The Migration of Neural Crest Cells and the Growth of Motor Axons Through the Rostral Half of the Chick Somite

/. Embryol. exp. Morph. 90, 437-455 (1985) 437 Printed in Great Britain © The Company of Biologists Limited 1985

The migration of neural crest cells and the growth of motor axons through the rostral half of the chick somite

M. RICKMANN, J. W. FAWCETT The Salk Institute and The Clayton Foundation for Research, California division, P.O. Box 85800, San Diego, CA 92138, U.S.A. AND R. J. KEYNES Department of Anatomy, University of Cambridge, Downing St, Cambridge, CB2 3DY, U.K.

SUMMARY We have studied the pathway of migration of neural crest cells through the somites of the developing chick embryo, using the monoclonal antibodies NC-1 and HNK-1 to stain them. Crest cells, as they migrate ventrally from the dorsal aspect of the neural tube, pass through the lateral part of the sclerotome, but only through that part of the sclerotome which lies in the rostral half of each somite. This migration pathway is almost identical to the path which presumptive motor axons take when they grow out from the neural tube shortly after the onset of neural crest migration. In order to see whether the ventral root axons are guided along this pathway by neural crest cells, we surgically excised the neural crest from a series of embryos, and examined the pattern of axon outgrowth approximately 24 h later. In somites which contained no neural crest cells, ventral root axons were still found only in the rostral half of the somite, although axonal growth was slightly delayed. These axons were surrounded by sheath cells, which had presumably migrated out of the neural tube, to a point about 50 jan proximal to the growth cones. With appropriate antibodies we found that the extracellular matrix components fibronectin and laminin are evenly distributed between the rostral and caudal halves of the somite. Neither of these molecules therefore plays a critical role in determining the specific pathway of neural crest cells or motor axons through the rostral half of the somite.

INTRODUCTION The cells of the neural crest are remarkable for the length and complexity of their migration pathway, and the number of cell types into which they differentiate (reviewed in Le Douarin, 1982). Perhaps the only comparable structures in terms of complexity of migratory behaviour are the growth cones of nerve fibres. It is hardly surprising, therefore, that the mechanisms controlling neural crest migration and axon outgrowth have been two of the most intensively studied processes in embryogenesis. Over the years it has been possible to establish which tissues are derived from neural crest (Horstadius, 1950; Weston, 1963; Johnston, 1966; Noden, 1975), more

Key words: neural crest, somite, axonal guidance, fibronectin, laminin. 438 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES recently with the technique of chimaeric grafting, in which quail cells are grafted into chick embryos (Le Douarin, 1973; Le Douarin & Teillet, 1974). The approxi- mate pathway of neural crest migration has also been elucidated, using this and a variety of other anatomical techniques. Recently, it has been possible to analyse the migration of crest cells with greater precision using immunohistochemical methods and monoclonal antibodies which bind selectively to the migrating neural crest cell surface (Vincent, Duband & Thiery, 1983; Vincent & Thiery, 1984), and it is a series of experiments using this technique which we report on here. Some progress has also been made in defining mechanisms which might be responsible for causing neural crest cells to migrate along their chosen pathways. In this respect much attention has focused over the last few years on the extracellular matrix component fibronectin, which is present in high concentration in the pathways followed by neural crest cells (Newgreen & Thiery, 1980; Mayer, Hay & Hynes, 1981; Duband & Thiery, 19826), is a good substrate for crest cell migration in culture (Rovasio et al. 1983), and, when coated onto microbeads, affects their transportation down the neural crest migration pathway (Bronner- Fraser, 1982). Shortly after truncal crest cells start to migrate ventrally past the neural tube, the presumptive motor axons begin to grow out from the neural tube into the somites. It has recently been reported that these motor nerve fibres grow only through the rostral half of each somite, and do so initially from only those portions of the neural tube immediately adjacent to the rostral half of each somite (Keynes & Stern, 1984). The specificity for this selective outgrowth lies in the cells of the somite itself, since in embryos with rostrocaudal rotations of the segmental plate the outgrowth of the motor nerve fibres is still restricted to the half of the somite which would, in an unoperated animal, have lain rostrally (Keynes & Stern, 1984). Moreover, ablating the somitic mesoderm with X irradiation results in motor axons growing out evenly along the length of the neural tube (Lewis, Chevallier, Kieny & Wolpert, 1981). In this paper we describe in detail the path taken by the neural crest cells through the somites, as revealed by staining them with the two monoclonal antibodies NC-1 (Vincent etal 1983) and HNK-1 (Tucker etal 1984). We report that neural crest cells demonstrate the same specificity as motor axons, and, like them, migrate through the rostral half of each somite. We also report the results of experiments in which we have ablated the neural crest, and examined the subsequent pathway of the motor nerve fibres through crest-free somites. We show that the motor fibres are not guided through the rostral half of the somite due to the presence of crest cells within it, since they still grow specifically through the rostral half when it is completely free of neural crest cells. We also describe the distribution of the extracellular matrix components fibronectin and laminin, and speculate on the possible roles of these molecules in guiding neural crest cells and nerve fibres. Neural crest migration and axon outgrowth 439

MATERIALS AND METHODS Immunocytochemistry The embryos were transferred from the egg into a dish filled with PBS (phosphate-buffered saline without Ca2+ and Mg2"1"), where they were carefully freed from the surrounding membranes, stretched out and pinned down. Thereafter, the embryos were fixed for 1 h at room temperature, in a fixative consisting of 1-25 % glutaraldehyde, 1 % paraformaldehyde and 3-5 % sucrose in O-lM-phosphate buffer at pH7-3. After rinsing in PBS the embryos were encap- sulated by placing them into a solution of 20 % BSA (bovine serum albumin) in 0-1 M-sodium phosphate at pH7-2 which was polymerized with 25 % glutaraldehyde (1 % in final volume). The resulting blocks were then hardened for 1 h in the original fixative. Since the blocks were transparent they could be easily trimmed for transverse, horizontal or parasagittal sectioning of the embryos. Sections were cut either on a vibratome at a thickness between 100 and 150 jum or, after soaking in 20% phosphate-buffered glycerol, on a freezing microtome at 50 to 100 fjm. They were collected in wells containing Tris-buffered saline (TBS: 50mmol Tris, 150mmol NaCl, pH7-6). The immunocytochemical method utilized the avidin-biotin-peroxidase procedure. Free-floating sections were passed through the following incubation steps at room temperature, while being gently agitated. (1) Non-specific antibody binding was blocked by incubation for 2-3 h in 0-02 % sodium azide, 0-1 M-L-lysine., 1 % BSA and 1:10 serum (from the same species as the secondary antibody) in TBS. The same solution was used to dilute both the primary and secondary antibodies. (2) Without rinsing, the sections were transferred to the primary antibody for 12-24 h. The following dilutions were used for the various antibodies employed in this study. Goat anti-chicken fibronectin (Calbiochem, Lot 001428) was diluted 1:2000, rabbit anti-laminin serum (EY labs no. AT 2404, lot no. 020106) 1:100, prediluted hybridoma ascites containing anti-HNKl antibodies (Becton Dickinson anti Leu-7, no. 7390, lot no. 60914) 1:50 and monoclonal anti-NCI ascites 1:500. Normal serum and control ascites were used at the same concentrations as controls. (3) Sections were rinsed in TBS, three changes of 45min each, and then floated onto biotinylated secondary antibody (Vector laboratories), diluted 1:250, for 6-8h. (4) Rinsed three times in TBS and left in TBS overnight. (5) Endogenous peroxidase activity was blocked with 0-3% H2O2 in methanol for 30min. (6) Rinsed three times in TBS, 5min each change. The last two steps were omitted in some preparations in order to improve the structural preservation of the rather fragile sections. (7) Incubated with Avidin-coupled horseradish peroxidase complex (Vector laboratories) for 3 h at 1:100 dilution in 1 % BSA in TBS. (8) Rinsed four times, 1 h each change, in TBS. (9) 0-01 % 3,3'-diaminobenzidine tetrahydrochloride (DAB) and 0-03 % H2O2 in TBS for 10-15 min. (10) Rinsed four times, 10min each change, in TBS. (11) Transferred to PBS. At this stage of the procedure, some of the sections were mounted on gelatinized slides, air dried, dehydrated in ethanol and xylene and coverslipped in DPX. In order to obtain higher resolution and better tissue preservation, in some cases the reaction product was intensified and the sections were embedded in epoxy resin using the following procedure. (12) 0-01 % OsO4 in PBS for 15 min. (13) Rinsed four times, 10 min each, in PBS. Subsequently the sections were dehydrated in a graded series of ethanols and propylene oxide, and then infiltrated with Spurr's low viscosity embedding medium. The sections were flattened between glass slides, one of which was coated with a mould release compound, and polymerized at 70°C for one day. Selected sections were attached to the flat surface of a plastic block, then heated up and detached from the slide. These sections were resectioned in an ultramicrotome to give semithin sections, which allowed optimum identification of labelled cells and nerve fibres.

Crest ablations These were performed on embryos which had been incubated at 38°C for about 48 h, and which had between 13 and 19 somites. The egg was windowed, a few drops of Hanks balanced salt solution were dripped onto it, and then about 0-02 ml of india ink (Pelikan fount india diluted 1:1 with Hanks) was injected under it to render it visible. The vitelline membrane overlying the embryo was then cut. The operations were performed with electrolytically sharpened tungsten needles, one in the shape of a hook, and another in the shape of a flat knife blade. The hook was run up the inside of the neural tube, reopening it, and then the knife was 440 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES used to cut against it, so as to remove approximately the dorsal half of the neural tube. Tissue was removed in this way from the neural tube opposite the six to eight most caudal somites, and from about half this length of neural tube opposite the undivided segmental plate. The eggs were then resealed with Scotch tape and returned to the incubator until they had matured to Hamburger & Hamilton stage 18 to 21 (Hamburger & Hamilton, 1951). They were then processed in the same way as described above for unoperated embryos.

RESULTS HNK-1 and NC-1 staining We find that the antibodies directed against these antigens bind to the surface of neural crest cells, the extracellular area around the notochord and to outgrowing nerve fibres (Vincent etal. 1983; Vincent & Thiery, 1984). We have concentrated on embryos at three stages of development, stages 13, 15 and 17, which corresponds to 19, 24-27 and 29-32 somites respectively. In any given embryo, there is a gradient in segmental development along the longitudinal axis, the youngest stages being found where the segmental plate is dividing into somites, and the most advanced next to the otic vesicle. At these stages the distribution of neural crest cells is the same a given number of somites rostral to the region where somites are forming from the segmental plate, regardless of the actual age of the embryo. Therefore, we will give a generalized account of the changes in the pattern of the crest cells with time, using the rostrocaudal developmental gradient of the embryo as our time axis, instead of describing the appearance of each age of embryo individually. We first saw antibody binding to neural crest cells about three somites rostral to the most caudal somite. These cells are on either side of the midline, dorsal to the neural tube, and are evenly spread along the longitudinal axis of the embryo. About three somites rostral to this, the crest cells show the first signs of migration around the lateral sides of the neural tube. The very first cells to migrate pass laterally to, and in between, the somites, but the number of migrating crest cells at

Fig. 1. (A) A section, cut slightly obliquely from the horizontal, from a stage-15 chick embryo, stained with anti-HNK-1. Rostral is to the right, and lateral is above. The left- hand somite is the seventh rostral to the most recently formed somite. These are therefore young somites, which are not yet fully dissociated into dermomyotome and sclerotome, and in which there is no obvious morphological divide between the rostral and caudal halves. The early pattern of neural crest migration is seen here. There are some crest cells in the intersomitic space, particularly in the more caudal somites, but there are also some cells which have entered the sclerotome, and become intermixed with sclerotomal cells. However, all these cells which have entered the somite are restricted to its rostral half. Bar equals 200 jum. (B) A semithin section taken from the middle somite in the illustration above. The cells with dark reaction product around them are neural crest cells, and can be seen to have mingled with the cells of the somite. (C) Transverse semithin section from the rostral half of the somite 13 or 14 rostral to that most recently formed in a stage-13 embryo, stained with anti-HNK-1. Dorsal is upwards. Only half of the cross-section of the embryo is included in this frame; half of the neural tube can be seen on the upper left of the picture. Neural crest cells can be seen migrating around the neural tube, and through the somite. Some crest cells are intermixed with the cells of the sclerotome. Bar equals 50/mi. Neural crest migration and axon outgrowth 441

this stage is small; about a dozen cells per somite in any 150 jUm section. Very soon the crest cells start to penetrate into the somite. Despite the fact that the somite shows no morphological sign of division into rostral and caudal halves at this stage, the migrating crest cells are all restricted to the rostral half of each somite (see Fig. 1). As the somite ages, the number of crest cells seen within it increases rapidly. These cells are always restricted to the rostral half of the somite, where they

¥.i:':#^, 442 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES occupy the space beneath the dermomyotome and they progressively penetrate deeper and deeper into the sclerotome, while apparently avoiding the notochord. This pattern probably reflects the migratory pathway which neural crest cells take through the sclerotome. Neural crest cells are evenly spread along the dorsal aspect of the neural tube, but more ventrally they become progressively localized to the rostral half of the somite, until, around the area where the ventral root emerges, all the crest cells are restricted to the rostral halves of the somites (see Figs 2, 3). About 18 somites anterior to the most recently formed somite, the somite becomes split morphologically into two halves, separated by a small cleft (von Ebner's fissure). Slightly before this, about 12 to 15 somites rostral to the most recently formed somite, the first nerve fibres grow out from the ventral aspect of the neural tube. As they extend through the sclerotome ventrolaterally towards the myotome they grow through an environment occupied by both sclerotomal and neural crest cells. From this stage onwards, until the latest stages we have studied, the pattern of neural crest staining alters little. The only significant change is that many antibody-binding cells associate with the outgrowing nerve fibres. Having migrated through the somite, the neural crest cells accumulate in the various structures ventral to it, for instance around either side of the aorta. Here, however, the crest cells are evenly spread along the axis of the embryo. The pattern of crest migration is rather different in the somites immediately caudal to the otic vesicle. Here, as described previously (Le Douarin & Teillet, 1974), a large proportion of the neural crest cells migrate between the ectoderm and the dermomyotome, although some cells do find their way down the lateral sides of the neural tube, and into the sclerotome. In addition, at cranial levels the pattern of crest cells becomes complicated by the craniocaudal migration of vagal neural crest cells. Control sections, treated with either control ascites, control whole serum, or with monoclonal antibodies derived from the same parent myeloma line but with a different specificity, showed no immunoreactivity comparable to that seen with HNK-1.

Fig. 2. These four sections are taken from a single somite (about no. 18) of a stage-15 embryo, stained with anti-HNK-1; dorsal is above. The sections are roughly equally spaced through the somite, which is about 180 jtan in length. (A) is from the caudal part of the somite. Neural crest cells are seen around the dorsal aspect of the neural tube, and ventral to the sclerotome, but none are seen in between. In (B), the picture is similar, but there are a few neural crest cells around the region where the ventral root nerve fibres leave the neural tube, and also, on the right side, a few cells around the edge of the sclerotome. (C) is taken slightly rostral to the middle of the somite. Neural crest cells here are present in the lateral part of the sclerotome in larger numbers, and in (D), which is taken from the rostral part of the somite, there are many crest cells intermixed with the sclerotomal cells. Note that the restriction of crest cells to the rostral half of the somite applies only to the region of the sclerotome; crest cells are evenly spread all along the axis of the embryo both at the dorsum of the neural tube, and ventral to the somite. Some crest cells can be seen migrating around the aorta. Bar equals 100 jum. Neural crest migration and axon outgrowth 443

Embryos with neural crest ablations, stained with NC-1 and HNK-1 These animals were operated on between Hamburger & Hamilton stages 11 to 13 (which corresponds to between 13 and 19 somites), and were killed between stages 18 and 21, approximately 24 h later. By this time motor nerve fibres have

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JCl\ . ^7^. ^' 444 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES grown out of the neural tube along most of its length. At the time when they were killed, the embryos looked almost normal, except that many of them had a degree of kinking of the neural tube around the region of the operation, and usually a length of neural tube that had not fully closed dorsally. Antibody staining of horizontal sections of the embryos revealed that crest cells had migrated longitudinally from the edge of the ablated region so as to fill in partially the area from which neural crest had been removed. Thus, although the original operation had removed the dorsal half of the neural tube over a distance of more than 8 somites, dorsal root ganglia were only completely absent over a length of 2 to 4 somites, and often only 1 or 2 somites were entirely free of crest cells. In all we produced nine embryos with successful crest ablations, and these had between 1 and 4 somites which were both completely clear of crest cells, and also contained motor axons. These were usually surrounded by somites in which there were very few crest cells. There were also regions in four of the embryos in which there were no axons growing from the neural tube, usually where the tube had been largely cut away. In general it was not difficult to identify axons, crest cells and presumptive Schwann cells, since the crest cells have a very distinctive, irregular shape, whereas axons, and the cells surrounding them, have a smooth outline. The motor axons were always found in the rostral half of the somite, even in somites which were entirely free of crest cells (see Fig. 4). They appeared to be following an entirely normal pathway, which, in the region of the somites, is virtually identical to the migration pathway of the neural crest cells. The axons growing through crest-free somites were not entirely naked. From the neural tube to approximately 50 ^m behind their growing tips they were encased by cells with a smooth outline, which showed NC-1 and HNK-1 immunoreactivity. These are almost certainly presumptive Schwann cells, which are stained by both antibodies in normal animals. We think that these cells probably migrated out from the neural tube and down the nerve fibres, as described in previous publications (Harrison, 1924; Yntema & Hammond, 1954; and see Horstadius, 1950). Certainly, we could see no neural crest cells migrating from the dorsal aspect of the neural tube to the

Fig. 3. Horizontal section of a stage-17 embryo, stained with anti-HNK-1. Rostral is to the left, and the leftmost somite is two somites caudal to the otic vesicle. The section is taken at the level of the notochord, which can be seen running between the somites over most of the section. The more rostral somites are clearly divided in two, and the HNK-1 staining (neural crest cells and nerve fibres) is restricted to the more rostral of the two halves. More caudally, the plane of section goes through the neural tube, and here the ventral roots can be clearly seen. Bar equals 200 /an. (B) This is a section from the same embryo as in (A), but the section is more dorsal, so as to cut through the neural tube about half way through it. The plane of section is slightly off the horizontal, so the lower side is slightly more dorsal than the upper. Rostral is to the left. Neural crest cells are almost evenly distributed along the axis of the animal dorsally, but start to form into segmental groups more ventrally. Bar equals 200 jum. (C) A horizontal semithin section from a stage-18 embryo taken just ventral to the level of the notochord. The neural crest cells are clearly restricted to the rostral half of each somite. Crest cells are intermingled with unstained cells. Bar equals 100 [jm. Neural crest migration and axon outgrowth 445

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ventral root axons. The growth cones in crest-free somites were clearly naked, and not in contact with any antibody-staining cells; we saw growth cones in the sclerotome, and on the myotome. The fibres looked essentially normal, but we formed the impression that the growth cones were rather more spread out in crest- free somites than in somites which contained crest cells. The axons in operated somites were generally slightly retarded in their development (see Fig. 5). In several pairs of somites, there were no crest cells on one side and an incomplete

B Neural crest migration and axon outgrowth 447 crest ablation on the other; here the nerve fibres appeared to have grown slightly further from the neural tube in the somite which still contained some crest cells. The somites adjoining those which were completely free of neural crest cells often had only a very small number of crest cells in them, and no dorsal root ganglion. In these somites, the crest cells were generally closely associated with the nerve fibres.

Fig. 5. This is a transverse section taken from an animal which was operated on to remove its neural crest. All the neural crest was successfully removed from the right side, but some remained on the left. On the left, crest cells can be seen migrating along their normal pathway through the somite. On the right, there are no crest cells, and the nerve fibres growing out from the ventral aspect of the cord are seen. Bar equals 50 /«n.

Fig. 4. (A) Horizontal section from an embryo which had a neural crest ablation at the 15-somite stage, was killed at stage 19, and stained with anti-NC-1. Only the middle somite on either side is entirely free of neural crest cells; the surrounding somites have small numbers of stained cells within them (arrowed). Some erythrocytes have also shown up because they contain an endogenous peroxidase. The pattern of outgrowth of the ventral root nerve fibres is essentially normal in the crest-free somites, being restricted to the rostral half of the somite. The nerve fibres appear thick in this picture, because they are surrounded by sheath cells proximally, which stain with anti-NC-1. Bar equals l/im. (B) A horizontal section from another crest-ablated embryo. The neural tube is at the lower edge of the frame. The left-hand somite is entirely crest free, and the nerve fibres are all in its rostral half. The plane of focus is through the tips of the axons, which are not covered by sheath cells. The somite on the right has one neural crest cell in the plane of focus (arrowed). Bar equals 50jum. (C) Transverse section through the rostral half of a somite from an animal with a neural crest ablation. Only half of the cross-section is included in this picture. The nerve fibres are clearly shown growing ventrolaterally through the crest-free somite, taking much the same pathway as neural crest cells would normally follow. There are no crest cells in this somite migrating down from the dorsal aspect of the neural tube. Bar equals 50 fjm. 448 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES

Staining for fibronectin Our results on the distribution of fibronectin are essentially in agreement with previous studies (Newgreen & Thiery, 1980; Mayer et al 1981; Duband & Thiery, 19826). Fibronectin is present at the earliest developmental stage we studied. The molecule is rather ubiquitous in its distribution, but initially it is concentrated in the extracellular spaces between structures; thus there is a high concentration of fibronectin around the neural tube, and around the somites. Later, there is also fibronectin staining within the somite, throughout the sclerotome, although its concentration always remains highest around the neural tube, notochord, and between the somites (see Fig. 6). At no time is there any difference in staining between the rostral and caudal halves of the somite.

Staining for laminin The amount of laminin in the youngest embryos which we examined is probably rather low, since we were only able to see any staining by using high concentrations of anti-laminin, which resulted in a certain amount of non-specific back- ground antibody binding. Up to the stage when the myotome begins to differentiate, the distribution of laminin present resembles that of fibronectin. The molecule is mainly found in the extracellular space around the neural tube, and between the somites (see Fig. 7A,B). In later stage embryos, from stage 17 onwards, strong laminin staining is seen in three main regions: around the developing myotome, around motor axons, and around the dorsal root ganglia (see Fig. 7B,C). The staining associated with axons does not extend up to their growing tips, and only seems to appear as they mature. We think that this staining is not associated with the axons themselves, but is on the surfaces of differentiating Schwann cells. Certainly adult Schwann cells secrete laminin and other basal lamina components (Cornbrooks etal. 1982; Cornbrooks etal. 1983). The laminin staining associated with the dorsal root ganglia appears late, and is not really marked until stages 22-26, the oldest embryonic stages at which we have performed laminin immunohistochemistry. The laminin is found only in the capsule of the ganglion, and associated with the sensory nerve fibres. The myo- tomal cells appear to produce laminin, resulting in clear longitudinally oriented tubes of reaction product. Outgrowing motor axons come into contact with the myotome, but laminin seems to appear in large quantities at the myotome cell surfaces some time after the first growth cones have reached them, The myoblasts migrating from the myotome into the limb (Chevallier, Kieny & Mauger, 1977) do not stain for laminin, and neither do any other cells in the path of the first nerve fibres growing into the limb.

DISCUSSION In the chick embryo, truncal neural crest cells are first detectable on the dorsal aspect of the neural tube about three or four somites rostral to the region where somites begin to differentiate from the segmental plate. Here they pause briefly, Neural crest migration and axon outgrowth 449

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Fig. 6. These illustrations show the pattern of fibronectin staining in the chick embryo. (A) is from a stage-16 embryo stained with anti-fibronectin antibody. The strongest staining is seen around the neural tube, notochord, and around the dermomyotome, particularly laterally. There is also less intense fibronectin staining throughout the sclerotome The neural tube and dermomyotome are unstained. Bar equals 50/an. (B,C) are photomicrographs of the same section, taken at different planes of focus. The embryo was fixed at stage 15, and this is about the 10th somite rostral to the most recently formed somite; the somite is younger than that illustrated in (A), since it has not yet fully dissociated into sclerotome and dermomyotome. In (B) the plane of focus goes through the intersomitic cleft, showing the intense reticular staining for fibronectin here. In (C) the plane of focus is on the centre of the somite; there is dark staining around the somite, but little inside it. Bars equal 100/an. 450 M. RlCKMANN, J. W. FAWCETT AND R. J. KEYNES

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Fig. 7. These pictures illustrate the pattern of anti-laminin staining at different stages of somitic development. (A) is a horizontal section from a stage-13 embryo, around somite number 8. The staining pattern is very similar to that of fibronectin at this stage, being concentrated around the somites and neural tube. Bar equals 100/mi. (B) is a transverse section from a stage-17 embryo. There is intense laminin staining around the neural tube and the dermomyotome, and faint staining throughout the rest of the somite. However, there is no staining at all in the neural tube. Bar equals 50/mi. (C) This is a horizontal section from a stage-26 embryo, taken through the level of the developing dorsal root ganglia. Two ganglia are surrounded by laminin staining, as are fascicles of axons going leftward towards the neural tube. To the right of the picture, the myotomes are darkly stained. Bar equals 100 jian. (D) A section from the same embryo, through the level of the ventral roots. The ventral root axons, surrounded by anti-laminin-stained sheath cells, are coming out of the neural tube on the left of the frame, the outside of which is also stained. The heavily stained myotome is on the right. Bar equals 50/an. Neural crest migration and axon outgrowth 451

before beginning to migrate ventrally and ventrolaterally. Their subsequent migration takes them almost throughout the body to parent all the neuronal and glial cells of the autonomic and sensory ganglia, the adrenal medullary cells, the Schwann cells, and the melanocytes of the skin (reviewed by Le Douarin, 1982). All the crest cells which migrate from the dorsal aspect of the neural tube, except for the progenitors of the melanocytes, have to pass down the lateral aspect of the neural tube, and then through the region of the somites. Our findings on the earliest migratory pathway of neural crest cells through the somites are similar to those reported in previous publications on the subject (Weston, 1963; Le Douarin & Teillet, 1974; Thiery, Duband & Delouvee, 1982; Vincent & Thiery, 1984). We find that the very first crest cells to migrate ventrally, which are few in number, take a pathway down either side of the neural tube, and then go around the somites, either medially, laterally, or between them. However, shortly after this first phase of crest migration, the neural crest cells start to migrate between the dermomyotome and sclerotome, and through the sclerotome itself, in which regions they are restricted to the rostral half of each somite. They then progressively populate the rostral half of the sclerotome. The specific restriction of the migration of neural crest cells to the rostral half of the somite has not been noted by previous authors. There has been some controversy in the past as to the extent to which migrating crest cells pass through the sclerotome. On this point our results are in general agreement with those of Weston (1963), who described neural crest cells migrating through the sclerotome, but are somewhat at variance with those of Thiery and others, who have suggested that most neural crest cells migrate around the somite, circumventing the sclerotome. However, crest cells are only seen in the more lateral regions of the sclerotome; they never come close to the notochord, and in fact neural crest cells in culture seem to be repelled by explanted notochord (Newgreen, Scheel & Kastner, 1985). A few hours after neural crest migration has begun, the first motor nerves begin to grow out of the neural tube, and through the sclerotome of the somite towards their targets. The motor nerves do not grow out evenly along the length of the neural tube, but instead emerge opposite the rostral half of each somite, and it is specifically through this half that they grow (Keynes & Stern, 1984). At the time when the first axons grow out from the neural tube, there are many crest cells around the lateral aspect of the tube, aligned with the rostral half of the somite, in the sclerotome. Since the distribution of crest cells so exactly matches the pathway of motor axon growth, we were obviously interested to find out if there is any causal relationship between the two; are the axons guided specifically through the rostral half of the somite by neural crest cells? In order to test this hypothesis, we operated on a series of chick embryos to remove their neural crest. We allowed these embryos to survive to a stage when motor axon outgrowth is well under way in normal animals, and then stained them with HNK-1 or NC-1, in order to demonstrate both the crest cells and the axons. We saw several somites which were quite free of crest cells, but through which motor nerve fibres were growing. These 452 M. RICKMANN, J. W. FAWCETT AND R. J. KEYNES motor axons were always restricted to the rostral half of the somite. The presence of crest cells is not, therefore, a prerequisite for this specific axon outgrowth. However, this is not to say that there is no interaction between nerve fibres and crest cells. In somites in which the crest removal was incomplete, the few remaining crest cells tended to be closely associated with the axons, indicating that axons and crest cells may be mutually adherent. Moreover, the ventral root axons in crest-ablated segments seemed to be growing more slowly than those in somites which contained crest cells. One cannot be sure that this was not simply due to non-specific effects of the operation, but it could be that the outgrowing axons often do adhere to neural crest cells, and this hastens their outgrowth. However, whatever the possible interactions of presumptive motor axons and neural crest cells, it seems very likely that both crest cells and axons are able to respond to the same cues in the somitic environment; there may be one or more molecules which are distributed differently in the two halves of the somite and which are attractive or repulsive to both axons and crest cells. The antigen recognized by HNK-1 antibodies is found on a family of related molecules, among which are myelin-associated glycoprotein, LI and N-CAM (Kruse etal. 1984), and Jl (Kruse etal. 1985). However, in the published studies of the immunohistochemical localization of N-CAM (Thiery, Duband, Rutishauser & Edelman, 1982) there is no evidence of any specific staining of neural crest cells. Moreover, since there was no differential distribution of HNK-1 immunoreactivity in the sclerotome during the migration of neural crest cells, it appears unlikely that any molecule on the sclerotomal cells bearing the HNK-1 epitope determines the path of neural crest cells or motor axons through the rostral half-sclerotome. In recent years a good deal of evidence has accumulated to show that both neural crest cells and growing axons can have their pathways and rates of growth influenced by basal lamina components. The time of onset of neural crest migration closely follows the time of appearance of fibronectin at high concentration in the migratory pathway (Newgreen & Thiery, 1980; Mayer etal. 1981; Duband & Thiery, 1982/?; Mitrani & Farberov, 1982; Duband & Thiery, 1982a), and neural crest cells in tissue culture migrate particularly well on fibronectin, an effect which is blocked by anti-fibronectin antibodies (Rovasio et al. 1982). Moreover, latex microspheres, which will normally migrate with crest cells when injected into their pathway, will not migrate when coated with fibronectin or laminin (neural crest cells themselves are fibronectin and laminin negative) (Bonner-Fraser, 1982). Fibronectin, therefore, may well play at least a permissive role in the control of neural crest migration. Nerve fibres grow very well in vivo on basal lamina (McMahan, Edgington & Kuffler, 1980; Keynes, Hopkins & Huang, 1984; Scherer & Easter, 1984; Fawcett & Keynes, 1985), and in vitro both fibronectin and laminin are good growth substrates for many types of axon (Rogers et al. 1983; Manthorpe etal. 1983; Edgar, Timpl & Thoenen, 1984; Lander, Fujii & Reichardt, 1985). We therefore stained a series of chick embryos with anti-fibronectin and anti-laminin antibodies, to see whether either molecule was restricted to the rostral half of the somite. However, it appears that both are evenly distributed between rostral and Neural crest migration and axon outgrowth 453 caudal halves. Fibronectin is present in large amounts around the sclerotome, and also in smaller amounts within it, at the times when crest cells migrate and motor nerve fibres grow out; whereas laminin is initially distributed around and between the somites, and is subsequently associated with the presumptive Schwann cells and with the developing myotome. Certainly, neither laminin nor fibronectin shows any preferential localization in the rostral half of the somite. Our feeling, therefore, is that fibronectin, and possibly laminin, may need to be present in order for crest cells to migrate normally; but since both fibronectin and laminin are equally distributed in the rostral and caudal halves of the somite (as is N-CAM, another possible candidate for guiding axons and crest cells (Thiery et al. 1982)), there must be one or more other factors present which guide axons and crest cells specifically through the rostral half-somite.

We are indebted to Dr Jean-Paul Thiery for his gift of anti-NC-1 antibody. We would like to thank Steve Pfeiffer and Jim Rokos for their skilled technical assistance. We also thank Dr Marianne Bronner-Fraser for her help and encouragement, and Dr W. M. Cowan for his support, and his many helpful suggestions on the manuscript. R.J.K. was in receipt of a Wellcome Trust travel grant when this work was done.

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(Accepted 24 September 1985)