mec-7 is an alpha-tubulin gene which is required for the production of 15-protofilament microtubules in C. elegans (Chalfie and Thomson, 1982, J. Cell Biol. 93, 15-23; Savage et al., 1989, Genes and Dev. 3, 870-881). These microtubules are found only in six touch receptor neurons (ALML, ALMR, AVM, PVM, PLML and PLMR). To examine
mec-7 gene expression, we have developed a whole mount in situ hybridization technique (write for a detailed protocol). Adults and larvae were fixed with paraformaldehyde, permeabilized by Proteinase K digestion in the presence of -mercaptoethanol, and incubated with a 20-mer probe corresponding to the C end of the
mec-7 protein (a
mec-7 specific region). The oligonucleotide was labeled by 3'-tailing by terminal deoxytransferase with digoxigenin-dUTP and dATP. The hybridization signal was detected using an anti-digoxigenin antiserum conjugated with alkaline phosphatase. The staining is touch cell-specific, a result that is consistent with the previous observations using an anti-
mec-7 antibody (Savage and Chalfie, unpublished). To see whether the
mec-7 gene expression is affected by putative upstream genes, we examined the hybridization in mutants defective in
lin-32,
unc-86 (supposed to be involved in touch cell generation) and
mec-3 (supposed to be involved in touch cell specification). In
lin-32 animals, only the ALM cells were found and they were often displaced to the anterior direction. In
unc-86 and
mec-3 animals no signal was found. These results are consistent with the previous observation about the touch cell differentiation and the notion that
mec-7 is expressed specifically in six touch cells as above. Temporally
mec-7 gene expression is first detected at L2 stage, first in the ALM and the PLM cells and then in AVM and PVM. The signal increases as the animals grow until the L3 and L4 stages. The staining decreases after these stages first in AVM and PVM and then in ALM cells. In adults, often the only detectable signal is in the PLM cells. These findings are consistent with temperature shifts with mec- 7ts mutants which reveal a temperature sensitive period during larval development (Chalfie and Thomson, 1982). In addition, in
mec-17 mutants the staining in PLM cells disappears sooner (L4/adult stage), suggesting that
mec-17 is needed for the maintained differentiation of the touch cells (see Way and Chalfie, 1989, Genes and Dev., 3, 1823- 1833). Since 15-protofilament microtubules are found maximally at the adult stage (Chalfie and Thomson, 1982), we wondered whether this decrease in the mRNA level was due to autoregulation of -tubulin mRNA (degradation in response to an increased amount of free -tubulin protein or tubulin dimers: Cleveland, 1989, Current Opinion in Cell Biol. 1, 10-14). To test this possibility, we examined
mec-7 mutants with one of eleven strong recessive (putative null) alleles. Seven mutants showed no hybridization signal and two mutants had a spatio- temporal pattern similar to that of wild-type animals. However, mutants defective in either of alleles
u428 and
u429 showed a strong signal in all six touch cells in egg-bearing adults. These results suggest that alpha-tubulin autoregulation may limit the production of
mec-7 protein in the touch cells after the animals become adults. We are also using this in situ hybridization technique to see whether any known mutations affect touch cell differentiation. So far, we have examined about forty genes (and gene combinations). No unusual patterns of staining have been seen with many lineage mutants.
ced-3 and
ced-4 mutants have additional PLM-like cells in the tail. These cells have processes that stain with an anti-
mec-7 antibody. Examination of double mutants by in situ hybridization reveals that the appearance of these extra cells requires the wildtype
lin-32, unc- 86, and
mec-3 genes. Double mutants
ced-3;
mec-4d and
ced-4;
mec-4d have more degenerating cells in the tail than in
mec-4d (see Chalfie and Sulston, 1981, Dev. Biol., 82, 358-370). Two cells die in each wild-type PLM lineage (Sulston et al., 1983, Dev. Biol. 100, 64-119), but we do not know which of these (if either) is the source of the additional staining (MC bets it is ABp(l/r)apapppa). One hypothesis we are entertaining, given these results, is that
mec-3 is expressed quite early in the PLM lineage (or in part of it) and is possibly repressed. Approximately 10% of
lin-4 and
lin-14sd mutations have what appears to be an extra set of AVM and PVM cells. Since in mutants with
lin-14(o) alleles, the Q cell gives a V5.pa lineage ( Ambros and Horvitz, 1984, Science, 226, 409-416), the opposite transformation (V5.pa to Q) results in these extra
mec-7-positive cells. Presumably this change occurs only 10% of the time because previous retarded steps in the V5 lineage prevent the appearance of the V5.pa cells. We are currently lineaging these mutants to determine whether this hypothesis is correct.
lin-4, aps other heterochronic genes may be important regulators of the Q and V5.pa fates.