Alterations in Neural Crest Migration Antibody that Affects Cell Adhesion by a Monoclonal

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hydridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and l-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex. Several important developmental events are accompanied by temporal or spatial changes in the composition of the extracellular matrix (ECM). ~ For example, hyaluronate accumulates in the corneal stroma during the invasion of the corneal fibroblast and is subsequently removed by increased production of hyaluronidase (25). Fibronectin has been implicated in guiding cell movement during gastrulation (1) and neural crest migration (10, 24) because it lines pathways followed by migrating cells. Such correlations have led to the proposal that adhesive interactions between the extracellular matrix and the embryonic cell surface may play an important role in morphogenesis (13). The neural crest is an interesting model for examining the role of cell surface-ECM interactions because these cells migrate extensively during development along pathways that Abbreviations used in this paper. CFSE, carboxyfluorescein diacetate suecinimyl ester; ECM, extracellular matrix. 610 are lined with ECM molecules. Neural crest cells arise during neurulation and depart from the newly formed neural tube shortly after tube closure. The pathways followed by neural crest cells contain numerous matrix molecules including fibronectin, hyaluronic acid, type I collagen, and probably various other molecules. In tissue culture, neural crest cells can migrate in a directed fashion along fibronectin rich matrices (22) and can also migrate on laminin (17). When transplanted into premigratory regions of axolotl embryos, microcarrier filters coated with embryonic matrices from older embryonic regions can promote premature migration of neural crest cells (14). These studies indicate that the ECM may be both permissive and stimulatory for neural crest migration. Recently, several monoclonal antibodies were described that recognize cell surface proteins involved in adhesion ( l l, 16, 20). Two of these monoclonals, called JG22 and CSAT, perturb the adhesion of embryonic muscle cells to some THE JOURNAL OF CELL BIOLOGY VOLUME 101 AUGUST 1985 610-617 © The Rockefe[ler University Press • 0021-9525/85/08/610J08 $1.00 on O cber 0, 2017 jcb.rress.org D ow nladed fom substrates in vitro. JG22 and CSAT apparently recognize the same antigen, a 140-kD complex of proteins localized in the region of cell-substratum attachment (6, 1 l, 16). The 140kD complex has recently been shown to co-distribute with both a-actinin and flbronectin, which suggests that the antigens may represent a cell surface linkage between ECM molecules and the cytoskeleton (6). In this study I examined some effects of the monoclonal antibody JG22 on neural crest cells in vitro and in vivo. To test the effects of JG22 in vivo, a novel perturbation approach has been developed in which antibody-containing medium together with the hybridoma cells themselves are microinjected onto neural crest pathways. The hybridoma cells are labeled by a new fluorescent staining method that renders the cells identifiable in living preparations and histologically processed tissue sections. Both in tissue culture and in the embryo, JG22 affects the adhesion and migration of neural crest cells. In contrast, two other monoclonal antibodies, I-B and I-N, that bind to bands of the 140-kD complex precipitated by JG22, affect neither cell adhesion or neural crest migration. The results suggest that the adhesive interaction blocked by JG22 is important for normal neural crest migration. MATERIALS AND METHODS Neural Crest Cultures Primary cultures of neural crest cells were prepared from the neural tubes of Japanese quail embryos (coturnix coturnix japonica) (7, 8). Embryos were incubated for 48 h at 38"C, at which time their developmental age was comparable to chick stages 13-15 (12). The region of the trunk consisting of the six to nine most recently formed somites and some unsegmented mesenchyme was dissected away from the embryo. The neural tubes were isolated from surrounding tissues by proteolytic digestion with 150 U/ml of collagenase (CLS I1; Cooper Biomedical Inc., Malvern, PA) for 15 min at 4"C and for an additional 6-9 minutes at 37'C. The reaction was stopped with complete tissue culture medium of Eagle's minimum essential medium containing 15% horse serum and 10% chick embryo extract, since the serum components contain endogenous protease inhibitors. The isolated neural tubes, with notochords sometimes attached to the ventral surface, were rinsed in complete medium and then plated onto fibronectinor laminin-coated substrates. After 2-3 h, neural crest cells begin migrating away from the dorsal aspect of the neural tube and form a monolayer of cells on the culture dish. At this time, the cultures were fed complete medium (Eagle's minimum essential medium, 10% embryo extract, 15% horse serum). In some cases, Eagle's minimum essential medium, a defined medium lacking serum and embryo extract, was substituted for complete medium during rinsing, plating, and feeding of the cells to eliminate any possible effects of serum or embryo extract components. Most cultures were used for adhesion assays 24 h after plating. In those cultures maintained for longer periods, neural tubes were scraped away after 24 h, leaving a monolayer of neural crest cells. Cultures were fed fresh medium every other day. For some experiments, somites isolated in the same manner as the neural tubes described above were plated onto fibronectinor laminin-coated