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Further tales of the midline
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Further tales of the midline Alain Che´dotal1,2,3 In the vertebrate central nervous system (CNS), specialized glial and neuronal cells positioned at the dorsal and ventral midline act as intermediate targets for commissural axons by secreting a variety of attractants and repellents. Despite the diversity of commissural projections, recent findings suggest that the same basic set of molecules controls midline crossing at all level of the CNS. Midline crossing is associated with an important switch of the combinatorial expression of several axon guidance receptors on the growth cone of commissural axons. I will review here novel studies that reveal how the expression of these receptors and the activity of their ligands are modulated by transcriptional, translational, and post-translational modifications. This also uncovers extensive cross talks between axon guidance pathways. Addresses 1 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), UMR S968, Institut de la Vision, F-75012 Paris, France 2 Universite´ Pierre et Marie Curie (UPMC) Paris VI, UMR S968, Institut de la Vision, F-75012 Paris, France 3 Centre National de la Recherche Scientifique (CNRS) UMR 7210, Institut de la Vision, F-75012 Paris, France Corresponding author: Che´dotal, Alain ([email protected])
Current Opinion in Neurobiology 2011, 21:68–75 This review comes from a themed issue on Developmental neuroscience Edited by Silvia Arber and Graeme Davis Available online 17th August 2010 0959-4388/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2010.07.008
Introduction From the spinal cord to the base of the olfactory bulb, millions of commissural neurons project their axon across the midline and are essential for the integration of sensory information and for eliciting proper motor and sensory motor behaviors [1]. In the forebrain, commissural axons cross the midline at specific and restricted locations such as the anterior and posterior commissures or the corpus callosum, each of which gathers axons originating from various regions. For instance, the anterior commissure includes axons from the amygdala, anterior olfactory nucleus, and cortex, while the corpus callosum is a heterogeneous collection of axons from cortical layers II/III and V/VI. In the hindbrain and spinal cord, there exists a higher diversity of commissural neurons that cross the midline at all levels, rarely forming well defined tracts. Current Opinion in Neurobiology 2011, 21:68–75
Despite this apparent heterogeneity, the past two decades have revealed that the molecular mechanisms controlling midline crossing in the CNS are highly similar and that the same basic set of attractive and repulsive cues are used throughout developing brains. In the past two years there has been a decrease in the discovery of new axon guidance molecules, but in the meantime, there has been considerable progress made in assembling the molecular pieces of the midline guidance puzzle. I will discuss here the current understanding of the molecular mechanisms underlying midline crossing in the vertebrate brain, taking the corpus callosum and spinal cord as model systems. In both cases new findings confirmed that semaphorins are key players at the midline acting as repellents or attractants and also started to reveal how their activity is modulated in commissural axons. I will end reviewing a series of studies that provide new insights into the transcriptional and translational regulation of the expression of axon guidance receptors.
Crossing the corpus callosum In eutherians, the two hemispheres are connected by callosal axons that establish reciprocal connections between cortical areas processing similar information. Although it is estimated that the corpus callosum contains about half of all commissural axons, its ablation, performed in ‘split-brain’ patients, or its absence in a variety/large number of mutant mice does not clearly result in major disorders of brain function [2,3]. Still, the corpus callosum has become a major model system for understanding axon guidance at the midline. In rodents, the specification of all callosal neurons requires the transcription factor Satb2 [4], but there is growing evidence supporting a molecular diversity of cortical neurons projecting through the corpus callosum [5]. Callosal axons are known to extend across specific dorsal territories that are permissive for their growth and delineated by specific glial (glial wedge, indusium griseum) and neuronal (subcallosal sling) cell populations (Figure 1). Slits secreted by the glial wedge and by indusium griseum cells were shown to prevent callosal axons from entering ventral and dorsal domains [6]. These cells also secrete the morphogen Wnt5 that repels post-crossing callosal axons away from the midline. Accordingly, knockout mice lacking the Wnt5a receptor Ryk, Slit1/Slit2 or their receptors Robo1/Robo2 all exhibit major defects of corpus callosum development [7–9]. Recently, a third major glial chemorepellent for callosal axons has been identified [10]. This secreted molecule named Draxin (dorsal repulsive axon guidance protein) is www.sciencedirect.com
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Figure 1
Axon guidance at the cortex midline. Schematic coronal section of a mouse brain at the level of the corpus callosum summarizing known receptors (left) and their ligands. Callosal neurons are guided at the midline by attractive (Netrin, Sema3C) and repulsive (Slit2, Draxin, Wnt5a) proteins secreted by glial cells in the induseum griseum (IG) and glial wedge (GW) and two transient subsets of migrating gabaergic and glutamatergic neurons. How commissural axons switch from attraction to repulsion is unknown. The attractant secreted by Gabaergic neurons is also unknown (?).
highly expressed by glial cells surrounding the corridor followed by cortico-cortical axons and repels these axons in vitro. Moreover, Draxin/ mice are acallosal. Draxin function in patterning commissural axons extends to the anterior commissure and spinal cord [10]. It will now be important to identify Draxin receptor and try to understand why despite this extensive repulsive redundancy, switching off a single ligand/receptor complex is sufficient to fully prevent crossing. One possibility is that these repellents and their receptors share common downstream cytoskeletal targets such as microtubules. Interestingly, in mice and human, mutations in TUBB3 (class III btubulin) also compromise corpus callosum development [11]. Until quite recently, another intriguing issue was the apparent lack of attractant for callosal axons. Callosal axons express DCC and Netrin1 was detected at the cortex midline [5,12] (Figure 1). Netrin-1 and DCC mutants have no corpus callosum, but direct evidence that Netrin1/DCC attracts callosal axons is still lacking. Several recent studies have started to fill this gap by identifying the secreted semaphorin Sema3C as an attractant for callosal axons. In the hindbrain and spinal cord, floor plate cells secrete attractants and thus act as interwww.sciencedirect.com
mediate targets for commissural axons. By contrast, in the cortex such specialized midline guidepost cells were not known to exist. A recent study has shown that two populations of GABAergic and glutamatergic neurons, migrate to the cortical midline, above the septum, prior the arrival of callosal axons and act as bona fide intermediate targets for callosal pionneer axons [13]. This is reminiscent of previously described forebrain guidepost neurons, such as LOT cells [14] and corridor cells [15] that act on olfactory bulb axons and cortico-thalamic axons respectively. Forebrain explant cultures were used to show that the cortical midline releases attractants for callosal axons and that both transient populations of midline neurons are contributing to this activity. Interestingly, the secreted semaphorin Sema3C is expressed by the glutamatergic midline neurons and attracts callosal axons. Moreover, the corpus callosum is severely perturbed in Sema3C knockout mice [13]. Sema3C attractive activity appears to be mediated by the Neuropilin1 receptor both in vitro and in vivo, but other components of the Sema3C receptor complex in callosal axons (which express L1, PlexinA3 and PlexinA1 and PlexinD1 [5,13,16]) are still unknown. In addition to controlling Current Opinion in Neurobiology 2011, 21:68–75
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midline crossing in the corpus callosum, guidepost neurons could also play a role in the dorso-ventral sorting of the various subtypes of callosal axons (Figure 1).
Semaphorin 6A and the secret of the pyramids The corticospinal tract (CST) connects layer V pyramidal neurons, primarily from the motor cortex, to their target neurons (mostly interneurons) in the spinal cord [17]. CST axons grow ipsilaterally through the internal capsule, cerebral peduncle, and midbrain and penetrate the hindbrain ventrally, over the pons. CST axons next follow a ventral trajectory passing beneath the inferior olive where they abruptly reorient dorsally and cross the midline, forming the pyramidal decussation, before continuing their journey into the spinal cord. In humans, anomalies of CST decussation are found in several neurological diseases and result in abnormal motor behavior such as mirror movements [17,18]. Not much is known about the factors that guide CST axons and in particular those that control their decussation and ventro-dorsal switch. In mice, CST decussation is pertubed in Netrin1, DCC, and Unc5h3 knockouts suggesting that Netrin1 controls the midline crossing of CST axons [19]. Interestingly, a genetic study on humans has recently shown that, in two distinct families, all individuals with mirror movements carry autosomal dominant mutations in DCC [18]. In addition to Netrin1/DCC, two novel studies show that once again, semaphorin signalling is involved in CST guidance at the midline. Sema6A is a transmembrane semaphorin known to control axon guidance and neuronal migration via two receptors, PlexinA2 and PlexinA4. In Sema6A knockout, many CST axons fail to decussate upon leaving the hindbrain and keep extending in the ventral spinal cord. Similar CST defects were detected in PlexinA4/ and PlexinA4/;PlexinA3/ mice [20,21]. Surprisingly, despite a bilateral CST, the motor behavior of these mice is seemingly unaffected. Sema6A is highly expressed in the inferior olive, suggesting that it could either restrain the spreading of CST axons laterally or repels CST axons toward the dorsal spinal cord. However, why midline crossing is prevented in these mice is still unclear. A cue may come from zebrafish, where there is genetic evidence suggesting that PlexinA4 is coexpressed with Robo and contributes to Slit2 axon branching function [22]. It will be important to determine if CST crossing defects in PlexinA4 knockout mice are due to defects in Slit/Robo activity.
The semaphorin double switch One of the most challenging questions in the field is to understand how commissural axons switch from midline attraction to repulsion. In vertebrates, a first step seems to be the silencing of Netrin1 attraction triggered by the binding of Robo1 receptor to DCC in the presence of Slit (Figure 2) [23]. Secondly, stem cell factor (SCF) Current Opinion in Neurobiology 2011, 21:68–75
released by the floor plate is required to expel commissural axons expressing its receptor Kit from the midline [24]. A third step is a gain of repulsion to Slits that is to some extent correlated with an upregulation of Robo receptors expression on post-crossing axons and a downregulation of one of the two Robo3 splice variants (Robo3.1) [25]. How the expression level of Robo receptors is regulated in vertebrates is largely unknown but there is recent data suggesting that they may be, like drosophila Robo (see below), subjected to ubiquitination and protein degradation [26]. However, Slits and Netrin1 are not the only midline-derived factors acting on commissural axons and other signals have also to be tightly regulated in time and space. Previous studies have shown that midline crossing is associated with a gain in responsiveness to several secreted semaphorins that mediate repulsion [27]. Two recent studies have started to unravel the molecular mechanisms behind this switch in commissural sensitivity to semaphorins [28,29]. Spinal cord commissural axons express the Neuropilin2 receptor before and after crossing; its ligand Sema3B is highly expressed by the floor plate. Various in vitro assays have shown that only post-crossing commissurals respond to Sema3B repulsive/collapsing activity [27,28,29]. These post-crossing commissurals also gain responsiveness to Sema3F that, however, is absent from the floor plate. Accordingly, midline crossing is strongly perturbed in Neuropilin2 knockout mice [27]. It is well established that neuropilins are just the binding subunit of the receptor complex for most secreted semaphorins and that several transmembrane proteins such as Plexins [30], VEGF receptor [31], and IgCAMs (Immunoglobulin Cell adhesion molecules) [32,33] form multi-molecular receptor complexes with neuropilins and transduce the semaphorin signal. A novel study reveals that a key event triggering the gain of responsiveness to Sema3B is the upregulation of the expression of PlexinA1 on post-crossing axons triggered by floor plate-derived factors (Figure 2). A biochemical screen showed that one of the floor plate factors that initiate the switch is a soluble form of neuronal cell adhesion molecule (sNrCAM). Further pharmacological dissection of the mechanism regulating PlexinA1 expression level on commissural axons identified the cysteine protease calpain1 as a key regulator of PlexinA1 processing. Calpain1 is strongly expressed in commissural axon and cleaves the extracellular domain of PlexinA1, thereby inactivating it (not known if cleavage occurs inside or outside the cell). Silencing calpain1 function induces commissural axon stalling at the floor plate by precociously switching on Sema3B repulsion before crossing. In the current working model, when commissural enter the floor plate, sNrCAM, alone or together with additional floor plate factors, inactivates calpain1, which in turn prevents PlexinA1 processing thus allowing it to www.sciencedirect.com
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Figure 2
Axon guidance at the spinal cord midline. Schematic coronal sections of the embryonic spinal cord illustrating the axon guidance receptor expressed by pre-crossing (left) and post-crossing (right) commissural axons. Pre-crossing axons are repelled from the dorsal part of the spinal cord by Draxin and BMPs. They are attracted to the floor plate by Netrin1 and Shh. In pre-crossing axons, Slit/Robo repulsion is inactivated by Robo3.1. At the floor plate, SCF promotes midline exit and DCC/Netrin1 attraction is silenced by Robo1 in a Slit dependent manner. The expression of the other Robo receptors is upregulated and Slit repulsion is activated. In parallel, the inhibition of Calpain1 induced by soluble NrCAM leads to an upregulation of PlexinA1 expression on post-crossing commissural axons allowing it to form a complex with Neuropilin2 that mediates Sema3B repulsion. Shh is also involved in switching Sema3B repulsion and, in chick, may also directly repel post-crossing axons.
mediate Sema3B repulsion, together with Neuropilin2 [28]. How sNrCAM inhibits calpain is still unknown. Importantly, the phenotypic analysis of PlexinA1, NrCAM, and Sema3B knockouts brings further support to this model as in all three mutants, midline crossing by spinal cord commissural axons is strongly perturbed and mimics the defects found in Neuropilin2 knockout. Likewise, forcing expression of PlexinA1 in pre-crossing axons induces premature repulsion by floor plate. It remains to determine if the same mechanisms regulate semaphorin function in other parts of the CNS such as the cortex and if sNrCAM alone can activate the switch. A second study suggests that this might not be the case [29]. Floor plate cells produce the morphogen Sonic hedgehog (Shh) that was previously suggested to play a dual role at the midline: Shh attracts commissural axons toward the midline upon binding the Boc receptor (this requires Smoothened, Smo, but not Gli family members [34–36]) and after crossing, Shh repels post-crossing axons, an effect mediated by the Hhip receptor independently of Smo [37]. One should keep in mind that these two contrasted findings were obtained in different www.sciencedirect.com
species, rodents and chick respectively. The possible contribution of Patched (Ptc), the other major Shh receptor, to Shh chemoattractive function has not yet been studied, but Ptc is not expressed by chick commissural neurons and therefore does not mediate Shh chemorepulsive activity [37]. The loss of responsiveness to Shh chemoattraction after crossing is another unsolved mystery. Previous in vitro studies have shown that hindbrain commissural axons become fully unresponsive to floor plate attraction after crossing it [38] therefore rendering it likely that post-crossing commissurals lose sensitivity to Shh. The silencing of Shh attraction could be explained by a downregulation of Boc whose expression pattern in commissurals matches Robo3 [35]. A recent study shows that Shh has a third activity on commissural axons that is to act as a regulator of the semaphorin switch [29]. Adding Shh to dorsal spinal cord explants in the presence of Netrin1 is sufficient to trigger Sema3B and Sema3F repulsion. Likewise, applying Shh blocking antibodies to spinal cord explants prevents midline crossing and induces additional guidance errors. Electroporation of dominant negative forms of Ptc or of Current Opinion in Neurobiology 2011, 21:68–75
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its partner Smo in the rat neural tube severely perturbs midline crossing. Interestingly, similar axon guidance defects are present in explants treated with compounds that modify the level of cAMP in commissural axons. As adding Shh to the explants inhibits PKA, it has been proposed that the semaphorin switch is associated to a decrease of cAMP level in commissural axons. It is unclear how these molecules will initiate the switch from attraction to repulsion as Shh, Ptc, and Smo all seem
to be expressed in pre-crossing and post-crossing axons. However, the first study suggests that the upregulation of PlexinA1, without which Sema3B is silent, is the main trigger [28]. Shh could also be another floor platederived factor that can induce calpain inhibition, although, based on unpublished data [28], this does not seem to be the case. This apparent discrepancy could be explained by technical differences between the two in vitro studies such as the species (chick/mouse vs rat) or the assay (growth cone collapse vs repulsion). This could
Figure 3
Translational and Transcriptional modulation of axon guidance receptors. (a) In absence of Netrin1, DCC directly binds to many components of the translational machinery, thereby preventing translation. (b) Upon Netrin1 binding, DCC releases the components that can assemble in polysomes and activate the translation of unknown target mRNAs. (c) In Drosophila, Frazzled, in a Netrin1-independent manner, activates via an unknown pathway, the transcription of Commissureless (Comm) mRNA. In turn, Comm targets Robo receptors to endosomes and maintains a low level of Robo at the membrane. (d) The MiR-218.1 gene is localized in intron 15 of the Slit2 gene. Robo1 mRNA is one of the target of MiR-218.1. High Slit2 expression leads to low Robo1 expression. The second MiR218 gene, MiR218.2, is encoded by intron 14 of Slit3, but it is not known if it targets Robo receptors. Current Opinion in Neurobiology 2011, 21:68–75
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also just reflect the diversity and heterogeneity of spinal cord commissural neurons. Yet, there is strong evidence supporting a cross talk between Shh and Sema3B signalling pathways. First, in drosophila, PKA regulates PlexinA activity [39]. Second, Shh chemoattractive action on commissural axons requires the Src family kinases, Src, and Fyn) that were previously shown [40] to associate with PlexinA1 and promote its phosphorylation in response to Sema3A. Moreover, inhibiting Src kinases was previously shown to convert Sema3B attraction of anterior commissure axons to repulsion, and Sema3B stimulates the recruitment of Src kinases to the membrane [33]. Although these two studies lift a corner of the veil, there is still plenty of room for other guidance molecules and other molecular interactions to be uncovered as being important for switching the response of spinal cord commissurals from attraction to repulsion. For example, one recent study showed that Robo cleavage by the metalloprotease Kuzbanian is involved in the regulation of midline crossing, and provided preliminary evidence suggesting that vertebrate robos are also proteolitically processed by Adam10 [41]. Another interesting candidate is VEGF (vascular endothelial growth factor) that is expressed by floor plate cells, at least in the hindbrain [42] and whose receptor VEGFR forms a complex with Neuropilin1 and PlexinD1 to mediate Sema3E attractive action on forebrain axons [31]. As both Sema3E and VEGF are present at the floor plate [27,42], it will be interesting to know whether a VEGF/Sema3E cross talk occurs during midline crossing.
Newcomers for midline found in transcription and translation As mentioned before, in the past few years, there has been little new receptors and ligands added to the list of molecules acting on midline crossing. By contrast, there is a recent burst of findings that reveals that known molecules and biochemical pathways can modulate the activity and expression of axon guidance receptors in commissural axons. Three reports show that transcription plays a central role in axon guidance at the midline by controlling the expression level of several receptors. In drosophila, the commissureless protein (Comm) controls midline crossing by preventing Robo receptors from reaching the cell surface [43]. High Comm expression in pre-crossing axons silences Slit repulsion and allows Frazzled (Fra; the DCC ortholog in fly) expressing axons to grow toward the midline in response to Netrin. After crossing, Comm is downregulated, Slit/Robo repulsion is activated, and this steers growth cones away from the midline. Bashaw and colleagues recently found that in www.sciencedirect.com
commissural axons, Fra activates the transcription of Comm mRNA independently of Netrin (Figure 3) [44]. How Fra activates Comm transcription is unknown. As there is still no known Comm orthologs in vertebrates it is also unclear if a similar mechanism exists in higher organisms. However, previous studies have shown that in vertebrates Netrin/DCC signalling can stimulate the import of NFAT transcription complex to the nucleus [45]. In addition, following cleavage by g-secretase, the intracytoplasmic domain of Neogenin, one of Netrin1 receptors in vertebrates, is cleaved and transferred to the nucleus where it activates transcription [46]. As there is also evidence for cleavage of DCC cytoplasmic domain [47], this could be one way to link DCC (or Fra) to gene transcription. It has previously been shown that in Xenopus, axon turning toward a Netrin source requires local protein synthesis in the growth cone [48] and that signals from the floor plate may also influence local translation during midline crossing [49]. A recent study shows that DCC directly controls translation by interacting through its cytosplamic tail (in particular the P1 domain) with many components of the translation machinery such as initiation factors and ribosome subunits (Figure 3) [50]. DCC activation by Netrin1 releases the translation complex and it can then assemble into active polysomes and perform protein synthesis. More relevantly for this review, electroporation in the chick spinal cord of DCC constructs lacking the P1 domain severely perturbs commissural axon growth toward the midline suggesting that DCC regulation of translation is functionally relevant. DCC translational targets remain to be identified. Although such mechanisms have not yet been described for Robo receptors, a study conducted in tumor cells also revealed some previously unsuspected links between Slits, Robos, and transcription/translation. Microribonucleic acids (MiRNAs) are 21–24 nucleotide long posttranscriptional modulators that bind to the 30 UTR of their target mRNAs, preventing their translation [51]. MiR-218 is a miRNA whose expression is downregulated in some invasive cancer cell lines [52]. A bioinformatic screen and in vitro experiments showed that one of miR-218 target is Robo1. More surprisingly, it was found that one of the two miR-218 precursors is produced from a DNA sequence localized within an intron of the Slit2 gene (Figure 3). Therefore Slit2 and miR-218 are co-transcribed from a common promoter and MiR-218 represses Robo1 expression. Moreover, the second miR-218 precursor is localized in the Slit3 sequence but is not synthetized in tumor cells. There is as of yet no evidence that this negative feedback loop has a function at the midline but this could represent another regulatory mechanism controlling the combinatorial expression of Robo receptors and thereby Slit/Robo function in midline crossing [53]. Current Opinion in Neurobiology 2011, 21:68–75
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Conclusion Although there still is a pile of questions to answer, there has been some constant progress toward a better understanding of midline crossing in vertebrates. An emerging picture is that in every part of the CNS, commissural axons are guided by the same basic set of molecules: netrin1/DCC, a morphogen (Wnt5a or Shh), Slits and Robos, some secreted semaphorins, one Neuropilin, and PlexinA. This suggests that the signalling pathways and cross talk are highly conserved between commissural axons. However there are also some noticeable differences between forebrain and hindbrain neurons: Robo3 does not control commissure development in the forebrain and no repulsive BMPs are known to pattern commissurals at this level. In addition, the molecular mechanisms regulating the response of forebrain commissural axons to midline attractants and repellents before, during and after crossing are unknown.
Acknowledgements I thank Athena Ypsilanti and Vale´rie Castellani for critical reading of the manuscript. This work is supported by the Agence Nationale pour la Recherche (ANR), and the Fondation pour la Recherche Me´dicale (Equipes labelise´es FRM).
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exit from the midline intermediate target. Neuron 2008, 57:501-510. 25. Chen Z, Gore BB, Long H, Ma L, Tessier-Lavigne M: Alternative splicing of the Robo3 axon guidance receptor governs the midline switch from attraction to repulsion. Neuron 2008, 58:325-332. 26. Yuasa-Kawada J, Kinoshita-Kawada M, Wu G, Rao Y, Wu JY: Midline crossing and Slit responsiveness of commissural axons require USP33. Nat Neurosci 2009, 12:1087-1089. 27. Zou Y, Stoeckli E, Chen H, Tessier-Lavigne M: Squeezing axons out of the gray matter: a role for slit and semaphorin proteins from midline and ventral spinal cord. Cell 2000, 102:363-375. 28. Nawabi H, Briancon-Marjollet A, Clark C, Sanyas I, Takamatsu H, Okuno T, Kumanogoh A, Bozon M, Takeshima K, Yoshida Y et al.: A midline switch of receptor processing regulates commissural axon guidance in vertebrates. Genes Dev 2010, 24:396-410. A very thorough in vitro and in vivo study unraveling one the mechanism regulating Sema3B repulsion of post-crossing commissural spinal cord axons. It is shown that pre-crossing axons are kept unresponsive to floor plate-derived Sema3B due to the cleavage of the PlexinA1 receptor by calpain1. Calpain1 activity is silenced during midline crossing by floor platederived factors such as a soluble form of NrCAM. This allows Sema3B to repel post-crossing commissural axons away from the midline. 29. Parra LM, Zou Y: Sonic hedgehog induces response of commissural axons to Semaphorin repulsion during midline crossing. Nat Neurosci 2010, 13:29-35. Shh is known to attract spinal cord commissural axons toward the midline upon binding the Boc receptor. This novel study suggests that Shh is also able to trigger Sema3B repulsion of post-crossing commissural axons. This effect is mediated by Ptc and Smo. Shh switching activity is dependent on PKA activation and cAMP level. 30. Tamagnone L, Artigiani S, Chen H, He Z, Ming GI, Song H, Chedotal A, Winberg ML, Goodman CS, Poo M et al.: Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates. Cell 1999, 99:71-80. 31. Bellon A, Luchino J, Haigh K, Rougon G, Haigh J, Chauvet S, Mann F: VEGFR2 (KDR/Flk1) signaling mediates axon growth in response to semaphorin 3E in the developing brain. Neuron 2010, 66:205-219. 32. Castellani V, Chedotal A, Schachner M, Faivre-Sarrailh C, Rougon G: Analysis of the L1-deficient mouse phenotype reveals cross-talk between Sema3A and L1 signaling pathways in axonal guidance. Neuron 2000, 27:237-249. 33. Falk J, Bechara A, Fiore R, Nawabi H, Zhou H, Hoyo-Becerra C, Bozon M, Rougon G, Grumet M, Puschel AW et al.: Dual functional activity of semaphorin 3B is required for positioning the anterior commissure. Neuron 2005, 48:63-75. 34. Charron F, Stein E, Jeong J, McMahon AP, Tessier-Lavigne M: The morphogen sonic hedgehog is an axonal chemoattractant that collaborates with netrin-1 in midline axon guidance. Cell 2003, 113:11-23. 35. Okada A, Charron F, Morin S, Shin DS, Wong K, Fabre PJ, TessierLavigne M, McConnell SK: Boc is a receptor for sonic hedgehog in the guidance of commissural axons. Nature 2006, 444:369-373. 36. Yam PT, Langlois SD, Morin S, Charron F: Sonic hedgehog guides axons through a noncanonical. Src-family-kinasedependent signaling pathway. Neuron 2009, 62:349-362. 37. Bourikas D, Pekarik V, Baeriswyl T, Grunditz A, Sadhu R, Nardo M, Stoeckli ET: Sonic hedgehog guides commissural axons along the longitudinal axis of the spinal cord. Nat Neurosci 2005, 8:297-304. 38. Shirasaki R, Katsumata R, Murakami F: Change in chemoattractant responsiveness of developing axons at an intermediate target. Science 1998, 279:105-107. 39. Terman JR, Kolodkin AL: Nervy links protein kinase a to plexinmediated semaphorin repulsion. Science 2004, 303:1204-1207.
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40. Sasaki Y, Cheng C, Uchida Y, Nakajima O, Ohshima T, Yagi T, Taniguchi M, Nakayama T, Kishida R, Kudo Y et al.: Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex. Neuron 2002, 35:907-920. 41. Coleman HA, Labrador JP, Chance RK, Bashaw GJ: The Adam family metalloprotease Kuzbanian regulates the cleavage of the roundabout receptor to control axon repulsion at the midline. Development 2010, 137:2417-2426. 42. Schwarz Q, Gu C, Fujisawa H, Sabelko K, Gertsenstein M, Nagy A, Taniguchi M, Kolodkin AL, Ginty DD, Shima DT et al.: Vascular endothelial growth factor controls neuronal migration and cooperates with Sema3A to pattern distinct compartments of the facial nerve. Genes Dev 2004, 18:2822-2834. 43. Keleman K, Ribeiro C, Dickson BJ: Comm function in commissural axon guidance: cell-autonomous sorting of Robo in vivo. Nat Neurosci 2005, 8:156-163. 44. Yang L, Garbe DS, Bashaw GJ: A frazzled/DCC-dependent transcriptional switch regulates midline axon guidance. Science 2009. It had been previously shown that in xenopus axons Robo can silence DCC attractive function. This nice genetic study in Drosophila shows that DCC can silence Robo repulsion by controlling the transcription of Comm mRNA. This leads to high levels of Comm that in turn block Robo targeting to the membrane thereby preventing Slit repulsion. DCC acts on Comm expression independently of Netrin1. 45. Graef IA, Wang F, Charron F, Chen L, Neilson J, TessierLavigne M, Crabtree GR: Neurotrophins and netrins require calcineurin/NFAT signaling to stimulate outgrowth of embryonic axons. Cell 2003, 113:657-670. 46. Goldschneider D, Rama N, Guix C, Mehlen P: The neogenin intracellular domain regulates gene transcription via nuclear translocation. Mol Cell Biol 2008, 28:4068-4079. 47. Mehlen P, Rabizadeh S, Snipas SJ, Assa-Munt N, Salvesen GS, Bredesen DE: The DCC gene product induces apoptosis by a mechanism requiring receptor proteolysis. Nature 1998, 395:801-804. 48. Campbell DS, Holt CE: Chemotropic responses of retinal growth cones mediated by rapid local protein synthesis and degradation. Neuron 2001, 32:1013-1026. 49. Brittis PA, Lu Q, Flanagan JG: Axonal protein synthesis provides a mechanism for localized regulation at an intermediate target. Cell 2002, 110:223-235. 50. Tcherkezian J, Brittis PA, Thomas F, Roux PP, Flanagan JG: Transmembrane receptor DCC associates with protein synthesis machinery and regulates translation. Cell 2010, 141:632-644. This important work uncovers an unexpected function of DCC in the iniation of translation. Co-immunoprecipitation experiments show that DCC cytoplasmic domain binds through its P1 and P3 domains to many components of the translation machinery such as ribosomes and initiation factors. Adding Netrin1 releases these components from DCC and allows the translation of unknown target mRNAs. Spinal cord commissural neurons expressing a DCC receptor lacking the P1 domain fail to reach the midline. The authors suggest that other axon guidance receptors may act similarly. This mechanism could allow the local translation of specific proteins in restricted domains of the growth cone of commissural axons. 51. Schratt G: microRNAs at the synapse. Nat Rev Neurosci 2009, 10:842-849. 52. Tie J, Pan Y, Zhao L, Wu K, Liu J, Sun S, Guo X, Wang B, Gang Y, Zhang Y et al.: MiR-218 inhibits invasion and metastasis of gastric cancer by targeting the Robo1 receptor. PLoS Genet 2010, 6:e1000879. 53. Spitzweck B, Brankatschk M, Dickson BJ: Distinct protein domains and expression patterns confer divergent axon guidance functions for Drosophila Robo receptors. Cell 2010, 140:409-420.
Current Opinion in Neurobiology 2011, 21:68–75
Further tales of the midline Alain Che´dotal1,2,3 In the vertebrate central nervous system (CNS), specialized glial and neuronal cells positioned at the dorsal and ventral midline act as intermediate targets for commissural axons by secreting a variety of attractants and repellents. Despite the diversity of commissural projections, recent findings suggest that the same basic set of molecules controls midline crossing at all level of the CNS. Midline crossing is associated with an important switch of the combinatorial expression of several axon guidance receptors on the growth cone of commissural axons. I will review here novel studies that reveal how the expression of these receptors and the activity of their ligands are modulated by transcriptional, translational, and post-translational modifications. This also uncovers extensive cross talks between axon guidance pathways. Addresses 1 Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), UMR S968, Institut de la Vision, F-75012 Paris, France 2 Universite´ Pierre et Marie Curie (UPMC) Paris VI, UMR S968, Institut de la Vision, F-75012 Paris, France 3 Centre National de la Recherche Scientifique (CNRS) UMR 7210, Institut de la Vision, F-75012 Paris, France Corresponding author: Che´dotal, Alain ([email protected])
Current Opinion in Neurobiology 2011, 21:68–75 This review comes from a themed issue on Developmental neuroscience Edited by Silvia Arber and Graeme Davis Available online 17th August 2010 0959-4388/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2010.07.008
Introduction From the spinal cord to the base of the olfactory bulb, millions of commissural neurons project their axon across the midline and are essential for the integration of sensory information and for eliciting proper motor and sensory motor behaviors [1]. In the forebrain, commissural axons cross the midline at specific and restricted locations such as the anterior and posterior commissures or the corpus callosum, each of which gathers axons originating from various regions. For instance, the anterior commissure includes axons from the amygdala, anterior olfactory nucleus, and cortex, while the corpus callosum is a heterogeneous collection of axons from cortical layers II/III and V/VI. In the hindbrain and spinal cord, there exists a higher diversity of commissural neurons that cross the midline at all levels, rarely forming well defined tracts. Current Opinion in Neurobiology 2011, 21:68–75
Despite this apparent heterogeneity, the past two decades have revealed that the molecular mechanisms controlling midline crossing in the CNS are highly similar and that the same basic set of attractive and repulsive cues are used throughout developing brains. In the past two years there has been a decrease in the discovery of new axon guidance molecules, but in the meantime, there has been considerable progress made in assembling the molecular pieces of the midline guidance puzzle. I will discuss here the current understanding of the molecular mechanisms underlying midline crossing in the vertebrate brain, taking the corpus callosum and spinal cord as model systems. In both cases new findings confirmed that semaphorins are key players at the midline acting as repellents or attractants and also started to reveal how their activity is modulated in commissural axons. I will end reviewing a series of studies that provide new insights into the transcriptional and translational regulation of the expression of axon guidance receptors.
Crossing the corpus callosum In eutherians, the two hemispheres are connected by callosal axons that establish reciprocal connections between cortical areas processing similar information. Although it is estimated that the corpus callosum contains about half of all commissural axons, its ablation, performed in ‘split-brain’ patients, or its absence in a variety/large number of mutant mice does not clearly result in major disorders of brain function [2,3]. Still, the corpus callosum has become a major model system for understanding axon guidance at the midline. In rodents, the specification of all callosal neurons requires the transcription factor Satb2 [4], but there is growing evidence supporting a molecular diversity of cortical neurons projecting through the corpus callosum [5]. Callosal axons are known to extend across specific dorsal territories that are permissive for their growth and delineated by specific glial (glial wedge, indusium griseum) and neuronal (subcallosal sling) cell populations (Figure 1). Slits secreted by the glial wedge and by indusium griseum cells were shown to prevent callosal axons from entering ventral and dorsal domains [6]. These cells also secrete the morphogen Wnt5 that repels post-crossing callosal axons away from the midline. Accordingly, knockout mice lacking the Wnt5a receptor Ryk, Slit1/Slit2 or their receptors Robo1/Robo2 all exhibit major defects of corpus callosum development [7–9]. Recently, a third major glial chemorepellent for callosal axons has been identified [10]. This secreted molecule named Draxin (dorsal repulsive axon guidance protein) is www.sciencedirect.com
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Figure 1
Axon guidance at the cortex midline. Schematic coronal section of a mouse brain at the level of the corpus callosum summarizing known receptors (left) and their ligands. Callosal neurons are guided at the midline by attractive (Netrin, Sema3C) and repulsive (Slit2, Draxin, Wnt5a) proteins secreted by glial cells in the induseum griseum (IG) and glial wedge (GW) and two transient subsets of migrating gabaergic and glutamatergic neurons. How commissural axons switch from attraction to repulsion is unknown. The attractant secreted by Gabaergic neurons is also unknown (?).
highly expressed by glial cells surrounding the corridor followed by cortico-cortical axons and repels these axons in vitro. Moreover, Draxin/ mice are acallosal. Draxin function in patterning commissural axons extends to the anterior commissure and spinal cord [10]. It will now be important to identify Draxin receptor and try to understand why despite this extensive repulsive redundancy, switching off a single ligand/receptor complex is sufficient to fully prevent crossing. One possibility is that these repellents and their receptors share common downstream cytoskeletal targets such as microtubules. Interestingly, in mice and human, mutations in TUBB3 (class III btubulin) also compromise corpus callosum development [11]. Until quite recently, another intriguing issue was the apparent lack of attractant for callosal axons. Callosal axons express DCC and Netrin1 was detected at the cortex midline [5,12] (Figure 1). Netrin-1 and DCC mutants have no corpus callosum, but direct evidence that Netrin1/DCC attracts callosal axons is still lacking. Several recent studies have started to fill this gap by identifying the secreted semaphorin Sema3C as an attractant for callosal axons. In the hindbrain and spinal cord, floor plate cells secrete attractants and thus act as interwww.sciencedirect.com
mediate targets for commissural axons. By contrast, in the cortex such specialized midline guidepost cells were not known to exist. A recent study has shown that two populations of GABAergic and glutamatergic neurons, migrate to the cortical midline, above the septum, prior the arrival of callosal axons and act as bona fide intermediate targets for callosal pionneer axons [13]. This is reminiscent of previously described forebrain guidepost neurons, such as LOT cells [14] and corridor cells [15] that act on olfactory bulb axons and cortico-thalamic axons respectively. Forebrain explant cultures were used to show that the cortical midline releases attractants for callosal axons and that both transient populations of midline neurons are contributing to this activity. Interestingly, the secreted semaphorin Sema3C is expressed by the glutamatergic midline neurons and attracts callosal axons. Moreover, the corpus callosum is severely perturbed in Sema3C knockout mice [13]. Sema3C attractive activity appears to be mediated by the Neuropilin1 receptor both in vitro and in vivo, but other components of the Sema3C receptor complex in callosal axons (which express L1, PlexinA3 and PlexinA1 and PlexinD1 [5,13,16]) are still unknown. In addition to controlling Current Opinion in Neurobiology 2011, 21:68–75
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midline crossing in the corpus callosum, guidepost neurons could also play a role in the dorso-ventral sorting of the various subtypes of callosal axons (Figure 1).
Semaphorin 6A and the secret of the pyramids The corticospinal tract (CST) connects layer V pyramidal neurons, primarily from the motor cortex, to their target neurons (mostly interneurons) in the spinal cord [17]. CST axons grow ipsilaterally through the internal capsule, cerebral peduncle, and midbrain and penetrate the hindbrain ventrally, over the pons. CST axons next follow a ventral trajectory passing beneath the inferior olive where they abruptly reorient dorsally and cross the midline, forming the pyramidal decussation, before continuing their journey into the spinal cord. In humans, anomalies of CST decussation are found in several neurological diseases and result in abnormal motor behavior such as mirror movements [17,18]. Not much is known about the factors that guide CST axons and in particular those that control their decussation and ventro-dorsal switch. In mice, CST decussation is pertubed in Netrin1, DCC, and Unc5h3 knockouts suggesting that Netrin1 controls the midline crossing of CST axons [19]. Interestingly, a genetic study on humans has recently shown that, in two distinct families, all individuals with mirror movements carry autosomal dominant mutations in DCC [18]. In addition to Netrin1/DCC, two novel studies show that once again, semaphorin signalling is involved in CST guidance at the midline. Sema6A is a transmembrane semaphorin known to control axon guidance and neuronal migration via two receptors, PlexinA2 and PlexinA4. In Sema6A knockout, many CST axons fail to decussate upon leaving the hindbrain and keep extending in the ventral spinal cord. Similar CST defects were detected in PlexinA4/ and PlexinA4/;PlexinA3/ mice [20,21]. Surprisingly, despite a bilateral CST, the motor behavior of these mice is seemingly unaffected. Sema6A is highly expressed in the inferior olive, suggesting that it could either restrain the spreading of CST axons laterally or repels CST axons toward the dorsal spinal cord. However, why midline crossing is prevented in these mice is still unclear. A cue may come from zebrafish, where there is genetic evidence suggesting that PlexinA4 is coexpressed with Robo and contributes to Slit2 axon branching function [22]. It will be important to determine if CST crossing defects in PlexinA4 knockout mice are due to defects in Slit/Robo activity.
The semaphorin double switch One of the most challenging questions in the field is to understand how commissural axons switch from midline attraction to repulsion. In vertebrates, a first step seems to be the silencing of Netrin1 attraction triggered by the binding of Robo1 receptor to DCC in the presence of Slit (Figure 2) [23]. Secondly, stem cell factor (SCF) Current Opinion in Neurobiology 2011, 21:68–75
released by the floor plate is required to expel commissural axons expressing its receptor Kit from the midline [24]. A third step is a gain of repulsion to Slits that is to some extent correlated with an upregulation of Robo receptors expression on post-crossing axons and a downregulation of one of the two Robo3 splice variants (Robo3.1) [25]. How the expression level of Robo receptors is regulated in vertebrates is largely unknown but there is recent data suggesting that they may be, like drosophila Robo (see below), subjected to ubiquitination and protein degradation [26]. However, Slits and Netrin1 are not the only midline-derived factors acting on commissural axons and other signals have also to be tightly regulated in time and space. Previous studies have shown that midline crossing is associated with a gain in responsiveness to several secreted semaphorins that mediate repulsion [27]. Two recent studies have started to unravel the molecular mechanisms behind this switch in commissural sensitivity to semaphorins [28,29]. Spinal cord commissural axons express the Neuropilin2 receptor before and after crossing; its ligand Sema3B is highly expressed by the floor plate. Various in vitro assays have shown that only post-crossing commissurals respond to Sema3B repulsive/collapsing activity [27,28,29]. These post-crossing commissurals also gain responsiveness to Sema3F that, however, is absent from the floor plate. Accordingly, midline crossing is strongly perturbed in Neuropilin2 knockout mice [27]. It is well established that neuropilins are just the binding subunit of the receptor complex for most secreted semaphorins and that several transmembrane proteins such as Plexins [30], VEGF receptor [31], and IgCAMs (Immunoglobulin Cell adhesion molecules) [32,33] form multi-molecular receptor complexes with neuropilins and transduce the semaphorin signal. A novel study reveals that a key event triggering the gain of responsiveness to Sema3B is the upregulation of the expression of PlexinA1 on post-crossing axons triggered by floor plate-derived factors (Figure 2). A biochemical screen showed that one of the floor plate factors that initiate the switch is a soluble form of neuronal cell adhesion molecule (sNrCAM). Further pharmacological dissection of the mechanism regulating PlexinA1 expression level on commissural axons identified the cysteine protease calpain1 as a key regulator of PlexinA1 processing. Calpain1 is strongly expressed in commissural axon and cleaves the extracellular domain of PlexinA1, thereby inactivating it (not known if cleavage occurs inside or outside the cell). Silencing calpain1 function induces commissural axon stalling at the floor plate by precociously switching on Sema3B repulsion before crossing. In the current working model, when commissural enter the floor plate, sNrCAM, alone or together with additional floor plate factors, inactivates calpain1, which in turn prevents PlexinA1 processing thus allowing it to www.sciencedirect.com
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Figure 2
Axon guidance at the spinal cord midline. Schematic coronal sections of the embryonic spinal cord illustrating the axon guidance receptor expressed by pre-crossing (left) and post-crossing (right) commissural axons. Pre-crossing axons are repelled from the dorsal part of the spinal cord by Draxin and BMPs. They are attracted to the floor plate by Netrin1 and Shh. In pre-crossing axons, Slit/Robo repulsion is inactivated by Robo3.1. At the floor plate, SCF promotes midline exit and DCC/Netrin1 attraction is silenced by Robo1 in a Slit dependent manner. The expression of the other Robo receptors is upregulated and Slit repulsion is activated. In parallel, the inhibition of Calpain1 induced by soluble NrCAM leads to an upregulation of PlexinA1 expression on post-crossing commissural axons allowing it to form a complex with Neuropilin2 that mediates Sema3B repulsion. Shh is also involved in switching Sema3B repulsion and, in chick, may also directly repel post-crossing axons.
mediate Sema3B repulsion, together with Neuropilin2 [28]. How sNrCAM inhibits calpain is still unknown. Importantly, the phenotypic analysis of PlexinA1, NrCAM, and Sema3B knockouts brings further support to this model as in all three mutants, midline crossing by spinal cord commissural axons is strongly perturbed and mimics the defects found in Neuropilin2 knockout. Likewise, forcing expression of PlexinA1 in pre-crossing axons induces premature repulsion by floor plate. It remains to determine if the same mechanisms regulate semaphorin function in other parts of the CNS such as the cortex and if sNrCAM alone can activate the switch. A second study suggests that this might not be the case [29]. Floor plate cells produce the morphogen Sonic hedgehog (Shh) that was previously suggested to play a dual role at the midline: Shh attracts commissural axons toward the midline upon binding the Boc receptor (this requires Smoothened, Smo, but not Gli family members [34–36]) and after crossing, Shh repels post-crossing axons, an effect mediated by the Hhip receptor independently of Smo [37]. One should keep in mind that these two contrasted findings were obtained in different www.sciencedirect.com
species, rodents and chick respectively. The possible contribution of Patched (Ptc), the other major Shh receptor, to Shh chemoattractive function has not yet been studied, but Ptc is not expressed by chick commissural neurons and therefore does not mediate Shh chemorepulsive activity [37]. The loss of responsiveness to Shh chemoattraction after crossing is another unsolved mystery. Previous in vitro studies have shown that hindbrain commissural axons become fully unresponsive to floor plate attraction after crossing it [38] therefore rendering it likely that post-crossing commissurals lose sensitivity to Shh. The silencing of Shh attraction could be explained by a downregulation of Boc whose expression pattern in commissurals matches Robo3 [35]. A recent study shows that Shh has a third activity on commissural axons that is to act as a regulator of the semaphorin switch [29]. Adding Shh to dorsal spinal cord explants in the presence of Netrin1 is sufficient to trigger Sema3B and Sema3F repulsion. Likewise, applying Shh blocking antibodies to spinal cord explants prevents midline crossing and induces additional guidance errors. Electroporation of dominant negative forms of Ptc or of Current Opinion in Neurobiology 2011, 21:68–75
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its partner Smo in the rat neural tube severely perturbs midline crossing. Interestingly, similar axon guidance defects are present in explants treated with compounds that modify the level of cAMP in commissural axons. As adding Shh to the explants inhibits PKA, it has been proposed that the semaphorin switch is associated to a decrease of cAMP level in commissural axons. It is unclear how these molecules will initiate the switch from attraction to repulsion as Shh, Ptc, and Smo all seem
to be expressed in pre-crossing and post-crossing axons. However, the first study suggests that the upregulation of PlexinA1, without which Sema3B is silent, is the main trigger [28]. Shh could also be another floor platederived factor that can induce calpain inhibition, although, based on unpublished data [28], this does not seem to be the case. This apparent discrepancy could be explained by technical differences between the two in vitro studies such as the species (chick/mouse vs rat) or the assay (growth cone collapse vs repulsion). This could
Figure 3
Translational and Transcriptional modulation of axon guidance receptors. (a) In absence of Netrin1, DCC directly binds to many components of the translational machinery, thereby preventing translation. (b) Upon Netrin1 binding, DCC releases the components that can assemble in polysomes and activate the translation of unknown target mRNAs. (c) In Drosophila, Frazzled, in a Netrin1-independent manner, activates via an unknown pathway, the transcription of Commissureless (Comm) mRNA. In turn, Comm targets Robo receptors to endosomes and maintains a low level of Robo at the membrane. (d) The MiR-218.1 gene is localized in intron 15 of the Slit2 gene. Robo1 mRNA is one of the target of MiR-218.1. High Slit2 expression leads to low Robo1 expression. The second MiR218 gene, MiR218.2, is encoded by intron 14 of Slit3, but it is not known if it targets Robo receptors. Current Opinion in Neurobiology 2011, 21:68–75
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Further tales of the midline Che´dotal 73
also just reflect the diversity and heterogeneity of spinal cord commissural neurons. Yet, there is strong evidence supporting a cross talk between Shh and Sema3B signalling pathways. First, in drosophila, PKA regulates PlexinA activity [39]. Second, Shh chemoattractive action on commissural axons requires the Src family kinases, Src, and Fyn) that were previously shown [40] to associate with PlexinA1 and promote its phosphorylation in response to Sema3A. Moreover, inhibiting Src kinases was previously shown to convert Sema3B attraction of anterior commissure axons to repulsion, and Sema3B stimulates the recruitment of Src kinases to the membrane [33]. Although these two studies lift a corner of the veil, there is still plenty of room for other guidance molecules and other molecular interactions to be uncovered as being important for switching the response of spinal cord commissurals from attraction to repulsion. For example, one recent study showed that Robo cleavage by the metalloprotease Kuzbanian is involved in the regulation of midline crossing, and provided preliminary evidence suggesting that vertebrate robos are also proteolitically processed by Adam10 [41]. Another interesting candidate is VEGF (vascular endothelial growth factor) that is expressed by floor plate cells, at least in the hindbrain [42] and whose receptor VEGFR forms a complex with Neuropilin1 and PlexinD1 to mediate Sema3E attractive action on forebrain axons [31]. As both Sema3E and VEGF are present at the floor plate [27,42], it will be interesting to know whether a VEGF/Sema3E cross talk occurs during midline crossing.
Newcomers for midline found in transcription and translation As mentioned before, in the past few years, there has been little new receptors and ligands added to the list of molecules acting on midline crossing. By contrast, there is a recent burst of findings that reveals that known molecules and biochemical pathways can modulate the activity and expression of axon guidance receptors in commissural axons. Three reports show that transcription plays a central role in axon guidance at the midline by controlling the expression level of several receptors. In drosophila, the commissureless protein (Comm) controls midline crossing by preventing Robo receptors from reaching the cell surface [43]. High Comm expression in pre-crossing axons silences Slit repulsion and allows Frazzled (Fra; the DCC ortholog in fly) expressing axons to grow toward the midline in response to Netrin. After crossing, Comm is downregulated, Slit/Robo repulsion is activated, and this steers growth cones away from the midline. Bashaw and colleagues recently found that in www.sciencedirect.com
commissural axons, Fra activates the transcription of Comm mRNA independently of Netrin (Figure 3) [44]. How Fra activates Comm transcription is unknown. As there is still no known Comm orthologs in vertebrates it is also unclear if a similar mechanism exists in higher organisms. However, previous studies have shown that in vertebrates Netrin/DCC signalling can stimulate the import of NFAT transcription complex to the nucleus [45]. In addition, following cleavage by g-secretase, the intracytoplasmic domain of Neogenin, one of Netrin1 receptors in vertebrates, is cleaved and transferred to the nucleus where it activates transcription [46]. As there is also evidence for cleavage of DCC cytoplasmic domain [47], this could be one way to link DCC (or Fra) to gene transcription. It has previously been shown that in Xenopus, axon turning toward a Netrin source requires local protein synthesis in the growth cone [48] and that signals from the floor plate may also influence local translation during midline crossing [49]. A recent study shows that DCC directly controls translation by interacting through its cytosplamic tail (in particular the P1 domain) with many components of the translation machinery such as initiation factors and ribosome subunits (Figure 3) [50]. DCC activation by Netrin1 releases the translation complex and it can then assemble into active polysomes and perform protein synthesis. More relevantly for this review, electroporation in the chick spinal cord of DCC constructs lacking the P1 domain severely perturbs commissural axon growth toward the midline suggesting that DCC regulation of translation is functionally relevant. DCC translational targets remain to be identified. Although such mechanisms have not yet been described for Robo receptors, a study conducted in tumor cells also revealed some previously unsuspected links between Slits, Robos, and transcription/translation. Microribonucleic acids (MiRNAs) are 21–24 nucleotide long posttranscriptional modulators that bind to the 30 UTR of their target mRNAs, preventing their translation [51]. MiR-218 is a miRNA whose expression is downregulated in some invasive cancer cell lines [52]. A bioinformatic screen and in vitro experiments showed that one of miR-218 target is Robo1. More surprisingly, it was found that one of the two miR-218 precursors is produced from a DNA sequence localized within an intron of the Slit2 gene (Figure 3). Therefore Slit2 and miR-218 are co-transcribed from a common promoter and MiR-218 represses Robo1 expression. Moreover, the second miR-218 precursor is localized in the Slit3 sequence but is not synthetized in tumor cells. There is as of yet no evidence that this negative feedback loop has a function at the midline but this could represent another regulatory mechanism controlling the combinatorial expression of Robo receptors and thereby Slit/Robo function in midline crossing [53]. Current Opinion in Neurobiology 2011, 21:68–75
74 Developmental neuroscience
Conclusion Although there still is a pile of questions to answer, there has been some constant progress toward a better understanding of midline crossing in vertebrates. An emerging picture is that in every part of the CNS, commissural axons are guided by the same basic set of molecules: netrin1/DCC, a morphogen (Wnt5a or Shh), Slits and Robos, some secreted semaphorins, one Neuropilin, and PlexinA. This suggests that the signalling pathways and cross talk are highly conserved between commissural axons. However there are also some noticeable differences between forebrain and hindbrain neurons: Robo3 does not control commissure development in the forebrain and no repulsive BMPs are known to pattern commissurals at this level. In addition, the molecular mechanisms regulating the response of forebrain commissural axons to midline attractants and repellents before, during and after crossing are unknown.
Acknowledgements I thank Athena Ypsilanti and Vale´rie Castellani for critical reading of the manuscript. This work is supported by the Agence Nationale pour la Recherche (ANR), and the Fondation pour la Recherche Me´dicale (Equipes labelise´es FRM).
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