Figure 5. CO designation in the absence of ZHP-1/2.

(A) Projection images of representative late pachytene nuclei stained for GFP-COSA-1 (green) and SYP-1 (red). In the absence of DSB-2 at 24 hr post-L4, an average of 3 bright GFP-COSA-1 foci are detected in each nucleus. However, very few bright GFP-COSA-1 foci are observed when ZHP-2 is co-depleted. By 48 hr post-L4, only ~2 bright GFP-COSA-1 foci are observed per nucleus in the absence of DSB-2, and most nuclei display 0–1 bright GFP-COSA-1 focus when ZHP-2 is also depleted. (B) Quantification of bright GFP-COSA-1 foci at late pachynema, 24 hr post-L4. Worms of the indicated genotypes were maintained in the presence or in the absence of 1 auxin for 24 hr and then dissected for analysis. Data were derived from 4 to 8 gonads for each genotype or condition. n = 165, 348, 172 and 377 nuclei, respectively. The number of GFP-COSA-1 foci differed significantly between dsb-2::AID and zhp-2::AID; dsb-2::AID following auxin treatment (***p<0.0001 by Chi-square test for trend). (C) Quantification of bright GFP-COSA-1 foci in late pachytene nuclei 48 hr post-L4. Data were derived from 4 to 8 gonads for each genotype or condition. n = 252, 558, 263 and 416 nuclei, respectively. Depletion of ZHP-2 significantly reduces the number of GFP-COSA-1 foci in the absence of DSB-2 (***p<0.0001 by Chi-square test for trend). The distribution of GFP-COSA-1 foci is also significantly different between 24 hr and 48 hr post-L4 for auxin treated zhp-2::AID; dsb-2::AID hermaphrodites (***p<0.0001 by Chi-square test for trend). (D) Representative images of mid-pachytene (MP) and late pachytene (LP) nuclei stained for RAD-51 (yellow), GFP-COSA-1 (green), SYP-1 (red), and DNA (blue). GFP-COSA-1 foci in the absence of ZHP-1/2 was visualized following exposure of SPO-11-depleted worms to varying doses of ionizing radiation. L4 worms were maintained in the presence or in the absence of 1 mM auxin for 24 hr, followed by irradiation at the indicated dosage, and then incubated for additional 8 hr to allow irradiated nuclei to progress to late pachynema. Scale bars, 5 µm.

Figure 5—figure supplement 1. Validation of protein depletion, and further characterization of ZHP-1/2.

(A) Western blot showing efficient auxin-mediated depletion of DSB-2 alone, or co-depletion of ZHP-2 and DSB-2. Blots were probed with anti-FLAG and anti-tubulin antibodies, respectively. Tubulin was blotted as a loading control. 3F: 3xFLAG. (B) Low magnification images of gonads from worms at 48 hr post-L4 stained for RAD-51 (yellow), GFP-COSA-1 (green), SYP-1 (red) and DNA (blue). RAD-51 staining in the upper panel indicates that few DSBs are induced in the absence of DSB-2. The lower panel shows GFP-COSA-1 and SYP-1 staining in the same gonad. A few bright GFP-COSA-1 foci are detected in the absence of ZHP-2 when the number of DSBs is limited by DSB-2 depletion. Insets show enlargements of corresponding regions of the gonad. (C) SPO-11 activity is effectively abrogated by AID-mediated depletion. Projection images of pachytene nuclei stained for RAD-51 (yellow), GFP-COSA-1 (green), SYP-1 (red), and DNA (blue). DSBs were induced by ionizing radiation following depletion of SPO-11. No COSA-1 foci were observed in unirradiated animals, but CO designation was rescued following 10 Gy of irradiation. Scale bars, 5 µm.

Figure 5—figure supplement 2. Partial depletion fails to separate the roles of ZHP-1/2 in limiting late recombination intermediates and promoting crossover maturation.

(A) Western blots showing partial depletion of AID-tagged ZHP-2 in animals exposed to low concentrations of auxin. Blots were probed with anti-FLAG and anti-tubulin antibodies, respectively. Tubulin was blotted as a loading control. 3F: 3xFLAG. (B) Quantification of ZHP-2 depletion. The intensity of ZHP-2 bands were normalized against the corresponding tubulin band, and expressed as percentage of the intensity in a control sample. The graph shows the mean ±SD protein levels from three independent experiments. (C) Low-magnification images of late pachytene nuclei stained for GFP-COSA-1 (green) and SYP-1 (red). Insets on the right show representative oocyte nuclei at late diakinesis under the same conditions. In the presence of 3 µM auxin for 24 hr, zhp-2:AID; P_sun-1:TIR1 germline still show six designated CO sites per nucleus marked by bright GFP-COSA-1 foci. At 10 µM auxin, a reduced number of bright GFP-COSA-1 were detected. Nuclei with more than six bright foci were not observed under any depletion conditions. Similar results were obtained when ZHP-1-AID was depleted (data not shown) Scale bars, 5 µm.