Worm Breeder's Gazette 12(2): 83 (January 1, 1992)

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.

Faintly-staining mec-7 -positiveCells

Hongping Du, Marty Chalfie

Department of Biological Sciences, Columbia University, New York, NY 10027

mec-7 encodes an alpha-tubulin that is needed for the function of the six touch receptor neurons ALM(L/R), PLM(L/R), AVM and PVM. In situ hybridization by Shohei Mitani in our lab (WBG 11 #3 103) suggested that detectable message was only found in these six cells. A mec-7 lacZfusion investigated by Hamelin et al. (WBG 11 #3 102) showed a similar pattern of expression except that these workers saw some staining in the FLP cells. The significance of this additional staining is not clear since Shohei Mitani found that egl-44 and egl-46 act as negative regulators of mec-7 expression in the FLP cells and the integrated mec-7 lacZstrain undoubtedly has several copies of the construct per cell (and so could, in theory, dilute out these negative regulators). Given these observations we have been reexamining the expression pattern of mec-7 protein using a rabbit antisera originally generated against a C-terminal peptide of the mec-7 alpha-tubulin by Cathy Savage in our lab.

We have been using the worm staining procedure that Mike Finney distributed at the Madison Worm Meeting. The only problem we had with this method was that the background was quite high (thus making faintly staining cells difficult to see). We have greatly reduced the background by pre-absorbing the primary and secondary antibodies using the procedure of G. D. Johnson (Antibodies. A Practical Approach, Vol. 2, D. Catty, ed., IRL Press, Oxford, pp. 179-200, 1989). In this procedure antibodies are mixed with acetone powders of tissues. We have used a powder of a mec-7 ( u443 )strain to pre-absorb the primary antibody [ mec-7 ( u443 )is a deletion of the mec-7 gene] and a powder of wild-type worms to pre-absorb the secondary antibody.

The staining pattern of N2 animals with the cleaned antibodies shows some surprises. The touch receptor neurons stain quite intensely, but a few other neurons also appear to stain (none of this staining is seen when the mec-7 deletion strain is examined). Both the FLP and the PVD cells have a low level of expression (these are the two other cell types that express the mec-3 gene). The FLP staining is seen in approximately 50% of the animals; PVD staining is even more frequent. (FLP staining in an egl-46 ( n1076 )is much stronger, similar to that seen within the touch receptor neurons.) In addition, a few cells in the ventral ganglion and several cells in the tail stain, but we have not yet determined the identity of these cells.

These data suggest that mec-7 is strongly expressed only in the six touch receptor neurons, but can be weakly expressed in other cells. Genes such as egl-44 and egl-46 might control cell fate, not so much by determining whether a gene is expressed or not, but by regulating the correct level of expression of target genes in different cells.

The mec-7 alpha-tubulin is needed for the production of the 15-protofilament microtubules in the touch receptor neurons. We do not know whether mec-7 protein expressed in other cells is found in microtubules in these cells and, if so, whether these microtubules have 15 protofilaments (a second gene, mec-12 ,is also required for 15-protofilament microtubules and its expression pattern is unknown). Further examination of the cell processes may resolve this issue.