Spinal nerve segmentation in higher vertebrates: axon guidance by repulsion and attraction Roger J. Keynes, Alan R. Johnson, Adrian Pini*, David Tannahill and Geoffrey M.W. Cook The development of spinal nerve segmentation in higher vertebrate embryos provides a convenient experimental system for the analysis of axon guidance mechanisms. We review evidence from chick embryo experiments that segmentation of motor and sensory axons results from a combination of contact repulsion of axon growth cones by posterior somite cells and chemoattraction of growth cones by anterior cells. We also review progress in identifying the underlying molecular mechanisms in this system, and suggest a prominent role for carbohydrate groups in mediating growth con e repu lsion . Key words: axon guidance / contact repulsion / glycoconjugate / segmentation / somite ©1996 Academic Press Ltd AS INCREASING NUMBERS of candidate axon guidance molecules are identified, the need remains for experi- mentally convenient, anatomically simple model sys- tems with which to evaluate their function in vivo . This need is enhanced, moreover, by the realization that axon growth cones are probably guided by a variety of influences, both attractive and repulsive, operating locally at the cell surface and over longer distances by diffusing within the extracellular matrix [see ref 1 for review]. One promising in-vivo system concerns the development of spinal nerve segmentation in higher vertebrate embryos. In this article we review progress in identifying the mechanisms and candidate mole- cules involved. We argue that the simultaneous operation of contact repulsion/ inhibition and chem- oattraction are required to explain the full pattern of axon outgrowth in the axial-trunk region of the embryo. We also suggest a prominent role for carbohydrate moieties in mediating repulsion. Spinal nerve segmentation We originally observed that spinal motor and sensory axons are confined to the anterior (A, rostral) half of each somite-derived sclerotome during their lateral outgrowth from the chick spinal cord region, avoiding posterior (P) half-sclerotome even after its surgical displacement in ovo . 2 This restriction, which also applies to migrating trunk neural crest cells, 3 ensures that the spinal nerves eventually acquire the correct anatomical relationship with the flexible vertebral column. In principle it could result from attractive influences in the anterior half-somite and/ or repul- sive influences in the posterior half-somite. 2 The latter possibility is strengthened by the finding that somite detergent extracts, incorporated into liposomes, induce reversible collapse of sensory axon growth cones extending on a laminin substrate in vitro . We envisage that somite-derived proteins present in the liposomes trigger collapse by interaction with appro- priate growth cone receptors. 4 Consistent with the repulsion hypothesis, chick motor axon growth cones also avoid posterior sclerotome cells in vitro by turning or branching after filopodial contact. 5 A means of identifying candidate repulsion mole- cules was suggested by the observation that peanut agglutinin (PNA, a lectin with specificity for Gal 1–3 GalNAc residues, see below) binds to posterior half- sclerotome cells but not to those of the anterior half. 6 Using PNA-affinity chromatography we isolated a PNA-binding glycoprotein fraction from chick somites, and identified two major components (M r 48,000 and 55,000) on one-dimensional SDS PAGE separations that are absent from anterior half- sclerotomes but present in posterior halves. Poly- clonal antibodies raised against these components were used to immunodeplete all the growth cone collapse-inducing activity in somite detergent extracts, and were found to stain only the posterior half- sclerotomes in tissue sections. 4 We hypothesize that expression of this material in the posterior half- From the Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY and *Department of Genetics and Biometry, University College London, Wolfson House, 4 Stephenson Way, London NW1 2HE, UK seminars in THE NEUROSCIENCES, Vol 8, 1996: pp 339–345 ©1996 Academic Press Ltd 1044-5765/96/060339 + 07 $25.00/0 339