Worm Breeder's Gazette 11(3): 38

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Touch Cell-Specific Expression and Developmental Regulation of the mec-7 beta-tubulin Gene in C. elegans

Shohei Mitani and Marty Chalfie

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and Chalfie, unpublished).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.