Gap junctions (GJs) occur in all animals from very early stages of development. Yet, despite their ubiquity, gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. GJs are difficult to detect. In vivo, microinjection is required which limits the technique to large and/or unusually accessible cells. We propose to develop a molecular biological method for the in-vivo detection of both enduring and transient GJs without the need for intracellular injection. ? Transgenic animals will be made with tissue specific expression of B-galactosidase. The animal will be injected with a B-gal substrate (C12FDG) which is taken up by cells and hydrolysed to a small fluorescent reporter molecule. B-gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing B-gal can be detected with antibodies; any cell not expressing 6-gal, but exhibiting the reporter color must have received its color via GJs. Another similar method will be tested which makes use of tissue specific expression of transporters to load the presynaptic cells with a small tracer which can pass through GJs and be detected in post-junctional cells. Controls include the demonstration that GJ mutants block the transmission. ? As a first use, we wish to apply the method, to the following problem. During the last 30 years, many authors have demonstrated temporary gap junctions (GJs) occurring in nervous system development just prior to the period when chemical synapses are formed. GJ communication has been hypothesized to play a direct causal role in the establishment of functional chemical synapses. However, it has not been possible to block the GJs and thus prove a causal link. ? In the adult eye, there are no retina-to-lamina gap junctions. Yet, we showed that mutations in two gap junction genes disrupt chemical synaptic transmission there. In mutants, transmission can be restored by GJ transgenes, but only if they are expressed during the period of eye development; expression of the genes in the adult does not rescue. We hypothesize that lamina gap junctions during development are essential for the later development of chemical synaptic transmission. We wish to use the method described herein to directly demonstrate such transient GJs ? ?
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| Curtin, Kathryn D; Wyman, Robert J; Meinertzhagen, Ian A (2007) Basigin/EMMPRIN/CD147 mediates neuron-glia interactions in the optic lamina of Drosophila. Glia 55:1542-53 |
