Worm Breeder's Gazette 10(3): 31
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.
mec-3 is a homeobox-containing gene that is required for expression of differentiated characteristics of the touch receptor neurons (Way and Chalfie, Cell 54, 5-16). To identify the cells in which mec-3 is expressed, we constructed a mec-3-lacZ gene fusion by inserting a lacZ XhoI-SalI fragment into the XhoI site in the mec-3 homeobox, leaving 6 kb of upstream C. elegans sequence. This construction was injected into C. elegans along with Andy Fire's 'twitcher' plasmid (pD10.41, which transcribes the antisense strand of unc-22 from the unc-54 promoter). We identified one injectant from which the two plasmids formed a large, heritable extrachromosomal tandem array (termed uEx4). Animals carrying uEx4 can be recognized as twitchers. When animals are stained with X-gal according to Andy Fire's procedure, the touch receptors (ALMs, PLMs, AVM and PVM) are stained, as predicted. In any given animal, the complete set of cells is usually not stained, suggesting that these animals are genetic mosaics. To our surprise, the FLP and PVD neurons also stain. The identification of these cells is based on observing the pattern of DAPI-stained nuclei in X-gal stained animals, and is supported by the fact that staining is not seen in unc-86(e1416) animals, which lack FLP and PVD (as well as the touch cells) due to lineage alterations. Ed Hedgecock suggested to us that these cells might be members of a single class (perhaps stretch receptors), since both send out undifferentiated anterior and posterior processes that make essentially no synapses, as well as sending a process into the ventral cord (see White et al., Phil. Trans. Royal Soc. London 314, 1-340 [1986]). At a low frequency (about 5% of all animals), staining is observed in other cells including mostly neurons in the ventral cord but also a few intestinal cells and cells within the pharynx. In stained gut cells, the stain is colocalized with the nuclear DAPI-stain. It is not possible to distinguish between staining of cell bodies or nuclear staining for neurons, since they have so little cytoplasm. Processes do not stain. In one preparation, we observed staining of two cells in the region of the vulva in several egg laying adults. Prompted by the staining result, we examined PVD in clr-1 and clr-1; PVD is located just anterior to the post- deirid and relatively isolated. In clr-1 animals, the PVD cell body is much larger than the cell bodies of the post-deirid neuron (PDE), its sheath or socket cells, and its anterior and ventral process can clearly be seen. In clr-1; the PVD cell is often not seen (50% of all animals). When it is present, it is no larger than the post-deirid cells and lacks the anterior process but retains the ventral process. It appears that PVD is always altered in a mec-3 mutant, most often expressing the fate of its sister, which undergoes programmed cell death. This idea is being tested by examination of a ced-1; The uEx4 element was crossed into strains containing unc-86, s to study the regulation of expression of the fusion construction. In unc-86(e1416); uEx4, no staining is seen at any stage of development except in occasional, random cells. In mec-3::Tc1(u298); uEx4 the fusion is expressed in embryos, L1 animals, and in the post-embryonic AVM, PVM and PVD cells within about 12 hours after these cells are born. The construction is not expressed at all in L4 or adult animals, although unstained adults containing stained embryos are often seen. Thus, in a mec-3 mutant, the expression of the fusion can be initiated but not maintained. This result suggests that mec-3 positively regulates itself. Since mec-3 is likely to be a DNA binding protein, it may directly induce its own transcription. This result implies that mec-3 is not only expressed but is active in FLP and PVDS, since the regulation of the fusion by mec-3 is seen in the FLP and PVD cells. These data and the clr-1 observations suggest that mec-3 regulates the differentiation of these cells as well as the touch receptors. mec-4(d) causes degeneration of the touch receptors. In mec-4(d); uEx4 strains, the FLP and PVD cells stain normally, indicating that they do not degenerate and that the mec-4(d)-induced degeneration is indeed touch cell specific. This fact, along with the ultrastructure of the touch cells vs. FLP and PVD, confirms that these really are two different cell types, and raises the question as to how mec-3 acts in each type to promote two different cell fates. Trimming the mec-3 gene. By injection of deleted forms of the intact gene into a mec-3 mutant, we have further limited the sequences necessary for mec-3 function. DNA can be deleted from the published sequence (Cell 54, 5-16) up to bp 1500, between 2250 and about 2580, and beyond 4900 and still rescue a mec-3 mutant in the transformation assay. The presence of a Tc1/EcoRV deletion remnant at bp 1818-1819, deletion of bp 2300 to 2630, deletion of sequences beyond bp 4000 all lead to loss of gene function. From these results, we believe that the coding sequence probably starts with the Met codon at bp 2686 (and that the first two putative exons in the published sequence do not seem to be required, since the second can be deleted and the first can then not be spliced to the other exons). Sequence gazing has revealed an unusual pattern of cysteine and histidine residues within the first three exons of mec-3. There are three repeats that each resemble: Cys/His X X Cys/His X X Cys/His X X Cys X X Cys/His. This motif is distinct from the zinc finger but reminiscent of the patterns of cysteines in metallothionein, the HIV tat protein (which requires metal ions to dimerize [Frankel et al., Science 240, 70-73]), a variety of viral regulatory proteins identified by Berg as possible metal-binding proteins (Science 232, 485-487). This similarity opens the possibility that mec-3 might be a metal-binding protein. We have no idea why. To our knowledge, no other homeobox proteins have this type of motif.