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Title: Gap junctional communication during patterning of the limb and neuromuscular junction formation
Author: Allen, Francesca Le Gros
Awarding Body: University College London (University of London)
Current Institution: University College London (University of London)
Date of Award: 1988
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Intercellular communication has been investigated between embryonic Xenopus neurones and myocytes in vitro and during digit patterning in the Chick wing bud. Lucifer yellow and Huridine were used to identify intercellular transfer and metabolic cooperation in both systems, and communication inhibition by a specific antibody raised against gap junctional protein confirmed the nature of this transfer. The molecular tracers and immunogenic reagents were loaded into tissues in bulk using DMSO permeabilisation. Dye transfer from embryonic Xenopus myocytes to neurones was observed during coculture. 24 hours after plating 77% of the loaded myocytes and 23% of the recipient neurones contained dye. The presence of gap junctional antibody in either cell type reduced the neuronal label to 4%, indicating cell coupling occurred. Metabolic cooperation experiments using the transfer of HNucleotide in both directions between the neurone and myocyte confirmed these findings. Blocking these gap junctions had no significant effect on neuronally induced AChR clustering on the myocytes. The signalling of the chick limb polarising region (PR) was studied by loading pellets of PR diluted with anterior mesenchyme (AM) with gap junctional antibody prior to grafting the tissue into the anterior margin of the host limb bud, and observing the resulting duplications. When PR cells alone were inhibited from intercellular communication more respecification of the host AM occurred, with greater duplications. In contrast, if both the diluting AM cells and the PR cells were loaded with antibody, reduced levels of duplication were obtained. Metabolic cooperation between dissociated PR and AM cells in culture confirmed that the antibody was active throughout the respecification period, and blocked gap junctions in the chick. Possible roles of intercellular communication in both systems are discussed. At the developing synapse gap junctions are probably not involved in the local structural rearrangements but may enable the neurones to recognise suitable targets for innervation in vivo. The chick results suggest that morphogenetic patterning in the limb bud may be more complex than previously recognised.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available