is observed toward ctrl and Sema3B or Slit2-secreting aggregates. (B) Ectopic graft of COS cells along the lateral side of the spinal cord open-book. Netrin1-secreting aggregate induces a re-routing of pre-crossing commissural fibers toward the ectopic cells. Sema3B and Slit2-secreting ectopic aggregates do not deflect pre-crossing commissural axons. Lateral grafting of roof-plate tissue (RP) re-routes commissural axons away from the ectopic tissue. (C) Collapse assay on dissociated neurons dissected from the dorsal spinal cord and grown in cultures. Growth cones responsive to repulsive cues collapse after short-term application of the cues, whereas unresponsive growth cones remain intact. (D) In the presence of the endogenous FP in the open-book, the growth of commissural axons emerging from the tissue toward the Sema3B or Slit2 sources is inhibited.

Figure 3 .

 3 Figure 3. Mechanisms reported to mediate pre-crossing silencing to midline repellents. (A) In drosophila, endosomal protein Comm silences Slit responsiveness in pre-crossing axons, by sorting the majority of Robo receptors to the lysosomal degradation. Robos that escape the degradation and reach the growth cone surface are inhibited via a trans-interaction by Robo2 expressed by midline glial cells. (B) (a) In the mouse, the isoform Robo3.1 of the Robo3 gene is expressed in the pre-crossing axons and the resulting protein antagonizes Slit-Robo signaling. (b) In the chick, Robo1 is trafficked to vesicles to maintain it at low levels at the pre-crossing stage. (c) In the mouse, pre-crossing commissural axons express Nrp2 at their surface but only low levels of PlexinA1, whose cell surface expression is prevented through processing by Calpain proteases. (d) In the chick, PlexinA2 and Sema6B form cis complex in pre-crossing commissural axons and PlexinA4 traffics in vesicles and is excluded from the growth cones.

Figure 4 .

 4 Figure 4. Models. for the regulation of the semaphorin signaling from pre-crossing to post-crossing. (A) In this model, PlexinA1 and Nrp2 are both expressed at the growth cone surface of pre-crossing commissural axons. Their sensitivity to Sema3B is prevented by trapping of Sema3B by FP-Nrp2. After the crossing, Nrp2 is transcriptionnaly downregulated in the FP at E13, which releases Sema3B and allows repulsion. (B) In this model, cell surface PlexinA1 is kept at low levels in pre-crossing commissural axons, to desensitize them to Sema3B. Upon crossing, calpain activity is suppressed by FP GDNF, PlexinA1 reaches the growth cone surface and can associate with Nrp2. The receptor complex activity is blocked by FP Nrp2 and PlexinA1, until the crossing is achieved. After the crossing, the complex is functional for Sema3B repulsion.

Figure 5 .

 5 Figure 5. Mechanisms releasing the pre-crossing silencing and mediating sensitization midline repellents. (A) In drosophila, after the crossing, Comm protein is down-regulated and Robo proteins are sorted at the growth cone surface to transduce the Slit repulsive signal. (B) (a) In the mouse, upon crossing, Robo3.1 is replaced by Robo3.2, locally synthesized under local FP triggers, which acts as an agonist of the Slit-Robo signaling. In the chick, exocytosis of Robo1 is activated by transcriptional up-regulation of RABgdi, and the receptor is sorted at the cell surface. (b)In the mouse, upon the crossing, GDNF secreted by FP glial cells inhibits Calpain activity and allows PlexinA1 to reach the surface, to associate with Nrp2 and to mediate Sema3B repulsive response. Gain of cell surface PlexinA1 also switches on repulsion by Slit-C fragments. (c) In the chick, the pre-crossing PlexinA2/Sema6B cis complex is released, and replaced by a Sema6B/PlexinA2 trans interaction, releasing commissural PlexinA2 which become available for semaphorin repulsion. PlexinA4 might be sorted at the cell surface to also mediate semaphorin repulsion.

Fig. 1 .

 1 Fig. 1. Modes of action of repulsive forces in axon guidance.As they grow, axons are oriented by a combination of attractive and repulsive forces. Among the repulsive ones, we can distinguish 3 main modes of action. A. Propelling. Axons perceive diffusible chemorepulsive cues emanating from a group of guidepost cells and turn away from this source. B. Confinement. group of guidepost cells act as a barrier and confine the axons within a territory. They not necessarily deflect them away but prevent them from exiting it, partly by the emission of diffusible cues. C. Channeling. Several groups of physically separated guidepost cells constrain commissural axons within a narrow path, by releasing repulsive cues.

Fig. 2 .

 2 Fig. 2. Guidepost territories instructing commissural axons navigation in the spinal cord.Spinal commissural axon trajectory is highly stereotyped. dI1c axons (in black) arise from a dorsal territory. They extend ventrally, cross the midline through the floor plate (FP), and then turn rostrally without ever crossing the midline again to connect their final target. This trajectory is influenced by various guidepost cells along the way. First, axons are pushed away by chemorepulsive cues emanating from the roof plate (RP) (in blue) and follow the pial surface (in green). Meninges (in grey) and the DREZ/DRBZ (in red) keep the axons away from the CNS/PNS boundary through diffusible cues. At around mid-distance from the ventral side, axons turn towards the FP (in purple), then run along the motoneurons domain (in red). Axons never enter the motoneuron domain nor the ventricular zone (in light green), these territories channeling the commissural tract towards the FP. As they reach the FP, axons interact with the basal lamina (in green) and navigate through the FP glial cells radial processes. Upon crossing, they gain sensitivity to repulsive cues emanating from the FP, that they did not perceive before and thus exit the FP, accomplish a sharp turning in the rostral direction and navigate longitudinally in bundles, guided by various gradients of guidance cues, including repulsive ones.

Fig. 3 .

 3 Fig. 3. Kick off repulsive forces at the roof plate. The BMP family members GDF7 and BMP7 are secreted by roof plate glia cells and bind to the axonal BMPR-IB receptor and repel axons toward the ventral part of the spinal cord. In parallel, the secreted factor Draxin propels axons via axonal DCC.

Fig. 4 .

 4 Fig. 4. Confinement and channeling of commissural axons. A. Repulsive forces confine axons within the spinal cord. dorsal root entry zone (DREZ) and the dorsal root bifurcation zone (DRBZ) confine axons in the spinal cord. Netrin-1 acts via DCC and DCC/UNC5 heterodimer in the DRBZ and in the DREZ respectively. DSCAM may modulate Netrin-1 attractive response with or without DCC. Neogenin, whose expression was reported in chick commissural axons may also transduce the signal initiated by Netrin-1. In addition, Draxin at the pial surface and in the DREZ has a repulsive effect on axons via DCC. Signals originating from the meninges and the pial surface are still largely unknown but may involve the glycoprotein Dystroglycan. Dystroglycan scaffolds Laminin, which has been shown in other context to switch Netrin-1 attraction into repulsion. B. Repulsive forces channel axons between the ventricular zone (VZ) and the motoneuron domain. The VZ is a territory non-permissive to the entry of axons. The mechanisms mediating this barrier are still unknown but might involve Semaphorins and Eph/Ephrins. Netrin-1 is produced by progenitors. The motoneuron domain expresses Slits, Semaphorins and ROBO3 ligand, NELL2. NELL2/ROBO3 signaling mediates repulsion on commissural axons.

Fig. 5 .

 5 Fig. 5. Repulsive forces in the floor plate. While crossing the floor plate (FP), axons gain sensitivity to repulsive cues they did not perceive at the pre-crossing stage. PlexinA2 (PLEXA2), EphrinB3 (EphrinB3) and NOGO, expressed at the membrane of FP glial cells, interact respectively with Semaphorin6B (SEMA6B), EphB3 receptor and NOGO receptor (NOGOR) expressed by commissural axons. In parallel, the FP glial cells also secrete repulsive cues. A truncated, diffusible version of Nogo can also bind to the axonal Nogo receptor. Sema3B binds to a heterodimer of Neuropilin2 (NP2) and PlexinA1 (PLEXA1) axonal receptors. Slit produced by FP glia is cleaved into a C-terminal (SLITC) and an N-terminal fragments (SLITN). SLITC binds to PLEXA1, while SLITN interacts with ROBO.

Fig. 6 .

 6 Fig. 6. Repulsive forces in command of post-crossing axon navigation.After crossing the floor plate (FP), commissural axons turn rostrally in response to gradients of attractive and repulsive cues. Diffusible Shh from the FP glial cells interacts with the axonal receptor HHIP. In vertebrates, post-crossing commissural axons form two main tracts, the first turns in the ventral funiculus, while the second turns in the lateral funiculus. Slit contributes to this sorting through interaction with axonal ROBO1 and ROBO2. An interplay of ROBO and N-cadherin was also found to contribute to the lateral sorting of post-crossing commissural axons. Trans-interactions also occur between glial Ephrin-B3 (Eph-B3) and axonal Eph-B receptor to assign the medio-lateral position of post-crossing tracts.

Acknowledgments

We thank Pr. Edmund Derrington, Dr. Servane Tauszigdelamasure and Dr. C eline Delloye-Bourgeois for proofreading the manuscript and helpful comments. Funding VC is supported by funding from the LABEX CORTEX and Labex DevWeCAN of Universit e de Lyon, within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgments VC is supported by the LABEX CORTEX and DEVWECAN of the "Université de Lyon", within the programme 'Investissements d'Avenir' (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR), ANR-16-CE16-0023-01, and French Muscular Dystrophy Association (AFM).