Worm Breeder's Gazette 9(2): 55
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.
We have previously described the GABA immunoreactive neurons of C. elegans and the immunocytochemical identification of mutants affecting VD and DD axonal outgrowth or GABA expression (CSH Abstracts, 1985). Through further screening of existing locomotory mutants, we have identified additional genes affecting these processes. We summarize our results here: Putative GABAergic Neurons Antibodies to GABA label six embryonic and thirteen postembryonic cell bodies in the ventral nervous system, a corresponding set of commissures, and the ventral and dorsal nerve cords. The morphology of these cells and their positions in the nerve cord, as ascertained by double labeling with DAPI in adults and embryos, have led us to conclude that they are VD and DD motorneurons. Notable swellings occur at lateral and sublateral positions along the commissures. An additional set of four embryonic neurons just anterior to the nerve ring are also labelled. Based on their location and the disposition of their processes, we strongly suspect these to be the RME neurons. Analogous sets of GABA immunoreactive neurons have been described in Ascaris (See Gazette 8(2), p.33). A single postembryonic neuron, which is likely to be DVB, is labelled in the dorsal rectal ganglia of the hermaphrodite. Our results with certain mutants (described below) are consistent with this assignment. Two unpaired postembryonic neurons occur in the ventral ganglia. We suspect these to be the RIR and RIS neurons. A faintly labelled cell has been noted in the lateral ganglia just posterior to the nerve ring. Likely candidates are AVL or RID. A number of additional unidentified cells are labelled in the male tail. Mutants with Abnormal VD or DD Axonal Outgrowth We have identified ten genes that affect the outgrowth of VD and DD axons. Mutations in four genes [unc-5(e53), unc-6(e78), 69), and unc-69( e587)] result in a complete failure of the dorsal extensions of these neurons to reach the dorsal midline. Labelled processes appear in various lateral positions where they frequently interconnect or reenter the ventraI cord. Ectopic branches are observed. The disposition of labelled processes in the ventral cord can also appear abnormal in these mutants. The normal spacing between immunoreactive cell bodies in the ventral cord is variably disrupted and the DD or VD cell bodies have been observed in positions dorsal or lateral to the ventral cord. unc-5 was previously identified as being 'dorsal cordless.' Hedgecock et al. have identified aberrant amphid, phasmid, and PDE processes in unc-51. (Developmental Biology 111, 158 (1985)) Partial reconstructions of unc-6 have revealed a general disorganization of the dorsal cord and displacement of the excretory cell canal in three of nine cut animals (Leon Nawrocki, personal communication). Mutations in another four genes [unc-34(e315), unc-40(e271), 11), and unc-62(e644)] result in less complete defects in commissure formation. In these mutants, the dorsal extensions of DD and VD processes often run laterally for considerable distances before turning again to meet the dorsal cord. The commissures occasionally branch before reaching the dorsal midline and ectopic extensions are observed. Cell bodies can again appear dorsal or lateral to the ventral cord. Processes from these cell bodies tend to reach the dorsal midline by an atypical path. The placement of labelled processes in the ventral and dorsal cords can also appear abnormal. Axonal growth defects have been visualized in other neurons in unc-76 by FITC uptake Hedgecock et al., Dev. Bio. 111, 158 (1985) ]. Mutations in two genes, unc-71(e541) and unc-73(e936), result in frequent (25% and 35% respectively) formation of commissures on the incorrect side of the animal. Again, the labelled processes in the ventral cord appear disorganized. The commissures themselves are generally normal although they occasionally run anteriorly or posteriorly in a lateral position before continuing to the dorsal midline. Mutants with Altered GABA Expression We have identified possible defects in GABA expression in four genes with a shrinker phenotype. In unc-25(e156), there appears to be no immunoreactivity in any of the neurons that are normally labelled with antisera to GABA. Double labelling with Hoechst or DAPI reveals a normal number of neuron-like nuclei in the ventral cord. In unc-30(e191), there is normal staining in the head and tail of the hermaphrodite. The VD and DD motorneurons, however, consistently fail to stain. EM reconstructions of unc-30 had shown that the placement of VD and DD processes is abnormal; other ventral cord neurons appear unaffected (J. White, personal communication). In the shrinker, unc-47(e307), the intensity of labelling appears considerably enhanced which may represent a defect in the release of GABA from nerve terminals. Finally, in unc-43(e408), the VD neurons have a reduced intensity of labelling, larvae move better than adults in this mutant. Lineage Mutants We looked at several lineage mutants, many of which were suspected to involve lineages generating the GABAergic neurons. The mutation lin-4(e912) results in reiterations of K, which generates the postembryonic DVB neuron. In approximately 50% of lin-4 hermaphrodites, multiple labelled cells occurred in the position of DVB and had a similar morphology. We also noted from one to three additional labelled cell bodies in varying positions of the ventral cord in approximately 15% of lin-4 hermaphrodites. Additional DAPI stained nuclei were observed in the ventral cords of these animals. In unc-86(e1416), two RIR like cells were observed in 70% of adults and L1s. (RIR is the sister of the interneuron AVG.) One to two additional stained nuclei were observed in the tail of 60% of unc-86 hermaphrodites. Finally, enlarged stained cell bodies have been observed in unc-59, cesses of these cells were not, however, definable. Further analysis of some of the uncoordinated mutants discussed here may prove useful to an understanding of axonal guidance and the metabolism of GABA or neurotransmission at GABAergic synapses in C. elegans.