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Axon guidance to and from choice points
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Axon guidance to and from choice points Geoffrey Cook, David Tannahill and Roger Keynes* Significant progress has been made recently in understanding axon guidance to and from choice points. Netrins have been shown to function as conserved midline chemoattractants in vertebrates and insects, and receptors for netrins and semaphorins/collapsins have been identified. More evidence has accumulated that repulsion plays a key role in guidance, including the involvement of the ephrin/Eph receptor system in contact repulsion.
Addresses Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK *e-mail: rjkl [email protected] Current Opinion in Neurobiology 1998, 8:64-?2 htt p://biomednet.com/elecref/0959438800800064 © Current Biology Ltd ISSN 0959-4388 Abbreviations AP alkaline phosphatase CGRP calcitoningene related peptide Dec deleted in colon carcinoma DRG dorsal root ganglion HGF/SF hepatocyte growth factor/scatter factor Ig immunoglobulin NGF nerve growth factor NT-3 neurotrophin 3 Sema semaphorin
Introduction
At first sight, axon growth cones are set a formidable task in navigating to their often distant targets in the embryo. The task is simplified, however, by the fragmentation of their journey into shorter steps interrupted by intermediate targets, or choice points, at which other cells provide critical guidance cues that direct growth cones on the next stage of their trajectory'. A well-known example is provided by the specialized cells comprising the CNS midline in both insects and vertebrates, as discussed below. Choice points are also provided by smaller, isolated groupings of guidepost cells, which may be neurons themselves; in their absence, the earliest pioneer growth cones often make pathfinding errors [1-3]. Later-navigating axons may be guided to and from choice points by selective fasciculation on earlier-developing axons (as discussed in an accompanying review by Van Vactor, in this issue, pp 80-86). We focus here on the nature and function of the external cues that orient axons before and after their encounters with choice points, and that set the overall trajectories of axons and their fascicles. Netrins
The vertebrate floor plate is an intermediate, midline target for commissural axons traversing the spinal cord, and provides a prototype for axon guidance towards a choice
point region by chemoattraction. With the isolation of the netrins as floor plate chemoattractants [4] (Figure la), and the revealed homology between the netrins and UNC-6, a laminin-related protein required for circumferential growth of axons in the body wall of Caenorhabditis e/egans, there is now good evidence that netrins provide highly conserved midline guidance cues for axons, tlomologs have also been identified in the human genome [5] and in flies. In the latter case, two netrin genes map in tandem on the X chromosome, and embryos deficient for this region show defective commissures that can be rescued by expression of either netrin at the midline. Pan-neural expression of either gene also disrupts commissural and longitudinal tracts, showing that the pattern of netrin expression is critical for guidance. The presence of defects in motor axon projections in the double mutant further shows that netrins are required for motor axons to reach their correct target muscles [6",7°]. As predicted by earlier studies, netrin-l-deficient mice show abnormal spinal commissural axon projections I8"']. More evidence that netrin guides spinal commissural axons comes from analysis of the zebrafish mutant floating head, which lacks a notochord; as a result, netrin-la expression is absent in patches of caudal spinal cord, and commissural neurons sited in between these patches extend their axons aberrantly along the anterior-posterior axis towards the nearest available region of normal netHn-la expression, presumably through chemoattraction [9°]. Such results are explicable exclusively in terms of netrin, but it is possible that other, as yet unidentified attractants are also involved in guiding axons to the midline, especially as the trajectories of substantial numbers of commissutal axons are unperturbed in netrin-deficient mice. Netrin-1 is important for axon guidance to the midline in the brain, as nettin-deficient mice show additional defects in the corpus callosum as well as hippocampal and anterior commissures [8"], and it has been shown to attract ventrally decussating axons in the developing brainstem [10]. The internal capsule may provide a further source of netrin-1 in developing brain, acting as a chemoattractant for neocortical axons navigating towards the thalamus [ 11,12]. The possibility of extensive involvement of netrins in axon guidance within the brain has also been revealed by a detailed study of netrin gene (netHn-la) expression in the developing zebrafish [9°]. The macroscopic pattern of netrin-la expression in the early zebrafish brain comprises a ventral longitudinal stripe, inclusive of the floor plate, intersected at discrete locations (such as the isthmus and rhombomere boundaries) by orthogonal dorsoventral stripes. This correlates well with the orthogonal pattern of pioneer axon tracts in the brain,
Axon guidance to and from choice points Cook, Tannahill and Keynes
65
Figure 1
(a)
(b) Netrins
Netrinfamily receptors
(c)
Semaphorins
{
Ig Ig
Neuropilins
Ephrin-A Ephrin-B EphA/B
r--~
l--
Extracellular
s
DCC Frazzled UNC-40
UNC-5
~
I I
Sema III G-sema I SemD Collapsin-I
I
GP
I I I
Plasma membrane Intracellular
CurrentOpinionin Neurobiotogy
Outline structure of families of axon guidance molecules discussed in the text. (a) Netrins and their receptors; (b) semaphorins (secreted and transmembrane forms shown) and their receptors (neuropilins); and (c) ephrins (class A showing GPI linkage to the cell surface; class B showing transmembrane linkage) and their cognate Eph receptors. B +, basic domain; C, coagulation factor homology domain; CUB, complement/sea urchin-EGF/BMP1 domain; CYS, cysteine-rich domain; Egf, epidermal growth factor repeat; FN, fibronectin type III domain; GPI, glycophosphatidylinositot linkage; Ig, immunoglobulin domain; Lam, region with homology to amino terminal domains V and VI of laminin chains; MAM, meprin/A5/PTPmu domain; Sema, semaphorin domain; TK, tyrosine kinase domain; Ts, thrombospondin type I domain.
which develop at the borders of, or within, the domains of netrin-la expression [9°]. Lastly; netrin-1 expression by cells at the developing optic disc provides a local, short-range guidance cue for retinal axons. Nctrin-1 is expressed in the developing eye in several vertebrates [13,14°,15,16]. Retinal growth cones have been shown to turn towards a point source of netrin-1 in v,itro [17°], and in netrin-l-deficient mice (as well as DCC nulls, see below), many axons fail to exit the retina into the optic nerve, reaching the disc but growing past it into other retinal regions [14"]. In this case, the range of netrin action is more restricted than in the spinal cord; while the factors that modulate netrin diffusion in vivo are unknown, one possibility would be the binding of netrins to extracellular matrix molecules expressed in the vicinity of the netrin source. Netrin
receptors
The function of netrin signaling in vivo has been impressively confirmed by studies on the putative netrin receptors. The homology of netrins with UNC-6 suggested that the vertebrate homolog of UNC-40, a candidate reccptot involved in ventral migrations in C. e/egans, might be the netrin receptor. Two such genes, DCC (deleted in colon carcinoma) and neogenin, have now been identified [18"1. DeC, originally characterized as a tumour suppressor gcne that is frequently lost in human colorectal carcinomas, encodes a group of structurally related transmembrane
proteins with four immunoglobulin and six fibronectin type IIl repeats in their extracellular domains (Figure la). While a definitive role for DCC in colorectal carcinoma remains to be shown, it does have a clear function during neural development. DCC protein is expressed on commissural axons as they project towards the floor plate, binds netrin-I in vitro with high affinity, and a monoclonal antibody directed against its extracellular domain selectively blocks netrin-l-dependent outgrowth of commissural axons in collagen gels [18"]. Tile turning response in vitro of Xenopus retinal growth cones towards a micropipette ejecting nctrin-1 is also blocked by neutralizing DCC antibodies [17°]. The phenotype of mice lacking functional DCC has reinforced its status as a netrin receptor: like the netrin-I knockout phenotype, spinal commissural axons are misrouted, forebrain axons make aberrant ipsilateral projections and fail to form a corpus callosum and hippocampal commissure, while the anterior commissure is severely reduced [19"°]. Striking confirmation of the phylogenetic conservation of the netrin-receptor system comes from studies in flies and worms. The Drosophila DCC homolog frazzled is expressed by embryonic CNS axons and motor axons in the periphery, and the null phenotype appears identical to the netrin null phenotype with thin or absent commissures
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[20*']. uric-40 has also been shown to encode the nematode homolog of DCC, neogenin and frazzled [21*°]. T h r e e gene products, UNC-5, U N C - 6 and UNC-40, are essential for dorsoventral circumferential migrations in the body wall of C. e/egans. A model has becn suggested in which cclls and axons migrating ventrally orient towards a source of UNC-6 protein using UNC-40 as a receptor, whereas dorsal migrations orienting in the opposite direction use UNC-5 and UNC-40 as co-receptors for a repulsive response to U N C - 6 [22]. Consistent with this, U N C - 6 expression is restricted to the ventral cells within each region of the nervous system [231. UNC-40 is expressed by cells migrating in both ventral and dorsal directions, but dorsal migrations are less disrupted in UNC-40 nulls than in UNC-5 and U N C - 6 nulls, so this function of UNC-40 may bc redundant to a second pathway, pcrhaps involving a distinct DCC homolog [21"°1. Do netrins, like UNC-6, repel as well as attract axons? Netrin-secreting cells and floor plate explants repel trochlear motor axons in vitro [24], but so do floor plate explants taken from netrin-l-deficient mice [8°']. Moreover, although many axons growing from explants of the ventral half of the rostral midbrain are repelled by the floor plate, mimicking the behaviour in vivo of posterior commissure axons, they are not repelled by netrin-1 [101. It seems likely, therefore, that floor-plate-derived chemorepellents other than netrin-1 are involved in setting these dorsally directed axon trajectories, and Sema IIl/collapsin-1 (see below) has been suggested as a candidate [25]. T h e possibility nonetheless remains that netrins may repel axons. Cultured Xenopus spinal neurons will normally turn towards a micropipette releasing netrin-1, but this can be converted to a repulsive response by agents that reduce the level of cytoplasmic cAMP-dependent protein kinase A [26°]. Attractive and repulsive responses are both prevented by neutralizing antibody to D C C [26*], implying that the intracellular signaling pathways for these opposite responses are related (see accompanying review by Holland, Peles, Pawson and Schlessinger, in this issue, pp 117-127). T h e involvement of D C C in repulsion also raises the question of whether netrin-based repulsion in vertebrates is homologous to the UNC-40/UNC-S co-receptor system suggested for dorsal migrations in C. elegans. Vertebrate homologs of UNC-5 (Figure la) have been identified as netrin-binding proteins, and are expressed in vivo by axons that are repelled bv netrin-1 in vitro [27°]. T h e further finding that these homologs are expressed in the same regions of the developing C N S as DCC is at least consistent with thcir putative function as co-receptors [27°], and underlines the remarkable conservation of this guidance system from nematodes to chordates.
Semaphorins T h e semaphotin family, which now counts more than 30 members, has been strongly implicated in axon guidance in both flies and vertebrates [28-31]. Semaphorins are
related by a homologous stretch of some 500 amino acids, comprising the semaphorin domain, and can be grouped structurally into five types that include both secreted and membrane-bound forms (Figure lb). Secreted semaphorins possess a carboxy] C2-type immunoglobulin (Ig) domain followed by a highly basic tail, whereas membrane-bound forms have either a carboxyl Ig domain, several thrombospondin repeats or no discernible features carboxyl to the semaphorin domain. T h e large number of semaphorins, coupled with their complex expression patterns in the embryo, has meant that their potential biological function has been assessed in only a few cases. Studies in insects have suggested that secreted semaphorins may be involved in target recognition by motor axons (see accompanying review by Holt and Harris, in this issue, pp 98-105), while one transmembrane semaphorin, G-Sema I, has been implicated as both a repulsive and attractive/permissive guidance factor for different axon subtypes navigating within the grasshopper limb bud. G-Sema I, originally identified by a monoclonal antibody and designated fasciclin IV [32[, is expressed in the limb bud epithelium, and Til pioneer neurons projecting from the bud towards the C N S apparcntly respond to a stripe of G-Sema I expression as a repulsive cue, stalling and tt, rning ventrally to follow the boundary of G-Sema I expression [31,32] (Figure 2). Later in development, another group of axons, originating more distally in the bud, reach the C N S by growing across a second band of G-Sema I-expressing cells and then fasiculating upon the Til axons. In reaching these axons, growth cones appear to use the G-Sema I band as an attractive/permissive cue, as neutralizing antibody to G-Sema I prevents their extension across it [33 °] (Figure 2). T h e best studied vertebrate scmaphorin is known as collapsin-1 in the chicken, Sema III in humans and SemaD in rodents, and is referred to here as Sema III. It was originally purified from adult chicken brain as a secreted glycoprotein that induces the paralysis and growth cone collapse of cultured sensory dorsal root ganglion (DR(;) axons [29], and independently as a vertebrate homolog of G-Sema I [28]. Consistent with a role for Sema III in axon guidance, it has been shown that sensory axons can be steered away from immobilized sources of Sema III [34], and both sensory and sympathetic axons, but not retinal axons, grow away from COS cells expressing Sema I11 in collagen gcls [35,36]. Spinal motor axons and certain cranial motor axons have also been shown to be responsive i, vino [25]. Scma IIl has been implicated in the patterning of sensory axon projections in the developing spinal cord. It is likely that a ventral-to-dorsal gradient of Sema III protein is established in the spinal cord during these stages, as sustained expression of sema III is seen in the ventral half of the neural tube [35,37]. Detailed analysis of the sensitivity of D R G axon subtypes in vitro has shown
Axon guidance to and from choice points Cook, Tannahill and Keynes
67
Figure 2 (a) Wild-type O)
(b) Til ablation
(ii)
(c) Anti-G-Sema I
Current Opinion in Neurobiology
Role of G-Sema I in grasshopper limb bud. (a) In wild-type embryos, (i) Til neurons project axons from their cell bodies towards the CNS, responding to a repulsive stripe of epithelial G-Sema I expression by turning ventrally, whereas (ii) the subgenual organ (SGO) cell body lies more distally in the bud, and its axon reaches the CNS by growing across a band of G-Sema-l-expressing cells and then fasiculating on the Til axons. (b) After Til ablation, the S G O axon stalls within the band of G-Sema I. (c) The S G O axon fails to sprout across the band of G-Sema I in the presence of neutralizing antibody to G-Sema I, suggesting that G-Sema I acts as a permissive/attractive guidance cue.
that both nerve growth factor (NGF)-dependent nociceptive afferents and neurotrophin 3 (NT-3)-dependent proprioceptive afferents are repelled by Sema III when explanted at early stages [38"]. This broad-spectrum in vitro sensitivity is associated in vivo with the transient early expression of Sema III adjacent to the site at which sensory afferents first enter the dorsal spinal cord, perhaps confining them locally by repulsion. Later, NT-3- but not NGF-dependent axons lose their responsiveness to Sema III, correlating with the ventral projection of proprioceptive afferents in the spinal cord towards their neuronal targets [35]. In support of the repulsion model, ventral neural tube explants are known to chemorepe] sensory axons in vitro [39], and this activity can be neutralized by an anti-Sema III antibody [38°]. Targeted disruptions of mouse sema I I I have, however, yielded conflicting results regarding the role of Sema III in sensory afferent ingrowth in the spinal cord. In one phenotype, some NGF-dependent sensor3" afferents (CGRP-positive) misproject to more ventral locations [40°], while in another, no aberrant ventral projections are observed [41°]. A possible explanation for the discrepancy concerns the different genetic backgrounds used in the two studies, complicating a direct comparison between them and raising the question of gene compensation. Alternatively,
the discrepancy may lie in the assessment of different subpopulations of NGF-dependent afferents in the two studies. Other defects seen in one of the sema I I I knockout studies [41"] suggest possible roles for Sema III in axon guidance in the craniofacial region. T h e cranial branchiomotor nerves, for example, defasciculate in their branchial arch territories, although their overall trajectory is unchanged and they do appear to reach their targets. As Sema III is normally expressed in the branchial arches as axons grow into them, it may drive axons to fasciculate by creating a repulsive surround (see also review by Van Vactor, in this issue, pp 80-86). A related explanation could account for a further feature of the knockout phenotype seen in the DRGs, which now sprout many of their axons from their lateral rather than ventral side. In this case there is a change in trajectory, consistent with the possibility that Sema III, secreted by the dermomyotome, participates in surround-repulsion of early DRG axons, as outlined below (Figure 3). Neuropilins
Progress has been made recently in the characterization of receptors for the semaphorins. A high affinity
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Development
Figure 3 (a) Mouse/chick DRG
(b) Sema III/neuropilin mouse DRG
"t
Current Opinion in Neurobiology
Surround repulsion of DRG axons. (a) Transverse section through the trunk region of a mouse/chick embryo, showing the trajectory of axons sprouting from a DRG cell body (filled black circle) within the sclerotome (Sc). The dermomyotome (Dm) lies lateral to the DRG neurons and secretes repellent molecules towards them (short arrows), supplemented by the ectoderm (arrowheads). The medially placed notochord (NC) and ventral neural tube (NT) also secrete repellents (long arrows), so DRG axons are flanked by two sources of chemorepulsion. (b) In sema III or neuropilin knockout mice, some DRG axons sprout aberrantly towards the dermomyotome. According to the surround-repulsion model, this would result from the imbalance created by the loss of lateral chemorepulsion and the continued operation of midline-derived chemorepulsion.
Sema-III-binding protein, neuropilin, has been identified using an alkaline phosphatase-tagged (AP) Sema III ligand to screen a COS cell expression library [42",43"], and neuropilin homologs have been found in a variety of vertebrates. Neuropilin was previously identified as a transmembrane glycoprotein expressed in the Xenopus tectum (Figure lb), and was suggested to participate in axon-axon or axon-target interactions [44]. Consistent with its potential rolc as a Sema III receptor, neuropilin is expressed on Sema-III-responsive axons, including sensory', sympathetic and spinal motor axons, and application of neutralizing anti-neuropilin antibodies blocks Sema-llI-mcdiated growth cone collapse and chemorepulsion in collagen gels [42",43"]. At least two distinct neuropilins have now been identified [43",45"], and these have largely nonoverlapping expression patterns in the developing nervous system. Neutopilin-1 appears to bind all secreted semaphorins that have been tested with high affinity, whereas neuropilin-2 binds Sema E (collapsin-3) and Sema IV, but not Sema III (collapsin-1) [45"]; the possibility remains, however, that neuropilin-2 binds Sema III with lower affinity: Although the carboxy-terminal doraain or the semaphorin domain is sufficient for neuropilin binding, the use of chimeric semaphorins has shown that the semaphorin domain appears to impart specific binding in embryo sections [46"]. Different semaphorins are also likely to have distinct biological specificities, as collapsin-1 can induce collapse of both DRG and sympathetic growth cones, collapsin-2 affects neither, whereas only sympathetic growth cones respond to collapsin-3 [47"]. As for receptor binding, the specificity of the collapse-inducing activity of collapsins 1
and 3 is also mediated by the semaphorin domain, which must be dimerized to function, and is conferred by a 70 amino acid stretch within its amino-terminal half. Although neuropilin-1 may be sufficient for ligand binding, these results raise the possibility that it is a common component of a larger receptor complex, binding semaphorins/collapsins through their carboxyl tails, while other, as yet unidentified, components of the complex impart biological specificity [47"]. The phenotype of neuropilin-deficient mice is consistent with the role of neuropilins as semaphorin receptors. Like the sema III knockout mice, neuropilin knockout mice exhibit defasciculation in certain cranial branchiomotor nerves, and in the segmental nerves innervating the limb buds, and the DRGs produce aberrant lateral projections towards the dermomyotome consistent with the surround-repulsion model [48"] (Figure 3). E p h r i n l i g a n d s a n d Eph r e c e p t o r s A variety of ephrins and their Eph receptors (Figure 1c) have been implicated in guiding axons to and from choice points, axon fasciculation and target selection (see [49] for a review). We focus here on recent studies showing that these molecules are involved at early stages in the formation of axon trajectories, guiding peripheral axons into the somites and limb buds. The roles of ephrins and receptors in guiding CNS axons across the midline, as well as in axon fasciculation, target selection and growth cone signal transduction are discussed in accompanying reviews (see Van Vactor pp 80-86, Holt and Harris pp 98-105, and Holland, Peles, Pawson and Schlessinger pp 117-127, in this issue).
Axon guidance to and from choice points Cook, Tannahill and Keynes
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The segmented pattern of peripheral axons in higher vertebrates results from the preference of motor and sensory axons, and earlier-migrating neural crest cells, for growth through the anterior rather than posterior half of the somite-derived sclerotome [50]. This preference correlates with the expression by posterior cells of molecules that cause collapse in vitro of motor and sensory axon growth cones [51], implying that repulsion of axons by posterior cells in vivo causes peripheral nerve segmentation by forcing axons to traverse the consecutive anterior half-sclerotomes. Several candidate repellents expressed in posterior half-sclerotome have now come to light, including peanut-lectin-binding glycoproteins [51], Sema III [52,53], T-cadherin [54], versican [55] and collagen IX [56]. A critical role for Sema III is unlikely, however, in view of the normal spinal nerve segmentation seen in both sema III and neuropilin knockout mice [40°,41°,48"].
While the factors mediating axon segmentation influence all classes of vertebrate spinal motor neuron, the expression patterns of Eph receptors by motor neuron subsets suggest a possible role for this system in more specific axon guidance decisions. In the chick embryo, the EphA4 (Cek8) receptor is expressed preferentially in motor neurons that innervate limb muscle rather than axial muscle [59], and the ephrins A2 and A5 are expressed in regions of the limb bud avoided by axons [60]. In contrast, the EphA3 (Tyro4) receptor in the rat is restricted to motor neurons innervating axial muscle [61]. Ephrin-A5 (AL-1), moreover, is expressed at higher levels in rostral rather than caudal mouse muscles and, reciprocally, inhibits the outgrowth of axons from caudal rather than rostral motor neurons [62]. Whether these findings relate to axonal recognition of target muscle subsets in vivo, or to earlier stages of axon guidance, is at present uncertain.
Two transmembrane ephrins, ephrin-B2 (HtkL/Lerk5/Elf-2) in the rat [57 "°] and ephrin-B1 (Lerk2/Elk-L/Cek5-L) in the chick [57",58"*], are also expressed by posterior but not anterior cells, whereas the predominant Eph receptor expressed by neural crest cells is EphB2 (Nuk) in the rat [57 °"] and EphB3 in the chick [58"°]. In vitro assays of neural crest migration and motor axon outgrowth have shown that clustered ephrin ligands can guide by repulsion, provided they are presented in a discontinuous or graded manner. For example, rat motor axons (which also express the EphB2 receptor) avoid stripes of ephrin-B1 and ephrin-B2 superimposed on a uniform laminin substrate, but will grow on a uniform ephrin/laminin substrate if given no alternative [57"]. Such behaviour is more consistent with growth-repulsion than growth-inhibition, and contrasts with the inhibition seen using uniform substrates of T-cadherin [541 and collagen IX [56]. Further evidence for the involvement of ephrin-induced repulsion in peripheral nerve segmentation comes from the observation that crest cells migrate aberrantly into the posterior half-sclerotome when soluble, unclustered ephrin-B1 (acting as a competitive antagonist of endogenous ephrins) is added to explants of chick embryo trunks [58"'].
Further chemoattractants and chemorepellents In the search for chemoattractants other than the netrins, the hepatocyte growth factor/scatter factor (HGF/SF) has been identified as a chemoattractant for motor axons secreted by limb bud mesenchyme, and a subset of motor neurons expresses c-Met, the receptor for HGF/SF [63"]. Motor axon branching patterns in the developing forelimb of HGF/SF knockout mice show abnormalities consistent with a chemoattractive role for HGF/SF, although this interpretation is complicated by the fact that the same protein is required for the migration of myoblast precursors into the limb [63"]. Assuming the cytoskeletal response within the growth cone to c-Met activation to be similar to that following DCC activation, it will be interesting to see how the respective signaling pathways converge (see accompanying review by Holland, Peles, Pawson and Schlessinger, in this issue, pp 117-127).
As for the other candidate molecular mediators of peripheral nerve segmentation, a critical role for the ephrin-Eph-receptor system remains to be shown directly in vivo. The normal patterns of crest migration and motor axon outgrowth seen in EphB2 (Nuk);EphB3 (Sek4) double-mutant mice could be attributable to functional compensation by other Eph family members [57"'], but could equally be attributable to the continued operation of other guidance systems. It will also be important to test whether chick sensory axon growth cones, which collapse in response to a somite-derived glycoprotein fraction [51], collapse in response to clustered ephrin-B1.
In addition to the floor plate (see above), the optic chiasm has been identified as a further choice point region at which axons may be subject to negative growth regulation (e.g. chemosuppression [64]), but the relevant molecules remain to be characterized. Models of chemorepulsion usually place the source of repellent either in front of axons (e.g. repulsion of spinal cord afferents by Sema III/collapsin-1 [35]) or behind them (e.g. repulsion of cranial branchiomotor and trochlear motor neurons by the floor plate [24,25]). A recent study has shown that repellents can also generate linear patterns of axon growth when secreted in gradients that flank neurons on either side. Chick or mouse DRGs sprout radially in three dimensions when cultured with neurotrophins in collagen gels, but are constrained to sprout in a bipolar manner when sandwiched between a length of notochord and a dermomyotome placed at a distance. These tissues flank the early DRGs in vivo, and the oriented bipolar trajectory of primary sensory axons extending from the DRGs in the
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Development
dorsoventral plane is suggested to be due to the resulting surround-repulsion exerted on axons [65*] (Figure 3). One of the repellent molecules may be Sema III/collapsin-1 (see above); it is expressed by the dermomyotome, and its loss of function in Sema III and neuropilin-deficient mice may, according to the surround-repulsion model, explain the abnormal sprouting of DRG axons towards the dermomyotome seen in these animals [41°,48 °] (Figure 3).
2.
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Conclusions Significant advances have been made recently in characterizing attractive and repulsive ligands and receptors that guide axons to and from choice points. Manipulating their expression in both mice and flies has generally confirmed their functional importance and, for the netrins and semaphorins, their phylogenetic conservation, while invertebrate homologs of the ephrins/Eph receptors have yet to be described. Both vertebrates and invertebrates use choice points for axon guidance, but the extent to which the underlying mechanisms have evolved independently at the molecular level is unknown, and will become clearer as further guidance molecules are identified. To this end, candidate genes have been identified by systematic genetic screens for axon pathfinding mutants. Recent examples come from screens in C. elegans [66] and the zebrafish [67"], and the molecular cloning and functional analysis of examples such as the zebrafish mutants bashful, grumpy and sleepy, in which retinal axons projecting to the opposite tectum make errors after crossing the midline [67"], should be revealing. Lastly, it is also relevant that, in modelling axon guidance by diffusible factors, the maximum range for guidance by chemoattraction or chemorepulsion has been estimated to be some 500-10001.tin for a stable gradient, expanding to much greater distances before stabilization [68]. It may be significant, then, that transplanted foetal rat CNS neurons can grow axons in the adult rat CNS to appropriate target structures at least 10 mm away from the implantation site [69], raising the intriguing possibility that long-distance guidance mechanisms in the embryo may also exist in the mature nervous system. This will be interesting to assess alongside the further characterization of guidance molecules and their signalling pathways.
Acknowledgements Our work is supportcd by grants from the Medical Research Council and the Wellcome Trust. G Cook is a Member of the External Scientific Staff of the Medical Research Council, I) Tannahill is a Royal SocietT t.Tniversity Research Fellow and R Keynes is an lntcrnational Research Scholar of the Howard Hughes Medical Institute.
6. •
Mitchell KJ, Doyle JL, Serafini T, Goodman CS, Dickson B J: Genetic analysis of netrin genes in Drosophila: netrins guide CNS commissural axons and peripheral motor axons. Neuron 1996, 17:203-215. Two Drosophila netrin genes are identified, their expression patterns analysed, and the results of loss- and gain-of-function assessed. Midline commissures are disrupted under both conditions, showing that the pattern of netrin expression is critical for netrin-based axon guidance. 7. •
Harris R, Sabatelli LM, Seeger MA: Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila netrin/UNC-6 homologs. Neuron 1996, 17:217228. This paper describes essentially the same strategy for the analysis of Drosophila netrin genes as [6"]; as in the latter study, ectopic expression of either netrin throughout the CNS causes similar axon guidance defects to those seen in loss-of-function mutants. 8. •.
Serafini 1", Colamarino SA, Leonardo ED, Wang H, Beddington R, Skarnes w e , Tessier-Lavigne M: Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Ceil 1996, 87:1001-1014. This paper describes the phenotype of netrin-l-deficient mice, highlighting the abnormal ventral cornrnissures in the spinal cord and brain. The data provide strong evidence confirming the chemoattractive role of netrin-1 in guiding axons across the vertebrate midline, as well as the phylogenetic conservation of netnn function from nematodes to chordates. 9. •
Lauderdale JD, Davis NM, Kuwada JY: Axon tracts correlate with netrin-la expression in the zebrafish embryo. Mol Cell Neurosci 1997, 9:293-313. A detailed study of netrin 9ene expression in zebrafish, with an analysis of the floating head mutant, providing further evidence that netrin attracts spinal comrnissural axons. 10.
Shirasaki R, Mirzayan C, Tessier-Lavigne M, Murakami F: Guidance of circumferentially growing axons by netrin-dependent and -independent floor plate chemotropism in the vertebrate brain. Neuron 1996, 17:1079-1088.
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Richards U, Koester SE, Tuttle R, O'Leary DDM: Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target. J Neurosci 1997, 17:2445-2458.
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Metin C, Deleglise D, Serafini T, Kennedy TE, Tessier-Lavigne M: A role for netrin-1 in the guidance of cortical efferents. Development 1997, 124:5063-5074.
13.
Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M: Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Ceil 1994, 78:425-435.
14. •
Deiner MS, Kennedy TE, Fazeli A, Serafini T, Tessier-Lavigne M, Sretavan DW: Netrin-1 and Dec mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia. Neuron 1997, 19:575-589. Further analysis of netrin-l-deficient mice shows a defect in guidance of retinal axons at the optic disc. In this case, netrin-1 appears to provide short-range guidance for axons. 15.
Livesey FJ, Hunt SP: Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral and cerebellar development. Mo/Ceil Neurosci 1997, 8:417-429.
16.
Str&hle U, Fischer N, Blader P: Expression and regulation of a netrin homolog in the zebrafish embryo. Mech Dev 1997, 62:147-160.
17 •
De la Torre JR, HSpker VH, Ming G-I, Poo M-m, Tessier-Lavigne M, Hemmati-Brivanlou A, Holt CE: Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 1997, 19:1211-1224.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: ,, =.
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Axon g u i d a n c e to and f r o m choice points Cook, Tannahill and Keynes
A clear demonstration that netrin-1 alone is sufficient to evoke axon turning in vitro, through a DeC-mediated response. The results also support those of Deiner et aL [14 °] in showing a role for netrin-1 in retinal axon guidance. 18. ••
Keino-Masu K, Masu M, Hinck L, Leonard• ED, Chan SSY, Culotti JG, Tessier-Lavigne M: Deleted in Colorectal Cancer (DCC) encodes a netrin receptor. Ceil 1996, 87:175-185. The identification of DCC as a netrin receptor by homology screening. The paper reports a combination of in vitro binding studies, analysis of protein expression in vivo, and results of functional perturbation using neutralizing antibody, Fazeli A, Dickinson SL, Hermiston ML, Tighe RV, Steen RG, Small CG, Stoeckli El-, Keino-Masu K, Masu M, Rayburn H et al.: Phenotype of mice lacking functional Deleted in colorectal cancer (Dec) gene. Nature 1997, 386:796-804. Reports the D C C knockout phenotype, showing strong similarity to the netrin-1 knockout phenotype.
33. •
Wong JTW, Yu WTC, O'Connor TP: Transmembrane grasshopper semaphorin I promotes axon outgrowth in vivo. Development 1997, 124:3597-3607. This paper shows that transmembrane semaphorins may have bifunctional properties in axon guidance, providing permissive/attractive cues for some axons alongside repulsive cues for others. 34.
Fan J, Raper JA: Localized collapsing cues can steer growth cones without inducing their full collapse. Neuron 1995, 14:263-274.
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Messersmith EK, Leonardo ED, Shatz C J, Tessier-Lavigne M, Goodman CS, Kolodkin AL: Semaphorin III can function as a selective chemorepellent to pattern sensory projections in the spinal cord. Neuron 1995, 14:949-959.
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POschel AW, Adams RH, Betz H: The sensory innervation of the mouse spinal cord may be patterned by differential expression of and differential responsiveness to semaphorins. Me/Ceil Neurosci 1996, 7:419-431.
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Shepherd I, Luo Y, Raper JA, Chang S: The distribution of collapsin-1 mRNA in the developing chick nervous system. Dev Biol 1996, 173:185-199.
19. ••
20. ••
Kolodziej PA, Timpe LC, Mitchell KJ, Fried SR, Goodman CS, Jan LY, Jan YN: frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Ceil 1996, 87:197-204. Identification and functional analysis of a Drosophila netrin receptor with homology to DCC. The midline phenotype of the frazzled null mutant is identical to that of netrin loss-of-function and can be rescued by pan-neural expression of frazzled. 21. o•
Chan SSY, Zheng H, Su MW, Wilk R, Killeen MT, Hedgecock EM, Culotti JG: UNC-40, a C. elegans h e m • l o g of DCC (Deleted in Col•rectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Ceil 1996, 87:187-195. Reports the positional cloning of unc-40, revealing homology with vertebrate DGC and neogenin. The paper also suggests that UNC-40 and UNC-5 are required as co-receptors to orient migrating cells away from a source of netrin/UNC-6. 22.
Culotti JG: Axon guidance mechanisms in Caenorhabditis elegans. Curt Opin Genet Dev 1994, 4:587-595.
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Wadsworth WG, Bhatt H, Hedgecock EM: Neuroglia and pioneer neurons express UNC-6 to provide global and local netrin cues for guiding migrations in C. elegans. Neuron 1996, 16:35-46.
24.
Colamarino SA, Tessier-Lavigne M: The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons. Ceil 1995, 81:621-629.
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Varela-Echavarria A, Tucker A, P~Jschel AW, Guthrie S: Motor axon subpopulations respond differentially to the chemorepellents netrin-1 and semaphorin D. Neuron 1997, 18:193-207.
71
38. •
Shepherd IT, Luo Y, Lefcort F, Reichardt LF, Raper JA: A sensory axon repellent secreted from ventral spinal cord explants is neutralized by antibodies raised against collapsin-1. Development 1997, 124:1377-1385. This study shows that antibodies raised against chick collapsin-1/Sema III neutralize the chemorepulsive activity of chick ventral spinal cord explants for sensory axons, supporting the possibility that repulsion by cotlapsin1/Sema III may guide sensory afferent targeting in the spinal cord in rive. 39.
Fitzgerald M, Kwiat GC, Middleton J, Pini A: Ventral spinal cord inhibition of neurite outgrowth from embryonic rat dorsal root ganglia. Development 1993, 117:1377-1384.
40. •
Behar O, Golden JA, Mashimo H, Schoen FJ, Fishman MC: Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature 1996, 383:525-528. Reports the phenotype of the sema III knockout mouse. The main abnormality in the nervous system is the extension of CGRP-positive sensory afferents beyond their normal territory in the spinal cord. This contrasts with the normal spinal cord phenotype described in [41"].
26. •
Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, Mishina M, Yagi T: Disruption of Semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron 1997, 19:519-530. Reports the phenotype of another sema I/I knockout mouse. In contrast to [40"], no abnormalities of sensory afferent targeting in the spinal cord are described, but abnormal axon branches are seen to sprout laterally from the DRGs, and aberrant peripheral axon trajectories are seen in the branchial arches, periocular region and limb buds.
27 •
42. He Z, Tessier-Lavigne M: Neuropilin is a receptor for the axonal • chemorepellent semaphorin III. Cell 1997, 90:739-751. Together with [43"], this paper reports the expression cloning and binding properties of neuropilin as a repulsion receptor for Sema III. Neuropilin is shown to be expressed on Sema Ill-responsive axons, and neutralizing antineuropilin antibodies block Sema Ill-mediated repulsive responses in vitro.
Ming G-I, Song H-j, Berninger B, Holt CE, Tessier-Lavigne M, Poe M-m: cAMP-dependent growth cone guidance by netrin-l. Neuron 1997, 19:1225-1235. Good in vitro evidence that netrin can both attract and repel the same population of axons, depending on cAMP levels within the growth cone. The two responses may therefore share similar signal transduction mechanisms. Leonard• ED, Hinck L, Masu M, Keino-Masu K, Ackerman SL, Tessier-Lavigne M: Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature 1997, 386:833-838. Reports the cloning of two rat homologs of UNC-5, a candidate receptor for netrin-based repulsive responses in C. e/egans (see [22]). The vertebrate homologs define a new subfamily of the immunoglobulin supeffamily, and are expressed in a variety of differentiating neurons in the developing brain and spinal cord. They are also shown to bind netrin-1 with high affinity, consistent with a role as netrin receptors with the potential to mediate repulsive responses. 28.
Kolodkin AL, Matthes DJ, Goodman CS: The Semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell 1993, 75:1389-1399.
29.
Luo Y, Raible D, Raper JA: Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Ceil 1993, 75:217-227.
30.
Luo Y, Shepherd I, Li M, Renzi M J, Chang S, Raper JA: A family of molecules related to collapsin in the embryonic chick nervous system. Neuron 1995, 14:1131-1140.
31.
Kolodkin A: Semaphorins: mediators of repulsive growth cone guidance. Trends Cell Bio/1996, 6:15-22.
32.
Kolodkin AL, Matthes DJ, O'Connor TP, Patel NH, Admen A, Bentley D, Goodman CS: Fasciclin IV: sequence, expression, and function during growth cone guidance in the grasshopper embryo. Neuron 1992, 9:831-845.
41. •
43. •
Kolodkin AL, Levengood DV, Rowe EG, Tai Y-T, Giger RJ, Ginty DD: Neuropilin is a semaphorin III receptor. Ce//1997, 90:753-762. Together with [42"], this paper describes the identification and characterization of neuropilin as a Sema III receptor. It also describes the cloning of neuropilin-2 (see also [45"]). 44.
Takagi S, Kasuya Y, Shimizu M, Matsuura T, Tsuboi M, Kawakami A, Fujisawa H: Expression of a cell adhesion molecule, neuropilin, in the developing chick nervous system. D~v Bio/1995, 170:207-222.
45. •
Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M: Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 1997, 19:547-559. Identification of a second member of the neuropilin family. The study reveals differential affinities of individual semaphorins for neuropilin-1 and -2, as well as largely nonoverlapping expression patterns for the two neuropilins in the developing nervous system. See also [43°]. 46. •
Feiner L, Koppel AM, Kobayashi H, Raper JA: Secreted chick semaphorins bind recombinant neuropilin with similar affinities but bind different subsets of neurons in situ. Neuron 1997, 19:539-545. Reports the use of AP-tagged collapsins/semaphorins to assess the distribution of their receptors during neural development. Individual members of
72
Development
the family show distinct anatomical patterns of binding in tissue sections, and the semaphorin domain is shown to determine binding specificity. 47. •
Koppel A, Feiner L, Kobayashi H, Raper JA: A 70 amino acid region within the semaphorin domain activates specific cellular response of semaphorin family members. Neuron 1997, 19:531-537. Domain swapping experiments show that a small region of the semaphorin domain confers specificity of binding and biological activity on individual members of the collapsin/semaphorin family. 48. •
Kitsukawa T, Shimizu M, Sanbo M, Hirata T, Taniguchi M, Bekku Y, Yagi T, Fujisawa H: Neuropilin-semaphorin Ill/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice. Neuron 1997, 19:995-1005. Reports the phenotype of neuropifin knockout mice, showing substantial sireilarity to the sema I//knockout phenotype described by Taniguchi eta/. [41 °]. 49.
Orioli D, Klein R: The Eph receptor family: axonal guidance by contact repulsion. Trends Genet 1997, 13:354-359.
50.
Keynes RJ, Johnson AR, Pini A, Tannahill D, Cook GMW: Spinal nerve segmentation in higher vertebrates: axon guidance by repulsion and attraction. Semin Neurosci 1996, 8:339-345.
51.
Davies JA, Cook GMW, Stern CD, Keynes RJ: Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones. Neuron 1990, 4:11-20.
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Wright DE, White FA, Gerfen RW, Silos-Santiago I, Snider WD: The guidance molecule semaphorin III is expressed in regions of spinal cord and periphery avoided by growing sensory axons. J Comp Neuro/1995, 361:321-333.
53.
Adams RH, Betz H, Puschel AW: A novel class of rnurine semaphorins with homology to thrombospondin is differentially expressed during early embryogenesis. Mech Dev 1996, 57:33-45.
54.
Fredette B J, Miller J, Ranscht B: Inhibition of motor axon growth by T-cadherin substrata. Development 1996, 122:3163-31 71.
55.
Landolt RM. Vaughan L, Winterhalter KH, Zimmerman DR: Versican is selectively expressed in embryonic tissues that act as barriers to neural crest cell migration and axon outgrowth, Development 1995, 121:2303-2312.
56.
Ring C, Hassell J, Halfter W: Expression pattern of collagen IX and potential role in the segmentation of the peripheral nervous system. Dev Biol 1996, 180:41-53.
57. ••
Wang HU, Anderson D J: Eph family transmembrane ligands can mediate repulsive guidance of trunk neural crest migration and motor axon outgrowth. Neuron 1997, 18:383-396. Ephrins are expressed in the posterior half-somite, and repel motor axons and migrating neural crest cells in a variety of in vitro assays. Together with [58"], this study identifies ephrins as candidates for mediating peripheral nerve segmentation in higher vertebrates. 58. oo
Krull CE, Lansford R, Gale NW, Collazo A, Marcelle C, Yancopoulos GD, Fraser SE, Bronner-Fraser M: Interactions of Eph-related receptors and ligands confer rostrocaudal pattern to trunk neural crest migration. Curt Biol 1997, 7:571-580.
This study describes the restriction of ephrin expression in the somites to the posterior half-sclerotomes. Cognate Eph receptors are expressed by migrating neural crest cells, and crest migration patterns are perturbed in explants of chick embryo trunks treated with soluble ephrin. Together with [57"], this study identifies ephrins as candidates for mediating peripheral nerve segmentation in higher vertebrates. 59.
Ohta K, Nakamura M, Hirokawa K, Tanaka S, Iwama A, Suda T, Ando M, Tanaka H: The receptor tyrosine kinase, Cek8, is transiently expressed on subtypes of motoneurons in the spinal cord during development. Mech Dev 1996, 54:59-69.
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Ohta K, Iwamasa I, Drescher U, Terasaki H, Tanaka H: The inhibitory effect on neurite outgrowth of motoneurons exerted by the ligands ELF-1 and RAGS. Mech Dev 199"7, 64:127-135.
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Kilpatrick TJ, Brown A, Lai C, Gassmann M, Goulding M, Lemke G: Expression of the Tyro4/Mek4/Cek4 gene specifically marks a subset of embryonic motor neurons and their muscle targets. Mo/ Ceil Neurosci 1996, 7:62-74.
62.
Donoghue M J, Lewis RM, Merlie JP, Sanes JR: The Eph kinase ligand AL-1 is expressed by rostral muscles and inhibits outgrowth from caudal neurons. Mo/Ceil Neurosci 1996, 8:185198.
63. •
Ebens A, Brose K, Leonardo ED, Hanson MG, Bladt F, Birchmeier C, Barres BA, Tessier-Lavigne M: Hepatocyte growth factor/scatter factor is an axonal chemoattractant and a neurotrophic factor for spinal motor neurons. Neuron 1996, 17:1157-1172. Identification of a novel function for hepatocyte growth factor/scatter factor as a chemoattractant for motor neurons. The mouse knockout phenotype shows abnormalities of limb motor innervation. 64.
Wang L-C, Rachel RA, Marcus RC, Mason CA: Chemosuppression of retinal axon growth by the mouse optic chiasm. Neuron 1996, 17:849-862.
65. •
Keynes R.I, Tannahill D, Morgenstern DA, Johnson AR, Cook GMW, Pini A: Surround repulsion of spinal sensory axons in higher vertebrate embryos. Neuron 1997, 18:889-897 This study uses explants of chick embryo DRGs and surrounding tissues in collagen gels to show that linear axon trajectories can be generated by gradients of diffusible repulsion molecules flanking axon pathways. 66.
Wightman B, Barab R, Garriga G: Genes that guide growth cones along the C. elegans ventral nerve cord. Development 1997, 124:2571-2580.
87. •
Karlstrom RO, ]'rowe T, Klostermann S, Baier H, Brand M, Crawford AD, Grunewald B, Haffter P, Hoffmann H, Meyer S U e t aL: Zebrafish mutations affecting retinotectal axon pathfinding. Development 1996, 123:427-438. Shows the utility of the zebrafish in the identification of candidate guidance genes. 68.
Goodhill G J: Diffusion in axon guidance. Eur J Neurosci t 997, 9:1414-1421.
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Isacson O, Deacon TW: Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat. Neuroscience 1996, 75:827-837.
Axon guidance to and from choice points Geoffrey Cook, David Tannahill and Roger Keynes* Significant progress has been made recently in understanding axon guidance to and from choice points. Netrins have been shown to function as conserved midline chemoattractants in vertebrates and insects, and receptors for netrins and semaphorins/collapsins have been identified. More evidence has accumulated that repulsion plays a key role in guidance, including the involvement of the ephrin/Eph receptor system in contact repulsion.
Addresses Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK *e-mail: rjkl [email protected] Current Opinion in Neurobiology 1998, 8:64-?2 htt p://biomednet.com/elecref/0959438800800064 © Current Biology Ltd ISSN 0959-4388 Abbreviations AP alkaline phosphatase CGRP calcitoningene related peptide Dec deleted in colon carcinoma DRG dorsal root ganglion HGF/SF hepatocyte growth factor/scatter factor Ig immunoglobulin NGF nerve growth factor NT-3 neurotrophin 3 Sema semaphorin
Introduction
At first sight, axon growth cones are set a formidable task in navigating to their often distant targets in the embryo. The task is simplified, however, by the fragmentation of their journey into shorter steps interrupted by intermediate targets, or choice points, at which other cells provide critical guidance cues that direct growth cones on the next stage of their trajectory'. A well-known example is provided by the specialized cells comprising the CNS midline in both insects and vertebrates, as discussed below. Choice points are also provided by smaller, isolated groupings of guidepost cells, which may be neurons themselves; in their absence, the earliest pioneer growth cones often make pathfinding errors [1-3]. Later-navigating axons may be guided to and from choice points by selective fasciculation on earlier-developing axons (as discussed in an accompanying review by Van Vactor, in this issue, pp 80-86). We focus here on the nature and function of the external cues that orient axons before and after their encounters with choice points, and that set the overall trajectories of axons and their fascicles. Netrins
The vertebrate floor plate is an intermediate, midline target for commissural axons traversing the spinal cord, and provides a prototype for axon guidance towards a choice
point region by chemoattraction. With the isolation of the netrins as floor plate chemoattractants [4] (Figure la), and the revealed homology between the netrins and UNC-6, a laminin-related protein required for circumferential growth of axons in the body wall of Caenorhabditis e/egans, there is now good evidence that netrins provide highly conserved midline guidance cues for axons, tlomologs have also been identified in the human genome [5] and in flies. In the latter case, two netrin genes map in tandem on the X chromosome, and embryos deficient for this region show defective commissures that can be rescued by expression of either netrin at the midline. Pan-neural expression of either gene also disrupts commissural and longitudinal tracts, showing that the pattern of netrin expression is critical for guidance. The presence of defects in motor axon projections in the double mutant further shows that netrins are required for motor axons to reach their correct target muscles [6",7°]. As predicted by earlier studies, netrin-l-deficient mice show abnormal spinal commissural axon projections I8"']. More evidence that netrin guides spinal commissural axons comes from analysis of the zebrafish mutant floating head, which lacks a notochord; as a result, netrin-la expression is absent in patches of caudal spinal cord, and commissural neurons sited in between these patches extend their axons aberrantly along the anterior-posterior axis towards the nearest available region of normal netHn-la expression, presumably through chemoattraction [9°]. Such results are explicable exclusively in terms of netrin, but it is possible that other, as yet unidentified attractants are also involved in guiding axons to the midline, especially as the trajectories of substantial numbers of commissutal axons are unperturbed in netrin-deficient mice. Netrin-1 is important for axon guidance to the midline in the brain, as nettin-deficient mice show additional defects in the corpus callosum as well as hippocampal and anterior commissures [8"], and it has been shown to attract ventrally decussating axons in the developing brainstem [10]. The internal capsule may provide a further source of netrin-1 in developing brain, acting as a chemoattractant for neocortical axons navigating towards the thalamus [ 11,12]. The possibility of extensive involvement of netrins in axon guidance within the brain has also been revealed by a detailed study of netrin gene (netHn-la) expression in the developing zebrafish [9°]. The macroscopic pattern of netrin-la expression in the early zebrafish brain comprises a ventral longitudinal stripe, inclusive of the floor plate, intersected at discrete locations (such as the isthmus and rhombomere boundaries) by orthogonal dorsoventral stripes. This correlates well with the orthogonal pattern of pioneer axon tracts in the brain,
Axon guidance to and from choice points Cook, Tannahill and Keynes
65
Figure 1
(a)
(b) Netrins
Netrinfamily receptors
(c)
Semaphorins
{
Ig Ig
Neuropilins
Ephrin-A Ephrin-B EphA/B
r--~
l--
Extracellular
s
DCC Frazzled UNC-40
UNC-5
~
I I
Sema III G-sema I SemD Collapsin-I
I
GP
I I I
Plasma membrane Intracellular
CurrentOpinionin Neurobiotogy
Outline structure of families of axon guidance molecules discussed in the text. (a) Netrins and their receptors; (b) semaphorins (secreted and transmembrane forms shown) and their receptors (neuropilins); and (c) ephrins (class A showing GPI linkage to the cell surface; class B showing transmembrane linkage) and their cognate Eph receptors. B +, basic domain; C, coagulation factor homology domain; CUB, complement/sea urchin-EGF/BMP1 domain; CYS, cysteine-rich domain; Egf, epidermal growth factor repeat; FN, fibronectin type III domain; GPI, glycophosphatidylinositot linkage; Ig, immunoglobulin domain; Lam, region with homology to amino terminal domains V and VI of laminin chains; MAM, meprin/A5/PTPmu domain; Sema, semaphorin domain; TK, tyrosine kinase domain; Ts, thrombospondin type I domain.
which develop at the borders of, or within, the domains of netrin-la expression [9°]. Lastly; netrin-1 expression by cells at the developing optic disc provides a local, short-range guidance cue for retinal axons. Nctrin-1 is expressed in the developing eye in several vertebrates [13,14°,15,16]. Retinal growth cones have been shown to turn towards a point source of netrin-1 in v,itro [17°], and in netrin-l-deficient mice (as well as DCC nulls, see below), many axons fail to exit the retina into the optic nerve, reaching the disc but growing past it into other retinal regions [14"]. In this case, the range of netrin action is more restricted than in the spinal cord; while the factors that modulate netrin diffusion in vivo are unknown, one possibility would be the binding of netrins to extracellular matrix molecules expressed in the vicinity of the netrin source. Netrin
receptors
The function of netrin signaling in vivo has been impressively confirmed by studies on the putative netrin receptors. The homology of netrins with UNC-6 suggested that the vertebrate homolog of UNC-40, a candidate reccptot involved in ventral migrations in C. e/egans, might be the netrin receptor. Two such genes, DCC (deleted in colon carcinoma) and neogenin, have now been identified [18"1. DeC, originally characterized as a tumour suppressor gcne that is frequently lost in human colorectal carcinomas, encodes a group of structurally related transmembrane
proteins with four immunoglobulin and six fibronectin type IIl repeats in their extracellular domains (Figure la). While a definitive role for DCC in colorectal carcinoma remains to be shown, it does have a clear function during neural development. DCC protein is expressed on commissural axons as they project towards the floor plate, binds netrin-I in vitro with high affinity, and a monoclonal antibody directed against its extracellular domain selectively blocks netrin-l-dependent outgrowth of commissural axons in collagen gels [18"]. Tile turning response in vitro of Xenopus retinal growth cones towards a micropipette ejecting nctrin-1 is also blocked by neutralizing DCC antibodies [17°]. The phenotype of mice lacking functional DCC has reinforced its status as a netrin receptor: like the netrin-I knockout phenotype, spinal commissural axons are misrouted, forebrain axons make aberrant ipsilateral projections and fail to form a corpus callosum and hippocampal commissure, while the anterior commissure is severely reduced [19"°]. Striking confirmation of the phylogenetic conservation of the netrin-receptor system comes from studies in flies and worms. The Drosophila DCC homolog frazzled is expressed by embryonic CNS axons and motor axons in the periphery, and the null phenotype appears identical to the netrin null phenotype with thin or absent commissures
66
Development
[20*']. uric-40 has also been shown to encode the nematode homolog of DCC, neogenin and frazzled [21*°]. T h r e e gene products, UNC-5, U N C - 6 and UNC-40, are essential for dorsoventral circumferential migrations in the body wall of C. e/egans. A model has becn suggested in which cclls and axons migrating ventrally orient towards a source of UNC-6 protein using UNC-40 as a receptor, whereas dorsal migrations orienting in the opposite direction use UNC-5 and UNC-40 as co-receptors for a repulsive response to U N C - 6 [22]. Consistent with this, U N C - 6 expression is restricted to the ventral cells within each region of the nervous system [231. UNC-40 is expressed by cells migrating in both ventral and dorsal directions, but dorsal migrations are less disrupted in UNC-40 nulls than in UNC-5 and U N C - 6 nulls, so this function of UNC-40 may bc redundant to a second pathway, pcrhaps involving a distinct DCC homolog [21"°1. Do netrins, like UNC-6, repel as well as attract axons? Netrin-secreting cells and floor plate explants repel trochlear motor axons in vitro [24], but so do floor plate explants taken from netrin-l-deficient mice [8°']. Moreover, although many axons growing from explants of the ventral half of the rostral midbrain are repelled by the floor plate, mimicking the behaviour in vivo of posterior commissure axons, they are not repelled by netrin-1 [101. It seems likely, therefore, that floor-plate-derived chemorepellents other than netrin-1 are involved in setting these dorsally directed axon trajectories, and Sema IIl/collapsin-1 (see below) has been suggested as a candidate [25]. T h e possibility nonetheless remains that netrins may repel axons. Cultured Xenopus spinal neurons will normally turn towards a micropipette releasing netrin-1, but this can be converted to a repulsive response by agents that reduce the level of cytoplasmic cAMP-dependent protein kinase A [26°]. Attractive and repulsive responses are both prevented by neutralizing antibody to D C C [26*], implying that the intracellular signaling pathways for these opposite responses are related (see accompanying review by Holland, Peles, Pawson and Schlessinger, in this issue, pp 117-127). T h e involvement of D C C in repulsion also raises the question of whether netrin-based repulsion in vertebrates is homologous to the UNC-40/UNC-S co-receptor system suggested for dorsal migrations in C. elegans. Vertebrate homologs of UNC-5 (Figure la) have been identified as netrin-binding proteins, and are expressed in vivo by axons that are repelled bv netrin-1 in vitro [27°]. T h e further finding that these homologs are expressed in the same regions of the developing C N S as DCC is at least consistent with thcir putative function as co-receptors [27°], and underlines the remarkable conservation of this guidance system from nematodes to chordates.
Semaphorins T h e semaphotin family, which now counts more than 30 members, has been strongly implicated in axon guidance in both flies and vertebrates [28-31]. Semaphorins are
related by a homologous stretch of some 500 amino acids, comprising the semaphorin domain, and can be grouped structurally into five types that include both secreted and membrane-bound forms (Figure lb). Secreted semaphorins possess a carboxy] C2-type immunoglobulin (Ig) domain followed by a highly basic tail, whereas membrane-bound forms have either a carboxyl Ig domain, several thrombospondin repeats or no discernible features carboxyl to the semaphorin domain. T h e large number of semaphorins, coupled with their complex expression patterns in the embryo, has meant that their potential biological function has been assessed in only a few cases. Studies in insects have suggested that secreted semaphorins may be involved in target recognition by motor axons (see accompanying review by Holt and Harris, in this issue, pp 98-105), while one transmembrane semaphorin, G-Sema I, has been implicated as both a repulsive and attractive/permissive guidance factor for different axon subtypes navigating within the grasshopper limb bud. G-Sema I, originally identified by a monoclonal antibody and designated fasciclin IV [32[, is expressed in the limb bud epithelium, and Til pioneer neurons projecting from the bud towards the C N S apparcntly respond to a stripe of G-Sema I expression as a repulsive cue, stalling and tt, rning ventrally to follow the boundary of G-Sema I expression [31,32] (Figure 2). Later in development, another group of axons, originating more distally in the bud, reach the C N S by growing across a second band of G-Sema I-expressing cells and then fasiculating upon the Til axons. In reaching these axons, growth cones appear to use the G-Sema I band as an attractive/permissive cue, as neutralizing antibody to G-Sema I prevents their extension across it [33 °] (Figure 2). T h e best studied vertebrate scmaphorin is known as collapsin-1 in the chicken, Sema III in humans and SemaD in rodents, and is referred to here as Sema III. It was originally purified from adult chicken brain as a secreted glycoprotein that induces the paralysis and growth cone collapse of cultured sensory dorsal root ganglion (DR(;) axons [29], and independently as a vertebrate homolog of G-Sema I [28]. Consistent with a role for Sema III in axon guidance, it has been shown that sensory axons can be steered away from immobilized sources of Sema III [34], and both sensory and sympathetic axons, but not retinal axons, grow away from COS cells expressing Sema I11 in collagen gcls [35,36]. Spinal motor axons and certain cranial motor axons have also been shown to be responsive i, vino [25]. Scma IIl has been implicated in the patterning of sensory axon projections in the developing spinal cord. It is likely that a ventral-to-dorsal gradient of Sema III protein is established in the spinal cord during these stages, as sustained expression of sema III is seen in the ventral half of the neural tube [35,37]. Detailed analysis of the sensitivity of D R G axon subtypes in vitro has shown
Axon guidance to and from choice points Cook, Tannahill and Keynes
67
Figure 2 (a) Wild-type O)
(b) Til ablation
(ii)
(c) Anti-G-Sema I
Current Opinion in Neurobiology
Role of G-Sema I in grasshopper limb bud. (a) In wild-type embryos, (i) Til neurons project axons from their cell bodies towards the CNS, responding to a repulsive stripe of epithelial G-Sema I expression by turning ventrally, whereas (ii) the subgenual organ (SGO) cell body lies more distally in the bud, and its axon reaches the CNS by growing across a band of G-Sema-l-expressing cells and then fasiculating on the Til axons. (b) After Til ablation, the S G O axon stalls within the band of G-Sema I. (c) The S G O axon fails to sprout across the band of G-Sema I in the presence of neutralizing antibody to G-Sema I, suggesting that G-Sema I acts as a permissive/attractive guidance cue.
that both nerve growth factor (NGF)-dependent nociceptive afferents and neurotrophin 3 (NT-3)-dependent proprioceptive afferents are repelled by Sema III when explanted at early stages [38"]. This broad-spectrum in vitro sensitivity is associated in vivo with the transient early expression of Sema III adjacent to the site at which sensory afferents first enter the dorsal spinal cord, perhaps confining them locally by repulsion. Later, NT-3- but not NGF-dependent axons lose their responsiveness to Sema III, correlating with the ventral projection of proprioceptive afferents in the spinal cord towards their neuronal targets [35]. In support of the repulsion model, ventral neural tube explants are known to chemorepe] sensory axons in vitro [39], and this activity can be neutralized by an anti-Sema III antibody [38°]. Targeted disruptions of mouse sema I I I have, however, yielded conflicting results regarding the role of Sema III in sensory afferent ingrowth in the spinal cord. In one phenotype, some NGF-dependent sensor3" afferents (CGRP-positive) misproject to more ventral locations [40°], while in another, no aberrant ventral projections are observed [41°]. A possible explanation for the discrepancy concerns the different genetic backgrounds used in the two studies, complicating a direct comparison between them and raising the question of gene compensation. Alternatively,
the discrepancy may lie in the assessment of different subpopulations of NGF-dependent afferents in the two studies. Other defects seen in one of the sema I I I knockout studies [41"] suggest possible roles for Sema III in axon guidance in the craniofacial region. T h e cranial branchiomotor nerves, for example, defasciculate in their branchial arch territories, although their overall trajectory is unchanged and they do appear to reach their targets. As Sema III is normally expressed in the branchial arches as axons grow into them, it may drive axons to fasciculate by creating a repulsive surround (see also review by Van Vactor, in this issue, pp 80-86). A related explanation could account for a further feature of the knockout phenotype seen in the DRGs, which now sprout many of their axons from their lateral rather than ventral side. In this case there is a change in trajectory, consistent with the possibility that Sema III, secreted by the dermomyotome, participates in surround-repulsion of early DRG axons, as outlined below (Figure 3). Neuropilins
Progress has been made recently in the characterization of receptors for the semaphorins. A high affinity
68
Development
Figure 3 (a) Mouse/chick DRG
(b) Sema III/neuropilin mouse DRG
"t
Current Opinion in Neurobiology
Surround repulsion of DRG axons. (a) Transverse section through the trunk region of a mouse/chick embryo, showing the trajectory of axons sprouting from a DRG cell body (filled black circle) within the sclerotome (Sc). The dermomyotome (Dm) lies lateral to the DRG neurons and secretes repellent molecules towards them (short arrows), supplemented by the ectoderm (arrowheads). The medially placed notochord (NC) and ventral neural tube (NT) also secrete repellents (long arrows), so DRG axons are flanked by two sources of chemorepulsion. (b) In sema III or neuropilin knockout mice, some DRG axons sprout aberrantly towards the dermomyotome. According to the surround-repulsion model, this would result from the imbalance created by the loss of lateral chemorepulsion and the continued operation of midline-derived chemorepulsion.
Sema-III-binding protein, neuropilin, has been identified using an alkaline phosphatase-tagged (AP) Sema III ligand to screen a COS cell expression library [42",43"], and neuropilin homologs have been found in a variety of vertebrates. Neuropilin was previously identified as a transmembrane glycoprotein expressed in the Xenopus tectum (Figure lb), and was suggested to participate in axon-axon or axon-target interactions [44]. Consistent with its potential rolc as a Sema III receptor, neuropilin is expressed on Sema-III-responsive axons, including sensory', sympathetic and spinal motor axons, and application of neutralizing anti-neuropilin antibodies blocks Sema-llI-mcdiated growth cone collapse and chemorepulsion in collagen gels [42",43"]. At least two distinct neuropilins have now been identified [43",45"], and these have largely nonoverlapping expression patterns in the developing nervous system. Neutopilin-1 appears to bind all secreted semaphorins that have been tested with high affinity, whereas neuropilin-2 binds Sema E (collapsin-3) and Sema IV, but not Sema III (collapsin-1) [45"]; the possibility remains, however, that neuropilin-2 binds Sema III with lower affinity: Although the carboxy-terminal doraain or the semaphorin domain is sufficient for neuropilin binding, the use of chimeric semaphorins has shown that the semaphorin domain appears to impart specific binding in embryo sections [46"]. Different semaphorins are also likely to have distinct biological specificities, as collapsin-1 can induce collapse of both DRG and sympathetic growth cones, collapsin-2 affects neither, whereas only sympathetic growth cones respond to collapsin-3 [47"]. As for receptor binding, the specificity of the collapse-inducing activity of collapsins 1
and 3 is also mediated by the semaphorin domain, which must be dimerized to function, and is conferred by a 70 amino acid stretch within its amino-terminal half. Although neuropilin-1 may be sufficient for ligand binding, these results raise the possibility that it is a common component of a larger receptor complex, binding semaphorins/collapsins through their carboxyl tails, while other, as yet unidentified, components of the complex impart biological specificity [47"]. The phenotype of neuropilin-deficient mice is consistent with the role of neuropilins as semaphorin receptors. Like the sema III knockout mice, neuropilin knockout mice exhibit defasciculation in certain cranial branchiomotor nerves, and in the segmental nerves innervating the limb buds, and the DRGs produce aberrant lateral projections towards the dermomyotome consistent with the surround-repulsion model [48"] (Figure 3). E p h r i n l i g a n d s a n d Eph r e c e p t o r s A variety of ephrins and their Eph receptors (Figure 1c) have been implicated in guiding axons to and from choice points, axon fasciculation and target selection (see [49] for a review). We focus here on recent studies showing that these molecules are involved at early stages in the formation of axon trajectories, guiding peripheral axons into the somites and limb buds. The roles of ephrins and receptors in guiding CNS axons across the midline, as well as in axon fasciculation, target selection and growth cone signal transduction are discussed in accompanying reviews (see Van Vactor pp 80-86, Holt and Harris pp 98-105, and Holland, Peles, Pawson and Schlessinger pp 117-127, in this issue).
Axon guidance to and from choice points Cook, Tannahill and Keynes
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The segmented pattern of peripheral axons in higher vertebrates results from the preference of motor and sensory axons, and earlier-migrating neural crest cells, for growth through the anterior rather than posterior half of the somite-derived sclerotome [50]. This preference correlates with the expression by posterior cells of molecules that cause collapse in vitro of motor and sensory axon growth cones [51], implying that repulsion of axons by posterior cells in vivo causes peripheral nerve segmentation by forcing axons to traverse the consecutive anterior half-sclerotomes. Several candidate repellents expressed in posterior half-sclerotome have now come to light, including peanut-lectin-binding glycoproteins [51], Sema III [52,53], T-cadherin [54], versican [55] and collagen IX [56]. A critical role for Sema III is unlikely, however, in view of the normal spinal nerve segmentation seen in both sema III and neuropilin knockout mice [40°,41°,48"].
While the factors mediating axon segmentation influence all classes of vertebrate spinal motor neuron, the expression patterns of Eph receptors by motor neuron subsets suggest a possible role for this system in more specific axon guidance decisions. In the chick embryo, the EphA4 (Cek8) receptor is expressed preferentially in motor neurons that innervate limb muscle rather than axial muscle [59], and the ephrins A2 and A5 are expressed in regions of the limb bud avoided by axons [60]. In contrast, the EphA3 (Tyro4) receptor in the rat is restricted to motor neurons innervating axial muscle [61]. Ephrin-A5 (AL-1), moreover, is expressed at higher levels in rostral rather than caudal mouse muscles and, reciprocally, inhibits the outgrowth of axons from caudal rather than rostral motor neurons [62]. Whether these findings relate to axonal recognition of target muscle subsets in vivo, or to earlier stages of axon guidance, is at present uncertain.
Two transmembrane ephrins, ephrin-B2 (HtkL/Lerk5/Elf-2) in the rat [57 "°] and ephrin-B1 (Lerk2/Elk-L/Cek5-L) in the chick [57",58"*], are also expressed by posterior but not anterior cells, whereas the predominant Eph receptor expressed by neural crest cells is EphB2 (Nuk) in the rat [57 °"] and EphB3 in the chick [58"°]. In vitro assays of neural crest migration and motor axon outgrowth have shown that clustered ephrin ligands can guide by repulsion, provided they are presented in a discontinuous or graded manner. For example, rat motor axons (which also express the EphB2 receptor) avoid stripes of ephrin-B1 and ephrin-B2 superimposed on a uniform laminin substrate, but will grow on a uniform ephrin/laminin substrate if given no alternative [57"]. Such behaviour is more consistent with growth-repulsion than growth-inhibition, and contrasts with the inhibition seen using uniform substrates of T-cadherin [541 and collagen IX [56]. Further evidence for the involvement of ephrin-induced repulsion in peripheral nerve segmentation comes from the observation that crest cells migrate aberrantly into the posterior half-sclerotome when soluble, unclustered ephrin-B1 (acting as a competitive antagonist of endogenous ephrins) is added to explants of chick embryo trunks [58"'].
Further chemoattractants and chemorepellents In the search for chemoattractants other than the netrins, the hepatocyte growth factor/scatter factor (HGF/SF) has been identified as a chemoattractant for motor axons secreted by limb bud mesenchyme, and a subset of motor neurons expresses c-Met, the receptor for HGF/SF [63"]. Motor axon branching patterns in the developing forelimb of HGF/SF knockout mice show abnormalities consistent with a chemoattractive role for HGF/SF, although this interpretation is complicated by the fact that the same protein is required for the migration of myoblast precursors into the limb [63"]. Assuming the cytoskeletal response within the growth cone to c-Met activation to be similar to that following DCC activation, it will be interesting to see how the respective signaling pathways converge (see accompanying review by Holland, Peles, Pawson and Schlessinger, in this issue, pp 117-127).
As for the other candidate molecular mediators of peripheral nerve segmentation, a critical role for the ephrin-Eph-receptor system remains to be shown directly in vivo. The normal patterns of crest migration and motor axon outgrowth seen in EphB2 (Nuk);EphB3 (Sek4) double-mutant mice could be attributable to functional compensation by other Eph family members [57"'], but could equally be attributable to the continued operation of other guidance systems. It will also be important to test whether chick sensory axon growth cones, which collapse in response to a somite-derived glycoprotein fraction [51], collapse in response to clustered ephrin-B1.
In addition to the floor plate (see above), the optic chiasm has been identified as a further choice point region at which axons may be subject to negative growth regulation (e.g. chemosuppression [64]), but the relevant molecules remain to be characterized. Models of chemorepulsion usually place the source of repellent either in front of axons (e.g. repulsion of spinal cord afferents by Sema III/collapsin-1 [35]) or behind them (e.g. repulsion of cranial branchiomotor and trochlear motor neurons by the floor plate [24,25]). A recent study has shown that repellents can also generate linear patterns of axon growth when secreted in gradients that flank neurons on either side. Chick or mouse DRGs sprout radially in three dimensions when cultured with neurotrophins in collagen gels, but are constrained to sprout in a bipolar manner when sandwiched between a length of notochord and a dermomyotome placed at a distance. These tissues flank the early DRGs in vivo, and the oriented bipolar trajectory of primary sensory axons extending from the DRGs in the
70
Development
dorsoventral plane is suggested to be due to the resulting surround-repulsion exerted on axons [65*] (Figure 3). One of the repellent molecules may be Sema III/collapsin-1 (see above); it is expressed by the dermomyotome, and its loss of function in Sema III and neuropilin-deficient mice may, according to the surround-repulsion model, explain the abnormal sprouting of DRG axons towards the dermomyotome seen in these animals [41°,48 °] (Figure 3).
2.
Ho RK, Goodman CS: Peripheral pathways are pioneered by an array of central and peripheral neurones in grasshopper embryos. Nature 1982, 297:404-406.
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Van Raay TJ, Foskett SM, Connors TD, Klinger KW, Landes GM, Burn TC: The NTN2L gene encoding a novel human netrin maps to the autosomal dominant polycystic kidney disease region on chromosome 16p13,3. Genomics 1997, 41:279-282.
Conclusions Significant advances have been made recently in characterizing attractive and repulsive ligands and receptors that guide axons to and from choice points. Manipulating their expression in both mice and flies has generally confirmed their functional importance and, for the netrins and semaphorins, their phylogenetic conservation, while invertebrate homologs of the ephrins/Eph receptors have yet to be described. Both vertebrates and invertebrates use choice points for axon guidance, but the extent to which the underlying mechanisms have evolved independently at the molecular level is unknown, and will become clearer as further guidance molecules are identified. To this end, candidate genes have been identified by systematic genetic screens for axon pathfinding mutants. Recent examples come from screens in C. elegans [66] and the zebrafish [67"], and the molecular cloning and functional analysis of examples such as the zebrafish mutants bashful, grumpy and sleepy, in which retinal axons projecting to the opposite tectum make errors after crossing the midline [67"], should be revealing. Lastly, it is also relevant that, in modelling axon guidance by diffusible factors, the maximum range for guidance by chemoattraction or chemorepulsion has been estimated to be some 500-10001.tin for a stable gradient, expanding to much greater distances before stabilization [68]. It may be significant, then, that transplanted foetal rat CNS neurons can grow axons in the adult rat CNS to appropriate target structures at least 10 mm away from the implantation site [69], raising the intriguing possibility that long-distance guidance mechanisms in the embryo may also exist in the mature nervous system. This will be interesting to assess alongside the further characterization of guidance molecules and their signalling pathways.
Acknowledgements Our work is supportcd by grants from the Medical Research Council and the Wellcome Trust. G Cook is a Member of the External Scientific Staff of the Medical Research Council, I) Tannahill is a Royal SocietT t.Tniversity Research Fellow and R Keynes is an lntcrnational Research Scholar of the Howard Hughes Medical Institute.
6. •
Mitchell KJ, Doyle JL, Serafini T, Goodman CS, Dickson B J: Genetic analysis of netrin genes in Drosophila: netrins guide CNS commissural axons and peripheral motor axons. Neuron 1996, 17:203-215. Two Drosophila netrin genes are identified, their expression patterns analysed, and the results of loss- and gain-of-function assessed. Midline commissures are disrupted under both conditions, showing that the pattern of netrin expression is critical for netrin-based axon guidance. 7. •
Harris R, Sabatelli LM, Seeger MA: Guidance cues at the Drosophila CNS midline: identification and characterization of two Drosophila netrin/UNC-6 homologs. Neuron 1996, 17:217228. This paper describes essentially the same strategy for the analysis of Drosophila netrin genes as [6"]; as in the latter study, ectopic expression of either netrin throughout the CNS causes similar axon guidance defects to those seen in loss-of-function mutants. 8. •.
Serafini 1", Colamarino SA, Leonardo ED, Wang H, Beddington R, Skarnes w e , Tessier-Lavigne M: Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system. Ceil 1996, 87:1001-1014. This paper describes the phenotype of netrin-l-deficient mice, highlighting the abnormal ventral cornrnissures in the spinal cord and brain. The data provide strong evidence confirming the chemoattractive role of netrin-1 in guiding axons across the vertebrate midline, as well as the phylogenetic conservation of netnn function from nematodes to chordates. 9. •
Lauderdale JD, Davis NM, Kuwada JY: Axon tracts correlate with netrin-la expression in the zebrafish embryo. Mol Cell Neurosci 1997, 9:293-313. A detailed study of netrin 9ene expression in zebrafish, with an analysis of the floating head mutant, providing further evidence that netrin attracts spinal comrnissural axons. 10.
Shirasaki R, Mirzayan C, Tessier-Lavigne M, Murakami F: Guidance of circumferentially growing axons by netrin-dependent and -independent floor plate chemotropism in the vertebrate brain. Neuron 1996, 17:1079-1088.
11.
Richards U, Koester SE, Tuttle R, O'Leary DDM: Directed growth of early cortical axons is influenced by a chemoattractant released from an intermediate target. J Neurosci 1997, 17:2445-2458.
12.
Metin C, Deleglise D, Serafini T, Kennedy TE, Tessier-Lavigne M: A role for netrin-1 in the guidance of cortical efferents. Development 1997, 124:5063-5074.
13.
Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M: Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord. Ceil 1994, 78:425-435.
14. •
Deiner MS, Kennedy TE, Fazeli A, Serafini T, Tessier-Lavigne M, Sretavan DW: Netrin-1 and Dec mediate axon guidance locally at the optic disc: loss of function leads to optic nerve hypoplasia. Neuron 1997, 19:575-589. Further analysis of netrin-l-deficient mice shows a defect in guidance of retinal axons at the optic disc. In this case, netrin-1 appears to provide short-range guidance for axons. 15.
Livesey FJ, Hunt SP: Netrin and netrin receptor expression in the embryonic mammalian nervous system suggests roles in retinal, striatal, nigral and cerebellar development. Mo/Ceil Neurosci 1997, 8:417-429.
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Str&hle U, Fischer N, Blader P: Expression and regulation of a netrin homolog in the zebrafish embryo. Mech Dev 1997, 62:147-160.
17 •
De la Torre JR, HSpker VH, Ming G-I, Poo M-m, Tessier-Lavigne M, Hemmati-Brivanlou A, Holt CE: Turning of retinal growth cones in a netrin-1 gradient mediated by the netrin receptor DCC. Neuron 1997, 19:1211-1224.
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Axon g u i d a n c e to and f r o m choice points Cook, Tannahill and Keynes
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Kolodziej PA, Timpe LC, Mitchell KJ, Fried SR, Goodman CS, Jan LY, Jan YN: frazzled encodes a Drosophila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance. Ceil 1996, 87:197-204. Identification and functional analysis of a Drosophila netrin receptor with homology to DCC. The midline phenotype of the frazzled null mutant is identical to that of netrin loss-of-function and can be rescued by pan-neural expression of frazzled. 21. o•
Chan SSY, Zheng H, Su MW, Wilk R, Killeen MT, Hedgecock EM, Culotti JG: UNC-40, a C. elegans h e m • l o g of DCC (Deleted in Col•rectal Cancer), is required in motile cells responding to UNC-6 netrin cues. Ceil 1996, 87:187-195. Reports the positional cloning of unc-40, revealing homology with vertebrate DGC and neogenin. The paper also suggests that UNC-40 and UNC-5 are required as co-receptors to orient migrating cells away from a source of netrin/UNC-6. 22.
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Shepherd IT, Luo Y, Lefcort F, Reichardt LF, Raper JA: A sensory axon repellent secreted from ventral spinal cord explants is neutralized by antibodies raised against collapsin-1. Development 1997, 124:1377-1385. This study shows that antibodies raised against chick collapsin-1/Sema III neutralize the chemorepulsive activity of chick ventral spinal cord explants for sensory axons, supporting the possibility that repulsion by cotlapsin1/Sema III may guide sensory afferent targeting in the spinal cord in rive. 39.
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40. •
Behar O, Golden JA, Mashimo H, Schoen FJ, Fishman MC: Semaphorin III is needed for normal patterning and growth of nerves, bones and heart. Nature 1996, 383:525-528. Reports the phenotype of the sema III knockout mouse. The main abnormality in the nervous system is the extension of CGRP-positive sensory afferents beyond their normal territory in the spinal cord. This contrasts with the normal spinal cord phenotype described in [41"].
26. •
Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, Mishina M, Yagi T: Disruption of Semaphorin III/D gene causes severe abnormality in peripheral nerve projection. Neuron 1997, 19:519-530. Reports the phenotype of another sema I/I knockout mouse. In contrast to [40"], no abnormalities of sensory afferent targeting in the spinal cord are described, but abnormal axon branches are seen to sprout laterally from the DRGs, and aberrant peripheral axon trajectories are seen in the branchial arches, periocular region and limb buds.
27 •
42. He Z, Tessier-Lavigne M: Neuropilin is a receptor for the axonal • chemorepellent semaphorin III. Cell 1997, 90:739-751. Together with [43"], this paper reports the expression cloning and binding properties of neuropilin as a repulsion receptor for Sema III. Neuropilin is shown to be expressed on Sema Ill-responsive axons, and neutralizing antineuropilin antibodies block Sema Ill-mediated repulsive responses in vitro.
Ming G-I, Song H-j, Berninger B, Holt CE, Tessier-Lavigne M, Poe M-m: cAMP-dependent growth cone guidance by netrin-l. Neuron 1997, 19:1225-1235. Good in vitro evidence that netrin can both attract and repel the same population of axons, depending on cAMP levels within the growth cone. The two responses may therefore share similar signal transduction mechanisms. Leonard• ED, Hinck L, Masu M, Keino-Masu K, Ackerman SL, Tessier-Lavigne M: Vertebrate homologues of C. elegans UNC-5 are candidate netrin receptors. Nature 1997, 386:833-838. Reports the cloning of two rat homologs of UNC-5, a candidate receptor for netrin-based repulsive responses in C. e/egans (see [22]). The vertebrate homologs define a new subfamily of the immunoglobulin supeffamily, and are expressed in a variety of differentiating neurons in the developing brain and spinal cord. They are also shown to bind netrin-1 with high affinity, consistent with a role as netrin receptors with the potential to mediate repulsive responses. 28.
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Kolodkin AL, Levengood DV, Rowe EG, Tai Y-T, Giger RJ, Ginty DD: Neuropilin is a semaphorin III receptor. Ce//1997, 90:753-762. Together with [42"], this paper describes the identification and characterization of neuropilin as a Sema III receptor. It also describes the cloning of neuropilin-2 (see also [45"]). 44.
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Chen H, Chedotal A, He Z, Goodman CS, Tessier-Lavigne M: Neuropilin-2, a novel member of the neuropilin family, is a high affinity receptor for the semaphorins Sema E and Sema IV but not Sema III. Neuron 1997, 19:547-559. Identification of a second member of the neuropilin family. The study reveals differential affinities of individual semaphorins for neuropilin-1 and -2, as well as largely nonoverlapping expression patterns for the two neuropilins in the developing nervous system. See also [43°]. 46. •
Feiner L, Koppel AM, Kobayashi H, Raper JA: Secreted chick semaphorins bind recombinant neuropilin with similar affinities but bind different subsets of neurons in situ. Neuron 1997, 19:539-545. Reports the use of AP-tagged collapsins/semaphorins to assess the distribution of their receptors during neural development. Individual members of
72
Development
the family show distinct anatomical patterns of binding in tissue sections, and the semaphorin domain is shown to determine binding specificity. 47. •
Koppel A, Feiner L, Kobayashi H, Raper JA: A 70 amino acid region within the semaphorin domain activates specific cellular response of semaphorin family members. Neuron 1997, 19:531-537. Domain swapping experiments show that a small region of the semaphorin domain confers specificity of binding and biological activity on individual members of the collapsin/semaphorin family. 48. •
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Ring C, Hassell J, Halfter W: Expression pattern of collagen IX and potential role in the segmentation of the peripheral nervous system. Dev Biol 1996, 180:41-53.
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Wang L-C, Rachel RA, Marcus RC, Mason CA: Chemosuppression of retinal axon growth by the mouse optic chiasm. Neuron 1996, 17:849-862.
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Wightman B, Barab R, Garriga G: Genes that guide growth cones along the C. elegans ventral nerve cord. Development 1997, 124:2571-2580.
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Karlstrom RO, ]'rowe T, Klostermann S, Baier H, Brand M, Crawford AD, Grunewald B, Haffter P, Hoffmann H, Meyer S U e t aL: Zebrafish mutations affecting retinotectal axon pathfinding. Development 1996, 123:427-438. Shows the utility of the zebrafish in the identification of candidate guidance genes. 68.
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Isacson O, Deacon TW: Specific axon guidance factors persist in the adult brain as demonstrated by pig neuroblasts transplanted to the rat. Neuroscience 1996, 75:827-837.