During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and ...
DEVELOPMENTAL
BIOLOGY
113,429-435
(1986)
Distribution of Laminin in the Developing Peripheral Nervous System of the Chick’ SHERRY
L. ROGERS,’ KATHRYN
Deptrrtmwt
of Anatomy,
J. EDSON, PAUL C. LETOURNEAU,
4-135 Jucksox Hull,
hi?-ersity
AND STEVEN C. MCLOON
of‘Minnesota, Mir~wupolis.
Minnesota 55455
During axonal elongation in the developing peripheral nervous system, the temporal and spatial distribution of adhesive molecules in extracellular matrices and on neighboring cell surfaces may provide “choices” of pathways for growth cone migration. The extracellular matrix glgcoprotein laminin appears in early embryos and mediates neuronal adhesion and neurite extension in vitro. In this study, we have examined the distribution of laminin at early periods of peripheral nervous system development. The distribution of laminin, demonstrated by immunostaining frozen sections of chick embryos, was compared to the distribution of fibronectin and of early peripheral neurites as revealed with an antibody to a neurofilament-associated protein. Laminin is present in the neural tube basement membrane, in early ganglia, and in developing dorsal and ventral roots, where the laminin staining pattern parallels that of neurofilaments. In early ganglia and nerve roots, laminin immunostaining defines loose “meshworks” rather than basement membranes, which seem to form slightly later in these structures. In contrast, fibronectin is absent in neural tube basement membrane, ganglia, and nerve roots, although it is present along neural crest migratory pathways and in intersomitic spaces. Our observations of laminin distribution are consistent with the possibility that laminin provides an adhesive surface for ‘c 1986 Academic Press, Inc neurite extension at some stages of early peripheral nervous system development. INTRODUCTION
Guidance of nerve fibers extending into the peripheral nervous system is likely to involve the influence of environmental cues. Growth cones of ventral horn axons, for example, have multiple interactions with their surroundings, depending upon their location along pathways to synaptic sites (Tosney and Landmesser, 1985). These interactions include the close proximity of growth cones to extracellular matrices, nonneuronal cells, and neighboring nerve fibers, but the molecular basis of such contacts is just beginning to be defined. Laminin is an extracellular matrix glycoprotein that mediates cellular adhesion to a variety of surfaces (see Yamada, 1983, for review). Synthesis of laminin, along with other matrix molecules, is developmentally regulated and may be involved in cell and tissue differentiation throughout embryogenesis (Wartiovaara et al., 1980; Trelstad, 1984). The appearance of laminin in early embryos and its interactions with neurons in vitro (BaronVanEvercooren et al., 1982; Rogers et al., 1983), raise the possibility that this molecule has a role(s) in nervous system development. ’ This work was supported by National Institutes of Health Grants NS17192 to P.C.L. and EY05371 and EY05372 to S.C.M. and National Science Foundation Grant PCM8203855 to P.C.L. ‘To whom correspondence should be addressed; Department of Anatomy, Basic Medical Sciences Bldg., University of New Mexico, Albuquerque, N. Mex. 87131.
Previous work has demonstrated that neuronal growth cones migrate preferentially along adhesive pathways (Hammarback et ah, 1985; Letourneau, 1975), suggesting that the precise distribution of adhesive molecules in embryos may be critical to directed axonal elongation. This is true of neural crest cells as well, which migrate through extracellular spaces prior to axonal growth in the peripheral nervous system. Potential roles of fibronectin in neural crest migration and subsequent peripheral nervous system development have been studied by several laboratories (Thiery et al, 1982; Erickson and Turley, 1983; Rovasio et ah, 1983; Weston et ah, 1984), but possible involvement of laminin in these events is still unclear (Bignami et al., 1984). W e have applied immunohistochemical techniques with antisera against laminin, fibronectin, and a putative neurofilament-associated protein to examine the relationship between neurons and these glycoproteins during the initial formation of dorsal root ganglia and dorsal and ventral roots of chick embryos. W e find that laminin appears very early in these structures, and the distribution of laminin immunoreactivity is strikingly similar to patterns of early nerve fiber elongation. MATERIALS
AND
METHODS
Chick embryos were removed to 0.1 M phosphate buffer, pH 7.4, and staged according to Hamburger and Hamilton (1951). Embryos between stages 13 and 25 were 429
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DEVELOPMENTAL BIOLOGY
used in this study. Embryos were fixed by immersion in 4% paraformaldehyde in 0.1 Mphosphate buffer, pH 7.4, for 2 hr at 4°C and stored in 20% sucrose in 0.1 Mphosphate buffer for 2-24 hr at 4°C. The embryos were embedded in OCT Compound (Lab-Tek Products) and frozen in liquid nitrogen. Serial sections (10 pm) were cut in longitudinal or transverse planes on a Slee cryostat, mounted on gelatin-coated slides, and stored at -20°C. The sections were processed for immunohistochemistry as follows: slides were warmed to room temperature, washed in PBS for 15 min, incubated with normal goat serum (Polysciences) diluted 1:lO in antibody buffer (0.1 Mphosphate buffer, 0.3% Triton X-100,0.05% sodium azide) for 15 min, incubated overnight in primary antibody in a humid chamber at 4”C, rinsed, and washed for 15 min in PBS. Following incubation in secondary antibody for 1 hr at 37°C slides were washed for 15 min in PBS, mounted in either buffered glycerin (pH 9) or Elvanol permanent mounting media, and stored at -20°C. Sections were examined with a Leitz Dialux 20 microscope equipped for epifluorescence. Primary antibodies used were affinity-purified rabbit anti-human plasma fibronectin (a generous gift of Dr. Leo Furcht) diluted 1:25 in antibody buffer, affinity purified rabbit anti-mouse EHS tumor laminin (gift of Dr. Leo Furcht) diluted 1:25, and E/C8, a mouse monoclonal antibody to a putative neurofilament-associated protein (generous gift of Dr. Gary Ciment and Dr. James Weston; see Ciment and Weston, 1982) diluted 1:50. To visualize neurites and laminin staining simultaneously, a mixture of E/C8 and anti-laminin was prepared at the concentrations indicated. Control slides were made by incubating adjacent sections with absorbed antiserum (anti-laminin passed over a laminin affinity column) or in antibody buffer alone during the primary antibody step. Secondary antibodies were fluorescein isothiocyanate (ITC) goat-anti-rabbit IgG (Cappel) and rhodamine ITC-labeled goat-anti-mouse IgG (Cappel), both diluted 1:lOO in antibody buffer. RESULTS
Stages 13-16. Cross sections used in this study were taken from the brachial region of the embryos. Sections of early embryos were examined for patterns of laminin, fibronectin, and E/C8 immunoreactivity. During this period migration of neural crest cells occurs, but neither peripheral ganglia nor dorsal or ventral roots have formed, and E/C8 staining is not yet evident. Laminin is present in the basement membrane surrounding the neural tube at stages 13-16 (Fig. 1A) and persists in this location at all stages examined (through stage 25). Meshworks, or punctate patterns of laminin staining can
VOLUME 113, 1986
be seen between the neural tube and dermomyotomes, an area occupied by sclerotome cells and neural crest cells (Thiery et al., 1982). Condensations of punctate laminin also appear in areas where crest cells coalesce to form dorsal root ganglia (Fig. 1A). Although laminin in these meshworks seems to be extracellular, it does not appear to be organized into discrete basement membranes as elsewhere in the embryo. Laminin is also present in basement membranes of the dermomyotome and blood vessels, around the notochord, and in the skin. In contrast to laminin, fibronectin staining around the neural tube, present at earlier stages, begins to disappear. Figure 1B shows fibronectin at the dorsal and lateral margins of the tube, probably in association with neural crest cells (Newgreen and Thiery, 1980; Thiery et ul., 1982), but it does not seem to be present in more ventral portions of the neural tube basement membrane. Fibronectin is present in extracellular spaces throughout the embryo and, like laminin, is present in skin, around the notochord, and in basement membranes of dermomyotomes and blood vessels. Stages 17-18. By stage 1’7, dorsal root ganglia are readily identifiable with both laminin and E/C8 immunostaining (Figs. 2A, B). As earlier, laminin in ganglia is punctate in appearance and well-defined ganglion capsules have not yet formed. In contrast to laminin, fibronectin is strikingly absent from the ganglia at all stages of development (see Fig. 3C). The distribution of laminin and fibronectin in basement membranes corresponds to that described for stages 13-16. Neurites emerge from the ventrolateral neural tube at stages 17-18 in the brachial region of the chick and from dorsal root ganglia at about the same time (Tosney and Landmesser, 1985). In longitudinal (craniocaudal) sections through the level of dorsal root ganglia, laminin can be seen in both ganglia and accumulations of sclerotome cells, which alternate along the length of the neural tube (Fig. 2A). Ganglia can be easily distinguished from sclerotome in sections double-stained for laminin and the E/C8 antigen, which clearly identifies neuronal processes. Processes of central nervous system neurons traveling along the inside of the neural tube basement membrane also stain with the antibody (Fig. 2B). As processes of sensory neurons extend from the ganglia centrally and peripherally, laminin is present along the paths that they trace (Figs. 2A, B). Laminin is also associated with ventral horn axons emerging from the neural tube (Figs. 2C; 3A, B). In both dorsal and ventral roots, neurites appear to extend through a loose cellular region containing laminin (box, Fig. 2A; open arrowhead, Fig. 2C). Basement membranes containing laminin (arrows, Fig. 2C), appear as the rootlets mature. The presence of laminin in the basement membrane of the neural tube may also be important, as growth cones migrate
431
FIG. 1. Cross sections through the brachial region of a chick embryo at approximately stage 16, stained with antibodies to laminin (A) and tibronectin (B). Laminin antibodies densely stain the basement membranes of the neural tube and dermomyotome (d), and define a meshwork in regions where dorsal root ganglia form (arrows, A). Fibronectin is present along paths of neural crest migration (long arrows, B) but begins to disappear from basement membrane of the neural tube (nt). Both fibronectin and laminin surround the notochord (n) and are present in blood vessel basement membranes (short arrows).
along this membrane before emerging into the periphery (Singer et ab, 1979). In some sections, as in Fig. 2C, basement membranes of the neural tube and ventral rootlets are seen to be continuous. Stage 25. By this stage, peripheral nerve roots are well developed (Fig. 3). As at earlier stages, laminin immu-
noreactivity is present in dorsal and ventral (Fig. 3A) roots, identified with the E/C8 antibody (Fig. 3B). Similar staining patterns were seen in heads of embryos where cranial nerves form. Laminin is also present in developing capsules of dorsal root ganglia. In contrast, fibronectin is strikingly absent from ganglia and nerve
DEVELOPMENTALBIOLOGY
VOLUME 113,1986
roots, and does not appear to be present in the neural tube basement membrane (Fig. 3C). In Fig. 3C, fibronectin immunoreactivity can still be seen in connective tissue throughout the embryo, excluding the nervous system and basement membrane of the dermomyotome. DISCUSSION
Nerve fibers require an adhesive surface for elongation, and the distribution of adhesive molecules probably plays a role in guiding nerve fibers in viva These observations of laminin distribution in chick embryos, combined with previous in vitro studies, suggest that laminin may have such a role. Both central and peripheral nervous system neurons attach to and extend neurites on laminin bound to tissue culture surfaces (Baronet al., 1982; Rogers et al., 1983; FaivreVanEvercooren Bauman et al., 1984). Growth cones of elongating neurites preferentially migrate in vitro along narrow pathways that are more adhesive than surrounding surfaces et al., 1985; Letourneau, 1975, 1979). In (Hammarback the present study, laminin has been demonstrated in the vicinity of early nerve fibers that extend into the periphery from dorsal root ganglia and the neural tube. This apparent association, so far as can be assessed by immunostaining tissue sections, invites speculation that laminin is an adhesive surface for axonal elongation during early phase(s) of peripheral nervous system development. First, laminin in the basement membrane surrounding the neural tube might provide an adhesive surface for growth cones migrating along its inner surface (Singer et al., 1979). Second, laminin on the surface of nonneuronal cells between the neural tube and dermomyotomes might be contacted by migrating growth cones. The identity of cells associated with individual nerve fibers is still unclear, but sclerotome cells or early Schwann cells are likely candidates. Sclerotome cells synthesize laminin (see Figs. 2A and 3A), as do Schwann cells (Cornbrooks et al., 1983; Palm and Furcht, 1983). Finally, laminin may be involved in coalescence of neural crest cells into ganglia and/or in elongation of early dorsal root ganglion neurites. In all of these events, it must be emphasized that a direct role for laminin iu vim has not yet been demonstrated and that additional adhesive molecules are likely to be involved, possibly in
conjunction with laminin or during distinct phases of development. In particular, N-CAM (Thiery et al., 1977; Rutishauser and Edelman, 1980) and Ng-CAM (Thiery et al., 1985) may mediate certain cell-cell interactions in the peripheral nervous system. Our observations of laminin immunoreactivity in the early peripheral nervous system of chicks conflict with a recent report by Bignami et al. (1984) that shows laminin appearing relatively later in developing peripheral nerves and ganglia of rat embryos. The authors suggest that the interactions between Schwann cells and nerve fibers, critical to basal lamina formation by Schwann cells (Bunge et al., 1982), occur relatively late in rats, at least in ventral roots and peripheral nerve trunks. These discrepencies may be due to differences in species, antisera, fixation, or staining procedures. In our study, laminin in early ganglia and nerve roots does not appear to be organized in basement membranes as at later times. Rather, the meshworks of laminin that we see appear to be associated with cell surfaces. Interestingly, laminin, along with proteoglycans, is a component of conditioned media produced by a variety of cell types i?~-c$ro; when bound to tissue culture surfaces, these molecular complexes promote neurite outgrowth (Davis et al., 1984; Lander ef ab, 1985). Thus, although laminin is commonly defined as a basement membrane glycoprotein, it may also be present in other configurations within adhesive surfaces. The absence of fibronectin immunoreactivity in peripheral ganglia and nerve roots was previously reported by Thiery et ul. (1982). Although fibronectin has been reported to appear transiently in some areas of the developing CNS (Hatten et al., 1982; Pearlman et al., 1984), it has not been noted in the spinal cord or in early ventral roots. It appears, therefore, that neither cell bodies nor axons of ventral horn neurons interact with fibronectin at stages that we have examined, similar to our in vitro findings. Fibronectin is present between somites (i.e., at intersomitic levels, data not shown) where crest cells migrate ventrally to form sympathetic ganglia (see Thiery ef ul., 1982) and also remains in dermal and hypodermal connective tissues of the limb (Tomasek et al., 1982) where it may interact with growth cones and neurites of sensory and sympathetic neurons. Our observations raise several questions concerning
FIG. 2. Horizontal sections through levels of dorsal root ganglia (A and 8) and ventral roots (C) of stage 17-18 chick embryos. (A) Laminin immunoreactivity is present in dorsal root ganglia (arrows) and accumulations of sclerotome cells (s) that alternate with the ganglia along the length of the neural tube (nt). Note laminin in dorsal rootlets (arrows) extending from ganglia to the neural tube, and in basement membranes of the neural tube and dermomyotome (d). Inset: Enlargement of boxed-in area, showing laminin staining (arrows) along path from ganglion to neural tube. (B) Same section as (A), double-labeled with the E/C8 antibody to visualize neurites. The pattern of staining in ganglia and dorsal rootlets parallels the laminin staining in (A). E/C8 also stains processes of central neurons at the lateral borders of the neural tube (curved arrow); s, sclerotome; boxed area is enlarged in inset in (A). (C) Laminin immunoreactivity in developing ventral roots defines both a meshwork (arrowhead) and basement membranes of maturing rootlets (arrows); s, sclerotome cells; curved arrow, edge of dermomyotome.
FIG. 3. Cross sections of a stage-25 chick embryo. A and B are the same section stained for laminin (A) and neurofilament-associated protein (B). (C) is an adjacent section, stained for fibronectin. By this time, ventral roots (arrows) and dorsal root ganglia (short arrows in (A)) are well developed. Laminin remains in the neural tube basement membrane and ventral roots, and has accumulated in ganglion capsules. Fibronectin is strikingly absent from ganglia, ventral roots and dermomyotome basement membranes (C); nt, neural tube; d, dermomyotome. 434
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the role(s) for laminin in peripheral nervous system development. Do growth cones interact directly with laminin in the locations examined? What is the identity of the laminin-producing cells, and is the molecule located on cell surfaces or in more complex basal laminae? These problems will be approached with immunolabeling techniques for transmission and scanning electron microscopy. In addition, if laminin is located where it can provide an adhesive surface for neurite elongation, does it, in fact, do so? Experiments that perturb normal development with antibodies should help to resolve this question. REFERENCES BARON-VANEVERCOOREN, A., KLEINMAN, H. K., OHNO, S., MARANGOS, P., SCHWARTZ, J. P., and DUBOIS-DALQ, M. (1982). Nerve growth factor, laminin, and fibronectin promote neurite growth in human fetal sensory ganglion cultures. J. Neurosci. Res. 8,170-193. BIGNAMI, A., CHI, H. N., and DAHL, D. (1984). First appearance of laminin in peripheral nerve, cerebral blood vessels and skeletal muscle of the rat embryo. Immunofluorescence study with laminin and neurofilament antisera. Znt. J. Dev. Neurosci. 2, 367-376. BIJNGE, M. B., WILLIAMS, A. K., and WOOD, P. M. (1982). NeuronSchwann cell interaction in basal lamina formation. Des. Biol. 92, 449-460. CIMENT, G., and WESTON, J. A. (1982). Early appearance in neural crest and crest-derived cells of an antigenic determinant present in avian neurons. Dev. Biol. 93, 355-369. CORNBROOKS,C. J., CAREY, D. J., MCDONALD, J. A., TIMPL, R., and BUNGE, R. P. (1983). Zrl I-ivo and in vitro observations on laminin production by Schwann cells. Proc. Natl. Acad. Sci. USA 80, 3850-3854. DAVIS, G. E., MANTHORPE, M., and VARON, S. (1984). Purification of rat schwannoma neurite promoting factor. Sot. Neuwosci. Abstr. 10, 40. ERICKSON, C., and TURLEY, E. A. (1983). Substrata formed by combinations of extracellular components alter neural crest motility in v&m J. Cell Sci. 61, 299-323. FAIVRE-BAUMAN, A., PUYMIRAT, J., LOUDES, C., BARRE?T, A., and TIXIERVIDAL, A. (1984). Laminin promotes attachment and neurite extension of fetal hypothalamic neurons grown in serum-free medium. Nwro.sci. L&f, 44, 83-89. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49-92. HAMMARBACK, J. A., PALM, S. L., FURCHT, L. T., and LETOURNEAU, P. C. (1985). Guidance of neurite outgrowth by pathways of substratum-bound laminin. J. Neurosci. Res. 13,213-220. HAITEN, M. E., FURIE, M. B., and RIFKIN, D. B. (1982). Binding of mouse developing cerebellar cells to fibronectin: a possible mechanism for the formation of the external granule layer. J. Neurosci. 2, 11951206. LANDER, A. D., FUJII, D. K., GOSPODARAWICZ, D., and REICHARDT, L. F. (1985). Neurite outgrowth-promoting factors in conditioned media are complexes containing laminin. Proc. Natl. Acad. Sci. USA 82,2183-2187.
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