Worm Breeder's Gazette 10(2): 18

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More About xol-1: A Gene that Controls the Male Mode of Sex Determination and Dosage Compensation

Leilani Miller and Barbara Meyer

Figure 1

In a previous WBG article (Miller, et al.,October, 1987), we 
proposed that wildtype xol-1 gene product negatively regulates sdc-1 
and sdc-2 to ensure that they, and other genes involved in XX sex 
determination and dosage compensation, remain inactive in XO animals.  
XO animals carrying mutations in xol-1 cause the inappropriate 
expression of sdc-1 and sdc-2, thus allowing the expression of the XX 
mode of sex determination and dosage compensation.  This results in 
lethality (presumably caused by under expression of X-linked genes) 
and feminization.  In this article, we will provide evidence for 
reduced X chromosome expression and feminization.
We postulated that the XO-specific lethality caused by xol-1 
mutations is due to inappropriately low X chromosome expression.  To 
test this, we looked at X-expression directly in dying embryos using 
the Northern hybridization assay (Meyer and Casson, Cell 47, 871-881) 
to measure X-specific mRNA levels.  We found that xol-1 XO animals 
under-express their X-linked genes.  Using two different X-linked 
probes (myo-2 and uvt-4), we showed that, relative to autosomal 
controls (myo-1 and act-1, respectively), the X-linked transcripts 
were significantly under-expressed in RNA prepared from dying xol-1 
embryos when compared to RNA prepared from normal embryos.  
Quantitation by densitometer tracing of many experiments (over 20) 
revealed that xol-1 dying embryos express their X-linked genes at 
levels less than half that of wild-type levels, supporting the theory 
that mutant XO animals are carrying out the XX mode of dosage 
Sex determination in xol-1 XO animals is also shifted to the XX mode.
Although xol-1 XO animals die as embryos or young larvae, some of 
the larvae are healthy enough to allow observation of cells that are 
sexually dimorphic at hatching.  To ensure that we were looking at XO 
dying larvae, the following cross was made: dpy-21 V; flu-2 
were mated with him-8 IV; unc-3 X 
hermaphrodites.  The dpy-21 mutation was used to rescue the xol-1 XO 
males; the X-linked flu-2 mutation (an intestinal autofluorescence 
marker scoreable in L1 larvae) was used to identify XO animals.  Flu 
cross progeny from the above cross must be xol-1 XO dying larvae.  The 
sexual phenotypes of three sexually dimorphic cells (B, Y, and HSN) 
were scored in these larvae and all three cells were found to be 
capable of undergoing hermaphrodite specific cell fates.  
Unfortunately, it was very difficult to score the majority of the 
animals due to the general sickness and extensive cellular 
disorganization in these animals.  As a further complication, the HSN 
was often impossible to see because the region around the gonad in 
these dying animals is very refractile.  Of 15 scoreable B cells (out 
of over 50 animals), 11 had chosen the hermaphrodite fate and 4 the 
male fate.  All 7 scoreable Y cells had undergone the hermaphrodite 
fate.  HSNs (hermaphrodite fate) were observed in 3 animals.  It is 
apparent that some cells in xol-1 mutant XO animals undergo 
hermaphrodite-specific developmental fates, implying that the wild 
type xol-1 gene product is required in XO animals for proper male 
sexual development.  However, due to the difficulty in scoring most of 
the animals, it is impossible to estimate the extent to which dying XO 
animals are transformed.
XO animals carrying mutations in xol-1 inappropriately execute the 
XX mode of both sex determination and dosage compensation.  Mutations 
in genes required for XX sex determination and dosage compensation (
sdc-1 and sdc-2) completely suppress all the defects associated with 
xol-1 mutations.  Doubly mutant animals are essentially 100% viable 
and all are males.  In contrast, mutations in genes required only for 
dosage compensation (dpy-21, dpy-27, and dpy-28) 
suppress only the dosage compensation defects (XO lethality and under-
expression) but not the sex determination defects (feminization).  
Doubly mutant animals are alive, but many are still feminized.
A typical experiment demonstrating the above points involved mating 
dpy-21(y47) V; xol-1 X males with rol-6 II; dpy-21(y47) V; xol-1(y9) 
hrodites.  A comparison of Dpy non-Rol non-
Unc (XX) cross progeny with Unc non-Dpy non-Rol (XO) cross progeny 
yields information about the extent of XO rescue.  100% rescue would 
give equal numbers of XX and XO cross progeny.  For this cross, we saw 
essentially complete rescue (97%).  In the case of the temperature-
sensitive, maternal effect mutation, dpy-28(y1), we observed (at 20 C) 
26% rescue by homozygous dpy-28(y1) progeny from heterozygous mothers 
and over 80% rescue by dpy-28(y1) homozygous progeny from homozygous 
mothers.  The sexual phenotypes of the XO cross progeny can also be 
observed and used to show that, with dpy-21, dpy-
27, and dpy-28, many rescued XO animals are still feminized.  For 
example, of the XO cross progeny (Unc non-Dpy non-Rol) in the dpy-21 
cross above, 35% were hermaphrodites, 6% were pseudomales, and 59% 
were males.  For XO animals rescued by mutations in dpy-26, 
ved a higher percentage of 
hermaphrodites (60%-100%) than with the dpy-21 mutations.
When males were mated with XO xol-1 hermaphrodites in the above 
crosses, two classes of XO cross progeny were observed: one class in 
which the XO animals carried an X chromosome from the mother and one 
in which the X chromosome came from the father.  Interestingly, the 
sexual phenotype of a rescued XO animal is dependent upon the parent 
from which it inherited its X chromosome.  XO animals that received a 
maternal X chromosome were more likely to develop as hermaphrodites, 
while those that received a paternal X chromosome tended to develop as 
males.  For example, 62% of the dpy-28(y1);  
rescued XO animals that received a maternal X developed as 
hermaphrodites, while only 16% of those that inherited a paternal X 
developed as hermaphrodites.
As a final note, it is possible to use a xol-1 mutation to design a 
WZ/ZZ system of sex determination in C.  elegans (similar to J.  
Hodgkin's system with tra-1).  As we have reported before, xol-1 
mutations have a masculinizing effect on XX animals that are already 
partially masculinized by a mutation in the sex determination pathway. 
For example, a tra-2; X animal can be a 
mating male.  Because mutations in xol-1 allow tra-2(lf) XX males to 
mate, the genotypes shown below depict a system whereby sex is 
determined by the activity state of one gene.
[See Figure 1]

Figure 1