During the L1 larval stage, the P blast cells divide to generate 12 Pn.p cells, of which six (P3.p - P8.p) become the Vulval Precursor Cells (VPCs) that go on to divide and form the vulva.
pvl-5(
ga87 ), a mutation which affects the generation of Pn.p cells, was isolated in a screen for mutations causing a P rotruding v u l va ( pvl ) phenotype. In
pvl-5 (
ga87 ) mutants there are an average of 7.0 large Pn.p cells [N=172; Range 4-10] in the ventral midline compared to 11 (P1.p - P11.p) in wild-type animals. Consequently, there are fewer than 6 VPCs and usually fewer than 22 cells available to form the vulva. The average number of Pn.p cells in
pvl-5 (
ga87 )/ mnDf39 animals is similar to that in
ga87 homozygous mutants [7.4 (N=48; range 4-11)], suggesting that
ga87 is likely a null allele.
de3, a second allele isolated in an F1 non-complementation screen, exhibits a similar range and severity of phenotypes as
ga87 [average number of large Pn.p cells = 8.6 (N=145; range 4-11)]. The decrease in the number of Pn.p cells could be attributed to a loss of P cells or Pn.p cells by cell death, a failure of P cells to migrate into the ventral midline, or a Pn.p to a Pn.a cell fate transformation. Lineage analysis shows that the 12 Pn.p cells are born correctly in
pvl-5 mutants, but later undergo abnormal cell death around the L1 molt. The nuclei of dying cells appear swollen, pitted and stain brightly with Acridine Orange (AO) and SYTO-12, nucleic acid stains that have been used to identify dying cells (1). This indicates that the Pn.p cells are dying in
pvl-5 mutants. If the cells are undergoing apoptosis in
pvl-5 mutants, then perturbing the machinery responsible for apoptosis should cause the cells to live. In
pvl-5 ;
ced-3 (lf) double mutants the average number of Pn.p cells is 10 (N=92; range 7-11). Further, in
pvl-5 ;
ced-9(gf) double mutants, the average number of Pn.p cells increases to 10.2 (N=87; range 7-11). This s uggests that the Pn.p cells are undergoing apoptosis in
pvl-5 mutants and that PVL-5 might function to prevent Pn.p cell death in wild-type animals. Curiously,
ced-4 does not appear to suppress the
pvl-5 phenotype. A single ORF, C56E6.6, rescues the Pn.p cell defect in
pvl-5 mutant animals, increasing the average number of Pn.p cells from 7.2 to 10.3 (N=95). C56E6.6 encodes a Leucine-Rich-Repeat (LRR) containing protein. The LRR is a protein-protein interaction motif and is present in some PCD regulating proteins. Experiments to determine the localization of PVL-5 protein are in progress and results will be reported. 1. Gumienny, T., Lambie, E., Hartweig, E., Horvitz, RH., Hengartner, M.(1999) Development 126(5) , 1011-1022 Keywords: Development: the vulva; Cell Death