Neuron, Vol. 32, 981–984, December 20, 2001, Copyright 2001 by Cell Press Signaling at the Growth Cone: Meeting Report The Scientific Progeny of Cajal Meet in Madrid

Peter W. Baas1,3 and Liqun Luo2,3 them to their targets (reviewed in Mueller, 1999; Tessier- 1Department of Neurobiology and Anatomy Lavigne and Goodman, 1996). During development, ax- MCP Hahnemann University ons undergo bouts of elongation and retraction, and 2900 Queen Lane their growth cones can turn dramatically toward or away Philadelphia, Pennsylvania 19129 from external cues such as chemical factors and guide- 2 Department of Biological Sciences post cells. Over the years, scientists interested in growth Stanford University cones have studied them from several different perspec- Stanford, California 94305 tives. There is great interest in the cytoskeletal machin- ery inside the growth cone that permits it to move for- ward, collapse, retract, and change direction. There is I had the good fortune to behold for the first time also great interest in the environmental factors that initi- that fantastic ending of the growing axon. In my ate such events and the cascade of biochemical events sections of the spinal cord of the three day chick that transduce these cues into cytoskeletal changes. embryo, this ending appeared as a concentration of The difficult challenge, as evidenced by the lively discus- protoplasm of conical form, endowed with sions at the meeting, is to understand how these biologi- amoeboid movements. It could be compared with a cal events are coordinated. living battering ram, soft and flexible, which advances, pushing aside mechanically the obstacles Cytoskeleton which it finds in its path, until it reaches the region Discussions on the cytoskeleton seemed to permeate of its peripheral termination. This curious virtually every session of the meeting. There was broad terminal club, I christened the growth cone. agreement that no matter what signaling cascades were Santiago Ramo´ n y Cajal, Recollections of My Life under study, the ultimate effect must be, at least in part, on the actin or microtubule cytoskeletal arrays within the growth cone (for a recent review, see Suter and In October 2001, over fifty scientists and students from Forscher, 2000). Actin filaments and microtubules are around the world met in Madrid to present their most configured into dense and highly organized arrays that recent insights and understanding of the neuronal are critical for determining the morphology of the growth growth cone. The auspicious occasion was funded and cone and for driving its motility. These cytoskeletal hosted by the Juan March Institute, and fittingly held arrays are also essential for orchestrating the intracellu- in the country where Cajal first observed specialized lar transport events that organize the neuronal cyto- formations at the tips of axons and dendrites, and plasm and for directing the endocytotic and exocytotic dubbed them “growth cones.” Cajal was equipped with traffic. Actin filaments are highly concentrated at the a light microscope, fixed samples of Golgi-stained ner- leading edge of the growth cone, forming bundles at vous tissue, and the artistic talent to translate what he the cores of filopodia and in the crosslinked meshwork saw with the microscope into remarkably accurate underlying lamellipodia (Figure 1). Actin filaments un- drawings. He had the extraordinary prescience to antici- dergo assembly and disassembly, and these dynamics pate from static images the range of dynamic functions have been mentioned for years as the potential target for which we now have firm evidence. The recent meet- for the biochemical cascades that cause axons to grow, ing included a vast array of genetic, molecular, biochem- retract, change direction, and branch. It was pointed ical, and imaging techniques, most of which were un- out in several spirited discussions that both the myosin- heard of in the days of Cajal, all aimed at improving our based contractility and dynamics of the actin cytoskele- knowledge of his “battering ram,” which he envisaged ton contribute to changes in growth cone morphology also to have “exquisite chemical sensitivity” (Cajal, and behavior. Microtubules, which also undergo dy- 1911). As much as scientists prefer to find simple and namic assembly and disassembly, form a dense bundle recurring themes in nature, the overall conclusions of along the length of the axon that extends into the growth the meeting were that growth cones are extraordinarily cone to “meet” the dense actin cytoskeleton (Figure 1). complex, that all growth cones are not alike, and that It has long been recognized that the coordination of the there are multiple pathways for orchestrating essential microtubules and actin are at the heart of how growth growth cone behaviors during neural development. Our cone behaviors are orchestrated, but the precise means purpose in this report is to briefly describe the findings by which this occurs has been elusive. Indeed, there presented at the meeting and thereby provide a flavor was broad agreement that ascertaining how these two for contemporary thinking on growth cone biology. filament systems engage one another could be the key Growth cones are present at the tips of axons and to understanding the machinery of growth cone navi- dendrites and at the tips of their developing branches. gation. Axonal growth cones, in particular, are capable of re- Reporting on work with vertebrate brain neurons in sponding to a host of environmental factors that guide culture, C. Dotti (Cavalieri Ottolenghi Scientific Institute, Italy) presented studies showing that the actin cytoskel- 3 Correspondence: [email protected] (P.W.B.), lluo@stanford. eton of the growth cone is remodeled during axogenesis. edu (L.L.) Specifically, he showed that the actin cytoskeleton be- Neuron 982

Figure 1. Summary of Signaling Mechanisms at the Growth Cone External cues result in activation of intracellular signaling pathways, which in turn cause alterations in cytoskeletal structures (actin filaments in red, microtubules in blue) and membrane dynamics. Ultimately, these effects cause changes in growth cone behaviors. Listed here is a sampling of some of the elements of this scenario. comes more labile to anti-actin drugs and shows dimin- Extracellular Cues and Their Receptors ished phalloidin staining compared to the growth cones Since the discovery of several classes of now “classical” of primitive neurites not undergoing axogenesis. These axonal guidance cues in the 90s—such as netrins, sema- changes in the actin cytoskeleton were posited to permit phorins, ephrins, and slits (Mueller, 1999; Tessier-Lavigne microtubules to invade different regions of the growth and Goodman, 1996)—it has become clear that these cone, thus accounting for the navigation of the growth molecules have a broad array of functions (Yu and Barg- cone in a particular direction. In a related presentation, mann, 2001). In addition, the list of extracellular cues K. Kalil (University of Wisconsin) showed fluorescent has grown, as evidenced by the array of different ligands microtubules and actin filaments imaged simultaneously and receptors discussed at the meeting. Ephrins and in living growth cones undergoing growth and branching. their Eph receptors, known for their role in establishing These studies suggested that a subset of the microtu- the anterior-posterior retinotectal topographic map, bules and actin filaments co-polymerize with one an- were discussed extensively. It was previously demon- other, providing another means by which the two poly- strated that ephrins, known as repulsive axonal guid- mer systems might interact. Two other presentations ance cues, can also act as receptors for “reverse signal- focused on changes in the microtubule array as growth ing” using the Eph-receptor as the ligand. U. Drescher cones respond to physiological cues. P. Baas (MCP (King’s College London, UK) presented evidence for Hahnemann University) showed studies indicating that ephrin-A’s function in the formation of the vomeronasal microtubules do not undergo detectable depolymeriza- (VNO) projection. Ephrin-A5 is more highly expressed tion as axons retract in response to nitric oxide, but on vomeronasal axons from the apical than the basal instead, undergo what appears to be a motor-driven VNO, and EphA6 is expressed higher in the anterior reconfiguration. Indeed, the reconfiguration of microtu- than the posterior parts of the target. High ephrin-A5- bules was very similar to that observed in axons induced expressing axons project to high EphA6-expressing an- to retract by manipulation of motor proteins. P. Salinas terior targets. This indicates that—in this case—ephrin- (Imperial College of Science, UK) showed that microtu- A5 serves as an attractive axon guidance receptor in bules splay apart in addition to showing alterations in the “reverse signaling” mode. Studies using the in vitro stability in response to environmental factors that utilize stripe assay and ephrin-A5 mutant mice support this GSK3 kinase (via the WNT-signaling pathway). These new role. Interestingly, W. Harris (University of Cam- studies demonstrate that alterations in the cytoskeleton bridge, UK) provided new data from C. Holt’s and his that occur within the growth cone are far more complex laboratory to show that Ephrin B signaling, both forward than can be explained simply by polymerization and and reverse, may be important for mapping along the depolymerization. There was great interest expressed dorsal ventral axis in the Xenopus retinotectal system. in the discussions in pursuing both the motor and non- Bi-directional signaling was also implicated in the motor microtubule-associated proteins and actin-regu- function of receptor tyrosine phosphatase (PTPase) latory proteins that are potential targets for the pathways Dlar. Dlar was identified previously as an axonal guid- that induce these changes in the cytoskeleton. ance molecule in the Drosophila embryonic nervous sys- Meeting Report 983

tem. B. Dickson (Research Institute of Molecular Pathol- where along the midline the crossing axons form the ogy, Austria) presented new data showing that Dlar is chiasm, whereas NrCAM functions during the crossing essential for Drosophilia photoreceptor R7 axon projec- step. P. Letourneau (University of Minnesota) presented tion into a specific layer of the medulla. In Dlar mutants, evidence that one class of extracellular cue could although R7 axons initially project to the correct layer, “prime” the response of growth cones to another cue: the axons cannot stabilize their connections and many pretreatment of NGF reduces growth cone collapse in retract to the R8 layer. Interestingly, single cell mosaic response to subsequent application of Sema 3A. Thus, analysis and single cell rescue experiments indicate that it will be important not only to explore the signaling Dlar has both a cell-autonomous function in R7 which mechanisms of individual guidance cues, but also how is dependent on PTPase function, and a cell-nonautono- multiple guidance cues act simultaneously or in se- mous function in R8 which is not dependent on PTPase quence to change growth cone behavior. How the ac- function. Thus, Dlar appears to serve as a receptor as tions of multiple guidance cues are integrated and what well as a ligand. How does this work? Could Dlar in mechanisms regulate spatially and temporally specific neighboring cells signal through homophilic interaction? expression of receptors for these cues remain to be This seems unlikely, as expression of Dlar in R7 alone explored. is largely sufficient for normal targeting even in the ab- sence of Dlar in either R8 or medulla cells. Homophilic Intracellular Signaling interactions may however contribute to LAR function How extracellular cues regulate the machinery inside in other systems. E. Macagno (University of California) the growth cone to achieve their purpose was a third presented evidence that the leech homolog of Lar focus of the meeting, aiming to link ligand-receptor inter- (HmLar) plays a critical role in a remarkable parallel actions to the cytoskeleton (for a recent review, see growth of multiple processes of comb cells. In normal Song and Poo, 2001). The Rho family of small GTPases comb cells, parallel growth cones lead to parallel growth are important intracellular signaling proteins that trans- of processes likely via contact-mediated retraction of duce signals from cell surface receptors to the cytoskel- filopodia from neighboring growth cones. Perturbation eton. Previous studies using dominant negative mutants of HmLar function via double strand RNA interference have shown that perturbation of Rho GTPases has dras- (RNAi) results in neighboring processes no longer tic effects on axonal growth and guidance (reviewed avoiding each other. in Mueller, 1999). L. Luo (Stanford University) provided A number of important morphogens that pattern early genetic evidence that endogenous Rac proteins are es- embryonic development appear to have additional roles sential for axonal growth, guidance, and branching in in axonal guidance, including household names such Drosophila mushroom body neurons. B. Dickson pre- as Sonic hedgehog (Shh), bone morphogenic protein sented data supporting the idea that Racs act down- (BMP), and WNT proteins. P. Bovolenta (Instituto Cajal, stream of guanine nucleotide exchange factor (GEF) Trio Spain) presented evidence that Shh serves as a repul- in Drosophila photoreceptor axons. Several RhoGEFs— sive axonal guidance cue for retinal ganglion cell (RGC) positive regulators of RhoGTPase signaling including axons expressing the Patched receptor. Expression of C. elegans Unc-73, Drosophila Trio, and vertebrate Shh in specific domains near the midline ensures RGC Ephexin—have previously been reported to play essen- axons form the optic chiasm at the appropriate location. tial roles for axonal guidance. Rho GTPases are also P. Salinas reported that WNT7a stops cerebellar granule regulated negatively by Rho GTPase activating proteins cell axonal growth and stimulates its synaptic matura- (RhoGAPs). L. Luo reported a systematic study of Dro- tion. A poster from the laboratory of P. Bovolenta sophila RhoGAPs using transgenic RNAi and identified showed that secreted frizzled related proteins (SFRP) stimulate RGC axon growth. SFRP in theory could func- p190 RhoGAP as essential for repressing a “retraction tion by titrating WNT signaling, thus supporting WNT as signaling pathway” involving RhoA, Drosophila Rho- an axonal stop signal. G. Marques (University of Minne- associated kinase (Drok), and phosphorylation of myo- sota) reported the function of a Drosophila BMP II recep- sin regulatory light chain. Inactivation of p190 RhoGAP tor in synapse maturation. J. Culotti (Samuel Lunenfeld results in activation of the RhoA pathway, resulting in Research Institute, Mount Sinai Hospital, Canada) pro- axonal retraction. vided genetic evidence that unc-129, encoding a TGF-␤ D. Van Vactor (Harvard Medical School) presented family member, is essential for C. elegans dorsal circum- genetic and biochemical evidence that a signaling com- ferential axonal guidance. plex composed of Abl tyrosine kinase, enabled protein, It is evident that growth cones listen to different extra- profilin, and the newly identified cyclase-associated cellular signals at different stages of their journey, or protein (CAP) may act downstream of multiple guidance even at the same time. In fact, unc-129 was identified receptors. cap exhibits genetic interactions with multi- in a genetic screen for suppressors of a guidance defect ple components in the slit repellent pathway at the mid- caused by misexpression of the worm netrin receptor line of Drosophila embryos. Cap itself is an actin mono- Unc-5 (J. Culotti), suggesting that netrin and the TGF- mer binding protein that antagonizes actin assembly ␤/BMP family of extracellular cues act in a concerted in different cellular contexts. C. Kla¨ mbt (University of manner. C. Mason (Columbia University) reported the Mu¨ nster, Germany) reported that kette is essential for collaborative roles of three different classes of guidance Drosophila motor axonal guidance. kette encodes a mul- cues/receptors, Slit/Robo, Nr-CAM, and Eph/Ephrins, in tidomain protein that on the one hand is in a complex ensuring the fidelity of RGC axon guidance at the mouse with Dock/Nck that potentially links to Rho GTPase sig- optic chiasm. Ephrin B2 works to prevent normally un- naling, and on the other hand binds to several actin crossed axons from crossing; Slit expression regulates binding proteins and actin itself. Elucidating the mecha- Neuron 984

nisms of how the Cap/Ena complex functions and how discussed the importance of protein degradation, and Kette links different binding proteins together promises A. Klar (Hebrew University, Israel) discussed the impor- to provide additional insights into how signals from cell tance of proteolytic processing by serine proteases in surface receptors are transduced into cytoskeletal alter- growth cone biology. Ferrus showed that Drosophila ations. ariadne mutants exhibit defects in axon guidance and Despite the strong focus of the meeting on the cyto- synapse maturation. ariadne encodes a protein with a skeleton, C. Dotti and M. Gonza´ lez-Gaita´ n (Max-Plank- RING-finger domain that likely serves as a ubiquitin li- Institute of Molecular Cell Biology and Genetics, Ger- gase and physically interacts with a ubiquitin conju- many) provided frequent reminders that membrane gating enzyme. Using nested deletion proteins, Klar dynamics are also profoundly important to growth cone showed evidence for the plasmin-mediated release of behaviors. Dotti noted that, early in axonal development, F-spondin from the extracellular matrix. This effect is there is a massive and indiscriminate flow of cytoplasm crucial for promoting the outgrowth of commissural ax- into the neuronal process that will become the axon, and ons while simultaneously suppressing the growth of mo- that this is subsequently replaced by a more selective tor axons. Future studies of the proteins that must be compartmentalization of membranous components into newly synthesized, degraded, or proteolytically pro- axons and dendrites. Gonza´ lez-Gaita´ n, focusing on syn- cessed for proper growth cone behavior will surely en- apse formation and function, discussed the “intermedi- rich our understanding of the complex biology of neural ate endosomal compartment” which acts as a buffer development. In addition, new axonal guidance genetic between the membrane released and retrieved during screens in zebrafish and worm reported in the meeting synaptic vesicle traffic. Specifically, he showed that the (M. Granato, University of Pennsylvania; S. Clark, Skir- small GTPase Rab5 determines the kinetics of recycling ball Institute) promise to identify new genes important and the release of the recycled vesicles. for growth cone signaling. As if to accentuate the vast array of pathways and mechanisms that can affect the growth cone, N. Spitzer Concluding Remarks (University of California) showed a series of remarkable Figure 1 is our attempt to integrate the topics discussed studies on the effects of calcium transients on growth at the meeting into a schematic summary. External cues cone behaviors, something not touched upon in any of (most of which involve specific receptors) activate sev- the other presentations. These studies showed that eral different but interrelated signaling pathways which rapid calcium transients correspond directly to pauses give rise to alterations in the cytoskeleton and mem- in growth cone advance, and that such pauses could brane dynamics. In turn, these alterations act within the be experimentally inhibited by locally chelating calcium. growth cone to cause axons to grow, retract, navigate, There was a great deal of curiosity during the discussion branch, and form synapses. The vastness of these cues, as to how these observations fit with the other messen- signals, and pathways is, at the same time, both bewil- ger systems of the growth cone. dering and exciting. It is our expectation that the next generation of growth cone biologists, represented by an array of excellent student poster presentations, will Local Protein Synthesis and Metabolism continue to piece together the puzzle of the growth Perhaps the most unexpected findings reported in the cone, the remarkable and yet enigmatic “battering ram” meeting dealt with new roles for mRNA protein synthesis of Cajal. and processing in growth cone guidance. G. Bassell (Albert Einstein College of Medicine) reported a role for Acknowledgments the RNA binding protein ZBP1 in the active transport of ␤-actin mRNA particles into developing axons and The meeting was organized by A. Ferrus, P. Bovolenta, and E. Ma- growth cones of cultured hippocampal neurons. Pertur- cagno and under the support of the Juan March Institute. On behalf bation of the transport of this mRNP complex impaired of all of the participants, we thank the organizers, the Juan March Institute, and our gracious host, Andre´ s Gonza´ lez. We thank Yan forward motility of growth cones. C. Holt (University of He of MCP Hahnemann University for assisting in the preparation Cambridge, UK) presented striking effects of inhibiting of Figure 1, Nick Spitzer and Bill Harris for critical comments on the protein synthesis on the Xenopus retinal ganglion cell manuscript, and the editors of Neuron for providing us with the growth cone “turning responses” to multiple attractive opportunity to share this meeting report with their readership. and repulsive guidance cues. Evidence for a potential References signaling pathway from guidance cues to the phosphor- ylation of regulators of protein synthesis was presented. Cajal, S.R. (1911). Histology of the Nervous System of Man and These studies suggest that growth cone structure and Vertebrates, 1995 translation (Oxford: Oxford University Press, Inc.). motility are regulated by mRNA transport and local Mueller, B.K. (1999). Growth cone guidance: first steps towards a translation mechanisms. Adding to the role of mRNA deeper understanding. Annu. Rev. Neurosci. 22, 351–388. transport and protein synthesis in growth cone biology, Song, H.-j., and Poo, M.-m. (2001). The cell biology of neuronal K. Zinn (Caltech) provided evidence that Pumilio and navigation. Nat. Cell Biol. 3, E81–E88. Nanos play key roles in the development of the Drosoph- Suter, D.M., and Forscher, P. (2000). Substrate-cytoskeletal cou- ila neuromuscular junction. Nanos and Pumilio are well pling as a mechanism for the regulation of growth cone motility and guidance. J. Neurobiol. 44, 97–113. known as regulators of mRNA translation in Drosophila Tessier-Lavigne, M., and Goodman, C.S. (1996). The molecular biol- early embryo patterning. Both loss-of-function and gain- ogy of axon guidance. Science 274, 1123–1133. of-function experiments now implicate these transla- Yu, T.W., and Bargmann, C.I. (2001). Dynamic regulation of axon tional regulators in determining the size of synapses. To guidance. Nat. Neurosci. Suppl. 4, 1169–1176. round out the story, A. Ferrus (Instituto Cajal, Spain)