Worm Breeder's Gazette 9(3): 33a
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The loci unc-13 and unc-15 are positioned very close together in the cluster of linkage group I. Both mutants have distinct phenotypes; unc- 15 (e73) worms are limp and paralyzed, while unc-13 (e450) worms are kinked and paralyzed. Mutations at the unc-15 locus affect the putative structural gene for paramyosin. Clones for unc-15 are available and have been used to map a contig that may be long enough to contain the unc-13 locus. A cosmid from the far end of this contig will be used to clone unc-13. Additional incentive to clone unc-13 has come from the isolation of a strain with a restriction site polymorphism from a TR679 background that affects both unc-13 and unc- 15 (see Roger Hoskins' report in this newsletter). In anticipation of cloning unc-13, we wished to identify the lesion causing its phenotype. In order to determine if the unc-13 phenotype was due to a lesion in the muscle, e450 adults were osmium fixed and sectioned for electron microscopy. All the fixation and ultramicrotomy was completed by Nichol Thomson to guarantee the highest quality. In both cross sections and transverse sections, the ultrastructural features of wild type muscle were observed with regular spacing of thick and thin filaments and the normal diagonal banding pattern due to overlapping of these filaments. To investigate our suspicions that unc-13 (e450) was a neural connectivity mutant, we produced a serial reconstruction of an adult ventral cord. Our series began about midway in the retroventricular ganglion and continued to include one repeat of motoneurones in the ventral cord. To facilitate the reconstruction, a neurone management program written by Richard Durbin was used. The lesion that we uncovered was subtle yet reproducible for each motoneurone affected. The largest pair of interneurones in the ventral cord, AVAL and AVAR, were found to have gap junctions to all the excitatory motoneurones of the A and B classes. All the wild type neural connections were preserved, but additional inputs were made from the AVA interneurones to the VB and DB motoneurones by gap junctions. A physiological interpretation based on this mutant circuitry would suggest that all the A and B classes of motoneurones may be electrically coupled together through the AVA interneurones. This would not allow forward locomotion (coordinated by the DB and VB motoneurones) to be separated from backward locomotion (coordinated by the DA and VA motoneurones). This lesion of neural connectivity is consistent with the observed phenotype of both larval and adult unc-13 worms. In L1 larvae, only DA and DB motoneurones would be present, yet both would have gap junctions to the AVA interneurones, prohibiting the separation cf forward and backward locomotion. In adults, the phenotype would be preserved with the addition of both VA and VB motoneurones, since both would have gap junctions to the AVA interneurones. Two further projects are presently being pursued concerning unc-13. The first is tc isolate the unc-13 gene by probing Northern blots of RNA from amber alleles using the cosmid C44E1 (believed to contain the unc-13 gene). In complementary experiments, Roger Hoskins will attempt to rescue the unc-13 mutant by transformation. The second is to reconstruct another unc-13 null allele to verify that this neural lesion is genuine.