Worm Breeder's Gazette 14(1): 36 (October 1, 1995)
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.
HHMI, Dept. Biology, MIT, Cambridge MA 02139, USA
Epithelial invagination is an important developmental process,
fundamental to gastrulation and organ formation. Invagination can involve
cell movement, changes in cell shape, and changes in cell-cell or
cell-extracellular matrix adhesion. We are interested in learning what
molecules are involved in this morphogenetic process and are using C.
elegans vulval formation as a model system. The hermaphrodite vulva is a
tube that connects the uterus to the outer epithelium and is formed by the
invagination of a specialized set of epithelial cells during the third and
fourth larval stages of development.
We screened for mutants with a defective vulval invagination but
normal vulval cell lineages and designed the screen to allow isolation of
mutations that cause sterility. From over 12,000 haploid genomes
screened, we isolated 26 mutations, which we placed into eight
complementation groups. Seven appear to define new genes, sqv-1 to 7
(squashed vulva), and alleles of the eighth fail to complement
spe-2(mn63). The strongest alleles of all eight genes result in
identical vulval phenotypes: a wild-type division pattern and detachment
from the cuticle, but indistinct separation between the anterior and
posterior halves of the invagination from the late L3 stage onward. We
found no mutations that caused any other abnormal vulval phenotype without
also affecting vulval cell lineages.
We have cloned sqv-3 and spe-2, both of which mapped to areas
sequenced by the genome project; we have entered their positions into the
Acedb database. sqv-3 is predicted to encode a homolog of mammalian
beta-1,4 galactosyltransferase, an enzyme localized to the trans Golgi,
where it adds galactose to proteins, many of which are then transported to
the cell surface. We have so far been unable to demonstrate that SQV-3
itself has galactosyltransferase activity. The predicted SPE-2 protein
has similarities to two classes of human expressed sequences from the
WashU-Merck EST project but has no similarity to any protein of known
function.
Each of our eight genes involved in vulval formation has alleles
that additionally cause hermaphrodites to be self-sterile. Homozygous
mutants generate eggs that have egg-shells but fail to hatch (and probably
even to divide). Genetic evidence suggests that spe-2 hermaphrodites have
inviable sperm but viable oocytes: they can have homozygous spe-2 progeny
if crossed with spe-2/+ males[1]. sqv-1 to 7 hermaphrodites, on the other
hand, cannot have progeny even if mated with wild-type males. We have so
far shown, using the technique of artificial insemination[2], that sqv-3
hermaphrodites do have viable sperm, implying that their oocytes or gonad
must be defective. It is therefore possible that SPE-2 may be required in
sperm and SQV-3 (or a SQV protein glycosylated by SQV-3) in another cell
with which the sperm must interact (e.g. the oocyte) for fertilization to
proceed beyond the cue for egg-shell formation. Alternatively, it is
possible that SPE-2 must simply be provided to the zygote via sperm and
that SQV-3 (or a SQV protein glycosylated by SQV-3) must be provided via
the oocyte and that both are required for an early embryonic process. We
are currently trying to define the infertility defect caused by these
genes more carefully and are hopeful that information about this defect
might also have implications for how the genes are affecting vulval
invagination.
We have raised antibodies against both SQV-3 and SPE-2 and, after
affinity-purification, have used them to stain whole worms. In each case,
we have so far been unable to see staining of wild-type worms but do see
reproducible staining patterns in worms overexpressing either SQV-3 or
SPE-2 by means of an integrated array containing the appropriate minimal
rescuing fragment. Our preliminary results indicate that SQV-3 may be
expressed in subcellular dots (Golgi perhaps) in the intestinal cells,
possibly spermatheca, head mesodermal cell, and vulva. The sqv-3
fertility defect may therefore result from a need for expression in the
intestine, which produces yolk and possibly other proteins that are
packaged into oocytes. Our preliminary results concerning SPE-2 indicate
that it may be expressed in subcellular dots in many cells in the head and
pharynx, in the tail, and, as expected, in adult sperm. We have not yet
seen SPE-2 staining in the vulva.
[1] Sigurdson et al. (1984). Genetics 108: 331. [2] LaMunyon and Ward
(1994).
Genetics 138: 689.