DEVELOPMENTAL BIOLOGY 82,86-94 (1981) Analysis of Developmentally Homogeneous Neural Crest Cell Populations ln Vitro I. Formation, Morphology and Differentiative Behavior JEANNE LORING,’ BENGT GLIMELIUS,’ CAROL ERICKSON,~ AND JAMES A. WESTO~~ Department of Biology, University of Oregon, Eugene, Oregon 9740.9 Received March 31, 1980; accepted in revised form May 2, 1980 When early embryonic quail neural tubes are dissected free from surrounding tissues and placed in culture, small stellate neural crest cells usually migrate from the explant onto the substratum. This outgrowth has been reported to consist of a mixture of cells, some of which undergo melanogenesis, while the rest remain unpigmented. We have, in contrast to earlier observations, obtained a spatial separation of the two phenotypes. In these cultures the primary outgrowth of migrating cells remained almost free of pigment-forming cells, whereas small spherical clusters con- taining several hundred pigment-forming cells appeared on the explanted neural tubes. Whether the clusters remained with the tube explants or were subcultured, all cluster cells differentiated into melanocytes. Prior to melanogenesis, the appearance of the cultured cells from a cluster was indistinguishable from the cells in the outgrowth. The clusters provide a source of neural crest cells, that (1) can be easily obtained in comparatively large numbers, (2) is not contaminated with any other cell type, (3) can be isolated before the onset of differentiation, and (4) is developmentally homogeneous. Thus, the cluster population is well suited for many types of experiments, such as the identification of specific environmental factors that might control neural crest cell differentiation. INTRODUCTION Neural crest cells migrate extensively during early stages of vertebrate embryogenesis, localize at specific sites in the embryo, and differentiate into many cell types including pigment cells, neurons, glial cells, skel- etal and connective tissue cells, and a variety of en- docrine derivatives (Weston, 19’70; LeDouarin et al., 1974; Noden, 1978). The neural crest, prior to migration, is thought to be a pluripotent population of cells whose differentiation is controlled by environmental factors encountered dur- ing migration and/or after localization (Weston, 1970; Cohen, 1972;Norr, 1973;Noden, 1978;Teillet et al., 1978). The pattern of differentiation of some crest derivatives in vitro can also be influenced by the culture environ- ment (for review, see Patterson, 1978; see also Hawrot, 1980), but the specific environmental factors have not yet been identified. To analyze how the choice and expression of partic- ular crest phenotypes is controlled, developmentally 1Present address: Department of Biology, University of Iowa, Iowa City, Iowa 52242. * Present address: Institute of Oncology, Akademiska Sjukhuset, Uppsala, Sweden. 3 Present address: Department of Zoology, University of California, Davis, California 95616. 4To whom reprint requests should be sent. homogeneous cell populations must be used. The crest cells that migrate onto a culture substratum away from explanted neural tubes, however (Cohen and Konigs- berg, 1975; Maxwell, 1976a,b; Cohen, 1977; Pintar, 1978), are developmentally heterogeneous (see Cohen and Konigsberg, 1975; Cohen, 1977) and are usually contam- inated with epithelial and fibroblastic ceils (Cohen and Konigsberg, 1975; Maxwell, 1976b; Pintar, 1978). The cloning techniques developed by Cohen and Kon- igsberg (1975) have provided important opportunities to analyze homogeneous neural crest cell populations, and these have begun to be exploited (Sieber-Blum and Cohen, 1978, 1980). However, since it is difficult to pre- dict the differentiative fate of a single clone before dif- ferentiation begins, investigation of other developmen- tal capabilities of the undifferentiated crest cells is limited. Moreover, time in culture is a critical consid- eration, since differentiation of crest cells occurs quickly under the culture conditions normally used. Therefore, cell cloning procedures, which require prolonged culture periods to produce populations large enough for de- tailed analysis, preclude analysis of early differentia- tive processes. We have recently observed that small spherical clus- ters of cells form in vitro on neural tubes explanted from quail embryos. When removed from the tube and cultured separately, all cells from these clusters dif- ferentiate into melanocytes. Under the conditions of 86 0012-1606/81/030086-09$02.00/O Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any farm reserved.