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.

mab-5 Levels Can Determine the Size of the Ray Domain

C. Kenyon

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.