Geoffrey M.W. Cook

Isolation from chick somites of a glycoprotein fraction that causes collapse of dorsal root ganglion growth cones

The segmented pattern of peripheral spinal nerves in higher vertebrates is generated by interactions between nerve cells and somites. Neural crest cells, motor axons, and sensory axons grow exclusively through anterior-half sclerotome. In chick embryos, posterior cells bind the lectins peanut agglutinin (PNA) and Jacalin. When liposomes containing somite extracts are applied to cultures of chick sensory neurons, growth cones collapse abruptly, recovering within 4 hr of liposome removal. Collapse activity is eliminated by immobilized PNA, and SDS-PAGE demonstrates two major components (48K and 55K), which are absent from anterior-half sclerotome. Rabbit polyclonal antibodies against these components recognize only posterior cells and may also be used to eliminate collapse activity. We suggest that spinal nerve segmentation is produced by inhibitory interactions between these components and growth cones.

AXON GUIDANCE MOLECULES

To survey axon guidance in 1995 is to cover a very different field from some 30 years ago, when Sperry elaborated his idea of chemoaffinity to explain the precision of regeneration of the amphibian retinotectal map (Sperry, 1963). With hindsight we know that Sperry was overenthusiastic in his attribution of molecular labels to define tectal addresses and that the role of electrical activity in refining connectivity was yet to be fully appreciated. But at least he could envisage the problem in molecular terms and, in so doing, rekindled interest in a spectacular feat of biology. That interest has been growing ever since. The biology of axon guidance has been reviewed by Goodman and Shatz (1993). Here we concentrate on recent progress in understanding the nature and mechanism of axon guidance molecules, defining these as molecules outside the growth cone that assist in its selection of the correct pathway and target by changing its direction of growth. After synapse formation, connections may be refined and remodeled (address selection) by a process that is more dependent on patterns of electrical activity (Goodman and Shatz, 1993). Since guidance molecules can be both pathway derived and target derived, this article complements the accompanying review on target recognition (Garrityandzipursky, 1995 [this issueof Cc//j). The identification of growth cone receptors for guidance molecules is also critical, but is still at an early stage (see below). Lastly, understanding how growth cones turn also requires the elucidation of the signaling pathways within them that transduce receptor activation into changes in the dynamics and arrangement of the cytoskeleton (reviewed by Doherty and Walsh, 1994; see also Tanaka and Sabry, 1995 [this issue of Cc//j), and this will not be considered here. The possibility that guidance molecules exert either short-range (cell-cell) or long-range (diffusion gradient) influences on growth cones has been recognized for many years. More recently, it has become clear that guidance may be both attractive or repulsive (Goodman and Shatz, 1993; Keynes and Cook, 1995) and that these influences may be exerted over both short and long ranges by membersof the same molecular family. And as increasing numbers of candidates have been characterized, the earlier concept of guidance by differential adhesion has been modified by a realization that the guidance molecules, whether attractive, repulsive, short-range, or long-range, may direct growth cones via signal transduction mechanisms (see also Tanaka and Sabry, 1995). Attractive and Review

Repulsive and inhibitory signals

Repulsive or inhibitory interactions between growth cones and their environment are now widely implicated in neural development and regeneration. Over the past year, descriptive studies of the various neuronal systems in which repulsion may participate have clarified its biology. Molecular and genetic studies have also provided the necessary entry point for further experimental manipulations, and are beginning to yield important clues regarding the function of repulsion in vivo. Although candidate second messengers underlying the growth cone response have been identified, they have yet to be incorporated into a comprehensive mechanism.

Cell-cell repulsion: Clues from the growth cone?

Since the classical studies of Holtfreter (1948), the generation and maintenance of tissue diversity during embryonic development have been explained on the basis of differential affinities between cells. More recently, it has been both reasonable and fashionable to describe such differences in terms of the selective expression of cell-cell adhesion systems (Nose et al., 1988; Jaffe et al., 1990). But variation by adhesion is not the only way to change cell affinities. It may, for example, be required in vivo to create and sustain discontinuities or boundaries between neighboring cell populations; but is it sufficient? Taking a cue from recent findings in the nervouqsystem, we argue here that active cell-cell repulsion should also be considered as a possible morphogenetic mechanism. Growth Cone Collapse Several studies on axon guidance have reported that the motile tip of growing axons, the growth cone, can undergo a dramatic change in shape (from a spread to collapsed state) and behavior (temporary cessation of motility) in response to particular environmental signals (see Figure 1). This was first described clearly by Kapfhammer and Raper (1987), who showed that growth cones derived from peripheral neurons collapse in vitro when they contact axons derived from central neurons, and vice versa. These authors recognized that it was difficult to explain the collapse solely by invoking changes in adhesive properties of the cells concerned. A direct extension of this elegant work has been the development of a quantitative in vitro assay for the presence in vivo of molecules that are responsible for mediating such effects (Raper and Kapfhammer, 1990). The application of the assay to the problem of peripheral nerve segmentation has reinforced the relevance of the collapse phenomenon for explaining a normal biological process. In higher vertebrate embryos, outgrowing spinal axons traverse exclusively the anterior half-somite (Keynes and Stern, 1984), and a strong case can be made for the existence of a molecular system that mediates repulsion between growth cones and cells of the posterior half-somite. In the chick, the lectin peanut agglutinin binds to the surfaces of posterior but not anterior cells. Exploiting this fact, Davies at al. (1990) have now isolated a peanut agglutinin binding glycoprotein fraction from chick somites, and shown that it has potent collapse-inducing activity in the assay. This activity may be mirrored, albeit on a lesser scale, in vivo. It may be enough, for example, for a single filopodium to respond to the repellent stimulus on posterior cells in order to inform the parent growth cone that it is approaching a “no-go” area. A further example of growth cone repulsion has been Minireview

Axon guidance to and from choice points

Significant progress has been made recently in understanding axon guidance to and from choice points. Netrins have been shown to function as conserved midline chemoattractants in vertebrates and insects, and receptors for netrins and semaphorins/collapsins have been identified. More evidence has accumulated that repulsion plays a key role in guidance, including the involvement of the ephrin/Eph receptor system in contact repulsion.

Repellent cues in axon guidance

There is increasing evidence that axons are guided by repulsion in several regions of the developing nervous system, although this has yet to be confirmed directly in vivo. As more candidate repulsion molecules are identified, it is becoming clear that collapse of the growth cone in vitro may be mediated by more than one intracellular mechanism. The present emphasis on molecular cloning of the ligands and their receptors should enable a proper definition of their function during development.

Dextrin Sulphate and Fucoidan are Potent Inhibitors of HIV Infection in vitro

Two sulphated polysaccharides, fucoidan (a derivative of seaweed) and the newly synthesised dextrin sulphate, were tested for their ability to inhibit human immunodeficiency virus (HIV) infection in vitro in comparison to dextran sulphate and azidothymidine. They were found to be potent inhibitors of diverse strains of HIV-1 in a variety of human cell lines and in peripheral blood lymphocytes (PBL) using a range of assays, including cell-free and cell-to-cell spread of infection. The drugs did not adversely affect cell proliferation or protein metabolism of PBL. As dextrin sulphate is less potent than dextran sulphate in prolonging thrombin-induced fibrin clotting time, it merits further development as an antiviral agent.

Differing Semaphorin 3A Concentrations Trigger Distinct Signaling Mechanisms in Growth Cone Collapse

Semaphorin-3A (Sema3A) is a major guidance cue in the developing nervous system. Previous studies have revealed a dependence of responses to Sema3A on local protein synthesis (PS) in axonal growth cones, but a recent study has called this dependence into question. To understand the basis of this discrepancy we used the growth cone collapse assay on chick dorsal root ganglion neurons. We show that the dependence of growth cone collapse on protein synthesis varies according to Sema3A concentration, from near-total at low concentration (<100 ng/ml) to minimal at high concentration (>625 ng/ml). Further, we show that neuropilin-1 (NP-1) mediates both PS-dependent and PS-independent collapse. Our findings are consistent with the operation of at least two distinct Sema3A signaling pathways: one that is PS-dependent, involving mammalian target of rapamycin, and one that is PS-independent, involving GSK-3β activation and operative at all concentrations of Sema3A examined. The results provide a plausible explanation for the discrepancy in PS-dependence reported in the literature, and indicate that different signaling pathways activated within growth cones can be modulated by changing the concentration of the same guidance cue.

Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin

An indirect affinity chromatography procedure utilizing biotinylated lectins and designed for the specific isolation of surface glycoproteins is described. The method is illustrated with intact acute leukemic lymphoblastic cells (ALL cells) with biotin-epsilon-aminocaproyl-concanavalin A (biocap-Con A) and streptavidin-Sepharose 4B. Biocap-Con A, containing on average 27 biotin residues per tetrameric lectin molecule, is used to isolate Con A-binding glycoproteins from the surface of [35S]methionine-radiolabeled intact cells. The biocap-Con A/glycoprotein complexes, after solubilization in detergent, are retrieved on immobilized streptavidin. The surface glycoproteins isolated from intact ALL cells by this method are subjected to two-dimensional gel electrophoresis and detected by autoradiography. More than fifty Con A-binding glycoproteins can be separated from the ALL cells. These glycoproteins retrievable from the cell surface were compared to those retrieved by the indirect affinity chromatography procedure from isolated plasma membrane fractions. Certain groups of glycoproteins present in the fraction isolated from intact cells were not detected in that from the plasma membrane preparations. The advantage of using the biocap-con A/streptavidin system with intact cells rather than isolated plasma membranes for the detection of surface glycoproteins is discussed.

Axons Turn as Netrins Find Their Receptor

A recurrent theme of recent years has been the similarity between the molecular mechanisms that control pattern formation in vertebrates and invertebrates. While in some cases the same regulatory proteins and pathways carry out divergent functions in different organisms, the new data illuminate an outstanding example of a conserved ligand–receptor system operating with little apparent redundancy at the body midline. They also show how the individual experimental advantages of worms, flies and vertebrates can be put to good effect. Many interesting questions are raised, for example whether DCC-like proteins constitute a complete receptor system or an essential ligand-binding component, the relation between UNC-40 and UNC-5 in mediating repulsion, the existence of fly and vertebrate homologs of UNC-5, and the molecular pathways downstream of netrin reception. Given the conservation of the biology, it will be surprising if the regulatory events connected with netrins and their receptors are not also retained from nematodes to chordates.