Worm Breeder's Gazette 14(3): 62 (June 1, 1996)
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.
Dept. of Developmental Biology, Stanford Medical Center, Stanford CA, 94305-5427
We identified pvl-1(ga80) X in a screen for mutations causing a
protruding vulva or Pvl phenotype (WBG 13.1, p69). In pvl-1(ga80)
hermaphrodites, one or more of P5.p, P6.p or P7.p remains undivided or
adopts a 3! fate, leading to the generation of fewer vulval cells than
normal, and a Pvl or Egl phenotype. In addition, P3.p, P4.p and P8.p
often remain undivided rather than adopting the 3! fate. pvl-1
hermaphrodites show a highly penetrant transformation of P12.p to a
P11.p fate, similar to that seen for mutations in components of the Ras
signaling pathway, lin-44 wingless , and mig-5 disheveled (Guo and
Hedgecock, 1995 Mtg Abs, p245). In addition, pvl-1 animals are slightly
uncoordinated and males mate poorly. A second allele of this gene,
sy324, was kindly provided by Andy Golden. Gene dosage experiments
using a deficiency suggest that neither of these mutations is a null
allele.
We used transformation rescue experiments to clone pvl-1. We
identified overlapping cosmids, T21H4 and F43E12, that rescue the pvl-1
mutant phenotypes, and then narrowed down the rescuing activity to a DNA
fragment predicted to contain a single gene. Comparison of the sequence
of full-length and partial cDNAs with genomic sequence from the Genome
Sequencing Project showed that this gene encodes a predicted 812 amino
acid protein with 30% identity to members of the armadillo/b-catenin
class of adhesion/signal transduction molecules. armadillo/b-catenin
proteins have two well-characterized functions. First, these proteins
are localized to adherens junctions where they play a role in cell-cell
adhesion through interactions with E-cadherins and a-catenin. Second,
armadillo/b-catenin proteins function in wingless signaling pathways.
In Drosophila, wg signal leads to the stabilization of arm protein and
its accumulation in cytoplasm, where it functions in signal
transduction. Recent experiments on vertebrate b-catenins have
suggested that they can interact with transcription factors, and may
translocate into the nucleus as a consequence of signaling. We are
currently doing experiments to try to determine if pvl-1 encodes a true
C. elegans homolog of armadillo/b-catenin, or is a novel, armadillo
-repeat-containing protein.
We have built a fusion gene that encodes residues 1-763 of PVL-1
fused to GFP, and that rescues the pvl-1 mutant phenotypes. Preliminary
results show expression of this fusion protein in a number of tissues,
including the Pn.p cells before induction, and hyp7 and other hypodermal
cells in larvae and adults. The GFP fusion protein appears to be
cytoplasmically-localized in these tissues. We soon hope to verify the
localization and expression pattern of Pvl-1 by staining with anti-Pvl-1
antibodies.
Genetic epistasis experiments with pvl-1(ga80) have shown that
the ga80 mutation can suppress the ectopic inductions at P3.p, P4.p and
P8.p caused by gain-of-function mutations in let-60 ras and mpk-1 MAP
Kinase. However, ga80 has much less of an effect on such inductions
caused by loss-of-function mutations in lin-1 and lin-31, which encode
transcription factors acting downstream of the ras pathway. One
interpretation of these results is that pvl-1 functions downstream of
let-60 and mpk-1 during vulval induction, but upstream or parallel to
lin-1 and lin-31. This would represent a novel function for an
armadillo/b-catenin-related protein, and we are interested in
determining if pvl-1 is a target of the Ras signaling pathway in the
vulva. For example, we would like to know if the amount or subcellular
localization of PVL-1 changes upon Ras signaling. Alternatively, pvl-1
might function in a novel signaling pathway that interacts with the Ras
pathway during vulval development, perhaps a wingless pathway. We are
testing if pvl-1 interacts with wingless pathway genes such as lin-44,
lin-17 frizzled (Sawa and Horvitz, 1995 Mtg Abs, p455) or mig-5
disheveled during vulval induction. In addition, we are doing yeast two
hybrid experiements to identify proteins that interact with PVL-1.