ABSTRACT
The distribution of type I, III and IV collagens and laminin during neural crest development was studied by immunofluorescence labelling of early avian embryos. These components, except type III collagen, were present prior to both cephalic and trunk neural crest appearance. Type I collagen was widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin shared a common distribution primarily in the basal surfaces of epithelia and in close association with developing nerves and muscles. In striking contrast with the other collagens and laminin, type III collagen appeared secondarily during embryogenesis in a restricted pattern in connective tissues.
The distribution and fate of laminin and type I and IV collagens could be correlated spatially and temporally with morphogenetic events during neural crest development. Type IV collagen and laminin disappeared from the basal surface of the neural tube at sites where neural crest cells were emerging. During the course of neural crest cell migration, type I collagen was particularly abundant along migratory pathways whereas type IV collagen and laminin were distributed in the basal surfaces of the epithelia lining these pathways but were rarely seen in large amounts among neural crest cells. In contrast, termination of neural crest cell migration and aggregation into ganglia were correlated in many cases with the loss of type I collagen and with the appearance of type IV collagen and laminin among the neural crest population. Type III collagen was not observed associated with neural crest cells during their development.
These observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities during neural crest ontogeny, (i) Type I collagen associated with fibronectins is a major component of the extracellular spaces of the young embryo. Together with other components, it may contribute to the three-dimensional organization and functions of the matrix during neural crest cell migration, (ii) Type III collagen is apparently not required for tissue remodelling and cell migration during early embryogenesis, (iii) Type IV collagen and laminin are important components of the basal surface of epithelia and their distribution is consistent with tissue remodelling that occurs during neural crest cell emigration and aggregation into ganglia.
Introduction
Neural crest cells constitute a unique embryonic cell population that originates from the dorsal edge of the neural epithelium and, after an extensive migration, gives rise to a large variety of cell types ranging from neurones of the peripheral nervous system to connective cells in the head and neck (for reviews, see Le Douarin, 1982, 1984; Weston, 1982). The development of the neural crest can be considered as a multistep process where cells may be found alternatively in a compacted or a dispersed state. Three phases can be distinguished, (i) In the first stage, termed emergence, neural crest cells that are integrated into the neural epithelium lose their epithelial structure and separate from the neural tube, (ii) In the second stage, called migration, neural crest cells actively migrate through extracellular spaces until they reach their sites of arrest in various areas of the embryo, (iii) In the third stage, called aggregation in the case of the peripheral ganglia, neural crest cells frequently form compacted structures and terminate differentiation.
Previous studies have suggested that the behaviour of neural crest cells during the ontogeny of the peripheral nervous system correlates with the modulation of their adhesive properties (for reviews, see Duband et al. 1985; Thiery et al. 1985; Thiery & Duband, 1986). In particular, the cell-substratum adhesion molecules, fibronectins, have been implicated in the migration of neural crest cells (Newgreen et al. 1982; Thiery, Duband & Delouvée, 1982a; Rovasio et al. 1983; Boucaut et al. 1984; Duband et al. 1986; Bronner-Fraser, 1986a; Tucker, Ciment & Thiery, 1986). The local disappearance of fibronectins can be correlated in some cases with cessation of movement of these cells (Thiery et al. 1982a; Duband et al. 1986). However, fibronectins are found in the environment of neural crest cells well before their emigration from the neural tube suggesting that their presence cannot account for the local disruption of the neural epithelium (Duband & Thiery, 1982; Thiery et al. 1982a). In addition, neural crest cells may aggregate in areas rich in fibronectins such as in the head and in the gut (Duband & Thiery, 1982; Tucker et al. 1986). In vitro, long-term-cultured neural crest cells progressively develop a higher binding for laminin than for fibronectins (Rovasio et al. 1983). It is thus important to analyse precisely the composition of the extracellular matrix associated with the developing neural crest and to determine which matrix molecules could possibly be involved in the events of emergence and aggregation of neural crest cells.
Several different extracellular matrix components including hyaluronic acid and chondroitin sulphate (Pratt, Larsen & Johnston, 1975; Pintar, 1978; Derby, 1978; Brauer, Bolender & Markwald, 1985; Tucker & Erickson, 1986) have already been discovered along the migratory routes of neural crest cells but their precise distribution and their possible role during each stage of neural crest development has not yet been clearly established. On the basis of ultrastructural studies, it has been proposed that collagens are important elements of the extracellular matrix encountered by neural crest cells (Cohen & Hay, 1971; Bancroft & Bellairs, 1976; Tosney, 1978, 1982; Lôfberg, Ahlfors & Fàllstrom, 1980; Newgreen et al. 1982). However, few striated fibres typical of collagens are evident in birds (Tosney, 1978, 1982; Newgreen et al. 1982), thus raising the possibility that the observed fibres could not be solely collagenous. In addition, these studies could not determine which among the various types of collagen were present in the extracellular spaces in the early embryo and thus could not reveal possible matrix heterogeneity resulting from such a variety. Laminin, frequently associated with entactin, has been detected in the basal surfaces of the epithelia lining the pathways of migration of neural crest cells (Sternberg & Kimber, 1986; Rogers, Edson, Letourneau & McLoon, 1986; Krotoski, Domingo & Bronner-Fraser, 1986) but its distribution and fate at the sites of emergence and aggregation of crest cells remains to be determined.
So far, at least ten distinct types of collagen have been characterized (for a review, see Martin, Timpl, Müller & Kühn, 1985). However, only a few types are ubiquitous; these include type I, III and IV collagens. Type I and III collagens are major structural components in many connective tissues, where they form large, banded fibres. Type IV collagen is a nonfibrillar form of collagen specific for basement membranes. These collagens have been considered to provide mechanical stability of extracellular matrices including basement membranes. (Hay, 1973, 1981; Eyre, 1980; Kleinman, Klebe & Martin, 1981; Kleinman et al. 1982; Timpl et al. 1981; Yurchenko & Furthmayr, 1984). However, other potential biological properties of collagens remain to be established. Laminin is a high molecular weight glycoprotein which primarily promotes the adhesion of epithelial cells and stimulates their growth, migration and proliferation (Terranova, Rohrbach & Martin, 1980; Kleinman et al. 1985). Laminin may also promote the adhesion of myoblasts, Schwann cells and some types of fibroblasts and tumour cells (Terranova, Liotta, Russo & Martin, 1982; Kleinman et al. 1985). In addition, laminin exerts diverse biological activities such as favouring neurite extension (Rogers et al. 1983; Adler, Fujii & Reichardt, 1985a; Adler, Jerdan & Hewitt, 1985b), promoting the locomotion of various nonepithelial cells (Goodman & Newgreen, 1985) and increasing the metastatic potential of tumour cells (Terranova et al. 1982).
The critical role of laminin in the adhesion of epithelial cells and the importance of the various collagens in the scaffolding of both epithelial and mesenchymal tissues indicate that these components may be key elements in tissue remodelling and cell migration during embryogenesis. In the present study, we have studied the distribution of laminin and of some major collagens, i.e. types I, III and IV, at the time of neural crest development using immuno-fluorescent staining of cryostat sections of early avian embryos. Our observations suggest that laminin and both type I and IV collagens may be involved with different functional specificities in the three stages of neural crest ontogeny.
Materials and methods
Embryos
Japanese quail embryos were used throughout the study. Eggs were incubated at 38±1°C in a humidified air chamber. The ages of the embryos were determined according to the number of somite pairs as well as to the duration of incubation.
Antibodies
Antibodies to mouse laminin had two origins: rabbit antiserum was produced according to a previously described procedure (Timpl et al. 1979) and affinity-purified IgGs were generously provided by Dr Furcht (University of Minnesota, Minneapolis, MN). The specificity of these antibodies was tested by immunoblotting (not shown). Rabbit antibodies to purified chicken skin type I and III collagens and to human placenta type IV collagen crossreacting with avian type IV collagen were purchased from the Pasteur Institute (Lyon, France). Their specificity was assessed by radioimmunoassay; these antisera did not crossreact with the other types of collagens (Grimaud et al. 1980). We have further characterized the specificity of these antibodies by immunofluorescence labelling of various tissues (i.e. skin, muscle, cartilage and blood vessels) obtained from a 9-day-old embryo. It was observed that type I and III collagens were abundant in the skin, muscles and connective tissues around blood vessels and were absent in cartilages (Fig. 1A,B,D,E,G,H). Type IV collagen was detected in the basal lamina of the epidermis along with both striated and smooth muscles; it was noticeably absent from connective tissues (Fig. 1C,F,I). These results are fully consistent with previously described data (for reviews, see Eyre, 1980; Martin et al. 1985). A monoclonal antibody, NC-1, staining migrating crest cells and their neural derivatives has been described elsewhere (Vincent, Duband & Thiery, 1983; Vincent & Thiery, 1984; Tucker et al. 1984, 1986).
Specificity of antibodies to collagens type I, III and IV as checked by immunofluorescence on connective tissues from late embryos. 9-day-old embryo stained for type I collagen (A,D,G), type III collagen (B,E,H), and type IV collagen (C,F,I). (A–C) Skin and feather bud. Large amounts of type I and III collagens are found in the dermis whereas type IV collagen is only present in the basal lamina of the epidermis. (D–F) Cartilage and striated muscles. No type I, III or IV collagen is found in the cartilaginous tissue but all of them can be detected in muscles. (G-I) Aorta,. Both type I and III collagens are present in the smooth muscle surrounding the endothelium of the aorta and also in connective tissues, while type IV collagen can be detected in high amounts in smooth muscles, a, aorta; c, cartilage; ct, connective tissue around blood vessels; d, dermis; ep, epidermis; m, striated muscle; sm, smooth muscle; A–C, X350; D–F, ×250; G–I, ×450.
Specificity of antibodies to collagens type I, III and IV as checked by immunofluorescence on connective tissues from late embryos. 9-day-old embryo stained for type I collagen (A,D,G), type III collagen (B,E,H), and type IV collagen (C,F,I). (A–C) Skin and feather bud. Large amounts of type I and III collagens are found in the dermis whereas type IV collagen is only present in the basal lamina of the epidermis. (D–F) Cartilage and striated muscles. No type I, III or IV collagen is found in the cartilaginous tissue but all of them can be detected in muscles. (G-I) Aorta,. Both type I and III collagens are present in the smooth muscle surrounding the endothelium of the aorta and also in connective tissues, while type IV collagen can be detected in high amounts in smooth muscles, a, aorta; c, cartilage; ct, connective tissue around blood vessels; d, dermis; ep, epidermis; m, striated muscle; sm, smooth muscle; A–C, X350; D–F, ×250; G–I, ×450.
Preparation of specimens and histological sections
Embryos were fixed in a 3-7 % formaldehyde solution in a phosphate-buffered saline (PBS) solution for 1–4 h at 4°C. After extensive washes in PBS, embryos were embedded in a graduated series of sucrose solutions in PBS (12–18% w/v) and frozen in Tissue Tek (Lab-Tek Products) in liquid nitrogen. Sections were cut at 5–10 μm on a cryostat (Bright Instrument Co. Ltd., Huntington, UK) at −20°C.
Immunofluorescence labelling
Immunofluorescent staining was performed using fluorescein- or rhodamine-conjugated goat antibodies to rabbit IgGs (Nordic, Tilburg, The Netherlands) and fluorescein-conjugated streptavidin and biotinylated sheep antibodies to mouse IgGs (Amersham International, Amersham, UK). In control experiments, the first antibodies were replaced by IgG from preimmune rabbits. Sections were examined and photographed on a Leitz Orthoplan epifluorescent microscope.
Results
The distribution of laminin and type I, III and IV collagens was studied during avian neural crest development using immunofluorescence labelling on cryostat sections. Particular emphasis was put on the distribution and fate of these extracellular matrix molecules in relation to processes of tissue remodelling that occur during neural crest cell emergence, migration and aggregation. In general, type I collagen was abundantly and widely distributed throughout the embryo in the basement membranes of epithelia as well as in the extracellular spaces associated with mesenchymes. Type IV collagen and laminin were found primarily in the basal laminae of epithelia. In contrast to type I and IV collagens and to laminin, which appeared very early before neural crest cell emigration (not shown, but see Mitrani, 1982; Sanders, 1982), type III collagen was first detected at 3 days of incubation around blood vessels in the extraembryonic tissues, in the developing digestive tract and around the mesonephric tubules (Fig. 2). In the late embryo, type III collagen showed a restricted pattern to connective tissues (see Fig. 1B,E,H).
Areas of first appearance of type III collagen in the avian embryo. Immunofluorescent labelling on a 35-somite-stage embryo. (A) Perivascular connective tissues in the extraembryonic mesoderm. Type III collagen is seen as a thin staining around the omphalomesenteric vein and small blood vessels. (B) Wolffian duct. At the time of kidney tubules condensation, type III collagen can be evidenced for the first time in the basal surface of the Wolffian duct and along the somatopleure. (C) Digestive tract at the level of the lung rudiment. Type III collagen distributes in the basal surface of the splanchnic mesoderm, oesophagus and lung rudiment, a, aorta; e, oesophagus; en, endoderm; kt, kidney tubule; Ir, lung rudiment; ov, omphalomesenteric vein; som, somatopleure; spm, splanchnic mesoderm; Wd, Wolffian duct; A–C, ×300.
Areas of first appearance of type III collagen in the avian embryo. Immunofluorescent labelling on a 35-somite-stage embryo. (A) Perivascular connective tissues in the extraembryonic mesoderm. Type III collagen is seen as a thin staining around the omphalomesenteric vein and small blood vessels. (B) Wolffian duct. At the time of kidney tubules condensation, type III collagen can be evidenced for the first time in the basal surface of the Wolffian duct and along the somatopleure. (C) Digestive tract at the level of the lung rudiment. Type III collagen distributes in the basal surface of the splanchnic mesoderm, oesophagus and lung rudiment, a, aorta; e, oesophagus; en, endoderm; kt, kidney tubule; Ir, lung rudiment; ov, omphalomesenteric vein; som, somatopleure; spm, splanchnic mesoderm; Wd, Wolffian duct; A–C, ×300.
Separation of the neural crest from the neural tube
In the head of avian embryos, neural crest cell emigration was directly correlated with neural tube closure (see Fig. 3A–C and Di Virgilio, Lavenda & Worden, 1967; Karfunkel, 1974; Duband & Thiery, 1982; Tosney, 1982). Before separation of the neural crest from the neural tube, the laminin and type IV collagen staining was almost continuous under the ectodermal and neural epithelia (Fig. 3D). This staining underwent temporal changes in distribution which correlated with changes in the spatial relationships among tissues. The first change occured with the apposition of the neural folds. In medial areas of apposition, the staining for the basal lamina-specific components, laminin and type IV collagen, was fragmented, forming patches, in contrast to the more lateral areas of apposition where it was still continuous (Fig. 3E,F). The presumptive crest population then lost its epithelial organization. Patches of laminin and type IV collagen appeared on the basal surface of ectodermal and neural tube cells in progressively more medial positions as crest cells emigrated (Fig. 3H,I). A continuous staining progressively developed under the ectoderm and over the neural tube. As laminin and type I and IV collagens expanded medially under both the ectoderm and neural tube, a space appeared between the previously apposed neural tube and ectoderm and was rapidly filled by migrating crest cells (Fig. 3G–I). Patches of laminin were found among the crest population close to their site of emergence, while more laterally these patches disappeared (Fig. 3I). After neural crest emigration, both the closed neural tube and the overlying ectoderm were delimited by continuous laminin and collagen stainings (Fig. 5A,C). During this whole process of crest cell emigration, type I collagen present in the basement membranes of the ectodérm and neural tube followed the same changes as laminin and type IV collagens (Fig. 3G). However, as soon as migrating crest cells occupied the cell-free space lateral to the neural tube, the distribution of type I collagen was completely different from that of laminin and type IV collagen; these cells were surrounded by abundant amounts of type I collagen (Figs 3G, 5 A) whereas very little laminin and type IV collagen could be detected among them (Figs 3H,I, 5C).
Emigration of neural crest cell at the mesencephalic level in the head. A–C are schematic representations of the mesencephalon at three typical steps of neural tube closure and neural crest emigration, i.e. 4-somite, 6-somite, and 10-somite embryos. Immunofluorescent stainings for laminin (LN), type IV collagen (C4), and type I collagen (Cl). (D) 4-somite embryo; (E,F) 6-somite embryo; (G–I) 10-somite stage. Prior to neural tube closure, the ectoderm and the neural plate are in continuity and are underlined by a continuous laminin staining (D). At the onset of neural crest emigration, as the neural folds come into apposition, neural crest cells start to emigrate and laminin staining becomes fragmented along the neural tube in the neural crest area (E,F). During the expansion of the neural crest population, the ectoderm progresses medially and becomes underlined by a matrix that contains both laminin and type IV collagen (arrows in H,I). Sparse patches of laminin can be seen among the neural crest population that leaves the neural tube (I) but not among cells that occupy the space lateral to the neural tube (small arrow in I). Type I collagen is found in the basal surfaces of the ectoderm and neural tube, as are laminin and type IV collagen. Type I collagen is also found among the migrating neural crest cells (small arrow in G). Note the presence of a large cell-free space devoid of collagens ahead of migrating crest cells (arrowhead in G,H). cm, cephalic mesenchyme; e, ectoderm; n, notochord; nc. neural crest cells; nf, neural folds; np, neural plate; nt, neural tube; D, ×350; E,F, ×450; G–I, ×350.
Emigration of neural crest cell at the mesencephalic level in the head. A–C are schematic representations of the mesencephalon at three typical steps of neural tube closure and neural crest emigration, i.e. 4-somite, 6-somite, and 10-somite embryos. Immunofluorescent stainings for laminin (LN), type IV collagen (C4), and type I collagen (Cl). (D) 4-somite embryo; (E,F) 6-somite embryo; (G–I) 10-somite stage. Prior to neural tube closure, the ectoderm and the neural plate are in continuity and are underlined by a continuous laminin staining (D). At the onset of neural crest emigration, as the neural folds come into apposition, neural crest cells start to emigrate and laminin staining becomes fragmented along the neural tube in the neural crest area (E,F). During the expansion of the neural crest population, the ectoderm progresses medially and becomes underlined by a matrix that contains both laminin and type IV collagen (arrows in H,I). Sparse patches of laminin can be seen among the neural crest population that leaves the neural tube (I) but not among cells that occupy the space lateral to the neural tube (small arrow in I). Type I collagen is found in the basal surfaces of the ectoderm and neural tube, as are laminin and type IV collagen. Type I collagen is also found among the migrating neural crest cells (small arrow in G). Note the presence of a large cell-free space devoid of collagens ahead of migrating crest cells (arrowhead in G,H). cm, cephalic mesenchyme; e, ectoderm; n, notochord; nc. neural crest cells; nf, neural folds; np, neural plate; nt, neural tube; D, ×350; E,F, ×450; G–I, ×350.
Emergence and early migration of neural crest cell in the trunk. (A) Immunofluorescent staining for laminin at the level of the 23rd somite in a 24-somite-stage embryo. Both the ectoderm and neural tube are limited by an almost continuous laminin staining except in the axis of symmetry where it is frequently interrupted (arrowhead). (B,D,F) Immunofluorescent staining for laminin (B), type IV collagen (D), and type I collagen (F) at the level of the 21st somite in a 24-somite-stage embryo; C and E represent the phase-contrast pairs of figures B and D, respectively. At this level, a few neural crest cells start to emigrate from the neural tube, the basal lamina of which is fragmented at the sites of crest cell emergence (arrows in B–E). Type I collagen can be seen around these emigrating neural crest cells (arrow in F). (G,H) Immunofluorescent staining for laminin (G) and type IV collagen (H) at the level of the 18th somite in a 24-somite-stage embryo; I represents the corresponding phase contrast of figure G. Neural crest emigration has commenced. Staining for both laminin and type IV collagen is completely absent from the dorsal area of the neural tube. (J–L) Immunofluorescent staining for laminin (J), type I collagen (K), and type III collagen (L) at the level of the 15th somite in a 24-somite-stage embryo. In this region of the embryo, neural crest emigration has reached its maximum; a wide area of the top of the neural tube expanding up to the level of the somite is now devoid of laminin. Neural crest cells now migrate in narrow pathways mostly between the neural tube and the somitic epithelium and, to a lesser extent, between the ectoderm and the somite; laminin is present in the basal laminae of the epithelia delimiting the migratory pathways of neural crest cells but not within the neural crest population. Type I collagen is found both in the basal surface of the epithelia and among crest cells. Note that type III collagen cannot be detected in any area of the embryo, e, ectoderm; nc, neural crest cells;nt, neural tube; s, somite; A–L, ×350.
Emergence and early migration of neural crest cell in the trunk. (A) Immunofluorescent staining for laminin at the level of the 23rd somite in a 24-somite-stage embryo. Both the ectoderm and neural tube are limited by an almost continuous laminin staining except in the axis of symmetry where it is frequently interrupted (arrowhead). (B,D,F) Immunofluorescent staining for laminin (B), type IV collagen (D), and type I collagen (F) at the level of the 21st somite in a 24-somite-stage embryo; C and E represent the phase-contrast pairs of figures B and D, respectively. At this level, a few neural crest cells start to emigrate from the neural tube, the basal lamina of which is fragmented at the sites of crest cell emergence (arrows in B–E). Type I collagen can be seen around these emigrating neural crest cells (arrow in F). (G,H) Immunofluorescent staining for laminin (G) and type IV collagen (H) at the level of the 18th somite in a 24-somite-stage embryo; I represents the corresponding phase contrast of figure G. Neural crest emigration has commenced. Staining for both laminin and type IV collagen is completely absent from the dorsal area of the neural tube. (J–L) Immunofluorescent staining for laminin (J), type I collagen (K), and type III collagen (L) at the level of the 15th somite in a 24-somite-stage embryo. In this region of the embryo, neural crest emigration has reached its maximum; a wide area of the top of the neural tube expanding up to the level of the somite is now devoid of laminin. Neural crest cells now migrate in narrow pathways mostly between the neural tube and the somitic epithelium and, to a lesser extent, between the ectoderm and the somite; laminin is present in the basal laminae of the epithelia delimiting the migratory pathways of neural crest cells but not within the neural crest population. Type I collagen is found both in the basal surface of the epithelia and among crest cells. Note that type III collagen cannot be detected in any area of the embryo, e, ectoderm; nc, neural crest cells;nt, neural tube; s, somite; A–L, ×350.
Migration of neural crest cells at cephalic levels. (A–D) Mesencephalon of a 12-somite-stage embryo stained for type I collagen (A), type III collagen (B), type IV collagen (C) and NC-1 (D). Neural crest cells stained for NC-1 migrate along the basal lamina of the ectoderm in a milieu that contains type I collagen. Type IV collagen is found in the basal surface of the ectoderm and not among migrating neural crest cells whereas type III collagen is totally absent from the embryo, e, ectoderm; en, endoderm; m. cephalic mesenchyme; nt, neural tube; A–D, ×250.
Migration of neural crest cells at cephalic levels. (A–D) Mesencephalon of a 12-somite-stage embryo stained for type I collagen (A), type III collagen (B), type IV collagen (C) and NC-1 (D). Neural crest cells stained for NC-1 migrate along the basal lamina of the ectoderm in a milieu that contains type I collagen. Type IV collagen is found in the basal surface of the ectoderm and not among migrating neural crest cells whereas type III collagen is totally absent from the embryo, e, ectoderm; en, endoderm; m. cephalic mesenchyme; nt, neural tube; A–D, ×250.
In the trunk of avian-embryos, neural crest emigration is delayed with respect to the neural tube closure (Tosney, 1978; Newgreen & Gibbins, 1982; Thiery et al. 1982a). Laminin and type IV collagen associated with basal laminae were distributed all over the neural .tube in the presumptive neural crest region but discontinuities could be frequently detected in some areas (Fig. 4A). In contrast, the staining was continuous under the ectoderm (Fig. 4A). As opposed to what was observed in the head, premigratory trunk crest cells did not form folds protruding over the neural tube and no strict delineation between premigratory crest cells and the neural tube could be observed. The first indication of crest emigration was the expansion of the discontinuities in laminin and type IV collagen staining in the mediodorsal part of the neural tube (Fig. 4B–E). The interruptions of staining corresponded with the presence of emerging neural crest cells (Fig. 4B–E). Simultaneously, type I collagen strands appeared between the premigratory crest cells (Fig. 4F). The area where the dorsal neural tube was devoid of both laminin and type IV collagen extended laterally almost up to the limit of apposition of the ectoderm to the neural tube, while crest cells emerged from this entire region (Fig. 4G–J). Continuous staining appeared along the ectoderm and the neural tube after the emigration of the last crest cells from the neural tube (Fig. 6A,C,E–H).
Neural crest migration in the rostral and caudal part of the 15th somite in a 33-somite-stage embryo. (A,B,E,G) and (C,D,F,H) show the rostral and caudal halves of the somite, respectively, and staining for laminin (A,C), NC-1 (B,D), type I collagen (E,F), and type IV collagen (G,H). In the rostral part of the sclerotome neural crest cells expand ventrally up to the ventral side of the neural tube and along the ventral side of the dermamyotome to the aorta; in the caudal part, their remnants accumulated dorsally between the neural tube, dermamyotome and sclerotome. The distribution of both laminin and collagen is essentially the same in the two halves of the sclerotome, a, aorta; d, dermamyotome; n, notochord; nt, neural tube; sc, sclerotome; A–H, ×250.
Neural crest migration in the rostral and caudal part of the 15th somite in a 33-somite-stage embryo. (A,B,E,G) and (C,D,F,H) show the rostral and caudal halves of the somite, respectively, and staining for laminin (A,C), NC-1 (B,D), type I collagen (E,F), and type IV collagen (G,H). In the rostral part of the sclerotome neural crest cells expand ventrally up to the ventral side of the neural tube and along the ventral side of the dermamyotome to the aorta; in the caudal part, their remnants accumulated dorsally between the neural tube, dermamyotome and sclerotome. The distribution of both laminin and collagen is essentially the same in the two halves of the sclerotome, a, aorta; d, dermamyotome; n, notochord; nt, neural tube; sc, sclerotome; A–H, ×250.
Neural crest cell migration
Soon after their emergence from the neural tube, neural crest cells undergo an extensive migration to various areas of the embryo. It has been shown previously that most of the migration of the neural crest cells in the head and trunk occurs in restricted extracellular spaces limited by epithelia (see in particular Duband & Thiery, 1982; Thiery et al. 1982a; Rickmann, Fawcett & Keynes, 1985; Krotoski et al. 1986; Bronner-Fraser, 1986b; Teillet, Kalcheim & Le Douarin, 1987).
At cephalic levels and particularly in the mesencephalic region (Fig. 5), neural crest cells occupy a large cell-free space located between the ectoderm and the underlying loose mesenchyme. Prior to neural crest cell arrival, this space was devoid of type I collagen, contrasting with the heavy labelling in the basal surface of the ectoderm and among the mesenchyme (Fig. 3G). As soon as neural crest cells invaded this space and migrated along the basal surface of the ectoderm (Figs 3G,H; 5C,D), they became surrounded by a dense network of type I collagen (Fig. 3G); this component persisted among neural crest cells as a uniform staining during the entire process of the migration (Fig. 5A).
In trunk regions, type I collagen was also abundant and uniformly distributed along neural crest migration pathways and the presence of neural crest cells in their migratory phase could always be correlated with its presence (Figs 4F,K, 6B,D–F). For example, it was found along the ventral pathway between the neural tube and the somitic epithelium as well as in the lateral pathway between the ectoderm and the dermatome (Figs 4K, 6E,F). In contrast to what was observed at cranial levels, type I collagen could be detected among the neural crest presumptive pathways well before they reached these sites (Fig. 4F). It should be stressed that type I collagen could also be found in sites of the embryo not invaded by neural crest cells; in particular, it was distributed around the notochord, an area which was never occupied by neural crest cells (Fig. 6B,D–F; see also Newgreen, Scheel & Kastner, 1986).
In contrast to type I collagen, type III collagen was never detected among neural crest cells migrating in the head and trunk (Figs 4L, 5B). The labelling of laminin and type. IV collagen provided more evidence for the restricted migration pathways of neural crest cells at cephalic and trunk levels: these two components were always found in the basal surface of the epithelial tissues that delineated neural crest pathways, but they were not found in noticeable amounts within the matrix surrounding migrating neural crest cells (Figs 3H,I, 4D,G–J, 5C,D, 6A–D,G,H).
Because neural crest cells do not distribute evenly along the somites (Rickmann et al. 1985; Thiery & Duband, 1986; Bronner-Fraser, 1986b; Teillet et al. 1987), the location of laminin and collagens in both the rostral and the caudal parts of the somites was examined in detail in connection with that of neural crest cells. At each step of neural crest cell migration, first between the somitic epithelium, then along the ventral side of the dermamyotome, as well as at each step of the formation and subsequent reorganization of the somitic epithelium, immunofluorescent staining for both type I and type IV collagens and for laminin were similar in intensity and distribution in the rostral and caudal halves of the somites (Fig. 6).
Aggregation of neural crest cells into ganglia
In the trunk of avian embryos, most crest cells located lateral and ventral to the neural tube aggregate to form the sensory and sympathetic ganglion rudiments. The sensory or dorsal root ganglia (DRG) form at the level of the anterior part of the somite at the border of the neural tube, the dermamyotome and the sclerotome (see also Thiery et al. 1982a; Erickson, 1985; Rickmann et al. 1985; Bronner-Fraser, 1986b; Teillet et al. 1987). Prior to their aggregation, neural crest cells accumulated along the neural tube in an area rich in type 1 collagen and devoid of laminin and type IV collagen (Fig. 6). Type I collagen progressively and completely disappeared among the crest cells forming the DRG primordium (Fig. 7A). Conversely, a very faint, patchy staining for laminin and type IV collagen could be detected within the ganglion primordium (Fig. 7B,C). These two components were progressively distributed around the whole contour of the ganglion, neatly delimiting it from the surrounding sclerotome (Fig. 7D).
Formation of the dorsal root ganglia at the brachial level. (A–C) 38-somite-stage embryo stained for type i collagen (A), laminin (B), and type IV collagen (C). Neural crest cells that are accumulating along the neural tube are progressively compacted into dorsal root ganglia. Staining for type 1 collagen is weak both among these neural crest cells and in the sclerotome. Type IV collagen and laminin exhibit a punctate staining among neural crest cells and along the outgrowing motor nerve. (D) 4-day-old embryo stained for laminin. Laminin distributes around the dorsal root ganglion. Note that laminin is organized into a basal-lamina-like structure along the motor nerve but only in its proximal aspect. (E–H) 9-day-old embryo stained for type I, III and IV collagens and laminin, respectively. Both type I and III collagens are found in connective tissues surrounding the dorsal root ganglion but not within the ganglion itself. In contrast, type IV collagen and laminin are clearly present in the ganglion particularly in its lateroventral aspect where neurones are fully differentiated; in the mediodorsal region where neurones are not entirely differentiated, type IV collagen and mostly laminin are much less abundant or absent, d, dermamyotome; drg, dorsal root ganglion; Iv, lateroventral region of the ganglion; md, mediodorsal region of the ganglion; mn, motor nerve; nt, neural tube; sc, sclerotome; A–C, ×400; D, ×450; E–H, ×350.
Formation of the dorsal root ganglia at the brachial level. (A–C) 38-somite-stage embryo stained for type i collagen (A), laminin (B), and type IV collagen (C). Neural crest cells that are accumulating along the neural tube are progressively compacted into dorsal root ganglia. Staining for type 1 collagen is weak both among these neural crest cells and in the sclerotome. Type IV collagen and laminin exhibit a punctate staining among neural crest cells and along the outgrowing motor nerve. (D) 4-day-old embryo stained for laminin. Laminin distributes around the dorsal root ganglion. Note that laminin is organized into a basal-lamina-like structure along the motor nerve but only in its proximal aspect. (E–H) 9-day-old embryo stained for type I, III and IV collagens and laminin, respectively. Both type I and III collagens are found in connective tissues surrounding the dorsal root ganglion but not within the ganglion itself. In contrast, type IV collagen and laminin are clearly present in the ganglion particularly in its lateroventral aspect where neurones are fully differentiated; in the mediodorsal region where neurones are not entirely differentiated, type IV collagen and mostly laminin are much less abundant or absent, d, dermamyotome; drg, dorsal root ganglion; Iv, lateroventral region of the ganglion; md, mediodorsal region of the ganglion; mn, motor nerve; nt, neural tube; sc, sclerotome; A–C, ×400; D, ×450; E–H, ×350.
The sympathetic ganglia (SG) first appeared as small clusters of cells along the aorta in an area rich in type I collagen and totally devoid of laminin and type IV collagen (Fig. 6). Type I collagen slowly disappeared from the mass of the ganglion but, in contrast to the DRG, type IV collagen and laminin were never detected either within or around the SG at the time of its formation (Fig. 8A–D).
Formation of the sympathetic ganglia at the brachial level. (A–D) 38-somite-stage embryo stained for type I collagen (A), type IV collagen, (B), laminin (C), and NC-1 (D). Neural crest cells destined to be the sympathetic cell progenitors form a cluster along the aorta where small amounts of type I collagen are found; neither type IV collagen nor laminin can be detected among neural crest cells. Note that the growing tip of the motor nerve is not stained for these two components. (E-H) 9-day-old embryo stained for type I, III and IV collagens and laminin, respectively. None of these extracellular matrix components are seen in the sympathetic ganglion at this stage, a, aorta; c, cartilage; drg, dorsal root ganglion; kt, mesonephric kidney tubules; mn, motor nerve; sc, sclerotome; sg, sympathetic ganglion; v, blood vessel; A–D, ×400; E–F, ×350.
Formation of the sympathetic ganglia at the brachial level. (A–D) 38-somite-stage embryo stained for type I collagen (A), type IV collagen, (B), laminin (C), and NC-1 (D). Neural crest cells destined to be the sympathetic cell progenitors form a cluster along the aorta where small amounts of type I collagen are found; neither type IV collagen nor laminin can be detected among neural crest cells. Note that the growing tip of the motor nerve is not stained for these two components. (E-H) 9-day-old embryo stained for type I, III and IV collagens and laminin, respectively. None of these extracellular matrix components are seen in the sympathetic ganglion at this stage, a, aorta; c, cartilage; drg, dorsal root ganglion; kt, mesonephric kidney tubules; mn, motor nerve; sc, sclerotome; sg, sympathetic ganglion; v, blood vessel; A–D, ×400; E–F, ×350.
The pattern of laminin and type IV collagen changed gradually with further differentiation of the DRG and SG. By 9 days of incubation, they were present around the whole DRG and in close association with the differentiated lateroventral neurones (Fig. 7G,H). However, immunofluorescence labelling did not permit the localization of these components in association with neurones or satellite cells. At the 9-day stage, both type I and III collagens were almost totally absent from the mass of the DRG (Fig. 7E,F). By 13 days of incubation, all the neurones were differentiated in the DRG and both LN and type IV collagens could be seen throughout the ganglion (not shown). Laminin and type I, III and IV collagens were not seen in the sympathetic ganglion at 9 days of incubation (Fig. 8E–H) but, at 13 days, they appeared as strands within the ganglion and at its periphery (not shown). Similar results were obtained with the various cranial sensory ganglia as well as with the parasympathetic ciliary ganglia. (Fig. 9).
Peripheral ganglia in the head. Trigeminal (A) and ciliary (B) ganglia of an 8-day-old embryo stained for laminin. Laminin is found between most if not all cells in the ganglia and also along nerves emerging from them. eg, ciliary ganglion; tg, trigeminal ganglion; A, ×300; B, ×400.
The case of the parasympathetic ganglia of the digestive tract was somewhat special. There occur, along the length of the digestive tract, important differences in the environment among which neural crest cells migrated and aggregated (see Tucker et al. 1986). In the foregut, this environment is rich in both laminin and type I, III and IV collagens and no obvious changes in the composition of the matrix could be detected at the time of aggregation of cells into clusters (Fig. 10).
Enteric ganglia in the gizzard of a 4-day-old embryo stained for collagen (A), laminin (B), and NC-1 (C). Neural crest cells in the process of aggregation are scattered among the splanchnic mesoderm in an extracellular matrix containing laminin and type I collagen. Note that these two components share the same distribution both in the basal surface of the epithelia and in the mesenchyme, eg, enteric ganglia; en, endoderm; spe, splanchnic epithelium; spm, splanchnic mesoderm; A–C, ×400.
Enteric ganglia in the gizzard of a 4-day-old embryo stained for collagen (A), laminin (B), and NC-1 (C). Neural crest cells in the process of aggregation are scattered among the splanchnic mesoderm in an extracellular matrix containing laminin and type I collagen. Note that these two components share the same distribution both in the basal surface of the epithelia and in the mesenchyme, eg, enteric ganglia; en, endoderm; spe, splanchnic epithelium; spm, splanchnic mesoderm; A–C, ×400.
Discussion
In the present study, we have analysed the distribution of three major collagen types and of laminin in the early avian embryo at the time of neural crest development. Our observations have revealed that the pattern of distribution of laminin and collagens can be correlated spatially and temporally with tissue remodelling occuring during the ontogeny of the neural crest. Laminin and type IV collagen, first associated in a basal lamina overlying premigratory neural crest cells, disappeared locally at the onset of the migration of these cells. Type I collagen was associated with neural crest cells during the whole process of their migration. When neural crest cells collected into sensory ganglia of the peripheral nervous system, type IV collagen and laminin reappeared among the ganglia whereas type I collagen became excluded from them. In contrast, no such drastic reorganization of the extracellular matrix composition was observed during the formation of the sympathetic and enteric nervous systems. Type III collagen was not associated with the different phases of neural crest development at either cranial or truncal levels.
General distribution of collagens and laminin during early embryogenesis
In the avian embryo, type I and IV collagens and laminin appeared very early during embryogenesis. They could be detected as early as the onset of gastrulation (our unpublished results; Sanders, 1979; Mitrani, 1982). At this stage, these components were exclusively restricted to the basal surface of the epiblast. Later on during neurulation and early organogenesis, the distribution of type I and IV collagens and of laminin changed according to the organization of the tissues. Type I collagen was found to be abundantly and widely distributed throughout the embryo in the basement membrane of all epithelia such as the ectoderm, the neural tube, the somites and the ectoderm as well as in the extracellular matrices associated with mesenchymes, including the sclerotome and all connective tissues in the head and the neck. The distribution of laminin and type IV collagen was almost exclusively restricted to the basal laminae of epithelia.
In embryos older than 4 days, type I collagen was still a predominant component of extracellular matrices, particularly in connective tissues. However, its prevalence and localization were dependent on the nature and state of differentiation of the tissue. For example, cartilage was completely devoid of type I collagen, containing type II collagen instead. In other cases, such as in muscles, blood vessels and dermis, type I collagen may be associated with type III and/or type IV collagens. Finally, type I collagen was completely lost and apparently not replaced by other collagen types in either the central or the peripheral nervous system.
Type IV collagens and laminin were still found at the basal surface of epithelia such as the epidermis, the kidney tubules and the endoderm. In addition to this well-known distribution, they were also detected in nonepithelial tissues. The most striking examples of such tissues were the ganglia of the peripheral nervous system where both type IV collagen and laminin were present around neurones and the Schwann cells along the nerves; the myotubes of skeletal and smooth muscles were also surrounded by these two basal laminae components.
The present observation confirms previous studies and provides further information on the distribution of laminin and type I and IV collagens during early avian embryogenesis (Trelstad, Hay & Revel, 1967; Cohen & Hay, 1971; von der Mark, von der Mark & Gay, 1976; von der Mark, von der Mark, Timpl & Trelstad, 1977; Wakely & England, 1977; Sanders, 1979; von der Mark, 1980; Mitrani, 1982; Kühl, Timpl & von der Mark, 1982; Liesi, Dahl & Vaheri, 1983; Liesi, 1985; Krotoski et al. 1986; Rogers et al. 1986; Sanes, Schachner & Covault, 1986). In addition, our study brings new data on the time of appearance and distribution of type III collagen. Indeed, in contrast to type I and IV collagens and laminin, the appearance of type III collagen was considerably delayed. This type of collagen was first detected at 3 days of incubation around blood vessels in the extraembryonic tissues, in the basal surface of the mesonephric tubules and in the digestive tract. It is noticeable that it was never found during the early stages of morphogenesis. In older embryos, type III collagen showed a restricted pattern in the dermis, skeletal muscles and in some connective tissues such as those surrounding the blood vessels (see also for a review Martin et al. 1985). Type III collagen has also been observed to appear late and in a restricted pattern during the development of the eye in the chicken embryo (von der Mark et al. 1977). Since type I and III collagens are deposited simultaneously by cultured skin fibroblasts (Gay et al. 1976) and are frequently associated in connective tissues in the late embryo and in the adult, it is of interest to note that, during early avian embryogenesis, type I and III collagens do not codistribute.
The time of appearance of collagens and laminin in the avian embryo appears to differ strikingly from that in the mouse. In the latter species, one of the polypeptide chains of laminin is already synthesized by oocytes and the complete molecule of laminin can be detected from the 16-cell stage on, i.e. much earlier than to the appearance of epithelial structures (Leivo, Vaheri, Timpl & Wartiovaara, 1980; Cooper & MacQueen, 1983). Type IV collagen is also detected very early at the blastocyst stage in the inner cell mass (Leivo, Vaheri, Timpl & Wartiovaara, 1980). Also, in contrast to our results in the chick, type I and III collagen are detected at 8 days of gestation closely distributed in tissues of mesodermal origin (Sherman, Gay, Gay & Miller, 1980; Leivo et al. 1980). However, later on during mouse organogenesis, laminin and type I and IV collagens show a distribution very similar to that in the chick (Adamson & Ayers, 1979; Sternberg & Kimber, 1986).
Collagens and laminin in connection with neural crest ontogeny
Emigration of neural crest cells
Prior to their emigration, neural crest cell forms an epithelium at the boundary between the ectoderm and the neural tube. They are limited on their basal surface by an extracellular matrix that contains type IV collagen and laminin. In contrast to the neighbouring tissues (i.e. ectoderm and neural epithelium), the matrix limiting the presumptive neural crest population in the trunk is characterized by discontinuities detectable well before the emergence of crest cells. This has also been reported in electron microscopy studies both in avians and in mammals (Tosney, 1978; Newgreen & Gibbins, 1982; Martins-Green & Erickson, 1986). In the cranial regions, the basal lamina along presumptive neural crest cells appears at first continuous and becomes fragmented only at the onset of their emigration from the neural tube (see also Tosney, 1982). These differences between cranial and truncal levels may reside in the delay between neural tube closure and crest cell emigration (Di Virgilio et al. 1967; Karfunkel, 1974). The mechanisms that provoke the local disruption of the basal lamina along neural crest cells are not known. This may result from the process of the neural tube closure which involves a complex folding of both the ectodermal and neural epithelia (Di Virgilio et al. 1967; Karfunkel, 1974; Jacobson, 1980, 1985). This process could induce physical constraints and create mechanical forces able to damage the basal lamina.
During neural crest cell emergence, the discontinuities in laminin and type IV collagen stainings expand significantly. This suggests that the disruption of the basal lamina under the neural epithelium is necessary to allow its local dislocation and neural crest emigration. Previous studies on corneal epithelium have shown that the disappearance of the basal lamina from under epithelial cells induces them to bleb, to send pseudopodia into the underlying extracellular matrix and to separate from their neighbours (Sugrue & Hay, 1981,1982; Greenburg & Hay, 1982).
The underlying mechanisms that trigger the expansion of the disruption of the basal lamina are not known. A wide variety of factors may participate in this phenomenon. Intense cell proliferation or expansion of the acellular space following the deposition of hyaluronate could cause local pressure which is able to damage the basal lamina. In this respect, it is interesting to note that neural crest cells synthesize large amounts of hyaluronate (Pratt et al. 1975; Pintar, 1978). Alternatively, local proteolytic activity (plasmin and collagenases) could digest laminin and type IV collagen of the basal lamina. Interestingly, crest cells synthesize plasminogen activator at least in vitro (Valinsky & Le Douarin, 1985). Finally, it has been shown that neural crest cells in the trunk leave the neural tube only after they reach a proper stage and this emigration depends upon calcium levels (Newgreen & Gibbins, 1982; Newgreen & Gooday, 1985). The precise schedule of the onset of crest cell migration could thus correlate with a decrease in the intercellular adhesion of crest cells mediated by the cell-adhesion molecules N-CAM and N-cadherin (Thiery, Duband, Rutishauser & Edelman, 1982b; Hatta, Takagi, Fujisawa & Takeichi, 1986; Duband et al. 1985; Duband, Volberg, Thiery & Geiger, 1987).
Migration of neural crest cells
During the course of neural crest development, type I collagen was found around the migratory cells and disappeared when the cells aggregated into ganglia of the peripheral nervous system. In contrast, type III collagen was not found in noticeable amounts in the migratory pathways in the head and in the trunk. These results provide additional data on the chemical composition of the extracellular matrix encountered by neural crest cells. This matrix contains fibronectins (Newgreen & Thiery, 1980; Mayer, Hay & Hynes, 1981; Duband & Thiery, 1982; Thiery et al. 1982a; Krotoski et al. 1986; Sternberg & Kimber, 1986), hyaluronate (Pratt et al. 1975; Pintar, 1978; Derby, 1978; Brauer et al. 1985; Tucker & Erickson, 1986), chondroitin sulphate proteoglycans (Derby, 1978; Brauer et al. 1985; Tucker & Erickson, 1986), cytotactin (Crossin et al. 1986) and type I collagen. Most importantly, this matrix appears uniform in its composition along the pathways; however, one cannot exclude possible local variations in the concentration or in the three-dimensional organization of each constituent that cannot be detected by immunofluorescent techniques.
Since ultrastructural studies did not reveal numerous striated fibres in neural crest pathways (Tosney, 1978, 1982; Newgreen et al. 1982), it is possible that type I collagen does not show the same threedimensional organization in the embryo as in the adult, thus allowing a greater plasticity necessary for rapid tissue remodelling. Moreover, the distribution of type I collagen in the head and trunk is reminiscent of that of fibronectins (Mayer, Hay & Hynes, 1981; Duband & Thiery, 1982; Thiery et al. 1982a; Krotoski et al. 1986). Fibronectins and type I collagen have been shown to codistribute in fibrils deposited in vitro by fibroblasts (Vaheri, Kurkinen, Lehto & Timpl 1978; Furcht et al. 1980). It is thus likely that both type I collagen and fibronectins are codistributed in the fibrillar meshwork present in the migratory pathways of the neural crest. As suggested by in vitro experiments, fibronectins should play a direct role in the formation of the fibrillar meshwork, possibly by organizing collagen fibres (McDonald, Kelley & Broekelmann, 1982). The cellular origin of type I collagen in these pathways has not been determined precisely. The neuroepithelium, the somites and the ectoderm are known to contribute to collagen and, unlike fibronectins (Newgreen & Thiery, 1980; Sieber-Blum, Sieber & Yamada, 1981), neural crest cells and melanocytes cultured in vitro and crest-derived corneal fibroblasts possess the ability to deposit type I and/or III collagens (Conrad, Dessau & von der Mark, 1980; Greenberg, Foidart & Greene, 1980).
Type I collagen was found in areas not invaded by neural crest cells such as around the notochord and in the posterior half of the sclerotome. Such is also the case for fibronectins (Rickmann et al. 1985; Krotoski et al. 1986; Thiery & Duband, 1986) and hyaluronic acid (Teillet et al. 1987). This would suggest that the presence of these extracellular matrix components is not solely responsible for the direction taken by neural crest cells during their migration. However, a detailed analysis of the three-dimensional organization of the matrix in both the rostral and caudal parts of the sclerotome would be necessary to confirm this hypothesis.
The role of type I collagen in neural crest cell migration has not yet been clearly established. In vitro, collagen deposited in two-dimensional gels or denatured collagen does not support neural crest cell displacement (Newgreen et al. 1982; Rovasio et al. 1983; Tucker & Erickson, 1984). However, collagen in a three-dimensional lattice promotes cell movement but only at low concentrations (Tucker & Erickson, 1984). In addition, neural crest cells are able to adhere to various types of collagen only in the presence of fibronectin (Greenberg, Seppâ, Seppâ & Hewitt, 1981). These results would suggest that type 1 collagen mostly serves as a scaffold which neural crest cells attach onto, and migrate through, due to their binding to fibronectins. However, in the mouse, a particular mutant (Movl3) has recently been obtained by the insertion of Moloney virus sequences into the second intron of the collagen alpha 1(1) gene, inactivating this gene. Mutants in the homozygous state die between the 13th and the 15th days of gestation long after neural crest migration (Schnieke, Harbers & Jaenisch, 1983; Lôhler, Timpl & Jaenisch, 1984). These results raise an important question about the role of type I collagen in cell migration. Additionally, branching of various epithelia has been obtained in vitro from cultures of primordia taken from these type-I-collagen-deficient mutants (Kratochwil et al. 1986). In these cases, however, one cannot exclude the possibility that type III collagen may replace type I collagen. Since, in avian embryos, type III collagen is absent in the migratory pathways of the neural crest, one must conclude that either none of the collagens are essential for cell migration or that there are species differences. One way to approach this question would be to produce transgenic chickens deficient in type I collagen. Another possible approach of the role of type I collagen in neural crest migration would be to perform perturbation experiments using antibodies to both collagen and collagen receptors.
Aggregation of neural crest cells
Type I collagen, fibronectins (Thiery et al. 1982a; Rickmann et al. 1985; Rogers et al. 1986) and hyaluronic acid (Derby, 1978) disappear from the vicinity of aggregating neural crests and, in many cases, are replaced by laminin and type IV collagen. This would indicate that the disappearance of the substratum used for migration associated with a complete change in the extracellular matrix composition provokes the arrest of crest cells. In addition, the appearance of laminin and type IV collagen in the vicinity of neural crest cells at the time of their aggregation into cranial and spinal sensory ganglia of the peripheral nervous system indicates that these two components participate in the aggregation process. This observation is consistent with the role that has been proposed for laminin in the model system of the kidney (Ekblom, 1981). These in vivo observations are corroborated by in vitro studies; indeed, long-term-cultured neural crest cells progressively develop a higher binding for laminin than for fibronectin (Rovasio et al. 1983). In addition to reorganizations of the extracellular matrix, modulations of cell-cell adhesion may play an important role in the aggregation of neural crest cells into ganglia (Thiery et al. 1982b; Duband et al. 1985, 1987; Hatta et al. 1987). Such is probably the case with the autonomic sympathetic ganglia where only the disappearance of type I collagen and fibronectins but no appearance of laminin or type IV collagen have been detected (see also Thiery et al. 1982a; Duband et al. 1985).
ACKNOWLEDGEMENTS
The authors are particularly indebted to Dr Leo Furcht for the generous gift of affinity-purified antibodies to laminin and to Dr Marc Vigny for the gift of the mouse EHS tumour. We thank Dr Evan Balaban for critical reading of the manuscript. This work was supported by the Centre National de la Recherche Scientifique (ASP 95109), the Institut de la Santé et de la Recherche Médicale (CRE 864015), the Association pour la Recherche contre la Cancer, and the March of Dimes (1-993).