Quantification of PGC death

The number of Vasa-positive PGCs in each embryo was manually counted following confocal microscope imaging. For each genotype, the average PGC number at ES 10 was set as 1 and the individual values at ES 10 and 13 were expressed as a percentage of the average value at ES 10. The values were then plotted using box and whisker charts, in which the median is shown as a line in the middle of the box and the minimum and maximum values are the bottom and the top of the whiskers respectively. Each dot in the box represents a single embryo clearly reflecting the sample number (n). On average, a sample number of 23 was used for each genotype. Note, whereas at ES 13 most of the dead PGCs are already eliminated, dying PGCs can be sometimes still detected by virtue of their highly condensed morphology and relatively faint Vasa staining signal, and are omitted from the counts.

PGC death index in Fig. 1d was calculated as follows: The number of embryos with at least one dying PGC (condensed/distorted) was divided by the total number of examined embryos giving a value A. The total number of dying PGCs in all the embryos was divided by the number of embryos with at least one dying PGC, giving a value B. Death index equals AxB.