Figure 6.

VAB-1/EphR and MAPK signaling. (a–d) Epifluorescence images of N2 wild type (a and b) and vab-1(dx31) (c and d) hermaphrodite gonads immunolabeled with anti-diphosphorylated MPK-1 (dpMPK-1) and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). dpMPK-1 labeling was comparable in the apoptotic region of the germline (brackets) of vab-1-null mutants as compared with the N2 wild type, as well as in the proximal region containing mature oocytes (arrowheads). (e) Quantification of mean fluorescence intensity within the oocytes and the apoptotic region of dpMPK-1-immunolabeled gonads, as shown in panels a–d. The data represent the mean pixel intensity in a fixed area after adjustment to an anti-CGH-1 control signal (n: N2, 31; vab-1, 28). (f) Length of the MPK-1 apoptotic signaling zone in N2 wild-type and vab-1(dx31) gonads, as defined by fluorescence thresholds relative to peak fluorescence (signal ≥50 or 75% of local peak value). vab-1(dx31) had modestly shorter zones of strong dpMPK-1 labeling (Student's t-test, ^*P=0.037), and fewer vab-1 mutants met the minimum fluorescence value for inclusion in the analysis. (g) Germ-cell corpse counts in N2 wild type, mpk-1/Erk, and let-60/Ras mutants with and without a transgene expressing a constitutively active, myristoylated VAB-1 receptor isoform (VAB-1^MYR). Apoptosis was increased upon VAB-1^MYR expression in the wild-type background (normal engulfment; n: no transgene, 30; VAB-1^MYR 39), and this increase was dependent upon both mpk-1/Erk (n: no transgene, 26; VAB-1^MYR 28) and let-60/Ras (n: no transgene, 28; VAB-1^MYR 26) function (Mann–Whitney test with ties adjustment, ^*P<0.001). Mutations used: mpk-1(oz140), let-60(dx16), and vab-1(dx31). Data were collected from at least three independent experiments and represent the mean±S.E.M.