The avian neural crest : behaviour and long-term survival in culture

Johnston, David Annandale (1986) The avian neural crest : behaviour and long-term survival in culture. University of Southampton, Doctoral Thesis.

Abstract

Avian mesencephalic neural crest cells (MNCCs) undergo an extensive migration within the developing head, following defined pathways to particular sites where they proliferate and subsequently differentiate. As tissue opacity prevents direct observation of these events, the behaviour of quail MNCCs has been studied in vitro. Time lapse video, phase contrast and anti-tubulin immunofluorescence photomicroscopy were used in quantitative analyses of the morphological, motile and structural polarity of cultured MNCCs, and, for comparison, cardiac fibroblasts (CFBs), a known, polarised cell population. At 48 hours in vitro; (1) isolated MNCCs and CFBs possessed similar, multipolar morphologies and showed random movement, (2) CFB explants comprised confluent, parallel arrays of elongated cells, possessing radially-polarised microtubule systems and showing directionally-persistent, radial movement, (3) mesencephalic neural crest (MNC) explants comprised a sub-confluent network of stellate cells, neither possessing radially-polarised microtubule systems, nor showing direct, radial movement. Thus, neither innate behaviour nor social interactions conferred directional persistence on MNCCs in these cultures. The radial polarisation of monolayered MNCCs, assessed by the orientation of their nucleus - microtubule-organising centre axes, did, however, increase with time in culture (24 - 72 hours). This correlated with progressive cell spreading and decreased cell-free space within the monolayer. The radial polarisation of monolayered CFBs remained marked throughout. The significance of these results for the control of MNC migration in vivo is discussed. To assess the role of cell cohesion in defining MNC migration pathways, the ability of MNC primary explants to spread over heterospecific (chick / quail) fragments of other mesencephalic tissues was examined. Histological examination of tissue fragments from reciprocal co-cultures, however, revealed inconsistent sorting behaviour and so this analysis proved inconclusive. Previously, analysis of MNC development in vitro has been limited by the rapid senescence of cultured MNCCs. During this study, crude bovine testicular hyaluronidase (CBTH) was found to maintain the proliferation of quail MNCCs, allowing repeated sub-culture over a period of several months. Purified hyaluronidase preparations, containing demonstrable hyaluronidase activity under tissue culture conditions, failed to reproduce this effect, suggesting that an impurity in the CBTH was responsible. Sub-cultured cells were both morphologically and antigenically similar to cells in MNC primary explants, sub-populations expressing E/C8, anti-glial fibrillary acidic protein, anti-vimentin and anti-fibronectin reactivities, NC-1 reactivity was, however, lost. Some sub-cultured cells retained a capacity for melanogenic or neuronal differentiation. The phenotypic similarities between sub-cultured, MNC-derived cells, in vitro, and MNC derivatives, in vivo are discussed. (D74720/87)

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