Worm Breeder's Gazette 12(1): 33 (September 1, 1991)

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.


Laird Bloom, Bob Horvitz

HHMI, Dept. Biology, MIT, Cambridge, MA 02139 (617) 253-5128

The gene unc-76 is required for normal outgrowth of axons of a number of neurons, primarily in regions of neuron-neuron contact. We have reported that the gene encodes alternatively-spliced proteins, 385 and 376 amino acids long, that are not similar to any proteins in the Genbank database (WBG, 11(5), p. 31, 1991; 1991 C. elegans meeting abstracts, p. 57). An unc-76 -ß-galactosidasefusion protein containing about two-thirds of the unc-76 coding region in Andy Fire's pPD16 .43vector was localized in neuronal bundles and a few neuronal cell bodies. Animals carrying this gene fusion on an extrachromosomal array showed intense staining in the ventral cord, nerve ring, head and tail ganglia, and in non-neuronal cells in the head hypodermis and the vulval region. Weak staining appeared in the dorsal cord and all other longitudinal neuronal bundles, including those in the head. Staining was first observed in embryos around the comma stage, after most embryonic neurons had been generated, and persisted through adulthood.

Similar results were obtained with animals carrying an epitope-tagged derivative of the unc-76 gene on an extrachromosomal array. This epitope, nine amino acids from influenza hemagglutinin recognized by the monoclonal antibody 12CA5 (Field et al., Mol. Cell. Biol. 8:2159, 1988), was attached to the C-terminus of the longer form of the unc-76 protein and did not interfere with the rescuing activity of the unc-76 clone. Antibody staining (using the method of Mike Finney, personal communication) was visible in neuronal cell bodies and in some axons but not in nuclei. Staining appeared in head and tail ganglia, in at least some members of all classes of ventral cord neurons, and in several lateral neurons, including the HSNs, CANs, and several cells in the postdeirid cluster. Fewer axons stained in animals carrying the epitope-tagged unc-76 clone than in animals carrying the unc-76 -lacZfusion, possibly because antibody staining was much weaker than ß-galactosidase staining or because the unc-76 -ß-galactosidasefusion protein might persist longer than the epitope-tagged unc-76 protein.

Although we have not yet identified all of the cells that express unc-76 ,a few conclusions are possible. First, at least some neurons express unc-76 while they are sending out axons. The HSNs stain as early as the L2 stage, when they first send out a process (Gian Garriga, personal communication). Also, Pn.a-derived ventral cord neurons were observed to stain shortly after their birth. Second, because large numbers of cells stain in young larvae but very few stain in L4 sand adults, it seems likely that most neurons do not need to express unc-76 at high levels to maintain their final morphology. However, several neurons in the head and tail express unc-76 in adults, well after axon outgrowth is believed to be complete, suggesting that unc-76 may be involved in processes other than axonal outgrowth. Third, some of the cells that express the epitope-tagged unc-76 protein (VD, DD HSN) are known to be affected by mutations in unc-76 .A major question that remains is whether unc-76 function is required in affected cells alone, in the neighbors of these cells, or in both. We hope that further analysis of the unc-76 expression pattern will suggest an answer.