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Neurite guidance molecules
Neurite guidance molecules P. Doherty and F.S. Walsh Department of Experimental Pathology, Guy's Hospital, London, UK Current Opinion in Cell Biology 1989, 1:1102-1106
Introduction
Interactions between neuronal growth cone receptors with specific guidance cues in the microenvironment are likely to contribute to the precise stereospecific manner in which neurons find and innervate their appropriate targets. Guidance or recognition cues may be diffusible or bound and, in the Lattercase, may be localized both in the extraceilular matrix and on the membranes of neurons and other cell types. As well as spatio-temporal changes in the expression of the cues, controlling mechanisms are likely to include temporal changes in the expression of the growth cone receptors. In the present article we review some of the recent studies that consolidate and extend our understanding of the general nature of the recognition cues, as well as identify some of the candidate molecules. Evidence that large families of biochemically distinct recognition molecules can be obtained from a single gene by the processes of alternative splicing and differential post-translational modification is also discussed. Finally, we will consider strategies developed recently that can test directly the function of the large number of isoforms of single cell adhesion molecules. Diffusible factors
Evidence obtained in vitro using peripheral nervous tissue supports the general hypothesis that target tissues may release diffusible chemoattractant molecules that contribute to axonal guidance (see Lumsden and Davies, Nature 1986, 323:538-539). In a recent study TessierLavigne et al. [1 ] used central nervous tissue to test this postulate further. During the development of the spinal cord, commissural neurons send axons from the dorsal horn to the ventral floor-plate region. On reaching this 'intermediate' target the axons cross the mid-line and subsequently show a marked change in the orientation of their projection. The authors have co-cultured the dorsal horn (which contains the commissural neurons) with other regions of the developing spinal cord. They observed that the floor-plate region is unique in its ability both to induce the specific outgrowth of commissural axons and also to orientate their direction of growth. Two lines of evidence suggest the presence of an active soluble molecule: firstly, in the co-culture studies the neurons and their 'target' were separated by up to 400 lam and, secondly, the neurite outgrowth promoting activity could be found in the media conditioned by the floorplate region. Although these data again focus attention
on the potential role that both the intermediate and final targets may play in attracting axons, a critical evaluation of the hypothesis will require the identification and characterization of the active molecules. A second class of soluble molecules that may contribute to axonal guidance are the neurotransmitters. Evidence that acetytcholine, dopamine and serotonin can all modulate growth cone motility has come from a large number of studies on the individual neurons of molluscs (Cohen and Kater. In The Cellular Basis of Neuronal PlasticiOJ edited by Bulloch. Manchester University Press, 1989, pp 79-96). More recently these findings have been extended to the vertebrate nervous system. In cultures of post-natal rat retina the blockade of acetylcholine receptor function by nicotinic but not muscarinic antagonists increased retinal ganglion cell neurite outgrowth by up to 50% [2]. This response was only found in cultures that contained a measurable basal level of endogenous acetytcholine, produced perhaps by amacrine cells. These data suggest that acetylcholine can induce a 'tonic' inhibition of neurite outgrowth that is released by the nicotinic block. The mechanism of action remains to be determined; however, the authors suggest that it is not solely due to depolarization, as other agents that depolarize the cells were without effect. Addition of micromolar concentrations of dopamine to cultures of embryonic chick retina resulted in an inhibition of neurite outgrowth from approximately 25% of neurons [3]. The inhibition was characterized by a reduction in filopodial activity followed by a flattening and retraction of growth cones. Selective antagonists were used to demonstrate that the response was linked to the activation of Dl-type dopamine receptors. These receptors are coupled to adenylate cyclase and, in this context, forskolin was also able to elicit a similar response, but only in dopamine-sensitive neurons. The physiological significance of neurotransmitter-mediated inhibition of neurite outgrowth remains to be determined. However, it is possible that neurotransmitters may modulate axonal guidance both during development and during periods of synaptic remodelling in the adult. A 'tonic' inhibition of axonal growth could be released by a decreased availability of transmitter (mediated by ceil uptake and/or enzymatic activity) and thereby provide a positive control. Conversely, direct inhibitory influences may restrict growth into areas that contain free neurotransmitters, or serve to inhibit growth cone motility prior to synapse formation.
Abbreviations
CAM~cell adhesion molecule; mRNA--messengerRNA;NCAM~neural CAM; ssd~small surface domain. 1102
(~) Current Science Ltd ISSN 0955-0674
Neurite guidance molecules Doherty and Walsh Cell and substrate associated molecules In vitro assays have also been used to identify bound molecular components that modulate neurite outgrowth either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. In an extensive series of studies, emanating primarily from the laboratory of Reicbardt [4-6], the molecules that mediate neurite outgrowth over a variety of cellular substrata have been identified. The studies have used two classes of blocking antibodies to perturb neurite outgrowth: firstly, antibodies that block the function of a large class of neuronal receptors, called integrins, which mediate adhesion to and neurite outgrowth over components of the extracellular matrix (e.g. laminin, collagens and fibronectin) and, secondly, antibodies that block the function of individual 'homophilic' cell adhesion molecules (CAMs). A 'homophilic' adhesion molecule is one that can act both as its own 'receptor' (for example when expressed in the growth cone membrane) and 'ligand' (for example when expressed by a cellular substratum). The specific reagents used were Fab fractions of antibodies that block the function of the neural CAMs (NCAM), N-cadherin (also known as N-Cal-CAM) and the L1 glycoprotein (also known as NILE, neuron-glial-CAM, G4 and the 8139 antigen). In the 'prototype' study Bixby et at (Proc NatlAcad Sci USA 1987, 84:2555-2559) demonstrated that when added individually, antibodies to integrins, NCAM and Ncadherin were without major effects on chick ciliary ganglion neurite growth over a myotube substratum. However, when added in combination all three were required for a maximal inhibition, suggesting that multiple mechanisms can act in concert to promote neurite outgrowth.
When the same neuronal population was cultured on an astrocyte monolayer, antibodies to N-cadherin (on their own) inhibited neurite outgrowth dramatically [4]. Integrin antibodies could act in concert with these to inhibit outgrowth from E8 neurons but had no effect on El4 neurons. There was no evidence for the involvement of NCAM at any developmental age. These data suggest that N-cadherin is the dominant recognition molecule in this system and that a relatively small contribution due to integrin function is down-regulated as development proceeds. Studying a different neuronal population on the same astrocyte monolayer, Neugebauer et at [5] found that all three of the above recognition molecules contributed to neurite outgrowth from embryonic chick retinal neurons. However, qualitative differences were again noted as a function of developmental age. Whereas integrins and Ncadherin contributed to neurite outgrowth from both E7 and E l l neurons, the role of NCAM changed dramatically over this period. There was no evidence for NCAM function at E7; however, by E l l NCAM was quantitatively as important as integrins and N-cadherin. Schwann cells are by far the best cellular substratum for supporting neurite outgrowth from both central and peripheral neurons. In addition to investigating the role of the same recognition molecules found on myotubes and astrocytes (as described above) the role of an ad-
ditional cell adhesion gtycoprotein called L1 has been determined for Schwann cells. The relative contribution of each of these components has recently been determined for mouse dorsal root ganglion neurons [7] and chick ciliary ganglion neurons [6] grown on monolayers of mouse and rat Schwann cells, respectively. Seilheimer and Schachner [7] have found that the addition solely of antibodies to L1 can inhibit dorsal root ganglion neurite outgrowth by approximately 90 and 80% for small and large diameter neurons, respectively. Antibodies to NCAM inhibited growth by 15--35%, with the larger neurons being more dependent on NCAM function. In contrast, although Bixby et al. [6] also found L1 to be the dominant Schwann cell neurite outgrowth promoting molecule for ciliary ganglion neurons, they found little inhibition when antibodies to L1 were added on their own. Maximal Inhibition ( > 85%) was only obtained when antibodies to integfin, N-cadhefin and L1 were added simultaneously. There was no evidence for NCAM function between these particular co-culture partners. The results of the above studies are summarized in Table 1. There are perhaps three important points. Firstly, four classes of recognition molecule can account for a substantial amount of neurite outgrowth over four complex cellular substrata, with fibroblasts stimulating this function solely via neuronal integrin receptors, and Schwann cells clearly activating up to four types of growth cone receptor. Secondly, individual neuronal populations can respond in a unique manner to an identical set of recognition glycoproteins present on one cell type. Thirdly, neuronal responsiveness to a fixed set of recognition cues can change as a function of the developmental age of the neuron. It has to be remembered that the above models describe relatively static systems. Although they clearly identify candidate neurite outgrowth-promoting molecules, there is little scope for observing the effects of 'spatiotemporal' changes in receptor expression. Thus the molecules described are clearly those that are 'constitutively' expressed in vitro. Further insights into how these molecules may contribute to regulating axonal growth in vivo require a full understanding of their expression patterns during development of the nervous system. A further class of membrane-associated molecules that the reader is referred to are those which can directly inhibit neurite outgrowth [8]. In addition to the 'constitutively' expressed glycoproteins described above, transiently expressed glycoproteins may also contribute to neurite guidance; however, the evidence for this remains indirect [9]. Although both may be of considerable importance in axonal guidance, space limits the scope of the present review.
Alternative splicing generates large families of adhesion molecules As discussed above, E8 ciliary ganglion neurons do not respond to NCAM on astrocytes whereas E l l retinal neurons do. The possibility that the neurons simply lack the machinery to recognize and respond can be discounted
1103
1104
Cell
differentiation
Table 1. The major molecules that mediate neurite outgrowth over cellular monolayers in vitro.
Monolayers Neuronal cell type Fibroblast E8 CG
Integrins
Astrocyte
Myotube
Schwann cell
Integrins, N-cad'
Integrins, N-cad'
Integrins, N-cad'
NCAM E14 CG
--
E7
RN
- -
Ell
RN
- -
PN1 DRG
N-cad' Integrin~ N-cadt Integfins, N-cad, NCAM
--
- -
L1 - -
- -
- -
- -
- -
--
L1, NCAM~t
"An essentially complete inhibition of neurite outgrowth obtained with antibodies to these molecules. 'fSubstantial residual growth in presence of maximally active concentrations of blocking antibodies~ tThese antibodies had no effect on neurite outgrowth over fibroblasts. CG, chick ganglion neurons; DRG, mouse dorsal root ganglion neurons; E, embryonic day; N-cad, N-cadherin; NCAM, neural cell adhesion molecule; PN, post-natal day; RN, chick retinal neurons. Data are derived from references cited in text.
as the same neurons respond to NCAM on myotubes. Two of the possible explanations for this selectivity are that responsiveness is controlled by the amount of NCAM expressed (prevalence modulation) or that structural difference (in terms o f primary sequence and post-translational modification) may determine recognition and responsiveness. Although there is good evidence for both prevalence and post-translational modulation operating at the level of adhesion (Hoffman and Edelman, Proc Natl A c a d Sci USA 1983, 80:5762-5766) the link between simple adhesive function and the complex cell-cell interactions that result in neurite outgrowth is at best tenuous. Over the past 2 years there has been a fundamental change in our understanding of the level of complexity that can arise at the level of a single cell adhesion molecule gene as a result of alternative splicing. NCAM is again at the forefront o f this research. The original description of the NCAM gene showed that three main size classes of protein were derived from a single gene by both alternative splicing and polyadenytation-site selection during RNA processing (Owens et al., Proc Natl A c a d Sci USA 1987, 84:294-298; Barbas et al, EAIBO J 1988, 7:625-632). The three size classes were believed to be identical in their extracellular domain and show variability only in their membrane-associated carboxy-terminal region. Two of the messenger RNA (mRNA) classes encode proteins with transmembrane domains that differ in the size of their intracellular carboxy-terminal tails (large domain or 180kD isoform and small domain or 140 kD isoform). The third class has a small surface domain (ssd; 120kD isoform)that associates the polypeptide with the cell membrane via covalent linkage to a glycosyl-phosphatidylinositol anchor. More recently evidence has been obtained for a secreted isoform of NCAM [I0]. The structure o f this novel isoform was established by sequence analysis of a complementary DNA cormsponding to an NCAM mRNA from human skeletal muscle. The mRNA has a stop codon inserted between the coding region for the NH2-terminal binding domain and the domain that encodes membrane association. North-
e m blot analysis using an isoform-spectfic probe revealed the presence of'sec' mRNA species of 5.2 kb in both muscle and brain. When the novel isoform was expressed in 3T3 fibroblasts it did not associate with the membrane, but was found in the media as a 115 kD species. The above four classes o f NCAM isoform all share a common five-unit immunoglobulin-like extracellular NHz-terminal binding domain of --, 500 amino acids that contains the homophilic binding site. The consequence of diversity in the above size classes may therefore be at the level of signal transduction rather than directly at the level o f recognition. A series of recent papers [11-14] has documented more subtle changes in the primary structure of the extracellular domain of NCAM that also arise from alternative exon usage. Human muscle has been shown to express a mRNA species with three additional exons encoding a 37 amino acid block (termed the MSD1 region) that is inserted between the NHz-terminal binding domain and the membrane-associated domain of the ssd and sec isoforms (Dickson et aL, Cell 1987, 50:1119-1130) [12]. A very similar 31 amino acid group, encoded by four exons, was found to be expressed in chick cardiac muscle, but not chick brain [11]. More recently, a 15-nucleotide exon that corresponds to the first of the three exons encoding the human muscle MSD1 region has been shown to be alternatively spliced in mouse brain [12,14]. Whereas all o f these events occur in the extracellular region of NCAM, they remain distal to the region that mediates homophilic binding. Polypeptide variation through alternative splicing has also been identified in the fourth of the five immunoglobulin-like folds that constitutes the region involved in recognition and binding [13]; the alternative use o f a 3 0 b p exon introduced a 10 amino acid insert. What was of particular interest was that the number of RNAs containing the exon increased dramatically during development, with immunocytochemical studies on cultured brain neurons showing that some, but not others, express the novel NCAM isoform.
Neurite guidancemoleculesDoherty and Walsh 1105 The above studies demonstrate that NCAM exists as a large family of isoforms. If all o f the potential alternative splice patterns that have been identified to date were used, the single NCAM gene could encode up to 18 different isoforms in the mouse brain [14], with $1 nuclease analysis predicting the presence of five isoforms in skeletal muscle [12]. Whereas the consequences of diversity in the COOH-terminal may affect transduction of a recognition signal (see above), the alternative use of exons in the extracellular domain has clearly the potential to alter the conformation of the homophilic recognition site and thereby directly modulate function via changes in the ability of NCAM to bind to itself. However, in this context, the only consequence o f diversity in the primary structure of the extracellular domain of NCAM found to date relates to differential post-translational processing. It has recently been shown that the MSD1 region, described above, directs the addition of O-linked carbohydrate to NCAM In a tissuespecific manner [15].
Molecular genetic approaches for understanding function Although blocking antibodies can be used to identify cell recognition molecules involved in neurite outgrowth, more direct assays are required to address questions conceming the relationship b e ~ ' e e n the level of expression and response, the effects of post-translational modifications on function, the relative efficacy of different isoforms and the post-recognition events that underlie signal tmnsduction and neurite outgrowth. Of the homophilic recognition molecules described above (NCAM, N-cadherin and L1), L1 is the only one which has been shown to stimulate neurite outgrowth, when purified and coated to an appropriate substratum. In a recent study Lemmon e t al. [16] have grown both mouse and chick neurons on purified mouse and chick L1. As well as confirming their earlier studies, which showed L1 to b e a potent neurite outgrowth promoting molecule, they show that this stimulatory effect worked across both species. This in turn allowed them to use species-specific antibodies to demonstrate that neurite outgrowth is indeed mediated via homophilic recognition mechanism. The strategy used by these workers is limited by three problems: firstly, the ability to purify and coat substrata with molecules in an active form; secondly, the ability to separate out different isoforms o f the one adhesion molecule and, thirdly, the fact that these molecules may behave q u i t e differently outside their natural membrane environment. The difficulties can be overcome by use of the 'molecular/genetic' approach. Cloned complementary DNA encoding unique isoforms of individual cell adhesion molecules can be tmnsfected into cells that do not normally express the function of that molecule. In this way panels of cells can b e generated that express distinct isoforn~ at varying levels. Using this approach Matsunaga e t al. [17] transfected Neuro 2a and L-cells with complementary DNA encoding chicken N-cadherin. When ex-
plants of E6 retina were cultured on monolayers of these cells there was a much more extensive neurite outgrowth over the monolayers expressing the transfected N-cadherin. The response could be specifically inhibited by Fab fractions of a blocking anti-N-cadherin immunoglobulin fraction. Our own laboratory has recently expressed five distinct human NCAM isoforms in 3T3 cells and L-cells and assayed the monolayers for their ability to support neurite outgrowth from both human and rat dorsal root ganglion neurons [18]. Our results demonstrated that both transmembrane (140kD) and lipid-linked (120 kD) isoforms significantly increased the morphological differentiation of both human and rat neurons. Modification of the extracellular structure o f both classes of NCAM, consequent to the expression of the O-gtycosylated MSD1 region described above, had no obvious effect on the neurons' ability to recognize and respond to NCAM. 3T3 cells that were transfected with a full length complementary DNA encoding the secreted NCAbi isoform did not differ from control cells in their ability to support neuronal differentiation. These two studies demonstrate the power of the molecular genetic approach in studying the function of individual CAMs. The last few years have led to an understanding of the nature of the soluble and bound recognition molecules that modulate growth cone motility and thereby control neurite outgrowth: the challenge for the future is to understand the signals that arise from recognition and h o w the multitude of signals interact to define pattern in the developing nen,ous system.
Annotated references and recommended reading • •• 1.
Of interest Of outstanding interest
TESSIER-LAVIGNEM, PLACZEKM, IAJMSDENAGS, DODD J, JESSELLTM: Chemotypic guidance of developing axons in the mammalian central nervous system. Nature 1988, 336:775--778. This paper describes an elegant sede.s of experiments which suggest that intermediate targets in the central nen~ous s~tem can release diffusible factors that guide axons to their final destination. 2. IAPTONSA, FROSCHMP, PHILUPSMD, TAUCKDL, AAZENMAN • E: Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture. Science 1988, 239:1293-1296. Agents that block nicotinic receptors of rat retina promote neurite outgro~xh from retinal ganglion cells. These data support the concept of neurotransmitter mediated 'tonic' inhibition of neurite outgrowth. 3. I.A.'~"RI:ORD KL, DEMELLOFG, KLEINWE: Dl-type dopamine • receptors inhibit growth cone motility in cultured retinal neurons: evidence that neurotransmitters act as morphogenic growth regulators in the des-eloping central nervous system. Proc Natl A c a d Sci USA 1988, 85:4567-4571. The effectsof dopamine on growth cone morphologywere studied using high resolution ,ddeo microscopy.Dopaminewas found to inhibit motility,probablyvia activationof a receptor-coupledaden)late cyclase. 4. TO.~SEtuKJ, NEUGEBAUERKM, BIXBYJL, LILIENJ, REICHARITr • LF: N-Cadherin and integrins: two receptor sTstems that mediate neuronal process outgrowth on astrocyte surfaces. Neuron 1988, 1:33--43. See [6]. 5. NEUGEBAUERKM, TOM&SELLIKJ, l~n:N J, REICHARDTIF: • N-cadherin,NCAM, and integrins promote retinal neu•
1106
Cell differentiation rite outgrowth on astrocytes in vitro. J Cell Biol 1988, 107:1177-1187.
See
[6].
6. •
BIXBYJL, l~r~N ~J, RIECHARIYrLF: Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro. J Cell Biol 1988, 107:353--362. This series of papers [4-7] uses the classic method of antibody perturbation to identify the molecules that mediate neurite outgrowth over a ~miety of cellular monola)~ers in culture. Interactions bet~xen g r o w t h cone receptors for extracellular matrix components, NCAM,N-cadhedn and L1 can account for most of the neurite outgro~d~ over fibroblasts, rrs"otubes , astrocytes and Schwann cells. The relative contribution of each recognition system is dependent on the monolayer as well as the type and developmental age of the neuron. 7. •
SEILHEL',tER B, SCttACt~'ERM: Studies of adhesion molecules mediating interactions between ceils of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann ceils in culture. J Cell Biol 1988, 107:341-351. See [6]. 8. •
CARO~ P, SCHWAB /~m: Antibody against myelin-associated lnh~itor of neurite growth neutralizes nonpermissNe substrate properties of CNS white matter. Neuron 1988, 1:85--96. This paper is essentia~ similar in its approach to [4--7]. However, in this instance a blocking antibody neutralizes a membrane-associated inhibitory ac~'ity and thereby facilitates neurite outgrowth_ This study (and others by the authors) clearly shows that, as with neurotransmitters [2~], inhibitory molecules may be as important as stimulatory molecules in axonal guidance. 9.
DODDJ, MORTONSB, KARAGOGEOUSD, YAMAMOTOM, JESSELL TM: Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron 1988, 1:105-116. This study uses antibodies against the neuronal cell surface to demonstrate that changes in glycoprotein expression correlate with well defined phases of axonal growth. Although the evidence is indirecL the clear association suggests that transiently expressed molecules may be important in axonal guidance. •
10.
GOWERHJ, BARTONCH, ELSOMVI., THOMPSONJ, MOORE SE, WALSHFS: Alternative splicing generates a secreted form of N-CAM in muscle and brain. Ceil 1988, 55:955--964. Analysis of a complementary DNA clone isolated from a human musd e library identified a puta~-e mRNA with a stop codon inserted before the region that normally coders membrane association of NCAM. The mRNA~ s shown to be expressed in both muscle and brain. The observation is of particular importance as it identifies a novel class of NCAM isoforms. •
DICKSON G,
I1. •
PREDIGERE.A, HOFFMAN S, EDELMANGM, CUNh'L'qGH2L',tBA: Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken h e a r t a n d skeletal muscle. Proc Natl Acad Sci USA 1988, 85:9616-9620. Analysis of a complementary DNA clone from chicken heart is the starting point of a study that shows all of the major NCAM isoforrus present in this tissue to have a muscle-specific domain encoded by four exons that is similar, but not identical, to that found in human skeletal muscle [12]. 12. •
THOMPSONJ, DICKSON G, MOORE SE, GOWER HJ, PUTt W, KENL'VIER JG, BARTONCH, WALSHFS: Alternative splicing of the neural cell adhesion molecule gene generates variant
extraceltular domain structure in skeletal muscle and brain. Genes Dev 1989, 3:348--357. This paper reports on the genomic organization of that portion of the NCAMgene that encodes the MSD1 region. It identifies three exons that encode this muscle-specific group, and suggests that the first exon can also be altema~ely spliced into brain mRNAs. 13.
SMALLSJ, HA~'ES SL, AKESON RA: Polypeptide variation in N-CAM extracellular immunoglobulln-like fold is de~'elopmentally regulated through alternative splicing. Neuron 1988, 1:1007-1017. Altema~'e splicing can introduce a small 10 amino-add grouping into the immunoglobulin-like domain of NCAMin a developmental and neuronal sub-set-specific manner. This paper is of particular importance as it constitutes direct evidence for diversity at the amino acid level within the region that mediates recognition and binding. Such alternative usage of this exon may alter directly NCAM function. • •
an
14.
SANTONIMJ, BARTHELSD, VOPPERG, BONEDA, GORIDISC, WRa£ Differential ¢xon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO J 1989, 8:385-392. This paper characterizes 2 alternative splice sites present in the extracellular coding domain of the NCAM gene. These are identical to the sites reported in [12] and [13]. The authors provide evidence for up to 8 isoforms in mouse brain, but specuhte that if'all of the altema~-e splice patterns were used there may be 18 different NCAM isoforms in this tissue. •
W:
15. •
WALSHFS, PAREKH RB, MOORE SE, DICKSON G, BARTON CH, GOWERltJ, DWEK RA, RADEMACHERTW: Tissue specific Olinked glycosylafion of the neural cell adhesion molecule (N-CAM). Development 1989, 105:803--813. A detailed biochemical anab~is of muscle and brain NCAMshowed the presence of O-linked carboh)xirate on the former but not the latter. A series of gene transfer studies using insertional mutants demonstrated that the MSD1 region directed this post-translational modification. 16. •
LEMMONV, FARRKI, LAGENAURC: Ll-mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron 1989, 2:1597-1603. Purified L1 can promote neurite outgro~da ~hen coated onto an appropriate substratum. Species-speclfic antibodies that bind to neuronal but not substratum-associated L1 can block this response, providing direct evidence for a homophilic mechanism.
17. • •
MA'rsuNAGAM, HATrA K, NAGAFUCHI-A,TAKEICHI M: [letter] Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 1988, 334:62--64. A complementary DNA encoding chick N-cadherin was transfected into Neuro-2a or L-cells. Chicken retinal neurons showed increased neurite outgrowth over monolayers of transfected cells. This paper is important as it demonstrates the potential of the molecular genetic approach for the study of neurite outgrowth. 18. ••
DOHERTYP, BARTONCH, DICKSONG, SEATONP, ROWETr LH, MOORESE, GOWER HJ, WALSH FS: Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms. J Cell Biol 1989, 109:789-798. This study uses an arbitrary" index of neurite outgrowth to quamitare neuronal differentiation on monola~rs of 3T3 cells and L-cells transfected with up to 5 distinct NCAM isoforms. Transmembrane and glycosTl-phosphatidylinositol-linked, but not secreted isofonns all promoted neurite outgrov,~Ja. Expression of the MSD1 region did not obviously affect the neurons' ability to recognize and respond to NCAM.The paper demonstrates the potential of the molecular genetic approach for testing subtle differences in isoform structure in relation to a complex function.
Introduction
Interactions between neuronal growth cone receptors with specific guidance cues in the microenvironment are likely to contribute to the precise stereospecific manner in which neurons find and innervate their appropriate targets. Guidance or recognition cues may be diffusible or bound and, in the Lattercase, may be localized both in the extraceilular matrix and on the membranes of neurons and other cell types. As well as spatio-temporal changes in the expression of the cues, controlling mechanisms are likely to include temporal changes in the expression of the growth cone receptors. In the present article we review some of the recent studies that consolidate and extend our understanding of the general nature of the recognition cues, as well as identify some of the candidate molecules. Evidence that large families of biochemically distinct recognition molecules can be obtained from a single gene by the processes of alternative splicing and differential post-translational modification is also discussed. Finally, we will consider strategies developed recently that can test directly the function of the large number of isoforms of single cell adhesion molecules. Diffusible factors
Evidence obtained in vitro using peripheral nervous tissue supports the general hypothesis that target tissues may release diffusible chemoattractant molecules that contribute to axonal guidance (see Lumsden and Davies, Nature 1986, 323:538-539). In a recent study TessierLavigne et al. [1 ] used central nervous tissue to test this postulate further. During the development of the spinal cord, commissural neurons send axons from the dorsal horn to the ventral floor-plate region. On reaching this 'intermediate' target the axons cross the mid-line and subsequently show a marked change in the orientation of their projection. The authors have co-cultured the dorsal horn (which contains the commissural neurons) with other regions of the developing spinal cord. They observed that the floor-plate region is unique in its ability both to induce the specific outgrowth of commissural axons and also to orientate their direction of growth. Two lines of evidence suggest the presence of an active soluble molecule: firstly, in the co-culture studies the neurons and their 'target' were separated by up to 400 lam and, secondly, the neurite outgrowth promoting activity could be found in the media conditioned by the floorplate region. Although these data again focus attention
on the potential role that both the intermediate and final targets may play in attracting axons, a critical evaluation of the hypothesis will require the identification and characterization of the active molecules. A second class of soluble molecules that may contribute to axonal guidance are the neurotransmitters. Evidence that acetytcholine, dopamine and serotonin can all modulate growth cone motility has come from a large number of studies on the individual neurons of molluscs (Cohen and Kater. In The Cellular Basis of Neuronal PlasticiOJ edited by Bulloch. Manchester University Press, 1989, pp 79-96). More recently these findings have been extended to the vertebrate nervous system. In cultures of post-natal rat retina the blockade of acetylcholine receptor function by nicotinic but not muscarinic antagonists increased retinal ganglion cell neurite outgrowth by up to 50% [2]. This response was only found in cultures that contained a measurable basal level of endogenous acetytcholine, produced perhaps by amacrine cells. These data suggest that acetylcholine can induce a 'tonic' inhibition of neurite outgrowth that is released by the nicotinic block. The mechanism of action remains to be determined; however, the authors suggest that it is not solely due to depolarization, as other agents that depolarize the cells were without effect. Addition of micromolar concentrations of dopamine to cultures of embryonic chick retina resulted in an inhibition of neurite outgrowth from approximately 25% of neurons [3]. The inhibition was characterized by a reduction in filopodial activity followed by a flattening and retraction of growth cones. Selective antagonists were used to demonstrate that the response was linked to the activation of Dl-type dopamine receptors. These receptors are coupled to adenylate cyclase and, in this context, forskolin was also able to elicit a similar response, but only in dopamine-sensitive neurons. The physiological significance of neurotransmitter-mediated inhibition of neurite outgrowth remains to be determined. However, it is possible that neurotransmitters may modulate axonal guidance both during development and during periods of synaptic remodelling in the adult. A 'tonic' inhibition of axonal growth could be released by a decreased availability of transmitter (mediated by ceil uptake and/or enzymatic activity) and thereby provide a positive control. Conversely, direct inhibitory influences may restrict growth into areas that contain free neurotransmitters, or serve to inhibit growth cone motility prior to synapse formation.
Abbreviations
CAM~cell adhesion molecule; mRNA--messengerRNA;NCAM~neural CAM; ssd~small surface domain. 1102
(~) Current Science Ltd ISSN 0955-0674
Neurite guidance molecules Doherty and Walsh Cell and substrate associated molecules In vitro assays have also been used to identify bound molecular components that modulate neurite outgrowth either directly by demonstrating their ability to promote neurite outgrowth, or indirectly by the ability of specific antibodies to inhibit neurite outgrowth. In an extensive series of studies, emanating primarily from the laboratory of Reicbardt [4-6], the molecules that mediate neurite outgrowth over a variety of cellular substrata have been identified. The studies have used two classes of blocking antibodies to perturb neurite outgrowth: firstly, antibodies that block the function of a large class of neuronal receptors, called integrins, which mediate adhesion to and neurite outgrowth over components of the extracellular matrix (e.g. laminin, collagens and fibronectin) and, secondly, antibodies that block the function of individual 'homophilic' cell adhesion molecules (CAMs). A 'homophilic' adhesion molecule is one that can act both as its own 'receptor' (for example when expressed in the growth cone membrane) and 'ligand' (for example when expressed by a cellular substratum). The specific reagents used were Fab fractions of antibodies that block the function of the neural CAMs (NCAM), N-cadherin (also known as N-Cal-CAM) and the L1 glycoprotein (also known as NILE, neuron-glial-CAM, G4 and the 8139 antigen). In the 'prototype' study Bixby et at (Proc NatlAcad Sci USA 1987, 84:2555-2559) demonstrated that when added individually, antibodies to integrins, NCAM and Ncadherin were without major effects on chick ciliary ganglion neurite growth over a myotube substratum. However, when added in combination all three were required for a maximal inhibition, suggesting that multiple mechanisms can act in concert to promote neurite outgrowth.
When the same neuronal population was cultured on an astrocyte monolayer, antibodies to N-cadherin (on their own) inhibited neurite outgrowth dramatically [4]. Integrin antibodies could act in concert with these to inhibit outgrowth from E8 neurons but had no effect on El4 neurons. There was no evidence for the involvement of NCAM at any developmental age. These data suggest that N-cadherin is the dominant recognition molecule in this system and that a relatively small contribution due to integrin function is down-regulated as development proceeds. Studying a different neuronal population on the same astrocyte monolayer, Neugebauer et at [5] found that all three of the above recognition molecules contributed to neurite outgrowth from embryonic chick retinal neurons. However, qualitative differences were again noted as a function of developmental age. Whereas integrins and Ncadherin contributed to neurite outgrowth from both E7 and E l l neurons, the role of NCAM changed dramatically over this period. There was no evidence for NCAM function at E7; however, by E l l NCAM was quantitatively as important as integrins and N-cadherin. Schwann cells are by far the best cellular substratum for supporting neurite outgrowth from both central and peripheral neurons. In addition to investigating the role of the same recognition molecules found on myotubes and astrocytes (as described above) the role of an ad-
ditional cell adhesion gtycoprotein called L1 has been determined for Schwann cells. The relative contribution of each of these components has recently been determined for mouse dorsal root ganglion neurons [7] and chick ciliary ganglion neurons [6] grown on monolayers of mouse and rat Schwann cells, respectively. Seilheimer and Schachner [7] have found that the addition solely of antibodies to L1 can inhibit dorsal root ganglion neurite outgrowth by approximately 90 and 80% for small and large diameter neurons, respectively. Antibodies to NCAM inhibited growth by 15--35%, with the larger neurons being more dependent on NCAM function. In contrast, although Bixby et al. [6] also found L1 to be the dominant Schwann cell neurite outgrowth promoting molecule for ciliary ganglion neurons, they found little inhibition when antibodies to L1 were added on their own. Maximal Inhibition ( > 85%) was only obtained when antibodies to integfin, N-cadhefin and L1 were added simultaneously. There was no evidence for NCAM function between these particular co-culture partners. The results of the above studies are summarized in Table 1. There are perhaps three important points. Firstly, four classes of recognition molecule can account for a substantial amount of neurite outgrowth over four complex cellular substrata, with fibroblasts stimulating this function solely via neuronal integrin receptors, and Schwann cells clearly activating up to four types of growth cone receptor. Secondly, individual neuronal populations can respond in a unique manner to an identical set of recognition glycoproteins present on one cell type. Thirdly, neuronal responsiveness to a fixed set of recognition cues can change as a function of the developmental age of the neuron. It has to be remembered that the above models describe relatively static systems. Although they clearly identify candidate neurite outgrowth-promoting molecules, there is little scope for observing the effects of 'spatiotemporal' changes in receptor expression. Thus the molecules described are clearly those that are 'constitutively' expressed in vitro. Further insights into how these molecules may contribute to regulating axonal growth in vivo require a full understanding of their expression patterns during development of the nervous system. A further class of membrane-associated molecules that the reader is referred to are those which can directly inhibit neurite outgrowth [8]. In addition to the 'constitutively' expressed glycoproteins described above, transiently expressed glycoproteins may also contribute to neurite guidance; however, the evidence for this remains indirect [9]. Although both may be of considerable importance in axonal guidance, space limits the scope of the present review.
Alternative splicing generates large families of adhesion molecules As discussed above, E8 ciliary ganglion neurons do not respond to NCAM on astrocytes whereas E l l retinal neurons do. The possibility that the neurons simply lack the machinery to recognize and respond can be discounted
1103
1104
Cell
differentiation
Table 1. The major molecules that mediate neurite outgrowth over cellular monolayers in vitro.
Monolayers Neuronal cell type Fibroblast E8 CG
Integrins
Astrocyte
Myotube
Schwann cell
Integrins, N-cad'
Integrins, N-cad'
Integrins, N-cad'
NCAM E14 CG
--
E7
RN
- -
Ell
RN
- -
PN1 DRG
N-cad' Integrin~ N-cadt Integfins, N-cad, NCAM
--
- -
L1 - -
- -
- -
- -
- -
--
L1, NCAM~t
"An essentially complete inhibition of neurite outgrowth obtained with antibodies to these molecules. 'fSubstantial residual growth in presence of maximally active concentrations of blocking antibodies~ tThese antibodies had no effect on neurite outgrowth over fibroblasts. CG, chick ganglion neurons; DRG, mouse dorsal root ganglion neurons; E, embryonic day; N-cad, N-cadherin; NCAM, neural cell adhesion molecule; PN, post-natal day; RN, chick retinal neurons. Data are derived from references cited in text.
as the same neurons respond to NCAM on myotubes. Two of the possible explanations for this selectivity are that responsiveness is controlled by the amount of NCAM expressed (prevalence modulation) or that structural difference (in terms o f primary sequence and post-translational modification) may determine recognition and responsiveness. Although there is good evidence for both prevalence and post-translational modulation operating at the level of adhesion (Hoffman and Edelman, Proc Natl A c a d Sci USA 1983, 80:5762-5766) the link between simple adhesive function and the complex cell-cell interactions that result in neurite outgrowth is at best tenuous. Over the past 2 years there has been a fundamental change in our understanding of the level of complexity that can arise at the level of a single cell adhesion molecule gene as a result of alternative splicing. NCAM is again at the forefront o f this research. The original description of the NCAM gene showed that three main size classes of protein were derived from a single gene by both alternative splicing and polyadenytation-site selection during RNA processing (Owens et al., Proc Natl A c a d Sci USA 1987, 84:294-298; Barbas et al, EAIBO J 1988, 7:625-632). The three size classes were believed to be identical in their extracellular domain and show variability only in their membrane-associated carboxy-terminal region. Two of the messenger RNA (mRNA) classes encode proteins with transmembrane domains that differ in the size of their intracellular carboxy-terminal tails (large domain or 180kD isoform and small domain or 140 kD isoform). The third class has a small surface domain (ssd; 120kD isoform)that associates the polypeptide with the cell membrane via covalent linkage to a glycosyl-phosphatidylinositol anchor. More recently evidence has been obtained for a secreted isoform of NCAM [I0]. The structure o f this novel isoform was established by sequence analysis of a complementary DNA cormsponding to an NCAM mRNA from human skeletal muscle. The mRNA has a stop codon inserted between the coding region for the NH2-terminal binding domain and the domain that encodes membrane association. North-
e m blot analysis using an isoform-spectfic probe revealed the presence of'sec' mRNA species of 5.2 kb in both muscle and brain. When the novel isoform was expressed in 3T3 fibroblasts it did not associate with the membrane, but was found in the media as a 115 kD species. The above four classes o f NCAM isoform all share a common five-unit immunoglobulin-like extracellular NHz-terminal binding domain of --, 500 amino acids that contains the homophilic binding site. The consequence of diversity in the above size classes may therefore be at the level of signal transduction rather than directly at the level o f recognition. A series of recent papers [11-14] has documented more subtle changes in the primary structure of the extracellular domain of NCAM that also arise from alternative exon usage. Human muscle has been shown to express a mRNA species with three additional exons encoding a 37 amino acid block (termed the MSD1 region) that is inserted between the NHz-terminal binding domain and the membrane-associated domain of the ssd and sec isoforms (Dickson et aL, Cell 1987, 50:1119-1130) [12]. A very similar 31 amino acid group, encoded by four exons, was found to be expressed in chick cardiac muscle, but not chick brain [11]. More recently, a 15-nucleotide exon that corresponds to the first of the three exons encoding the human muscle MSD1 region has been shown to be alternatively spliced in mouse brain [12,14]. Whereas all o f these events occur in the extracellular region of NCAM, they remain distal to the region that mediates homophilic binding. Polypeptide variation through alternative splicing has also been identified in the fourth of the five immunoglobulin-like folds that constitutes the region involved in recognition and binding [13]; the alternative use o f a 3 0 b p exon introduced a 10 amino acid insert. What was of particular interest was that the number of RNAs containing the exon increased dramatically during development, with immunocytochemical studies on cultured brain neurons showing that some, but not others, express the novel NCAM isoform.
Neurite guidancemoleculesDoherty and Walsh 1105 The above studies demonstrate that NCAM exists as a large family of isoforms. If all o f the potential alternative splice patterns that have been identified to date were used, the single NCAM gene could encode up to 18 different isoforms in the mouse brain [14], with $1 nuclease analysis predicting the presence of five isoforms in skeletal muscle [12]. Whereas the consequences of diversity in the COOH-terminal may affect transduction of a recognition signal (see above), the alternative use of exons in the extracellular domain has clearly the potential to alter the conformation of the homophilic recognition site and thereby directly modulate function via changes in the ability of NCAM to bind to itself. However, in this context, the only consequence o f diversity in the primary structure of the extracellular domain of NCAM found to date relates to differential post-translational processing. It has recently been shown that the MSD1 region, described above, directs the addition of O-linked carbohydrate to NCAM In a tissuespecific manner [15].
Molecular genetic approaches for understanding function Although blocking antibodies can be used to identify cell recognition molecules involved in neurite outgrowth, more direct assays are required to address questions conceming the relationship b e ~ ' e e n the level of expression and response, the effects of post-translational modifications on function, the relative efficacy of different isoforms and the post-recognition events that underlie signal tmnsduction and neurite outgrowth. Of the homophilic recognition molecules described above (NCAM, N-cadherin and L1), L1 is the only one which has been shown to stimulate neurite outgrowth, when purified and coated to an appropriate substratum. In a recent study Lemmon e t al. [16] have grown both mouse and chick neurons on purified mouse and chick L1. As well as confirming their earlier studies, which showed L1 to b e a potent neurite outgrowth promoting molecule, they show that this stimulatory effect worked across both species. This in turn allowed them to use species-specific antibodies to demonstrate that neurite outgrowth is indeed mediated via homophilic recognition mechanism. The strategy used by these workers is limited by three problems: firstly, the ability to purify and coat substrata with molecules in an active form; secondly, the ability to separate out different isoforms o f the one adhesion molecule and, thirdly, the fact that these molecules may behave q u i t e differently outside their natural membrane environment. The difficulties can be overcome by use of the 'molecular/genetic' approach. Cloned complementary DNA encoding unique isoforms of individual cell adhesion molecules can be tmnsfected into cells that do not normally express the function of that molecule. In this way panels of cells can b e generated that express distinct isoforn~ at varying levels. Using this approach Matsunaga e t al. [17] transfected Neuro 2a and L-cells with complementary DNA encoding chicken N-cadherin. When ex-
plants of E6 retina were cultured on monolayers of these cells there was a much more extensive neurite outgrowth over the monolayers expressing the transfected N-cadherin. The response could be specifically inhibited by Fab fractions of a blocking anti-N-cadherin immunoglobulin fraction. Our own laboratory has recently expressed five distinct human NCAM isoforms in 3T3 cells and L-cells and assayed the monolayers for their ability to support neurite outgrowth from both human and rat dorsal root ganglion neurons [18]. Our results demonstrated that both transmembrane (140kD) and lipid-linked (120 kD) isoforms significantly increased the morphological differentiation of both human and rat neurons. Modification of the extracellular structure o f both classes of NCAM, consequent to the expression of the O-gtycosylated MSD1 region described above, had no obvious effect on the neurons' ability to recognize and respond to NCAM. 3T3 cells that were transfected with a full length complementary DNA encoding the secreted NCAbi isoform did not differ from control cells in their ability to support neuronal differentiation. These two studies demonstrate the power of the molecular genetic approach in studying the function of individual CAMs. The last few years have led to an understanding of the nature of the soluble and bound recognition molecules that modulate growth cone motility and thereby control neurite outgrowth: the challenge for the future is to understand the signals that arise from recognition and h o w the multitude of signals interact to define pattern in the developing nen,ous system.
Annotated references and recommended reading • •• 1.
Of interest Of outstanding interest
TESSIER-LAVIGNEM, PLACZEKM, IAJMSDENAGS, DODD J, JESSELLTM: Chemotypic guidance of developing axons in the mammalian central nervous system. Nature 1988, 336:775--778. This paper describes an elegant sede.s of experiments which suggest that intermediate targets in the central nen~ous s~tem can release diffusible factors that guide axons to their final destination. 2. IAPTONSA, FROSCHMP, PHILUPSMD, TAUCKDL, AAZENMAN • E: Nicotinic antagonists enhance process outgrowth by rat retinal ganglion cells in culture. Science 1988, 239:1293-1296. Agents that block nicotinic receptors of rat retina promote neurite outgro~xh from retinal ganglion cells. These data support the concept of neurotransmitter mediated 'tonic' inhibition of neurite outgrowth. 3. I.A.'~"RI:ORD KL, DEMELLOFG, KLEINWE: Dl-type dopamine • receptors inhibit growth cone motility in cultured retinal neurons: evidence that neurotransmitters act as morphogenic growth regulators in the des-eloping central nervous system. Proc Natl A c a d Sci USA 1988, 85:4567-4571. The effectsof dopamine on growth cone morphologywere studied using high resolution ,ddeo microscopy.Dopaminewas found to inhibit motility,probablyvia activationof a receptor-coupledaden)late cyclase. 4. TO.~SEtuKJ, NEUGEBAUERKM, BIXBYJL, LILIENJ, REICHARITr • LF: N-Cadherin and integrins: two receptor sTstems that mediate neuronal process outgrowth on astrocyte surfaces. Neuron 1988, 1:33--43. See [6]. 5. NEUGEBAUERKM, TOM&SELLIKJ, l~n:N J, REICHARDTIF: • N-cadherin,NCAM, and integrins promote retinal neu•
1106
Cell differentiation rite outgrowth on astrocytes in vitro. J Cell Biol 1988, 107:1177-1187.
See
[6].
6. •
BIXBYJL, l~r~N ~J, RIECHARIYrLF: Identification of the major proteins that promote neuronal process outgrowth on Schwann cells in vitro. J Cell Biol 1988, 107:353--362. This series of papers [4-7] uses the classic method of antibody perturbation to identify the molecules that mediate neurite outgrowth over a ~miety of cellular monola)~ers in culture. Interactions bet~xen g r o w t h cone receptors for extracellular matrix components, NCAM,N-cadhedn and L1 can account for most of the neurite outgro~d~ over fibroblasts, rrs"otubes , astrocytes and Schwann cells. The relative contribution of each recognition system is dependent on the monolayer as well as the type and developmental age of the neuron. 7. •
SEILHEL',tER B, SCttACt~'ERM: Studies of adhesion molecules mediating interactions between ceils of peripheral nervous system indicate a major role for L1 in mediating sensory neuron growth on Schwann ceils in culture. J Cell Biol 1988, 107:341-351. See [6]. 8. •
CARO~ P, SCHWAB /~m: Antibody against myelin-associated lnh~itor of neurite growth neutralizes nonpermissNe substrate properties of CNS white matter. Neuron 1988, 1:85--96. This paper is essentia~ similar in its approach to [4--7]. However, in this instance a blocking antibody neutralizes a membrane-associated inhibitory ac~'ity and thereby facilitates neurite outgrowth_ This study (and others by the authors) clearly shows that, as with neurotransmitters [2~], inhibitory molecules may be as important as stimulatory molecules in axonal guidance. 9.
DODDJ, MORTONSB, KARAGOGEOUSD, YAMAMOTOM, JESSELL TM: Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron 1988, 1:105-116. This study uses antibodies against the neuronal cell surface to demonstrate that changes in glycoprotein expression correlate with well defined phases of axonal growth. Although the evidence is indirecL the clear association suggests that transiently expressed molecules may be important in axonal guidance. •
10.
GOWERHJ, BARTONCH, ELSOMVI., THOMPSONJ, MOORE SE, WALSHFS: Alternative splicing generates a secreted form of N-CAM in muscle and brain. Ceil 1988, 55:955--964. Analysis of a complementary DNA clone isolated from a human musd e library identified a puta~-e mRNA with a stop codon inserted before the region that normally coders membrane association of NCAM. The mRNA~ s shown to be expressed in both muscle and brain. The observation is of particular importance as it identifies a novel class of NCAM isoforms. •
DICKSON G,
I1. •
PREDIGERE.A, HOFFMAN S, EDELMANGM, CUNh'L'qGH2L',tBA: Four exons encode a 93-base-pair insert in three neural cell adhesion molecule mRNAs specific for chicken h e a r t a n d skeletal muscle. Proc Natl Acad Sci USA 1988, 85:9616-9620. Analysis of a complementary DNA clone from chicken heart is the starting point of a study that shows all of the major NCAM isoforrus present in this tissue to have a muscle-specific domain encoded by four exons that is similar, but not identical, to that found in human skeletal muscle [12]. 12. •
THOMPSONJ, DICKSON G, MOORE SE, GOWER HJ, PUTt W, KENL'VIER JG, BARTONCH, WALSHFS: Alternative splicing of the neural cell adhesion molecule gene generates variant
extraceltular domain structure in skeletal muscle and brain. Genes Dev 1989, 3:348--357. This paper reports on the genomic organization of that portion of the NCAMgene that encodes the MSD1 region. It identifies three exons that encode this muscle-specific group, and suggests that the first exon can also be altema~ely spliced into brain mRNAs. 13.
SMALLSJ, HA~'ES SL, AKESON RA: Polypeptide variation in N-CAM extracellular immunoglobulln-like fold is de~'elopmentally regulated through alternative splicing. Neuron 1988, 1:1007-1017. Altema~'e splicing can introduce a small 10 amino-add grouping into the immunoglobulin-like domain of NCAMin a developmental and neuronal sub-set-specific manner. This paper is of particular importance as it constitutes direct evidence for diversity at the amino acid level within the region that mediates recognition and binding. Such alternative usage of this exon may alter directly NCAM function. • •
an
14.
SANTONIMJ, BARTHELSD, VOPPERG, BONEDA, GORIDISC, WRa£ Differential ¢xon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO J 1989, 8:385-392. This paper characterizes 2 alternative splice sites present in the extracellular coding domain of the NCAM gene. These are identical to the sites reported in [12] and [13]. The authors provide evidence for up to 8 isoforms in mouse brain, but specuhte that if'all of the altema~-e splice patterns were used there may be 18 different NCAM isoforms in this tissue. •
W:
15. •
WALSHFS, PAREKH RB, MOORE SE, DICKSON G, BARTON CH, GOWERltJ, DWEK RA, RADEMACHERTW: Tissue specific Olinked glycosylafion of the neural cell adhesion molecule (N-CAM). Development 1989, 105:803--813. A detailed biochemical anab~is of muscle and brain NCAMshowed the presence of O-linked carboh)xirate on the former but not the latter. A series of gene transfer studies using insertional mutants demonstrated that the MSD1 region directed this post-translational modification. 16. •
LEMMONV, FARRKI, LAGENAURC: Ll-mediated axon outgrowth occurs via a homophilic binding mechanism. Neuron 1989, 2:1597-1603. Purified L1 can promote neurite outgro~da ~hen coated onto an appropriate substratum. Species-speclfic antibodies that bind to neuronal but not substratum-associated L1 can block this response, providing direct evidence for a homophilic mechanism.
17. • •
MA'rsuNAGAM, HATrA K, NAGAFUCHI-A,TAKEICHI M: [letter] Guidance of optic nerve fibres by N-cadherin adhesion molecules. Nature 1988, 334:62--64. A complementary DNA encoding chick N-cadherin was transfected into Neuro-2a or L-cells. Chicken retinal neurons showed increased neurite outgrowth over monolayers of transfected cells. This paper is important as it demonstrates the potential of the molecular genetic approach for the study of neurite outgrowth. 18. ••
DOHERTYP, BARTONCH, DICKSONG, SEATONP, ROWETr LH, MOORESE, GOWER HJ, WALSH FS: Neuronal process outgrowth of human sensory neurons on monolayers of cells transfected with cDNAs for five human N-CAM isoforms. J Cell Biol 1989, 109:789-798. This study uses an arbitrary" index of neurite outgrowth to quamitare neuronal differentiation on monola~rs of 3T3 cells and L-cells transfected with up to 5 distinct NCAM isoforms. Transmembrane and glycosTl-phosphatidylinositol-linked, but not secreted isofonns all promoted neurite outgrov,~Ja. Expression of the MSD1 region did not obviously affect the neurons' ability to recognize and respond to NCAM.The paper demonstrates the potential of the molecular genetic approach for testing subtle differences in isoform structure in relation to a complex function.