Worm Breeder's Gazette 12(5): 60 (February 1, 1993)

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 Use Of Unc-76 /lacZFusions to Label Specific C Elegans Axons

Laird Bloom, Erika Hartwieg, Bob Horvitz

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

The study of HSN axonal outgrowth relies on antisera against the neurotransmitter serotonin to show the morphology of the HSN axons in detail. Because serotonin is expressed only after HSN axon outgrowth is complete, these antisera cannot be used to observe events that occur during outgrowth. We have used the axonal localization of Unc-76 ß-galactosidase fusion proteins to develop a method for labeling axons of the HSNs and other neurons at earlier stages of growth, and we have constructed vectors into which any neuronally-expressed promoter can be inserted for labeling specific subsets of axons.

Translational fusions of the first 175 codons of unc-76 to the E. coli lacZ gene generated proteins localized in axons and the peripheries of cell bodies but not nuclei (visualized with an anti-ß-galactosidase monoclonal antibody, Promega Z378 A).To express such a fusion in a defined set of neurons, we used the unc-86 promoter, which is active in 57 neurons, including the HSNs (Finney and Ruvkun, Cell 63: 895-905,1990). Only five neurons expected to express an unc86 / unc-76 /lacZfusion have axons in the anterior half of the ventral cord (HSNL, HSNR, AVM, PVM, and PVR), and the positions of these axons in the cord suggested that the axon of the left HSN should be easily distinguishable from the others.

In collaboration with Gary Ruvkun and Mike Finney, we constructed p86 /76-1by fusing the unc-86 promoter and first 83 codons to codons 13-175 of unc-76 ,followed by lacZ. Animals carrying this plasmid and the rol-6 ( su1006 )-encodingplasmid pRF4 on an extrachromosomal array showed staining in axons and cell bodies of the cells expected to express unc-86 .Axons on the left and right sides of the ventral nerve cord were clearly distinguishable, and HSNs extending short processes with growth-cone-like structures at their tips were observed in L2 larvae. However, about half of the HSN cell bodies in these animals failed to migrate from the tail to their normal positions near the vulva, and the animals were insensitive to touch, suggesting defects in the mechanosensory neurons, which express unc-86 .These results suggested that a portion of the unc-86 / unc-76 /lacZconstruct was interfering with the normal HSN migration process, although migration defects were not observed in unc-86 or unc-76 loss-of-function mutants or in animals carrying nuclear-localized unc-86 /lacZor axonally-localized unc-76 /lacZfusions. Removal of unc-76 sequences from p86 /76-1did not alleviate these defects, nor did variation of the amounts of unc-86 coding sequences. We suspect that either the presence of the unc-86 promoter in high copy number or the presence of the N terminus of the Unc-86 protein outside its normal nuclear location interferes with HSN migration.

We believe that Unc-76 ß-galactosidase fusions might be generally useful for cell identification as well as for studying outgrowth. Cell bodies and axons were clearly labeled in fusions under the control of the unc-76 or unc-30 promoters (Yishi Jin, personal communication), and no obvious functional defects were observed in the neurons in which the fusions were expressed. Loss of unc-30 function blocks the expression of the neurotransmitter GABA in the VD and DD motorneurons, causing abnormal locomotion, but wild-type animals carrying an unc30 / unc-76 /lacZfusion (lacking all unc-30 coding sequences) moved normally; similarly, wildtype animals carrying unc-76 /lacZfusions showed no defects in locomotion or axonal outgrowth. We have constructed derivatives of Andy Fire's lacZ vectors in which a portion of unc-76 replaces the nuclear localization signal, and these vectors have been successfully used with the unc-30 promoter. These vectors are available upon request.

Because the blue reaction product of X-gal with ß-galactosidase is electron-dense, we have begun experiments to determine whether axonally-localized ß-galactosidase fusions can be used to label axons for electron microscopy. EM examination of X-gal-stained worms carrying p86 /76-1showed that the electron-dense label was usually restricted to neurons, but that it was not localized to the small number of expected axons, probably due to the diffusion of the reaction product evident even in the light microscope. However, cell bodies were clearly labeled, and so nuclear-localized ß-galactosidase fusions could be a useful way to label landmarks for electron microscopy.