Figure 1.

HP1a reduction during mid- and late-stage oogenesis leads to defects in ovarian morphology and subfertility. (A) HP1a expression is reduced significantly in HP1a RNAi ovaries. Immunoblot of wild-type, Su(var)2-5⁰⁴ heterozygous, HP1a RNAi, and control EGFP RNAi ovaries. HP1a RNAi #1 is obtained from the Transgenic RNAi Project and HP1a RNAi #2 is newly generated in this study (Supplemental Material). HP1a RNAi #3 is a third HP1a RNAi line that was generated to target a different site on HP1a and was not used in this study. Ctrl RNAi corresponds to EGFP RNAi with the same insertion sites. HP1a level is reduced to one-half the amount of w¹¹¹⁸ (wild-type) in Su(var)2-5⁰⁴ heterozygotes, while HP1a is reduced to an undetectable amount in HP1a RNAi ovaries. HP1a level is slightly higher than that of w¹¹¹⁸ control and the EGFP control RNAi flies. GAPDH expression serves as a loading control. (B) Fertility is significantly reduced in HP1a-deficient females. Eggs collected in females from each HP1a RNAi (#1 and #2) and control RNAi (#1 and #2) in the Su(var)2-5⁰⁴ heterozygous background were counted on days 2, 4, 6, 8, 10, and 12. The average number of eggs laid in the two HP1a RNAi lines and the control RNAi lines was calculated for each day. Starting on day 6, HP1a-deficient females laid significantly fewer eggs than the control females. * P-value ≤ 0.05. n, HP1a RNAi = 540; n, control RNAi flies: 1101. Error bars indicate the mean value ± SEM in three biological replicates. (C) HP1a RNAi ovaries exhibit various morphological and nuclear defects. HP1a expression was detected using anti-HP1a antibody labeling. (a–c) Confocal micrographs of the control EGFP RNAi ovariole in the Su(var)2-5⁰⁴ heterozygous background. The HP1a expression level shown in (b) is comparable to that of the w¹¹¹⁸ or Su(var)2-5⁰⁴ heterozygous ovarioles (data not shown), and no defects or abnormalities in the ovarian morphology are detected. (d–h) Confocal micrographs of HP1a RNAi ovarioles and egg chambers in the Su(var)2-5⁰⁴ heterozygous background. (e) shows a diminished level of HP1a expression throughout the HP1a RNAi ovariole. The insets in (d–f) highlight two joined egg chambers with multiple nuclei. (g) An egg chamber with overcondensed nuclei. (h) An ovariole with a series of early stage egg chambers. EGFP, enhanced GFP; fc, follicle cell; g, germarium; nc, nurse cell; o, oocyte; RNAi, RNA interference.