- Email: [email protected]
Neurogenesis and axonal pathfinding in invertebrates
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suggestions for improvement. Work in the author's laboratory is supported by NIH grant NS 15070.
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Selected references
Wessells, N. K. 11970) Proc. Natl Acad. Sci. USA 66, 1206-1212 Bamburg, J. R., Bray, D. and Chapman, K. (1986) Nature 321,788-790 Letourneau, P.C., Shattuck, T.A. and Ressler, A. H. (1986) J. Neurosci. 6, 19121917 Drubin, D. G., Feinstein, S. C., Shooter, E. M. and Kirschner, M. W. (1985) J. Cell Biol. 101, 1799-1807 Bray, D. and Chapman, K. (1985) J. Neurosci. 12, 321)4-3213 Letourneau, P. C. (1985) in Molecular Bases o f Neural Development (Edelman, G. M., Gall, W. E., and Cowan, W. M., eds), pp. 269-293, John Wiley & Sons Letourneau, P. C. (1981) Dev. Biol. 85, t13-122 Argiro, V., Bunge, M. B. and Johnson, M. I. 11984) J. Neurosci. 4, 3051-3062
1 Ellisman, M. H. and Porter, K. R. (1980) 34 J. Cell Biol. 87,464-479 2 Heuser, J. E., Reese, T. S. and Landis, D.M. (1976) Cold Spring Harbor Syrup. 35 Quant. Biol. 40, 17-24 3 Heuser, J. E. and Salpeter, S. R. (1979) 36 J. Cell Biol. 82, 150-173 4 Hirokawa,N. (1982)J. CellBiol. 94, 129-142 5 Schnapp, B. J. and Reese, T.S. (1982) 37 J. Cell. Biol. 94, 667-679 6 Tsukita, S., Usukura, J., Tsukita, S. and Ishikawa, H. (1982) Neuroscience 7, 2135- 38 2147 7 Hirokawa, N. (1986) Trends Neurosci. 9, 67-71 8 Hirokawa, N., Bloom, G. S. and Vallee, R. B. (1985) J. Cell Biol. 101,227-239 9 Matus, A., Bernhardt, R. and Hugh-Jones, T. (1981) Proc. Natl Acad. Sci. USA 78, 3010-3014 10 Hirokawa, N., Glicksman, M.A. and John Willard, M.B. (1984) J. Cell Biol. 98, t523-1536 Two o f the key aspects of neural 11 Levine, J. and Willard, M. (1981) J. Cell d e v e l o p m e n t upon which studies in Biol. 90, 631-643 i n v e r t e b r a t e s have shed considerable 12 Riederer, B. M., Zagan, I. S. and Goodman, light are n e u r o g e n e s i s (the g e n e r a t i o n S. R. 11986) J. Cell Biol. 102, 2088-2097 13 Grafstein, B. and Forman, D. S (1980) of nerve cells from n o n - n e u r a l precursors) and axonal pathfinding (the Physiol. Rev. 60, 1167-1283 14 Forman, D. S., Padjen, A. L. and Siggins, o r d e r e d growth of n e w axons t h r o u g h G. R. 11977) Brain Res. 136, 197-213 their e n v i r o n m e n t , be that within the 15 Papasozomenos, S. C., Yoon, M., Crane, R., CNS or t h r o u g h peripheral tissues). Autilio-Gambeni, L. and Gambetti, P. The study of both o f these topics has (1982) J. Cell Biol. 95, 672-675 16 Griffin, J. W., Fahnestock, K. E., Price, passed t h r o u g h an era of detailed D. L. and Hoffman, P. (1983) J. Neurosci. 3, description and of physical manipu557-566 lation (mostly cell ablation), and is n o w 17 Schnapp, B. J. and Reese, T.S. (1986) increasingy e n r i c h e d by genetic and Trends Neurosci. 9, 155-162 molecular approaches. 18 Brady, S. T., Lasek, R. J. and Alien, R. D. (1982) Science 218, 1129-1131 19 Allen, R. D., Allen, N. S. and Travis, J. L. Neurogenesis Possibly the most spectacular t o u r - d e 1198l) Cell Motil. 1,291-302 20 Allen, R. D. and Allen, N.S. 11983) f o r c e in the study of n e u r o g e n e s i s is the I. Microsc. 129, 3-17 cell-by-cell analysis of the d e v e l o p m e n t 21 Allen, R. D., Metuzals, J., Tasaki, I., Brady, o f the n e m a t o d e w o r m C a e n o r h a b d i t i s S. T. and Gilbert, S. P. (1982) Science 218, elegans. This animal is m a d e of exactly
O c t o b e r 1980
39 Marsh, L. and Letourneau, P. C J. Cell Biol. 99, 2041-2047 40 Shaw, G. and Bray, D. (1977) Exp. Cell Res. 104, 5~62 41 Letourneau, P. C. (1975) Dev. Biol. 44. 92-101 42 Wessells, N. K. and Nuttall, R. P. (1978) Exp. (?ell Res. 115, 111-122 43 Gundersen, R. W. and Barrett, J. N. (1980) J. Cell Biol. 87,546-554 44 Bray, D. (1979) J. ('ell Sci. 37, 391-410 45 Tosney, K. W. and Landmesser, L. T. (1985) J. Neurosci. 9, 2345-2358 46 Bray, D., Bunge, M. B. and Chapman, K. Exp. (?ell Res. (in press) Mare Bartlett Bunge is at the Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
Neurogenesls and axonal pat ng In invertebrates
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22 Schnapp, B. J., Vale, R. D., Sheetz, M. P. and Reese, T. S. (1985) Cell 40, 455-462 23 Vale, R. D., Reese, T. S. and Sheetz, M. P. (1985) Cell 42, 39-50 24 Adams, R. J. and Bray, D. (1983) Nature 303, 718-720 25 Vale, R. D., Sehnapp, B. J., Mitchison, T., Steuer, E., Reese, T. S. and Sheetz, M. P. 11985) Cell 43, 623-632 26 Miller, R. H. and Lasek, R. J. (1985) J. Cell. Biol. 101, 2181-2193 27 Lasek, R. J., Garner, J. A. and Brady, S. T. (t984) J. Cell Biol. 99, 212s--221s 28 Wujek, J. R. and Lasek, R.J. (1983) J. Neurosci. 3,243-251 29 Letourneau, P. C. (1983) J. Cell Biol. 97, 963-973 30 Tosney, K. W. and Wessells, N. K. (1983) J. Cell Sci. 61,389-411 31 Yamada, K. M., Spooner, B.S. and ~) 1986, Elsevier Science Publishers B V , , A m st erdam
810 somatic cells, and the origin of every single o n e has b e e n followed right f r o m the egg 1'2. O n e o f the m a j o r conclusions of the analysis is that the lineages giving rise to the adult are generally invariant: the exact pedigrees o f most cells, w h e t h e r in the larva or the m a t u r e adult, can be l o o k e d up in a published paper. H o w e v e r , t h e r e are i m p o r t a n t exceptions r e f e r r e d to as 'equivalence groups '2. In s o m e o f these cases, a given cell (often in the midline of the CNS) arises apparently rand o m l y f r o m o n e o f two potential lineages, o n e on either side of the animal. In o t h e r cases, a cell that normally has o n e fate (say A ) takes o n a n o t h e r (B) if the n o r m a l B cell is
0378 - 5912/86/$0200
Palka ablated, implying that (1) A is plurip o t e n t , (2) the difference b e t w e e n A and B d e p e n d s o n ceil-cell interaction, and (3) t h e B fate is primary. It is easier to study normal d e v e l o p m e n t than to p r o d u c e controlled p e r t u r b a t i o n s , so we may expect that m o r e examples, and p e r h a p s novel types, of cell-cell interactions remain to be b r o u g h t to light (Chalfie, M . , pers. c o m m u n . ) . T h e p a t t e r n o f cell divisions by which the e m b r y o o f a leech arises from the egg is very different from that in a n e m a t o d e , but the same general principles of neurogenesis apply2: specific n e u r o n s arise from specific precursor cells (probably by fixed lineages, t h o u g h t h e r e is no direct evidence as yet for the i n t e r m e d i a t e steps), except in specific cases of interchangeability that r e s e m b l e equivalence groups. T h e best studied case involves two of the early e m b r y o n i c p r o g e n i t o r cells that give rise to m a j o r parts o f the CNS in each s e g m e n t . T h e y are called the O/P teloblasts b e c a u s e prior to the interaction b e t w e e n their p r o g e n y their fate is not fixed. Again, a primary fate can be defined - P because if P p r o g e n y are eliminated, cells that otherwise would have had the O fate take their place. In grasshoppers, and p r o b a b l y all insects, the n e u r o n s of each s e g m e n t o f the CNS originate from a nearly fixed, bilaterally symmetric set of 67 precursor ceils, each o f which gives rise by an invariant series o f divisions to a specific set of n e u r o n s 3A (Fig. I A and B). H o w e v e r , each o f the precursor cells
483
T I N S - October 1986
l.,t
, ~ !~! ~
.,,
/
i l!I
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i ,
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The somatic cell lineage of the nematode Caenorhabditiselegans. Time runs verticallyfrom top to bottom; horizontal axis has no physical meaning. Entire lineage takes about 55 h at 20°C. (Figurekindlysuppliedby J. E. Sulston.)
arises not from a defined lineage, but rather from a small pool of apparently equipotent, experimentally indistinguishable cellsL In this equivalence group the neural fate is primary: if the forming neuroblast is ablated, a neighbor that would otherwise have formed an epidermal cell takes its place. The compound eye of Drosophila provides an extreme case of cell specification by interaction. The component cells of the single repeat units of the eye, the ommatidia, do not have a fixed lineage relationship with each other• Rather, they are recruited in two waves, each time apparently at random from a pool of indistinguishable precursor cells that only acquire an identity after they join a forming cell cluster6. The outcome of this process is so regular that the retina has aptly been called a 'neurocrystalline lattice'. Thus, we have learned that both fixed cell lineages and equivalence groups are prominent features of neurogenesis in all the well-studied invertebrate groups. The nematode may be dominated by lineage and the fly retina by cell-cell interactions, but their final cellular architectures are equally precise. Where are studies of neurogenesis headed in the near future? Much new work clearly embraces genetic and molecular approaches. In nematodes, many mutations affecting cell lineages have been isolated and the genes they define are being cloned1'7. In Drosophila two sets of genes, called neurogenic and antineurogenic, have been describeda. The phenotype of the
neurogenic mutants is overgrowth of the nervous system at the expense of the epidermis - 'all brain and no skin'. This has been taken to suggest, as did the ablation experiments on grasshopper neuroblasts, that the neural fate is primary over an epidermal one. Furthermore, it seems possible that the known neurogenic genes of Drosophila represent functions that mediate the cell-cell interactions indicated by the grasshopper results5 (Fig. 1B). The protein product of one neurogenic gene (Notch) has been deduced9 and, like the product of the nematode gene lin 12 (Ref. 10) (one of the group defined by lineage mutants), turns out to have significant sequence homology with mammalian epidermal growth factor. It appears to be a transmembrane protein, which is indeed consonant with a role in cell-cell interactions specifying precisely which cells in a neurogenic region will become neuroblasts and which dermatoblasts. Of course, this is only a preliminary interpretation, and it does not take into account the antineurogenic genes that interact in a complex way with the neurogenic ones (Campos-Ortega, J. A., pers. commun.). Still, it illustrates several points: (1) the profound influence that genetic and molecular results are likely to have on the formulation of problems in neural development; (2) the way in which the new data may contribute to the solution of the problems; and (3) the fact that the interpretation of molecular data will rest squarely on the details learned about cell lineage and interaction
obtained using more classical techniques. If anything, the molecular era will demand increasing sophistication from studies and models at higher levels of organization.
Axonal pathf'mding The process of neurogenesis takes us from the determination of the neurogenic regions of the embryo, through the selection of specific cells to become neuroblasts, to the orderly division of the blast cells to yield specific neurons. The next process is the formation of axons. The wiring diagram of the nervous system, is best known in nematodes; indeed, it has been completely reconstructed from serial electron micrographs 11. However, insects have provided the favored experimental material, so the following summary is based largely on data from grasshoppers and Drosophila; the findings are consistent with what is known about axonal pathfinding in crustaceans 12, leeches 13 and nematodes n as well. During the past few years we have learned that axonogenesis in the developing insect CNS is an astonishingly precise process (Fig. 1B), wherein the growth cone generated by a given cell makes a series of choices as it encounters pre-existing axons, following specific ones and avoiding the rest until the cell's characteristic morphology is produced 4. It appears that the main features of the CNS are assembled step-by-step on an initial scaffolding, a process that often requires the new growth cone to switch from one pre-
TINS- October 1986
484 existing axon to another. This formulation of the events of axonal pathfinding in the CNS has been called the labelled pathways hypothesis 4 because it implies the presence and recognition of molecularly distinct axons. How is the scaffolding itself laid down? What determines the precise trajectories of the very first axons? The answer is not clear, and while it is a difficult problem to approach in the crowded CNS, complementary data are available in the peripheral nervous system. Most sensory neurons of invertebrates arise in the periphery by a process in which the neurogenic and antineurogenic genes mentioned above also play a role. Their axons must reach the CNS, and the first ones (the pioneers) have been studied in some detail during their growth on nonneural substrata. Several factors seem to be important in guiding the pioneer axons 4'14'15 (Fig. 1C and D). Evidence has been offered that neurons have a special affinity for each other, so that the growth cone of a relatively distal neuron will seek out (via filopodial exploration) the soma of a more proximal one. In this way, a series of neurons can provide a chain of stepping stones leading to the CNS (the guide-post cell hypothesis). In addition, it appears that the epithelium between stepping stones can have a proximally increasing affinity for axons, thus drawing them in the direction of the CNS (the adhesiveness or affinity gradient hypothesis). Finally, at least one instance of a discrete, longitudinally oriented path of high affinity has been described; this has the property of drawing axons into a specific location in the periphery, rather than simply providing a general bearing towards the CNS (the peripheral highways hypo-
thesis) 16. A number of experimental questions have been asked concerning axonal pathfinding. For example, one may wonder whether only the first axon to traverse a given territory, the pioneer, is capable of correct navigation through it, or whether being a pioneer reflects merely the time of development and not any special navigational skills. In the CNS there are striking instances in which the growth of a follower axon will actually be arrested if the appropriate pioneer has previously been ablatedL However, sensory axons growing towards the CNS behave quite differently - virtually all that have been tested have proven to be capable of independent navigation 14A5. Secondly, it has been observed both
in the CNS and in the periphery that intimate relations are established between growing axons and the guidepost cells that they contact; these include electrical and dye coupling and the formation of deep invaginations that seem to bud off coated vesicles4. But are guide-post cells actually necessary for the correct guidance of the pioneers? Strong evidence both for 15A7 (Fig. 1D) and against 14As (Fig. IC) has been offered in different situations. If may well be that guidepost cells become necessary when other cues, perhaps simply the by-products of processes needed for non-neural aspects of development, are either lacking or give inappropriate guidance. However, in many cases several different cues give the same information, and this redundancy makes them individually non-essential (cf. Ref. 15). How specific are the factors that guide axons? Again, within the CNS they are specific, even at the single-cell level: growing axons follow specific predecessor axons, and if these have been eliminated new growth may either stop or become indiscriminate 4. In the periphery such specificity is not seen. Under experimental conditions axons of different modalities, segments, and developmental compartments are all able to fasciculate together and follow any path to the CNS, even though during normal development they follow only the nearest one 19 (compare with the description of peripheral 'highways' in vertebrates2°). Similarly, outgrowing motor axons travel in mixed peripheral nerves before exiting from them at specific muscles. Finally, is axon outgrowth directed from the beginning, or is there a significant component of random searching, overproduction of branches, and the subsequent retraction of incorrect ones? Filopodia certainly sample their environment, but axon outgrowth is well directed from the start, and long-distance searching seems not to be a prominent feature of axonal pathfinding either in the CNS or in the periphery 4A4`ls. This generalization applies to normal development; the events of regeneration may well be different. Overall, we find that within the CNS later axons follow earlier ones with extreme specificity; in the periphery fasciculation is opportunistic; cells that are positioned to be guide-posts may or may not actually be required; nonneural substrata provide cues that guide axons but that may have much more general roles in development as
well. These conclusions can all be subsumed under the adhesiveness hierarchy hypothesis TM. This hypothesis supposes that in the animal, as in culture, axons grow preferentially over the substrata to which they best adhere, so that the pathway choices that growth cones are seen to make actually reflect the geometrical distribution of maximum adhesiveness. However, it is not clear how this hypothesis would explain the striking cases of growth arrest following ablation of guiding axons in the CNS. Perhaps we need to think of more subtle forms of interaction as well 4"17 What are the prospects for the study of axon guidance in the near future? Again genetic and molecular approaches are coming into prominence. Mutants in which axons grow abnormally have recently been isolated in both nematodes 21 and Drosophila 22. Monoclonal antibodies that recognize specific subsets of axons have been obtained for leeches 23, grasshoppers 4 and Drosophila4; in some cases they bind selectively to axons that fasciculate together. Some progress has been made in isolating molecules that might be involved in selective cell adhesion in grasshoppers (Goodman, C. S., pers. commun.). Given an antibody, it is now possible to identify the corresponding antigen and the gene that codes for it6; given a gene, or initially even just a mutation, markers for its message(s) and protein(s) can be obtained 24. These technical capabilities should, in the long run, allow us to refine our now largely operational hypotheses concerning axon guidance into more mechanistic ones. The list of higher-level phenomena relating to pathfinding is surely not complete. In addition to the findings related in this essay we can point to the recent discovery of topographic projections in insects 25, the demonstration of direct effects of neurotransmitters and electrical activity on growth cones in molluscs 26'27, the novel case of axonal self-recognition in leeches 13, and a host of phenomena relating to plasticity. Nevertheless, the new element in studies on axonal pathfinding, as in those on neurogenesis, is the increasing use of genetic and molecular approaches.
Generality of findings A remarkable degree of developmental and phylogenetic conservatism has been demonstrated for the insect CNS 28. The basic set of segmental precursor cells occurs, with minor
T I N S - O c t o b e r 1986
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C
substantive m o d e l s for t h e study o f complex vertebrate and even human systems - in fact reflects biological reality.
B
Selectedreferences
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Fig. L The neural development of Insectum eompositum, a fruitfly-grasshopper hybrid believed to represent all insects. Like species identity, time is also scrambled in this figure in order to portray the events and processes about which the greatest amount of information is available. (&) The spatially restricted neurogenic region giving rise to the CNS is shown stippled in a schematic cross section of the embryo. (B) Neuroblasts (neural precursors) and dermatoblusts (epidermal precursors) are intermingled in the neurogenic region. The neuroblusts are found in a regular array of rows and columns, can be identified by position, and give rise to a defined set of progeny. Developing neuroblasts are thought to force their neigbors into the secondary, epidermal fate (this effect is indicated by the heavy arrow). Specific neurons (e.g. cells 1--4) always arise at specific points in the lineage generated by a given neuroblast. Their growth cones follow specific paths, either pre-existing axons (contained within fascicles a--c) or non-neural substrata. (C) In the periphery, neurons arise at specified locations and send axons towards the CNS. In this case (Drosophila wing), any of the first four neurons (cells 1--4) can be removed prior to axonogenesis without disturbing route-finding by the remaining axons; therefore, guide-post cells are not required. In genetically aneural host wings, the axons of implanted neurons generally grow towards and along the strip shown stippled, suggesting the presence of a longitudinally oriented region of high affinity ('highway'). (D) In the grasshopper leg, cell 2 is not required for correct navigation by the growth cone of cell 1, but cell 4 is required. In the absence of cell4, cell l's growth cone extends circamferentially as if it could not escape the region of high affinity shown stippled. Such regions seem to be associated with leg segment boundaries and their presence may create a requirement for guidepost cells on the proximal side. Thus, the epithelia of the flywing (C) and the grasshopper leg (D) both seem to have regional specializations that influence the growth of axons, but in neither case is it clear whether these are spatially distributed (as in some form of gradienO or have discrete boundaries as suggested in the figure.
modifications, in e v e r y s e g m e n t o f an individual animal; it occurs in widely divergent groups o f insects, and s o m e of the differentiated n e u r o n s are even recognizable in crustaceans a n d possibly o t h e r a r t h r o p o d s . Thus, we can claim to u n d e r s t a n d t h e basic m o r p h o logical events of n e u r o g e n e s i s in t h e CNS, exclusive o f t h e brain, throughout the largest animal p h y l u m . C o m parative studies are less extensive for leeches a n d n e m a t o d e s , but h e r e t o o t h e results f r o m favorable m o d e l systems s e e m to have wide phylogenetic application and interesting implications for t h e study o f evolution, especially w h e n c o u p l e d with genetic studies.
Finally, t h e g e n e r a l picture p r o v i d e d h e r e o f the g e n e r a t i o n o f specific n e u r o n s w h o s e axons grow out along specific paths, e v e n w h e n t h e s e take t h e m over previously u n i n n e r v a t e d territory, is by n o m e a n s u n i q u e to invertebrates. R e c e n t studies o n fish provide astonishingly close parallels: growing axons within t h e C N S m a k e specific choices a m o n g pre-existing axon fascicles 29, and the first m o t o r axons e m e r g i n g f r o m t h e C N S (just t h r e e p e r s e g m e n t ) follow entirely predictable p a t h s to target muscles 3°. It n o w a p p e a r s that t h e belief o f t e n voiced by s t u d e n t s o f m o d e l systems that in addition to their intrinsic interest they do i n d e e d serve as
1 Hedgecock, E. M. (1985) Trends Neurosci. 8, 288-293 2 Stent, G. S. and Weisblat, D.A. (1986) Annu. Rev. Neurosci. 8, 45-70 3 Goodman, C. S. and Bate, C. M. (1981) Trends Neurosci. 4, 163-169 4 Bastiani, M. J., Doe, C. Q., Helfand, S. L. and Goodman, C. S. (1985) Trends Neurosci. 8, 257-266 5 Doe, C. Q. and Goodman, C. S. (1985) Dev. Biol. 111, 206-219 6 Venkatesh, T. R., Zipursky, S.L. and Benzer, S. (1985) Trends Neurosci. 8, 251-257 7 Sternberg, P. W. and Horvitz, H. R. (1984) Annu. Rev. Genet. 18, 489-524 8 Campos-Ortega, J.A. (1985) Trends Neurosci. 8,245-250 9 Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonas, S. (1986) Cell 43, 567-581 10 Greenwald, I. (1986) Cell 43,583-590 11 White, J. G. (1985) Trends Neurosci. 8, 277-283 12 Macagno, E. (1984) BioScience 34, 308-312 13 Kramer, A. P. and Stent, G. S (1985) J. Neurosci. 5, 768-775 14 Blair, S. S. and Palka, J. (1985) Trends Neurosci. 8, 284-288
15 Bentley, D. and Caudy, M. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 573-585 16 Blair, S. S., Schubiger, M. and Palka, J. (1986) Soc. Neurosci. Abstr. 12, 196 17 Caudy, M. and Bentley, D. (1986) J. Neurosci. 6, 364-379 18 Berlot, J. and Goodman, C. S. (1984) Science 223,493-496 19 Palka, J. and Ghysen, A. (1982) .Trends Neurosci. 5,382-386 20 Landmesser, L. M. (1984) Trends Neurosci. 7, 336-339 21 Hedgecock, E. M., Culotti, J. G., Thomson, J. N., and Perkins, L. A. (1985) Dev. Biol. 111,158--170 22 Jan, Y. N., Bodmer, R., Jan, L. Y., Ghysen, A. and Dambly, C. UCLA Syrup. Molec. Biol. (in press) 23 McKay, R. D., Hockfield, S., Johansen, J. and Frederiksen, K. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 599--610 24 Rubin, G. M. (1985) Trends Neurosci. 8, 231-233 25 Murphey, R. K. (1985) Trends Neurosci. 8, 120-125 26 Haydon, P. G., McCobb, D. P. and Kater, S. B. (1984) Science 226, 561-564 27 Cohan, C. S. and Kater, S. B. (1986) Science 232, 1638-1640 28 Thomas, J. B., Bastiani, M., Bate, C. M. and Goodman, C. S. (1984) Nature 310, 203-207 29 Kuwada, J. Y. (1986) Science 233, 740-746 30 Eisen, J., Myers, P. Z. and Westerfield, M. (1986) Nature 320, 269-271 John Palka is at the Department of Zoology, University of Washington, Seattle, WA 98195, USA.
TINS-
suggestions for improvement. Work in the author's laboratory is supported by NIH grant NS 15070.
32 33
Selected references
Wessells, N. K. 11970) Proc. Natl Acad. Sci. USA 66, 1206-1212 Bamburg, J. R., Bray, D. and Chapman, K. (1986) Nature 321,788-790 Letourneau, P.C., Shattuck, T.A. and Ressler, A. H. (1986) J. Neurosci. 6, 19121917 Drubin, D. G., Feinstein, S. C., Shooter, E. M. and Kirschner, M. W. (1985) J. Cell Biol. 101, 1799-1807 Bray, D. and Chapman, K. (1985) J. Neurosci. 12, 321)4-3213 Letourneau, P. C. (1985) in Molecular Bases o f Neural Development (Edelman, G. M., Gall, W. E., and Cowan, W. M., eds), pp. 269-293, John Wiley & Sons Letourneau, P. C. (1981) Dev. Biol. 85, t13-122 Argiro, V., Bunge, M. B. and Johnson, M. I. 11984) J. Neurosci. 4, 3051-3062
1 Ellisman, M. H. and Porter, K. R. (1980) 34 J. Cell Biol. 87,464-479 2 Heuser, J. E., Reese, T. S. and Landis, D.M. (1976) Cold Spring Harbor Syrup. 35 Quant. Biol. 40, 17-24 3 Heuser, J. E. and Salpeter, S. R. (1979) 36 J. Cell Biol. 82, 150-173 4 Hirokawa,N. (1982)J. CellBiol. 94, 129-142 5 Schnapp, B. J. and Reese, T.S. (1982) 37 J. Cell. Biol. 94, 667-679 6 Tsukita, S., Usukura, J., Tsukita, S. and Ishikawa, H. (1982) Neuroscience 7, 2135- 38 2147 7 Hirokawa, N. (1986) Trends Neurosci. 9, 67-71 8 Hirokawa, N., Bloom, G. S. and Vallee, R. B. (1985) J. Cell Biol. 101,227-239 9 Matus, A., Bernhardt, R. and Hugh-Jones, T. (1981) Proc. Natl Acad. Sci. USA 78, 3010-3014 10 Hirokawa, N., Glicksman, M.A. and John Willard, M.B. (1984) J. Cell Biol. 98, t523-1536 Two o f the key aspects of neural 11 Levine, J. and Willard, M. (1981) J. Cell d e v e l o p m e n t upon which studies in Biol. 90, 631-643 i n v e r t e b r a t e s have shed considerable 12 Riederer, B. M., Zagan, I. S. and Goodman, light are n e u r o g e n e s i s (the g e n e r a t i o n S. R. 11986) J. Cell Biol. 102, 2088-2097 13 Grafstein, B. and Forman, D. S (1980) of nerve cells from n o n - n e u r a l precursors) and axonal pathfinding (the Physiol. Rev. 60, 1167-1283 14 Forman, D. S., Padjen, A. L. and Siggins, o r d e r e d growth of n e w axons t h r o u g h G. R. 11977) Brain Res. 136, 197-213 their e n v i r o n m e n t , be that within the 15 Papasozomenos, S. C., Yoon, M., Crane, R., CNS or t h r o u g h peripheral tissues). Autilio-Gambeni, L. and Gambetti, P. The study of both o f these topics has (1982) J. Cell Biol. 95, 672-675 16 Griffin, J. W., Fahnestock, K. E., Price, passed t h r o u g h an era of detailed D. L. and Hoffman, P. (1983) J. Neurosci. 3, description and of physical manipu557-566 lation (mostly cell ablation), and is n o w 17 Schnapp, B. J. and Reese, T.S. (1986) increasingy e n r i c h e d by genetic and Trends Neurosci. 9, 155-162 molecular approaches. 18 Brady, S. T., Lasek, R. J. and Alien, R. D. (1982) Science 218, 1129-1131 19 Allen, R. D., Allen, N. S. and Travis, J. L. Neurogenesis Possibly the most spectacular t o u r - d e 1198l) Cell Motil. 1,291-302 20 Allen, R. D. and Allen, N.S. 11983) f o r c e in the study of n e u r o g e n e s i s is the I. Microsc. 129, 3-17 cell-by-cell analysis of the d e v e l o p m e n t 21 Allen, R. D., Metuzals, J., Tasaki, I., Brady, o f the n e m a t o d e w o r m C a e n o r h a b d i t i s S. T. and Gilbert, S. P. (1982) Science 218, elegans. This animal is m a d e of exactly
O c t o b e r 1980
39 Marsh, L. and Letourneau, P. C J. Cell Biol. 99, 2041-2047 40 Shaw, G. and Bray, D. (1977) Exp. Cell Res. 104, 5~62 41 Letourneau, P. C. (1975) Dev. Biol. 44. 92-101 42 Wessells, N. K. and Nuttall, R. P. (1978) Exp. (?ell Res. 115, 111-122 43 Gundersen, R. W. and Barrett, J. N. (1980) J. Cell Biol. 87,546-554 44 Bray, D. (1979) J. ('ell Sci. 37, 391-410 45 Tosney, K. W. and Landmesser, L. T. (1985) J. Neurosci. 9, 2345-2358 46 Bray, D., Bunge, M. B. and Chapman, K. Exp. (?ell Res. (in press) Mare Bartlett Bunge is at the Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St Louis, MO 63110, USA.
Neurogenesls and axonal pat ng In invertebrates
1127-1129
22 Schnapp, B. J., Vale, R. D., Sheetz, M. P. and Reese, T. S. (1985) Cell 40, 455-462 23 Vale, R. D., Reese, T. S. and Sheetz, M. P. (1985) Cell 42, 39-50 24 Adams, R. J. and Bray, D. (1983) Nature 303, 718-720 25 Vale, R. D., Sehnapp, B. J., Mitchison, T., Steuer, E., Reese, T. S. and Sheetz, M. P. 11985) Cell 43, 623-632 26 Miller, R. H. and Lasek, R. J. (1985) J. Cell. Biol. 101, 2181-2193 27 Lasek, R. J., Garner, J. A. and Brady, S. T. (t984) J. Cell Biol. 99, 212s--221s 28 Wujek, J. R. and Lasek, R.J. (1983) J. Neurosci. 3,243-251 29 Letourneau, P. C. (1983) J. Cell Biol. 97, 963-973 30 Tosney, K. W. and Wessells, N. K. (1983) J. Cell Sci. 61,389-411 31 Yamada, K. M., Spooner, B.S. and ~) 1986, Elsevier Science Publishers B V , , A m st erdam
810 somatic cells, and the origin of every single o n e has b e e n followed right f r o m the egg 1'2. O n e o f the m a j o r conclusions of the analysis is that the lineages giving rise to the adult are generally invariant: the exact pedigrees o f most cells, w h e t h e r in the larva or the m a t u r e adult, can be l o o k e d up in a published paper. H o w e v e r , t h e r e are i m p o r t a n t exceptions r e f e r r e d to as 'equivalence groups '2. In s o m e o f these cases, a given cell (often in the midline of the CNS) arises apparently rand o m l y f r o m o n e o f two potential lineages, o n e on either side of the animal. In o t h e r cases, a cell that normally has o n e fate (say A ) takes o n a n o t h e r (B) if the n o r m a l B cell is
0378 - 5912/86/$0200
Palka ablated, implying that (1) A is plurip o t e n t , (2) the difference b e t w e e n A and B d e p e n d s o n ceil-cell interaction, and (3) t h e B fate is primary. It is easier to study normal d e v e l o p m e n t than to p r o d u c e controlled p e r t u r b a t i o n s , so we may expect that m o r e examples, and p e r h a p s novel types, of cell-cell interactions remain to be b r o u g h t to light (Chalfie, M . , pers. c o m m u n . ) . T h e p a t t e r n o f cell divisions by which the e m b r y o o f a leech arises from the egg is very different from that in a n e m a t o d e , but the same general principles of neurogenesis apply2: specific n e u r o n s arise from specific precursor cells (probably by fixed lineages, t h o u g h t h e r e is no direct evidence as yet for the i n t e r m e d i a t e steps), except in specific cases of interchangeability that r e s e m b l e equivalence groups. T h e best studied case involves two of the early e m b r y o n i c p r o g e n i t o r cells that give rise to m a j o r parts o f the CNS in each s e g m e n t . T h e y are called the O/P teloblasts b e c a u s e prior to the interaction b e t w e e n their p r o g e n y their fate is not fixed. Again, a primary fate can be defined - P because if P p r o g e n y are eliminated, cells that otherwise would have had the O fate take their place. In grasshoppers, and p r o b a b l y all insects, the n e u r o n s of each s e g m e n t o f the CNS originate from a nearly fixed, bilaterally symmetric set of 67 precursor ceils, each o f which gives rise by an invariant series o f divisions to a specific set of n e u r o n s 3A (Fig. I A and B). H o w e v e r , each o f the precursor cells
483
T I N S - October 1986
l.,t
, ~ !~! ~
.,,
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i l!I
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i ,
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The somatic cell lineage of the nematode Caenorhabditiselegans. Time runs verticallyfrom top to bottom; horizontal axis has no physical meaning. Entire lineage takes about 55 h at 20°C. (Figurekindlysuppliedby J. E. Sulston.)
arises not from a defined lineage, but rather from a small pool of apparently equipotent, experimentally indistinguishable cellsL In this equivalence group the neural fate is primary: if the forming neuroblast is ablated, a neighbor that would otherwise have formed an epidermal cell takes its place. The compound eye of Drosophila provides an extreme case of cell specification by interaction. The component cells of the single repeat units of the eye, the ommatidia, do not have a fixed lineage relationship with each other• Rather, they are recruited in two waves, each time apparently at random from a pool of indistinguishable precursor cells that only acquire an identity after they join a forming cell cluster6. The outcome of this process is so regular that the retina has aptly been called a 'neurocrystalline lattice'. Thus, we have learned that both fixed cell lineages and equivalence groups are prominent features of neurogenesis in all the well-studied invertebrate groups. The nematode may be dominated by lineage and the fly retina by cell-cell interactions, but their final cellular architectures are equally precise. Where are studies of neurogenesis headed in the near future? Much new work clearly embraces genetic and molecular approaches. In nematodes, many mutations affecting cell lineages have been isolated and the genes they define are being cloned1'7. In Drosophila two sets of genes, called neurogenic and antineurogenic, have been describeda. The phenotype of the
neurogenic mutants is overgrowth of the nervous system at the expense of the epidermis - 'all brain and no skin'. This has been taken to suggest, as did the ablation experiments on grasshopper neuroblasts, that the neural fate is primary over an epidermal one. Furthermore, it seems possible that the known neurogenic genes of Drosophila represent functions that mediate the cell-cell interactions indicated by the grasshopper results5 (Fig. 1B). The protein product of one neurogenic gene (Notch) has been deduced9 and, like the product of the nematode gene lin 12 (Ref. 10) (one of the group defined by lineage mutants), turns out to have significant sequence homology with mammalian epidermal growth factor. It appears to be a transmembrane protein, which is indeed consonant with a role in cell-cell interactions specifying precisely which cells in a neurogenic region will become neuroblasts and which dermatoblasts. Of course, this is only a preliminary interpretation, and it does not take into account the antineurogenic genes that interact in a complex way with the neurogenic ones (Campos-Ortega, J. A., pers. commun.). Still, it illustrates several points: (1) the profound influence that genetic and molecular results are likely to have on the formulation of problems in neural development; (2) the way in which the new data may contribute to the solution of the problems; and (3) the fact that the interpretation of molecular data will rest squarely on the details learned about cell lineage and interaction
obtained using more classical techniques. If anything, the molecular era will demand increasing sophistication from studies and models at higher levels of organization.
Axonal pathf'mding The process of neurogenesis takes us from the determination of the neurogenic regions of the embryo, through the selection of specific cells to become neuroblasts, to the orderly division of the blast cells to yield specific neurons. The next process is the formation of axons. The wiring diagram of the nervous system, is best known in nematodes; indeed, it has been completely reconstructed from serial electron micrographs 11. However, insects have provided the favored experimental material, so the following summary is based largely on data from grasshoppers and Drosophila; the findings are consistent with what is known about axonal pathfinding in crustaceans 12, leeches 13 and nematodes n as well. During the past few years we have learned that axonogenesis in the developing insect CNS is an astonishingly precise process (Fig. 1B), wherein the growth cone generated by a given cell makes a series of choices as it encounters pre-existing axons, following specific ones and avoiding the rest until the cell's characteristic morphology is produced 4. It appears that the main features of the CNS are assembled step-by-step on an initial scaffolding, a process that often requires the new growth cone to switch from one pre-
TINS- October 1986
484 existing axon to another. This formulation of the events of axonal pathfinding in the CNS has been called the labelled pathways hypothesis 4 because it implies the presence and recognition of molecularly distinct axons. How is the scaffolding itself laid down? What determines the precise trajectories of the very first axons? The answer is not clear, and while it is a difficult problem to approach in the crowded CNS, complementary data are available in the peripheral nervous system. Most sensory neurons of invertebrates arise in the periphery by a process in which the neurogenic and antineurogenic genes mentioned above also play a role. Their axons must reach the CNS, and the first ones (the pioneers) have been studied in some detail during their growth on nonneural substrata. Several factors seem to be important in guiding the pioneer axons 4'14'15 (Fig. 1C and D). Evidence has been offered that neurons have a special affinity for each other, so that the growth cone of a relatively distal neuron will seek out (via filopodial exploration) the soma of a more proximal one. In this way, a series of neurons can provide a chain of stepping stones leading to the CNS (the guide-post cell hypothesis). In addition, it appears that the epithelium between stepping stones can have a proximally increasing affinity for axons, thus drawing them in the direction of the CNS (the adhesiveness or affinity gradient hypothesis). Finally, at least one instance of a discrete, longitudinally oriented path of high affinity has been described; this has the property of drawing axons into a specific location in the periphery, rather than simply providing a general bearing towards the CNS (the peripheral highways hypo-
thesis) 16. A number of experimental questions have been asked concerning axonal pathfinding. For example, one may wonder whether only the first axon to traverse a given territory, the pioneer, is capable of correct navigation through it, or whether being a pioneer reflects merely the time of development and not any special navigational skills. In the CNS there are striking instances in which the growth of a follower axon will actually be arrested if the appropriate pioneer has previously been ablatedL However, sensory axons growing towards the CNS behave quite differently - virtually all that have been tested have proven to be capable of independent navigation 14A5. Secondly, it has been observed both
in the CNS and in the periphery that intimate relations are established between growing axons and the guidepost cells that they contact; these include electrical and dye coupling and the formation of deep invaginations that seem to bud off coated vesicles4. But are guide-post cells actually necessary for the correct guidance of the pioneers? Strong evidence both for 15A7 (Fig. 1D) and against 14As (Fig. IC) has been offered in different situations. If may well be that guidepost cells become necessary when other cues, perhaps simply the by-products of processes needed for non-neural aspects of development, are either lacking or give inappropriate guidance. However, in many cases several different cues give the same information, and this redundancy makes them individually non-essential (cf. Ref. 15). How specific are the factors that guide axons? Again, within the CNS they are specific, even at the single-cell level: growing axons follow specific predecessor axons, and if these have been eliminated new growth may either stop or become indiscriminate 4. In the periphery such specificity is not seen. Under experimental conditions axons of different modalities, segments, and developmental compartments are all able to fasciculate together and follow any path to the CNS, even though during normal development they follow only the nearest one 19 (compare with the description of peripheral 'highways' in vertebrates2°). Similarly, outgrowing motor axons travel in mixed peripheral nerves before exiting from them at specific muscles. Finally, is axon outgrowth directed from the beginning, or is there a significant component of random searching, overproduction of branches, and the subsequent retraction of incorrect ones? Filopodia certainly sample their environment, but axon outgrowth is well directed from the start, and long-distance searching seems not to be a prominent feature of axonal pathfinding either in the CNS or in the periphery 4A4`ls. This generalization applies to normal development; the events of regeneration may well be different. Overall, we find that within the CNS later axons follow earlier ones with extreme specificity; in the periphery fasciculation is opportunistic; cells that are positioned to be guide-posts may or may not actually be required; nonneural substrata provide cues that guide axons but that may have much more general roles in development as
well. These conclusions can all be subsumed under the adhesiveness hierarchy hypothesis TM. This hypothesis supposes that in the animal, as in culture, axons grow preferentially over the substrata to which they best adhere, so that the pathway choices that growth cones are seen to make actually reflect the geometrical distribution of maximum adhesiveness. However, it is not clear how this hypothesis would explain the striking cases of growth arrest following ablation of guiding axons in the CNS. Perhaps we need to think of more subtle forms of interaction as well 4"17 What are the prospects for the study of axon guidance in the near future? Again genetic and molecular approaches are coming into prominence. Mutants in which axons grow abnormally have recently been isolated in both nematodes 21 and Drosophila 22. Monoclonal antibodies that recognize specific subsets of axons have been obtained for leeches 23, grasshoppers 4 and Drosophila4; in some cases they bind selectively to axons that fasciculate together. Some progress has been made in isolating molecules that might be involved in selective cell adhesion in grasshoppers (Goodman, C. S., pers. commun.). Given an antibody, it is now possible to identify the corresponding antigen and the gene that codes for it6; given a gene, or initially even just a mutation, markers for its message(s) and protein(s) can be obtained 24. These technical capabilities should, in the long run, allow us to refine our now largely operational hypotheses concerning axon guidance into more mechanistic ones. The list of higher-level phenomena relating to pathfinding is surely not complete. In addition to the findings related in this essay we can point to the recent discovery of topographic projections in insects 25, the demonstration of direct effects of neurotransmitters and electrical activity on growth cones in molluscs 26'27, the novel case of axonal self-recognition in leeches 13, and a host of phenomena relating to plasticity. Nevertheless, the new element in studies on axonal pathfinding, as in those on neurogenesis, is the increasing use of genetic and molecular approaches.
Generality of findings A remarkable degree of developmental and phylogenetic conservatism has been demonstrated for the insect CNS 28. The basic set of segmental precursor cells occurs, with minor
T I N S - O c t o b e r 1986
485
C
substantive m o d e l s for t h e study o f complex vertebrate and even human systems - in fact reflects biological reality.
B
Selectedreferences
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Fig. L The neural development of Insectum eompositum, a fruitfly-grasshopper hybrid believed to represent all insects. Like species identity, time is also scrambled in this figure in order to portray the events and processes about which the greatest amount of information is available. (&) The spatially restricted neurogenic region giving rise to the CNS is shown stippled in a schematic cross section of the embryo. (B) Neuroblasts (neural precursors) and dermatoblusts (epidermal precursors) are intermingled in the neurogenic region. The neuroblusts are found in a regular array of rows and columns, can be identified by position, and give rise to a defined set of progeny. Developing neuroblasts are thought to force their neigbors into the secondary, epidermal fate (this effect is indicated by the heavy arrow). Specific neurons (e.g. cells 1--4) always arise at specific points in the lineage generated by a given neuroblast. Their growth cones follow specific paths, either pre-existing axons (contained within fascicles a--c) or non-neural substrata. (C) In the periphery, neurons arise at specified locations and send axons towards the CNS. In this case (Drosophila wing), any of the first four neurons (cells 1--4) can be removed prior to axonogenesis without disturbing route-finding by the remaining axons; therefore, guide-post cells are not required. In genetically aneural host wings, the axons of implanted neurons generally grow towards and along the strip shown stippled, suggesting the presence of a longitudinally oriented region of high affinity ('highway'). (D) In the grasshopper leg, cell 2 is not required for correct navigation by the growth cone of cell 1, but cell 4 is required. In the absence of cell4, cell l's growth cone extends circamferentially as if it could not escape the region of high affinity shown stippled. Such regions seem to be associated with leg segment boundaries and their presence may create a requirement for guidepost cells on the proximal side. Thus, the epithelia of the flywing (C) and the grasshopper leg (D) both seem to have regional specializations that influence the growth of axons, but in neither case is it clear whether these are spatially distributed (as in some form of gradienO or have discrete boundaries as suggested in the figure.
modifications, in e v e r y s e g m e n t o f an individual animal; it occurs in widely divergent groups o f insects, and s o m e of the differentiated n e u r o n s are even recognizable in crustaceans a n d possibly o t h e r a r t h r o p o d s . Thus, we can claim to u n d e r s t a n d t h e basic m o r p h o logical events of n e u r o g e n e s i s in t h e CNS, exclusive o f t h e brain, throughout the largest animal p h y l u m . C o m parative studies are less extensive for leeches a n d n e m a t o d e s , but h e r e t o o t h e results f r o m favorable m o d e l systems s e e m to have wide phylogenetic application and interesting implications for t h e study o f evolution, especially w h e n c o u p l e d with genetic studies.
Finally, t h e g e n e r a l picture p r o v i d e d h e r e o f the g e n e r a t i o n o f specific n e u r o n s w h o s e axons grow out along specific paths, e v e n w h e n t h e s e take t h e m over previously u n i n n e r v a t e d territory, is by n o m e a n s u n i q u e to invertebrates. R e c e n t studies o n fish provide astonishingly close parallels: growing axons within t h e C N S m a k e specific choices a m o n g pre-existing axon fascicles 29, and the first m o t o r axons e m e r g i n g f r o m t h e C N S (just t h r e e p e r s e g m e n t ) follow entirely predictable p a t h s to target muscles 3°. It n o w a p p e a r s that t h e belief o f t e n voiced by s t u d e n t s o f m o d e l systems that in addition to their intrinsic interest they do i n d e e d serve as
1 Hedgecock, E. M. (1985) Trends Neurosci. 8, 288-293 2 Stent, G. S. and Weisblat, D.A. (1986) Annu. Rev. Neurosci. 8, 45-70 3 Goodman, C. S. and Bate, C. M. (1981) Trends Neurosci. 4, 163-169 4 Bastiani, M. J., Doe, C. Q., Helfand, S. L. and Goodman, C. S. (1985) Trends Neurosci. 8, 257-266 5 Doe, C. Q. and Goodman, C. S. (1985) Dev. Biol. 111, 206-219 6 Venkatesh, T. R., Zipursky, S.L. and Benzer, S. (1985) Trends Neurosci. 8, 251-257 7 Sternberg, P. W. and Horvitz, H. R. (1984) Annu. Rev. Genet. 18, 489-524 8 Campos-Ortega, J.A. (1985) Trends Neurosci. 8,245-250 9 Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonas, S. (1986) Cell 43, 567-581 10 Greenwald, I. (1986) Cell 43,583-590 11 White, J. G. (1985) Trends Neurosci. 8, 277-283 12 Macagno, E. (1984) BioScience 34, 308-312 13 Kramer, A. P. and Stent, G. S (1985) J. Neurosci. 5, 768-775 14 Blair, S. S. and Palka, J. (1985) Trends Neurosci. 8, 284-288
15 Bentley, D. and Caudy, M. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 573-585 16 Blair, S. S., Schubiger, M. and Palka, J. (1986) Soc. Neurosci. Abstr. 12, 196 17 Caudy, M. and Bentley, D. (1986) J. Neurosci. 6, 364-379 18 Berlot, J. and Goodman, C. S. (1984) Science 223,493-496 19 Palka, J. and Ghysen, A. (1982) .Trends Neurosci. 5,382-386 20 Landmesser, L. M. (1984) Trends Neurosci. 7, 336-339 21 Hedgecock, E. M., Culotti, J. G., Thomson, J. N., and Perkins, L. A. (1985) Dev. Biol. 111,158--170 22 Jan, Y. N., Bodmer, R., Jan, L. Y., Ghysen, A. and Dambly, C. UCLA Syrup. Molec. Biol. (in press) 23 McKay, R. D., Hockfield, S., Johansen, J. and Frederiksen, K. (1983) Cold Spring Harbor Syrup. Quant. Biol. 48, 599--610 24 Rubin, G. M. (1985) Trends Neurosci. 8, 231-233 25 Murphey, R. K. (1985) Trends Neurosci. 8, 120-125 26 Haydon, P. G., McCobb, D. P. and Kater, S. B. (1984) Science 226, 561-564 27 Cohan, C. S. and Kater, S. B. (1986) Science 232, 1638-1640 28 Thomas, J. B., Bastiani, M., Bate, C. M. and Goodman, C. S. (1984) Nature 310, 203-207 29 Kuwada, J. Y. (1986) Science 233, 740-746 30 Eisen, J., Myers, P. Z. and Westerfield, M. (1986) Nature 320, 269-271 John Palka is at the Department of Zoology, University of Washington, Seattle, WA 98195, USA.