(A) Low-magnification images of whole mounted gonads depicting COSA-1::GFP and dpMPK-1 localization in wild type. Signals for the pro-crossover marker COSA-1::GFP and dpMPK-1 are both observed at the mid to-late pachytene region. Insets show higher magnification images of different regions in the germline: (green inset) Early to mid-pachytene, no COSA-1 localization and dpMPK-1 is off; (red inset) mid to late-pachytene, COSA-1::GFP starts to localize on the chromosomes concomitant with the appearance of the dpMPK-1 signal; (yellow inset) late pachytene, 6 COSA-1::GFP foci/nucleus are observed and strong dpMPK-1 signal is detected. 20 gonads were analyzed. Bars, 20μm for whole gonad and 2μm for insets. (B) Low-magnification images of wild type, cosa-1 and zhp-3 gonads co-stained with dpMPK-1 (red), and DAPI (blue). dpMPK-1 expression level is not turned off in the cosa-1 and zhp-3 mutants in late pachytene and diplotene in contrast to wild type. n>18 gonads were analyzed for each. Bar, 20 μm. (C) Graph showing the quantification of dpMPK-1 fluorescence signal intensity in different regions of the gonad arm in wild type, zhp-3 and cosa-1 mutants. **p<0.005, *p<0.02 (unpaired student’s t-test; n>18, 17 and 15 gonad arm were analyzed for wild type, zhp-3 and cosa-1 mutants, respectively). Regions where dpMPK-1 signal was quantified are indicated on the diagram depicting the hermaphrodite germline. TZ stands for transition zone (leptotene/zygotene). E, M and L stand for early, mid and late pachytene, respectively. Progression from mitosis into meiosis is displayed from left to right and the last three oocytes at diakinesis (-3 to -1) are indicated. (D) Co-staining with HTP-3 (green), SYP-1 (red) and DAPI (blue) of diplotene nuclei from the indicated genotypes. Illustrations depict the bivalent configuration at this stage. S indicates short arm and L indicates long arm. White boxes indicate the bivalent shown at a higher magnification on the right. Bivalents with both long and short arms clearly displayed were chosen for higher magnification. Histograms on the right indicate the percentage of diplotene and diakinesis stage nuclei with SYP-1 either only on the short arm (S, blue) or on both long and short arms (red, L&S) of the bivalents. All the bivalents were examined in every nucleus and the bivalents in the same nucleus either all exhibited SYP-1 staining on both the long and short arms or all exhibited staining only on the short arms. Numbers of nuclei scored are shown. Bar, 2 μm. Worms from all the genotypes indicated in (A–C) were grown at 20°C and analyzed 18–24 hr post-L4. Worms from all the genotypes indicated in (D) were grown at 15°C, shifted to 25°C at the L4 stage, and analyzed 18–24 hr post-L4.