Worm Breeder's Gazette 10(2): 15

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Analysis of the Temperature-sensitive Mutant sdc-1(y67)

Anne Villeneuve and Barbara Meyer

Figure 1

We have been performing temperature-shift experiments using the 
temperature-sensitive allele sdc-1 (y67) in an attempt to determine 
the time(s) at which sdc-1 acts in the sex determination and dosage 
compensation processes.  sdc-1 (y67) is sharply temperature-dependent, 
producing a strong mutant phenotype at 20 C (restrictive) and a nearly 
wild-type phenotype at 15 C (permissive).
Temperature-sensitive period for somatic sexual phenotype: In order 
to study specifically the effect of temperature on sex determination 
in the absence of the dosage compensation defect, we have used the 
temperature sensitive sdc-1 allele in combination with the suppressor 
mutation y63 (which appears to suppress the dosage compensation but 
not the sex determination defects of sdc-1 mutations [AMV and BJM, WBG 
Volume 10:1]).  A reciprocal temperature-shift experiment using this 
strain defines a brief and specific temperature-sensitive period 
during the first half of embryogenesis for the action of sdc-1 in 
somatic sex determination; the data for gonad sexual phenotype are 
depicted in the accompanying graph.  The tsps for both somatic gonad 
and tail/copulatory structures are coincident, extending from 
approximately the 100-cell stage to the end of the majority of 
embryonic cell division (approximately 320 minutes after the first 
cleavage), just prior to the onset of elongation.  This period is 
complete more than two hours before the first visible sexual 
dimorphism (HSN cell death in the male), approximately eight hours 
before hatching, and nearly a full day before the gonad itself becomes 
sexually dimorphic.
We have previously proposed that sdc-1 acts upstream of her-1 in the 
sex determination pathway, as a negative regulator of her-1 activity 
in XX animals.  Consistent with this proposal, the tsp for gonad 
sexual phenotype for sdc-1 is prior to that determined for her-1 (J.  
Hodgkin, JEEM 83, Supplement, 103-117).
As with any t-shift experiment, we cannot rule out the possibility 
that the tsp might reflect the time of synthesis of a thermostable 
product rather than the time of action of a thermolabile product.  The 
fact that the upshift curve has an inflection point during 
embryogenesis suggests that at least the maternal endowment of sdc-1 (
y67) may be thermolabile, since sdc-1 mutants are normally rescued by 
maternal sdc-1+ activity.  This is not a strong argument, however, 
since 1) 15 C sdc-1(y67) product may not be equivalent to wild-type 
sdc-1 for maternal rescue activity and/or 2) the maternal endowment of 
sdc-1 may be supplied as RNA.
Effects of temperature on the sdc-1 dosage compensation phenotypes:  
We have begun to assess the effects of temperature on the dosage 
compensation phenotypes of sdc-1(y67) .  This analysis is somewhat 
less straightforward than that for sexual phenotype, since the visible 
morphological manifestations of the dosage compensation defect (Sma, 
Egl, protruding vulva) are more ambiguous to score than is the case 
for sexual transformation.  Moreover, increased X-expression only late 
in development may not have any obvious scorable morphological 
consequences.  As a first step, we have performed reciprocal shift 
experiments defining the tsp for the 'Sma 
enotype using a her-1; ) 
strain.  The upshift curve in this case closely resembles that for 
somatic sexual phenotype; that is, remaining at permissive temperature 
through the end of embryonic cell proliferation is sufficient to yield 
a nearly wild-type phenotype.  The simplest interpretation of these 
data is that the presence of sdc-1 activity through the first half of 
embryogenesis is sufficient for establishment of the normal XX mode of 
dosage compensation.  The downshift curve, however, is quite different-
- shifting from restrictive to permissive temperature even as late as 
L2 can result in partial rescue of the mutant phenotype.  It seems 
that 'wild-type' sdc-1 activity may be capable of shifting animals 
back toward the XX mode of dosage compensation even when introduced at 
a time later than sdc-1 normally acts during wild-type development.
Conclusions about the function of sdc-1 in dosage compensation based 
on these experiments must of course remain tentative pending 
demonstration that these morphological phenotypes accurately reflect 
the state of X-linked gene expression.  We are currently assaying 
levels X-specific transcripts in RNA preparations from synchronized 
populations of sdc-1(y67) worms that have been raised at one 
temperature through embryogenesis and then subsequently shifted to the 
second temperature for the remainder of development.
Tail and gonad do not choose their sexual fates independently in sdc-
1 mutants: A striking observation has come out of careful analysis of 
the classes of sexually transformed animals produced by sdc-1 mutant 
strains in which animals range in sexual phenotype from fertile 
hermaphrodites to intersexes to pseudomales.  Specifically, there is a 
strong correlation between tail and gonad sexual phenotypes in 
individual animals even though these tissues diverge lineally at the 
first embryonic cleavage.  XX animals with male tails usually have 
male gonads, and animals with hermaphrodite tails usually have 
hermaphrodite gonads, while animals with tail and gonad of opposite 
sex are relatively rare.  Put more concisely, different cells in the 
embryo are not choosing their sexual fates independently.
How can we explain this phenomenon mechanistically? One possibility 
is that there is a strong influence of the maternally provided 
environment on the choice of sexual fate.  The complement of 
components supplied to the oocyte by the mother is likely to vary 
somewhat from oocyte to oocyte; in the absence of sdc-1 activity, the 
choice of sexual fate becomes sensitized to this naturally-occurring 
variability.  (This phenomenon probably cannot be accounted for simply 
by residual maternal sdc-1 activity resulting from mutations that do 
not entirely eliminate gene function, since it occurs with all sdc-1 
alleles including those likely to represent or approximate the null 
A second intriguing possibility is that the sex determination 
decision in C.  elegans is not cell autonomous, but rather involves 
cell-cell interactions.  This type of model suggests the existence of 
a 'master' cell or tissue in which the X/A ratio is assessed and the 
decision is made; that tissue or cell then communicates with the rest 
of the cells in the worm, perhaps via humoral factors, to regulate 
downstream switch genes such as tra-1 that control sexual identity in 
a cell-autonomous fashion (C.  Hunter and W.  Wood, WBG Volume 10:1).  
We are now preparing to do mosaic analysis of sdc-1 with the hope of 
shedding light on these issues.

Figure 1