Bill Harris

Stimulating New Turns

In this issue of Neuron, a paper by Ming et al. (2001)xMing, G.-l., Henley, J., Tessier-Lavigne, M., Song, H.-j., and Poo, M.-m. Neuron. 2001; 29: 441–452Abstract | Full Text | Full Text PDF | PubMed | Scopus (171)See all ReferencesMing et al. (2001) shows that stimulation of action potentials can dramatically alter the turning responses of growth cones to attractive and repulsive guidance factors in culture. Turning up a gradient of netrin-1 is enhanced, myelin-associated glycoprotein (MAG)–induced repulsive turning is converted to attraction, and Sema3A-induced repulsive turning is converted to full growth cone collapse.This study continues a line of investigation that opened with the remarkable 1997 discovery from Mu-ming Poos laboratory that attraction can be changed to repulsion in cultured Xenopus spinal neurons by changing cAMP levels pharmacologically (Song et al., 1997xNature 388, 275–279. Song, H.J., Ming, G.L., and Poo, M.M. Erratum: Nature. 1997; 389: 1997See all References(Song et al., 1997). In a series of subsequent papers, Poo and colleagues investigated the basis of this effect (Song and Poo, 1999xSong, H.J. and Poo, M.M. Curr. Opin. Neurobiol. 1999; 9: 355–363Crossref | PubMed | Scopus (368)See all References(Song and Poo, 1999). This has led to a balanced model of turning that divides attractants and repellents into two classes based on whether they are influenced by cAMP or cGMP levels. In this model, diffusible factors induce both polymerization (P) and depolymerization (D) of actin. The P/D ratio determines whether a factor is attractive or repulsive. Attractants generally stimulate P over D, and repellents stimulate D over P. But the P/D ratio is also influenced by baseline levels of cAMP (class 1) or cGMP (class 2). In the class 1 system, protein kinase A, stimulated by high cAMP, phosphorylates effectors of the P/D machine and pushes the P/D ratio toward P. In low cAMP, D is favored. Thus, growth cones turn toward a gradient of netrin-1 when cAMP is high and away when cAMP is low. MAG is a class 1 repellent that in normal cAMP levels stimulates D more than P, yet when cAMP is raised to high levels in the growth cone, the P/D ratio increases and MAG becomes attractive. Sema3A is a class 2 repellent that is affected by cGMP levels in a similar way.This model explains much of growth cone turning in vitro as influenced by pharmacological perturbations of cAMP and cGMP and raises the following questions. What normally causes changes in the levels of cyclic nucleotides in growth cones? Do these changes have an affect on guidance in vivo? Hopker et al. (1999) were the first to show that a natural substrate molecule, laminin, decreases cAMP in retinal growth cones, thereby switching netrin-induced attraction to repulsion and providing a mechanism for how these axons turn away from the laminin-rich surface of the optic fiber layer into the netrin-1-rich optic nerve head to exit the retina (Hopker et al., 1999xHopker, V.H., Shewan, D., Tessier-Lavigne, M., Poo, M., and Holt, C. Nature. 1999; 401: 69–73Crossref | PubMed | Scopus (352)See all References(Hopker et al., 1999). The Ming et al. (2001)xMing, G.-l., Henley, J., Tessier-Lavigne, M., Song, H.-j., and Poo, M.-m. Neuron. 2001; 29: 441–452Abstract | Full Text | Full Text PDF | PubMed | Scopus (171)See all ReferencesMing et al. (2001) study here provides another possible natural mechanism for modulating cAMP since calcium, which enters the growth cone in response to impulse activity, is known to stimulate certain adenylate cyclases and raise cAMP levels (Song and Poo, 1999xSong, H.J. and Poo, M.M. Curr. Opin. Neurobiol. 1999; 9: 355–363Crossref | PubMed | Scopus (368)See all References(Song and Poo, 1999). In this case, inhibiting the increase in either calcium or cAMP levels abolishes the dependence of growth cone turning responses on electrical stimulation.Do calcium rises induced by action potentials occur naturally in growth cones during pathfinding? Using calcium imaging, Spitzer and colleagues observed short single spikes of calcium in the growth cones of cultured Xenopus spinal neurons as a result of spontaneous action potentials invading the growth cone and opening voltage-gated calcium channels (Gu et al., 1994xGu, X., Olson, E.C., and Spitzer, N.C. J. Neurosci. 1994; 14: 6325–6335PubMedSee all References(Gu et al., 1994). These calcium spikes occur at a spontaneous rate of about three per hour and do not lead to changes in the rate of outgrowth (Gomez and Spitzer, 2000xGomez, T.M. and Spitzer, N.C. J. Neurobiol. 2000; 44: 174–183Crossref | PubMed | Scopus (99)See all References(Gomez and Spitzer, 2000). Ming et al. use higher frequency electrical stimulation of the cell body (ten pulses at 2 Hz) to produce a more sustained increase in growth cone calcium. Low-frequency stimulation does not lead to an alteration in growth cone turning just as the rather isolated spontaneous spikes do not affect axon growth. Spitzer and colleagues have also shown that slower spontaneous calcium transients, called waves, occur in growth cones in vitro and in vivo and that the rate of growth cone advancement is inversely proportional to the frequency of these transients (Gomez and Spitzer 1999xGomez, T.M. and Spitzer, N.C. Nature. 1999; 397: 350–355Crossref | PubMed | Scopus (343)See all References, Gomez and Spitzer 2000xGomez, T.M. and Spitzer, N.C. J. Neurobiol. 2000; 44: 174–183Crossref | PubMed | Scopus (99)See all References). However, these waves are the result of intracellular release from calcium stores and are not dependent on impulse activity (Gu et al., 1994xGu, X., Olson, E.C., and Spitzer, N.C. J. Neurosci. 1994; 14: 6325–6335PubMedSee all References(Gu et al., 1994). Thus, one might wonder if these axons really use impulse activity in growth cone guidance during normal development. While the literature clearly shows that electrical activity influences synapse formation and terminal arborization (Goodman and Shatz 1993xGoodman, C.S. and Shatz, C.J. Cell. 1993; 72: 77–98Abstract | Full Text PDF | PubMed | Scopus (876)See all References, Catalano and Shatz 1998xCatalano, S.M. and Shatz, C.J. Science. 1998; 281: 559–562Crossref | PubMed | Scopus (167)See all References, Dantzker and Callaway 1998xDantzker, J.L. and Callaway, E.M. J. Neurosci. 1998; 18: 4145–4154PubMedSee all References), there is little indication that impulse activity in the developing brain is needed for the formation of normal axon tracts or pathways. Indeed, in the absence of all normal impulse activity throughout development, the axonal connections in the nervous system look remarkably good (Harris, 1980xHarris, W.A. J. Comp. Neurol. 1980; 194: 303–317Crossref | PubMedSee all References(Harris, 1980).Because Ming et al. use a growth cone turning assay, it is tempting to assume that this behavior is relevant only for axon pathfinding. However, cultured neurons are limited in the behaviors they can express, and so it is possible that the “read out” seen as a directional turn may, in fact, represent something different such as an aspect of target innervation or terminal arbor formation. If bursts of spontaneous axon potentials in growing neurons were coordinated with target innervation, something that is not known (although there are suggestions that this might be so), the results could help explain target selection errors in growing axons deprived of activity.Finally, the potential significance of this work to axon regeneration should not be overlooked. It has been proposed that repulsion to molecules like MAG at injury sites plays a role in preventing damaged CNS axons from regenerating, because their growth cones, unlike those of embryonic axons, have low levels of cAMP (Cai et al., 1999xCai, D., Shen, Y., De Bellard, M., Tang, S., and Filbin, M.T. Neuron. 1999; 22: 89–101Abstract | Full Text | Full Text PDF | PubMed | Scopus (417)See all References(Cai et al., 1999). Thus, stimulation of injured CNS axons might return cAMP levels to more embryonic levels and thus help regenerating axons across repulsive barriers and make the correct pathfinding decisions that they did when they were young. The recent work of Al-Majed et al. (2000)xAl-Majed, A.A., Neumann, C.M., Brushart, T.M., and Gordon, T. J. Neurosci. 2000; 20: 2602–2608PubMedSee all ReferencesAl-Majed et al. (2000) suggests that electrical stimulation of motor nerves increases the speed and accuracy of peripheral regeneration, but in this case the effect is mediated through activity of the cell body and not the growth cone. Thus, we are left with a tantalizing quandary: the result is exciting but the in vivo relevance awaits resolution.

Chi-Bin Chien: A Tribute

As a child growing up in New Haven, CT and Palo Alto, CA, Chi-Bin Chien was so academically gifted that he skipped straight from the third to the eighth grade and, at the unbelievable age of 12, entered Johns Hopkins University as a Physics major. He was accepted to do graduate work in Physics at Caltech at the age of 15 but was considered too young to enter the program, so he took a fellowship at Cambridge University for a year. At 16, he began his PhD studies with the experimental physicist Jerry Pine, who had recently turned his attention to neurobiology and had pioneered the development of multielectrode arrays for studies of neuronal networks in vitro.Chi-Bins gift was not just his scintillating brilliance, because underneath he was a truly motivated scientist who was prepared to take practical and laborious steps to reach a distant goal. In the Pine laboratory, he designed an elegant apparatus that was sensitive enough to measure single action and synaptic potentials in cultured neurons using voltage-sensitive dyes (Chien and Pine, 1991xChien, C.B. and Pine, J. Biophys. J. 1991; 60: 697–711Abstract | Full Text PDF | PubMedSee all ReferencesChien and Pine, 1991). Consideration of how the neural networks in his experimental dishes made connections with each other sparked Chi-Bin to choose the area of research in which he made most of his major contributions to knowledge: how the nervous system wires up in development.That he was interested in exploring this problem in vivo was the main reason we were lucky enough to attract Chi-Bin to work with us at UCSD. Chi-Bin made a number of remarkable innovations in our laboratory. For example, he developed a viewing chamber in which it was possible to observe single Xenopus retinal axons growing in the brain while washing various pharmacological reagents in and out as a way of probing the signaling systems that growth cones use to navigate correctly. One of the first interesting findings that came from this was that growth cones deprived of filopodia by actin depolymerizers make navigational errors (Chien et al., 1993xChien, C.B., Rosenthal, D.E., Harris, W.A., and Holt, C.E. Neuron. 1993; 11: 237–251Abstract | Full Text PDF | PubMed | Scopus (194)See all ReferencesChien et al., 1993). He went on to produce some of the most stunning in vivo movies of navigating axons (Hutson and Chien, 2002xHutson, L.D. and Chien, C.B. Neuron. 2002; 33: 205–217Abstract | Full Text | Full Text PDF | PubMed | Scopus (129)See all ReferencesHutson and Chien, 2002) and morphogenetic eye movements (Kwan et al., 2012xKwan, K.M., Otsuna, H., Kidokoro, H., Carney, K.R., Saijoh, Y., and Chien, C.B. Development. 2012; 139: 359–372Crossref | PubMed | Scopus (27)See all ReferencesKwan et al., 2012) and some of the finest anatomical images of the developing visual system (see Figure 1Figure 1).Figure 1Topography of the Entire Retinofugal Pathway in Zebrafish Is Revealed with Red and Green Fluorescent DyesThis image produced by Chi-Bin graces the cover of the textbook Development of the Nervous System (San Diego, CA: Academic Press).View Large Image | View Hi-Res Image | Download PowerPoint SlideStriving to find the best system and approach to make progress into the molecular mechanisms of neural wiring in vivo, Chi-Bin did a second postdoc with Friedrich Bonhoeffer at the Max Planck Institute in Tubingen. Christianne Nusslein-Volhard and Friedrich had just done a major screen for developmental mutants of zebrafish, and Bonhoeffers laboratory concentrated on those that affect the retinotectal projection. At the time Chi-Bin went to the Bonhoeffer laboratory, they had already identified over a 100 mutants in genes that disrupted the retinotectal pathway. Some of these had pathfinding errors, and some had topographic mapping errors (Karlstrom et al., 1996xKarlstrom, R.O., Trowe, T., Klostermann, S., Baier, H., Brand, M., Crawford, A.D., Grunewald, B., Haffter, P., Hoffmann, H., Meyer, S.U. et al. Development. 1996; 123: 427–438PubMedSee all References, Trowe et al., 1996xTrowe, T., Klostermann, S., Baier, H., Granato, M., Crawford, A.D., Grunewald, B., Hoffmann, H., Karlstrom, R.O., Meyer, S.U., Muller, B. et al. Development. 1996; 123: 439–450PubMedSee all References). This, it seemed, was the opportunity for which Chi-Bin had long been preparing himself, and it was his work on the development of the zebrafish retinotectal system that shone so brightly on the developmental neurobiological community.In 1998, at the age of 32, Chi-Bin joined the Department of Neurobiology and Anatomy at the University of Utah. There he met and quickly fell in love with Niki Hack, who became his wife. But within a year he received devastating news. He had advanced colon cancer that required surgery and chemotherapy.This did not dissuade Chi-Bin from pursuing the most challenging scientific problems. In the Bonhoeffer laboratory, Chi-Bin had decided to focus on the astray mutant, which caused severe axon pathfinding defects in the brain. Identifying the molecule encoded by the astray gene was the task that Chi-Bin next set for himself. In 2001, he produced a landmark paper (Fricke et al., 2001xFricke, C., Lee, J.S., Geiger-Rudolph, S., Bonhoeffer, F., and Chien, C.B. Science. 2001; 292: 507–510Crossref | PubMedSee all ReferencesFricke et al., 2001) showing that the astray gene codes for the Robo2 receptor. Robo had recently been shown to act as a guidance receptor for Slit in Drosophila, and it had just been shown that mammalian Slit2 repelled RGC axons in vitro. Chi-Bins study brought together the in vitro studies in mammals and the genetic studies in Drosophila and showed that, in the vertebrate visual system, there was a conserved role for this ligand-receptor system. Importantly, Chi-Bin went beyond simply identifying the molecule; he did amazing eye transplants between normal and mutant fish embryos—the first person to get such incredibly difficult transplants to work, though several had tried before—to show that the Robo2 phenotype was autonomous to the navigating retinal axons. This extra effort is what made the paper a great achievement. It set a high standard for the zebrafish work in this area. Not only could one determine the molecules responsible for an axon guidance phenotype, but one could also combine the power of zebrafish genetics and embryology to determine the specific cells in which the function of that molecule is essential. It is gratifying to see that many zebrafish papers since have used such a chimeric approach, which is often critical if one is to differentiate the molecular systems that directly affect guidance from others that act earlier to pattern the brain or induce the guidance systems.For several years, Chi-Bin was in remission, and during those years he became more than a star in the developmental neurobiology world, publishing more than 50 papers that illuminated new molecular mechanisms of axon guidance and mapping. He also became a champion of zebrafish, inventing and freely distributing new molecular, optical, and computational tools to the entire fish community. He spent his summers with Niki and their young daughter, Molly, at the Woods Hole Marine Biological Laboratory, directing the Zebrafish Neural Development and Genetics course and collaborating generously and widely with colleagues around the world.His friend and colleague David Grunwald recounts that his cancer returned at age 38 and, though increasingly debilitated, Chi-Bin maintained “an indomitable optimism … resolutely resisting any limitation of his disease, holding to a worldview that included the future, even as he knew he was dying.”Chi-Bin was hugely admired and cherished by his colleagues, postdocs, and students. Although we all recognized his prodigious intellect, we never felt outclassed in his presence, because he listened and interacted with warmth and patience and on a level that the rest of us could understand. He was a superb mentor: approachable, humble, and gentle. He encouraged and inspired his students with his commendable style of generous and principled science. No one who ever met Chi-Bin will forget his kind blinking eyes and warm smile. His death on the 2nd of December, 2011, is a great loss to developmental neuroscience and the zebrafish communities, to which he leaves an enduring legacy as an innovator and a remarkable human being. The Chi-Bin Chien Award has been established through the zebrafish community and the Genetics Society of America to recognize the achievement of an outstanding graduate student or postdoc trainee from any country who has contributed to the advancement of the zebrafish research field and exhibited a spirit of generosity and openness, qualities that characterized and motivated Chi-Bin.Chi-Bin Chien: 1965–2011View Large Image | View Hi-Res Image | Download PowerPoint Slide

Viewpoints: contrasting opinions in Neural Development

We like to think of neurobiology as a field in which clear results lead inexorably to clear conclusions. Sometimes though, the systems we study are so complex that even large data sets are consistent with more than a single interpretation. When the issues are important ones it may be helpful to have proponents of distinct interpretations present their contrasting viewpoints side-by-side, so non-specialists can judge for themselves what all the fuss is about, and whether one view is closer to the mark than the others. In some cases, these pieces might also be didactically useful, for example in neuroscience courses that aim to give a more realistic view of how science progresses than is found in textbooks.

TRIBUTES TO JEFF HALL

I first met Jeff Hall in what must have been 1977, when I was interviewing for a job as an Assistant Professor at Brandeis. By todays standards, this was a most unconventional job interview, and q...