Molecular and cellular biology of an ecdysone-dependent cell-adhesion molecule in Drosophila

Imaginal discs of Drosophila undergo a dramatic change in shape in response to the steroid molting hormone 20-hydroxyecdysone (20-HE). This process, termed evagination, is brought about through small, directed movements of imaginal disc cells. When imaginal discs are cultured in-vitro, cell surface proteins show changes in response to 20-HE, suggesting that evagination could be caused partially by changes in cell surface and extracellular components. Certain Drosophila tissue culture cell lines respond to 20-HE by aggregating. In the Drosophila S3 cell line a number of 20-HE dependent cell surface and extracellular glycoproteins that are biochemically similar to proteins found in imaginal discs appear to play a role in hormone-dependent cell adhesion. A hormone-dependent extracellular glycoprotein with a molecular weight of 110 kD (P110) was purified and analyzed. Polyclonal antibodies to P110 (anti-P110) were generated and used to investigate the role of this molecule in cell adhesion. Fab fragments of anti-P110 effectively inhibited the reaggregation of hormone-treated S3 cells, while preimmune Fab fragments had no effect. Additionally, hormone-treated cells showed increased binding to the P110 band when incubated with nitrocellulose blots of glycoproteins from culture media of hormone-treated cells. A cDNA clone of the P110 transcript was recovered from a cDNA library prepared from mRNA of developing embryos and used as a probe to examine the levels of RNA coding for P110 in tissue culture cells and throughout the fly life-cycle. P110 message showed a hormone-induced increase in accumulation in tissue culture cells. During embryogenesis, P110 RNA was present in highest amounts between six to nine hr of development. P110 message was not detected in larvae until late third instar. During the larval/pupal transition, when imaginal disc morphogenesis occurs, signal dramatically increased as compared to levels in late third instar, then rapidly decreased over the next four hr. Extensive homology was found between the protein sequences of P110 and the Drosophila Aggregating Protein Activity I (DAPA I). It is proposed that the P110 glycoprotein complex functions as a hormone-dependent cell-adhesion molecule. This work provides a basis for further biochemical and genetic research on 20-hydroxyecdysone dependent morphogenesis in Drosophila.