Heterogeneity in neural crest development detected in monoclonal antibodies

Heath, Lindsay Ann (1990) Heterogeneity in neural crest development detected in monoclonal antibodies. University of Southampton, Doctoral Thesis.

Abstract

The vertebrate neural crest is a transient embryonic structure which arises as a morphologically homogeneous population at the tips of the neural folds. The cells of the neural crest undergo extensive migration and subsequently differentiate into a wide variety of adult cell types, including the autonomic nervous system, melanocytes and much of the skeletal and connective tissues of the head. Precisely how and when this apparently homogeneous population begins to diversify into this wide variety of adult cell types has not yet been fully determined. In order to investigate the question of cell heterogeneity within the premigratory and early migratory crest population, monoclonal antibodies were produced, following the injection of quail premigratory mesencephalic neural crest into mice. In order to overcome the problem introduced by the limited availability of immunogen, the intrasplenic route for immunization was chosen. Three monoclonal antibodies were subsequently isolated which recognised subpopulations within 24h cultures of both quail and chick mesencephalic neural crest during immunofluorescent studies. These antibodies are referred to as LH2D4, LH5D3 and LH6C2. Subsequent investigations using the antibodies raised during this study, and those of other workers, showed that this heterogeneity could be detected as early as 15h following mesencephalic crest explanation. However, the premigratory neural crest population of the same embryonic origin was homogeneous, as judged by the reactivity patterns obtained with these antibodies. Similar staining patterns were found within and on cells in 24h cultures (the earliest stages examined) of trunk neural crest cultures when compared to mesencephalic cultures of the same age. However, quantitative studies revealed a significant difference in the sizes of the antibody-positive and antibody-negative populations when such cultures were compared. By employing double immunofluorescence techniques, the number of subpopulations which could be detected within these cultures, using the antibodies raised here, was determined to be three or four, depending upon the antibody combination used. BrdU incorporation studies were carried out in order to confirm that the expression of the epitopes detected by these antibodies was not related to the cell cycle. The results of LH2D4, LH5D3 and LH6C2 staining on sections through quail embryos from stages 7-22 demonstrate a spatiotemporal pattern of regulation in the developmental and tissue expressions of the three epitopes during the early stages of avian ontogeny. The epitopes detected by these antibodies were found not to be confirmed to the neural crest or to cells of crest origin. Since the differential epitope expression described here occurred soon after neural crest explanation and took place in an in vitro culture environment, and in the absence of other cell types, it is concluded that this degree of heterogeneity can be generated very early in neural crest ontogeny (possibly while the cells are within the neural folds) without the need for embryonic tissue interactions.

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