Fig. 4. ZAM deletion from the flamenco piRNA cluster leads to ZAM reactivation and oocyte invasion.

a Density plots showing genome-unique piRNAs mapping over 20 kb of the flamenco piRNA cluster (without mismatch) where the ZAM insertion is located (Release 6: X:21,769,891..21,789,891) in the control (Iso1A) and flamΔZAM lines. The position of the flamenco ZAM copy (ZAM-flam) is indicated. The positions of the sgRNAs used for ZAM-flam deletion by CRISPR-Cas9 are shown in red. b Density plot of ZAM-mapping regulatory piRNAs along the ZAM sequence in control and flamΔZAM ovaries (up to 3 mismatches). c Scatter plot showing the normalized counts of antisense regulatory piRNAs mapping to individual internal TE sequences in control ovaries (Iso1A) versus flamΔZAM ovaries. Antisense piRNA counts, mapped allowing up to 3 mismatches, were normalized per million of genome-mapping piRNAs (RPM, here in logarithmic scale). TEs in red have a flamΔZAM/Iso1A ratio <0.3. d Color-inverted confocal images of ovarioles (upper panels) and stage 10 egg chambers (lower panels) from the indicated genotypes showing ZAM smRNA FISH signal. The experiment was independently repeated at least 5 times, with similar results obtained each time. e Ovaries of the flamΔZAM line and of the flamΔZAM line with three recent ZAM copies introduced by genetic crossing and X chromosome recombination with the RevI-H2 line. f Box plot displaying the number of eggs laid per fly per day by RevI-H2i2, flamΔZAM and flamΔZAM females with three recent ZAM copies. Each dot represents an individual female. n = 63 for RevI-H2i2, n = 60 for flamΔZAM, n = 60 for flamΔZAM with three recent ZAM copies. In the box plots, the midline corresponds to the median value; the lower and upper hinges correspond to the first and third quartiles; and the whiskers span the minimum and maximum values, excluding outliers. ***p value < 2.2e-16; ns, not significant (p value > 0.05) (Mann-Whitney two-sided test).