The likeness of being: Phylogenetically conserved molecular mechanisms of growth cone guidance

Cell, Vol. 76, 353-356,

August 12, 1994, Copyright

0 1994 by Cell Press

The Likeness of Being: Phylogenetically Conserved Molecular Mechanisms of G rowth Cone Guidance Corey S. Goodman Howard Hughes Medical Institute Division of Neurobiology Department of Molecular and Cell Biology University of California, Berkeley Berkeley, California 94720

At the turn of the century, Ramon y Cajal discovered neurons, the synapses of communication between them, and the growth cones that hook them up. Ever since, neurobiologists have struggled to understand how the nervous system gets wired up with such remarkable precision. Until recently, the importance of the question, coupled with the lack of conclusive data, kept the pendulum swinging back and forth between models in which specific synapses are thought to be encoded by unique molecular labels (Sperry, 1963), and models requiring little or no molecular specificity, but simply relying on random growth followed by functional validation of correct connections. Experiments over the past decade have taught us that neither extreme is correct. The events of growth cone guidance and target recognition appear to rely on molecular mechanisms that generate the initial specificity of synapticconnections. Patternsof neuronal activity then drive the refinement of these initial connections into highly tuned circuits (reviewed by Goodman and Shatz, 1993). Two questions have generated considerable debate concerning the mechanisms that control pathway and target recognition. First, what level of diversity of molecules and mechanisms is involved? Do a few cell and substrate adhesion molecules (CAMS and SAMs) and their receptors play the major roles in controlling these events, or are the molecules and mechanisms more complex and diverse? Second, are the nervous systems of phylogenetically divergent organisms such as nematode worms, fruit flies, and vertebrates constructed using different plans, or are the mechanisms and molecules of guidance conserved across species? Fortunately, several recent papers have provided insights into these questions. First, cell adhesion is not the sole force. At least two other mechanisms, repulsion and chemoattraction, play major roles in these events, and these mechanisms appear to be mediated by a diversity of molecules. Second, the molecules involved in each of these mechanisms appear to be highly conserved among worms, flies, and vertebrates. Understanding Neural Specificity: How Different Are Vertebrates from Invertebrates? During the 1960% the experimental manipulation of growth cone guidance in developing invertebrate and certain vertebrate nervous systems pointed to a high degree of precision in the ability of growth cones to make specific pathway choices and to find their correct targets (reviewed by Goodman and Shatz, 1993). As a result, some neurobiologists thought that a diversity of unknown molecules must exist to help confer pathway and target recognition.

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In contrast, others reasoned that a few CAMS (along with a few SAMs and their receptors) are sufficient to account for the specificity observed during the formation of synaptic connections in higher vertebrates. Little else was thought necessary, as the activity-dependent mechanisms of synapse rearrangement were invoked to do most of the work of establishing appropriate patterns of connections. To explain the discrepancies between this view and the evidence for specificity in invertebrates, the existence of phylogenetic differences in the mechanisms controlling neural specificity was suggested, contrasting what ap peared to be the rigid and predetermined patterns of guidance and connectivity in invertebrates (particularly in light of the invariant cell lineage and stereotyped synaptic wiring diagram described in the nematode) with the malleable and probabilistic nature of these events in vertebrates. Thus, neurobiologists engaged in a lively debate as to whether the discoveries arising from the two genetic systems (nematode and fruit fly) and other invertebrates are relevant for understanding the mechanisms generating neural specificity in vertebrates, especially in mammals. Growth Cones Guide Us to Some New Mechanisms Toward the end of the 1960% evidence emerged showing that many of the well known adhesion molecules and receptors are highly conserved from invertebrates to vertebrates. For example, fruit flies have CAMS of the immunoglobulin family (see below), SAMs of the laminin family, and receptors of the integrin family. Present evidence suggests that CAMS can play important roles in neurite outgrowth and growth cone guidance (e.g., Tang et al., 1992; Doherty and Walsh, 1994). However, genetic analysis of these molecules in Drosophila and mouse has revealed that cell adhesion is probably not the whole story. Loss-of-function mutations in the genes encoding Drosophila relatives of the vertebrate proteins Ll and NgCAM (Neuroglian; Bieberet al., 1969) NCAM (Fasciclin II; Grenningloh et al., 1991), and SClIDM-GRASP/BEN (Irrec; Ramos et al., 1993) and in the mouse NCAM gene (Tomasiewicz et al., 1993; Cremer et al., 1994) lead to much more subtle defects in guidance and connectivity (given their patterns of expression) than would be predicted if only a few CAMS played the major roles; in each case, specific defects are observed, but overall the mutant nervous systems look remarkably normal. During the 1960% evidence began to accumulate suggesting the existence of a wide array of other molecules and mechanisms that in many contexts may provide even stronger forces in pathway and target selection. Experiments using two different in vitro assays revealed that two of these other guidance mechanisms are repulsion and chemoattraction (reviewed by Goodman and Shatz, 1993). Repulsion was first demonstrated on the basis of the observation that the growth cones and axons of two different types of vertebrate neurons (sympathetic versus retinal) avoid one another (Kapfhammer et al., 1966). On contact with the inhibitory axons, the growth cones collapse, i.e., their filopodia and lamellipodia retract. The establishment

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of the growth cone collapse assay (Kapfhammer and Raper, 1987) led to several studies that revealed examples in which repulsion appears to play a major role in guidance. Another important discovery in the 1980s was the validation of an idea initially proposed by Ramon y Cajal at the turn of the century, whereby growth cones can be guided by gradients of diffusible chemoattractants. An early example of this mechanism was the response of sensory growth cones to a gradient of nerve growth factor (Gunderson and Barrett, 1979). Although the physiological relevance of this result is still unclear, this study initiated a resurgence of interest in chemotropism. What followed were several studies using the collagen gel matrix assay showing that different target tissues can attract appropriate growth cones from a distance (e.g., Lumsden and Davies, 1983; Tessier-Lavigne et al., 1988). In addition, although repulsion was initially identified in assays where contact was required, the use of the collagen gel matrix assay revealed that this mechanism also can function at a distance (Pini, 1993; Fitzgerald et al., 1993).

Growth Cones Discover Some Old Friends By the early 199Os, the search was on for molecules that can mediate growth cone collapse and chemoattraction in vitro. The first wave from this search has now come to fruition, and the results are quite revealing, both in what they potentially tell us about guidance, and in what they tell us about the phylogeny of these molecules. Recent papers report on the genes encoding the first growth cone collapse factor, collapsin, and the first chemoattractants for developing axons, the netrins. Although both proteins are new in vertebrates, both are homologous or highly related to molecules that have been shown previously to be involved in the control of growth cone guidance in vivo in invertebrates. The combination of the in vivo analysis in invertebrates and in vitro analysis in vertebrates provides complementary insights into the functions of these two families of guidance molecules. Collapsin (Luo et al., 1993) was purified from chick brain on the basis of its ability in vitro to cause the collapse of the growth cones from dorsal root ganglion neurons. Collapsin is highly related to Fasciclin IV (Fas IV), a guidance molecule previously described in grasshopper. Fas IV was later renamed Semaphorin I (Sema I) when it was found to be a member of a family of highly related transmembrane and secreted molecules, the Semaphorins, that are conserved from insects to humans (Kolodkin et al., 1993). Each of these proteins is about 750 amino acids in length and shares a highly conserved - 500 amino acid semaphorin domain; some members of the family are transmembrane, while others are secreted. Sema I functions in the grasshopper limb bud to stall and then steer a pair of growth cones as they encounter a stripe of epithelial cells expressing it; its expression on epithelial cells also appears to prevent axons that encounter it from defasciculating and branching. Tessier-Lavigne and colleagues now report on the molecular and functional characterization of the netrins (Serafiniet al., 1994[thisissueof M/j; Kennedyet al., 1994[this issue of (M/j), at least one of which appears to function in vertebrates as the circumferential chemoattractant emanating from the floor plate. The netrins are highly related

to UNC-8, which plays a role in circumferential guidance in the nematode (Hedgecocket al., 1990; lshii et al., 1992), as described below. Thus, we now have further evidence reinforcing the answer to the phylogenetic question: invertebrates and vertebrates appear to use many of the same mechanisms, and indeed in some instances the same or closely related molecules, for growth cone guidance. The phylogenetic conservation of molecular structure in the CAMS, SAM, semaphorins/collapsin, and netrinslUNC-8, and in those cases where information is available, in their apparent functions in vivo, in vitro, or both, is quite striking. Why then has there been such a lively debate about the relevance of invertebrates? One clue may lie in the relatively small number of neurons in the nervous systems of certain invertebrates and lower vertebrates as compared with mammals. As the number of interacting cells becomes increasingly small, the inductive interactions among these cells become so reproducible that it is easy to misinterpret the stereotyped patterns of cell fates and synaptic connections as reflecting a largely cell-autonomous program. All multicellular organisms appear to be constructed using highly conserved cell interaction and signal transduction mechanisms. Nevertheless, mammals, with orders of magnitude more neurons and synapses than most invertebrates, appear to use certain cell interaction mechanisms to a considerably greater extent to refine their initial patterns of synaptic connections and build highly tuned circuits. Molecular Basis of Chemotropkm Reveals

the Worm wlthln Us In vitro experiments have shown that the floor plate at the ventral midline of the developing spinal cord provides chemotropic guidance signals for the growth cones of commissural neurons whose axons extend toward and then across the floor plate (Tessier-Lavigne et al., 1988; Placzek et al., 1990). When a dorsal spinal cord explant is placed within a few hundred micrometers of a floor plate explant in a collagen gel matrix, commissural growth cones turn and extend toward the floor plate from a distance. Tessier-Lavigne and colleagues have now purified and microsequenced two highly related proteins called netrins from embryonic chick brain and cloned the cDNAs encoding them. The two netrins are encoded by different genes, have similar sequences, and are highly active (as purified and recombinant proteins) in inducing outgrowth from dorsal spinal cord explants (Serafini et al., 1994) and in attracting these growth cones from a distance (Kennedy et al., 1994). In the developing spinal cord, netrln-1 is expressed by the floor plate, and netrin-2 at lower levels by the ventral two-thirds of the spinal cord, but not by the floor plate (Figure 1). Netrfns have a high degree of sequence similarity ( - 50%) to the nematode UNC-8 protein (Hedgecock et al., 1990; lshii et al., 1992), a remarkable level of conservation considering the 800 million years separating worms and vertebrates. UNC-G/netrins are secreted proteins of -808 amino acids. Their N-termini (- 450 amino acids) are related to the N-termini of laminin subunits, in particular subunit 82, although B2 is much larger (>1800 amino acids).

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netrin-l

netrin-2

Figure 1. Schematic Diagram of Cross Section of Developing Vertebrate Spinal Cord The diagram shows behavior of commissural growth cone in relationship to expression of netrin-I, which is expressed at high levels by the floor plate and is postulated to form a diffusible gradient emanating from the floor plate, and netrin-2, which is expressed at lower levels by the ventral two-thirds of the spinal cord, but not by the floor plate.

The C-terminal - 150 amino acid domain diverges from laminins, but is highly conserved among the netrins and UNC-6. The netrins appear to be both diffusible and cell associated. The two bodies of data on nematode UNC-6 and vertebrate netrins provide complementary insights. The genetic analysis shows that UNC-6 can function to control circumferential guidance in the developing organism. The in vitro analysis shows that the netrins can function as diffusible chemoattractants exerting direct effects on growth cones. The present model is that netriWUNC-6 are secreted proteins that form a circumferential gradient in the extracellular environment. In vertebrates, the netrins appear to form a ventral to dorsal gradient in the developing spinal cord. Although there is no published report yet on UNC-6 expression in the nematode, the mutant phenotypes are compatible with a similar ventral to dorsal UNC-6 gradient. Such gradients could be generated either by a single molecule emanating from a point source (e.g., at the ventral midline), or by a series of molecules expressed in ventral to dorsal step functions. Both mechanisms might be used in the developing spinal cord, with netrin-1 being produced by the floor plate and netrin-2 at lower levels by the ventral spinal cord. One Signal May Get You Coming and Going Midline structures of nervous systems as diverse as nematodes, fruit flies, and vertebrates provide a variety of different ventral midline guidance signals. In principle, these might include signals that, first, attract some growth cones to extend toward the midline, second, guide some of these growth cones across the midline, third, prevent some growth cones from crossing the midline (while allowing them to extend rostrocaudally near it), and fourth, repel other growth cones away from the midline (and possibly other signals as well). One model to explain this diversity of growth cone behavior is to attribute the differential responsiveness of these different classes of growth cones to a variety of different midline cues, including, first, a possible diffusible chemoattractive cue, second, a contact-mediated attractive cue, third, a contact-mediated repulsive cue, and fourth, a diffusible chemorepulsive cue.

Figure 2. Growth Cone Guidance in Wild-Type and Mutant Nematodes (A) Behavior of circumferential growth cones in wildtype, uric-5 null loss-of-function, uric-5 dorsal defective hypomorphic l-f-function, urrc-5 ventral defective hypomorphic loss-of-function, ~0-5 lossoffunction, and uric-40 loss&function mutant alleles (after Hedgecock et al., 1990). (8) Behavior of growth cones of ALM mechanosensory neurons in wild type (in which they do not express UNC-5) when UNC-5 is ectopically expressed in the ALMS, and with UNC-5 ectopic expression in an uric-5 null mutant (after Hamelin et al., 1993).

In vertebrates, netrin-1 appears to be a diff usible chemoattractant emanating from the ventral midline, and nematode UNC6 might function in the same way. One possibility is that the netrinslUNC-6 might also function as a diffusible chemorepulsive midline cue. This model proposing the biinctionality of netrinsAJNC6 is based on the analysis of nematode mutants (Figure 2A; Hedgecock et al., 1996; Mclntire et al., 1992; Hamelin et al., 1993). First, there are three different classes of uric-6 mutations. Null (complete loss-of-function) mutations disrupt both dorsal and ventral migrations, whereas certain partial loss-of-function mutations disrupt either ventral (ventral defective) or dorsal (dorsal defective) migrations, but not both, suggesting that different domains of the protein control dorsal versus ventral growth cone guidance. Second, mutations in two other genes, uric-5 and unc40, also alter circumferential guidance. Mutations in uric-5 disrupt dorsal but not ventral migrations, whereas mutations in unc40 disrupt ventral but not dorsal migrations. The uric-5 gene encodes a transmembrane protein that has structural features of a receptor of the immunoglobulin superfamily (Leung-Hagesteijn et al., 1992). Mosaic analysis suggests that UNC-5 is required in the neurons that fail to extend properly in the uncd mutant. The function of UNC-46 is less well understood. The third line of evidence comes from experiments in which UNC-5 is ectopically expressed by mechanosensory neurons whose growth cones normally extend either laterally (the ALMS) or ventrally; the growth cones of these neurons ectopically expressing UNC-5 now instead extend dorsally away from the ventral midline (Figure 26; Hamelin et al., 1993). These altered dorsal trajectories depend upon the presence of UNC-6, as ectopic expression of UNC-5 in an uric-6 mutant leads to a wild-type trajectory. These results are compatible with a model in which ax-

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ons can respond in two ways to a presumptive UNC6 gradient, depending upon the particular UNC-6 receptor that they express. Assuming that UNCB is highest at the ventral midline, then neurons expressing UNC-5, which extend their axons dorsally away from the ventral midline, would move down the UNC6 gradient, whereas neurons expressing a different receptor (perhaps the UNC40 gene product), which extend their axons ventrally, would move up the UNCS gradient. If the UNC-6 gradient were in the opposite direction, then the roles of these two proteins would swap in relation to the gradient. Although the netrins/UNC-6, and presumably UNC-5 and UN&IO, are important components of a phylogenetitally conserved circumferential guidance system, it is likely that other gene products are also involved in the control of guidance near the ventral midline. First, mutations in uric-6, uric-5, and uric-40 do not completely disrupt the circumferential outgrowth of any one axon in every organism. For example, in null mutations in uric-6, there is considerable variability from animal to animal in whether any one particular circumferential axon appears normal or disrupted, with certain axons having a phenotypic penetrance as low as 300/b, suggesting that other genes are involved (Hedgecock et al., 1990; Mclntire et al., 1992). Moreover, in vertebrates, many commissural growth cones extend toward the ventral midline even in the absence of the floor plate (owing to surgical removal of the notochord, or by laser or genetic ablation of the floor plate), although they make mistakes near the midline (reviewed by Goodman and Shatz, 1993). Second, it is difficult to explain some important aspects of growth cone behavior near the ventral midline solely on the basis of what is known about the function of these genes. For example, while a netrin/UNCX-mediated mechanism appears to guide growth cones toward (and probably away from) theventral midline, once at the midline, adifferent set of mechanisms (probably involving contact-mediated signals) operate to determine which axons cross and which axons remain on their own side. Some axons, whether they have crossed the midline once or not at all, are capable of extending rostrocaudally for long distances right next to the midline without crossing it. Genetic screens in Drosophila have identified two genes (commissureless and round&out) that control important aspects of guidance near the midline (Seeger et al., 1993). Although there is no report yet of the sequence of these two genes, their mutant phenotypes suggest that they encode components of guidance mechanisms that function near the midline. Taken together, the vertebrate netrin and nematode UNC-6 data suggest

an intriguing

model whereby

certain

guidance molecules may not be exclusively attractive or repulsive, but rather both, playing different roles for different neurons. In this model, a given target expresses a particular signal, and growth cones that are attracted to the target express an “attractive” receptor, whereas growth cones that are repelled express a “repulsive” receptor. After all, if a particular

neurotransmitter

released

at a syn-

apse can be either excitatory or inhibitory depending upon the postsynaptic receptor, then it seems equally plausible that the signals that help these synaptic

partners

find one

another in the first place might also be multifunctional. Having these molecules serve as guidance and targeting signals (rather than as attractive versus repulsive molecules), with different receptors determining the particular growth cone response, seems like an economical and reliable way to design such a complex

targeting

system.

Now

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