Spatial and temporal changes in the distribution of proteoglycans during avian neural crest development

ROBERTO PERRIS 1 DANUTA KROTOSKI 0 1 THOMAS LALLIER 1 CARMEN DOMINGO 1 J. 1 MICHAEL SORRELL 1 2 MARIANNE BRONNER-FRASER 1 0 National Institute of Child Health and Human Development, Executive Plata North , Room 643, 6130 Executive Blvd., Rockville, MD 2085 , USA 1 'Developmental Biology Center, University of California , Irvine, CA 92717 , USA 2 Department of Biology, Case Western Reserve University , Cleveland, OH 44106 , USA 3 Department of Cell and Molecular Biology, University of California , Berkeley, CA 94720 , USA Spatial and temporal changes in the distribution of proteoglycans during * Author for correspondence - In this study, we describe the distribution of various classes of proteoglycans and their potential matrix ligand, hyaluronan, during neural crest development in the trunk region of the chicken embryo. Different types of chondroitin and keratan sulfate proteoglycans were recognized using a panel of monoclonal antibodies produced against specific epitopes on their glycosaminoglycan chains. A heparan sulfate proteoglycan was identified by an antibody against its core protein. The distribution of hyaluronan was mapped using a biotinylated fragment that corresponds to the hyaluronanbinding region of cartilage proteoglycans. Four major patterns of proteoglycan immunoreactivity were observed. (1) Chondroitin-6-sulfate-rich proteoglycans and certain keratan sulfate proteoglycans were absent from regions containing migrating neural crest cells, but were present in interstitial matrices and basement membranes along prospective migratory pathways such as the ventral portion of the sclerotome. Although initially distributed uniformly along the rostrocaudal extent of the sclerotome, these proteoglycans became rearranged to the caudal portion of the sclerotome with progressive migration of neural crest cells through the rostral sclerotome and their aggregation into peripheral ganglia. (2) A subset of chondroitin/keratan sulfate proteoglycans bearing primarily unsulfated chondroitin chains was observed exclusively in regions where neural crest cells were absent or delayed from entering, such as the perinotochordal and subepidermal spaces. (3) A subset of chondroitin/keratan sulfate proteoglycans was restricted to the perinotochordal region and, following gangliogenesis, was arranged in a metameric pattern corresponding to the sites where presumptive vertebral arches form. (4) Certain keratan sulfate proteoglycans and a heparan sulfate proteoglycan were observed in basement membranes and in an interstitial matrix uniformly distributed along the rostrocaudal extent of the sclerotome. After gangliogenesis, the neural crestderived dorsal root and sympathetic ganglia contained both these proteoglycan types, but were essentially free of other chondroitin/keratan-proteoglycan subsets. Hyaluronan generally colocalized with the first set of proteoglycans, but also was concentrated around migrating neural crest cells and was reduced in neural crest-derived ganglia. These observations demonstrate that proteoglycans have diverse and dynamic distributions during times of neural crest development and chondrogenesis of the presumptive vertebrae. In general, chondroitin/keratan sulfate proteoglycans are abundant in regions where neural crest cells are absent, and their segmental distribution inversely correlates with that of neural crest-derived ganglia. Neural crest cells migrate long distances along pathways containing an intricate extracellular matrix (ECM). As a consequence, the ECM is thought to play a central role in several aspects of neural crest development. In vitro, neural crest cells migrate avidly on numerous ECM molecules including fibronectin, laminin and collagens (Newgreen and Erickson, 1986; Perris and Johansson, 1987; Perris etal. 1989; Perris et al. 1990a), suggesting that individual matrix components may serve as permissive migratory substrates. Ultrastructural studies performed in situ reveal that neural crest cells form specialized contacts with this fibrillar matrix network encountered during migration (Lofberg et al. 1980; Newgreen and Erickson, 1986; Penis et al. 1990b). In ovo injections of antibodies against individual matrix molecules or their cell surface receptors results in abnormal neural crest development in vivo (Bronner-Fraser, 1985, 1986a; Bronner-Fraser and Lallier, 1988; Bronner-Fraser, 1988). Moreover, transplantations of regionally and temporally denned matrices adsorbed onto membrane microcarriers have provided evidence that the ECM can prematurely promote the onset of neural crest cell movement in vivo (Lofberg et al. 1985, 1988). A logical first step in establishing the role of the ECM in neural crest cell migration is to determine its structural and molecular composition at various phases of neural crest development. Ultrastructural and immunohistochemical studies have revealed that the interstitial matrix along trunk neural crest migratory pathways consists of a fibrillar collagenous network, which contains abundant amounts of fibronectin, tenascin/cytotactin and glycosaminoglycans (Newgreen and Erickson, 1986; Perris and Bronner-Fraser, 1989; Perris et al. 1990a; Newgreen et al. 1986, 1990). Basement membrane matrices, enriched in laminin and collagen type IV, also line some neural crest migratory routes (Newgreen and Erickson, 1986; Perris and Bronner-Fraser, 1989; Perris et al. 19906). Although a great deal of information has been compiled regarding the distribution and possible function of cell adhesion glycoproteins such as fibronectin, laminin, cytotactin/tenascin and various collagens during neural crest cell migration, far less is known about the role of proteoglycans in this process. Proteoglycans represent a heterogeneous population of molecules that contribute to the compositional diversity of the ECM. Indirect evidence that proteoglycans might be well-represented during neural crest development has emerged from a series of histochemical studies using cationic dyes, in situ metabolic labelling and differential enzymatic degradation (Kvist and Finnegan, 1970; Pintar, 1978; Lofberg et al. 1980; Newgreen et al. 1982, 1986; Perris et al. \99Qb). From these observations it was concluded that several distinct families of matrix and cell-associated proteoglycans were expressed at various phases of neural crest development. However, the nature and spatiotemporal distribution of specific populations of proteoglycans present at these early stages of development, as well as their relationship to their potential ligands, such as collagens and hyaluronan, has not been determined. In this study, we have examined the distribution of various proteoglycan subclasses in situ and have determined their spatial and temporal organization relative to the development of the trunk neural crest in the chick embryo. For this purpose, a panel of monoclonal antibodies that specifically detect native carbohydrate structures of chick proteoglycans (...truncated)