Supplementary Figure 7: Neonate Lgr5⁺ populations and their progeny are lifelong contributors of the adult OSE lineage in vivo.

(Top) Wholemount and histological analyses of LacZ activity in ovary at various times post P1-induction. A single limiting dose of 4-OHT induced LacZ activity at clonal density. Sparse isolated LacZ⁺ cells were first observed 2 days (2d) p.i. Uniform rate of expansion pre-puberty led to similar-sized LacZ⁺ clones 1 week (1w) and 4 weeks (4w) p.i., whilst variable rate of expansion post-puberty led to heterogenous-sized LacZ⁺ clones at 3 months (3m) p.i. Frequency of lineage tracing was maintained up to 16 months (16m). Note larger-sized clones adjacent to corpora lutea (red arrowheads) compared to follicles (white arrowheads). Dot plot shows LacZ⁺ OSE clone size quantification post P1-induction. n = 5 females/time-point, total of 461 clones counted. Each dot represents a disparate LacZ⁺ OSE clone. Red bars indicate s.d. ranges. Green circles denote mean clone size, which steadily increased over time (green line). F, follicle; CL, corpus luteum. (Bottom) Confocal z-stack images of Lgr5-KI/tdTomato ovary treated with an increased dose of 4-OHT at various times post P1-induction, revealing steady increase in the number of tdTomato⁺ progeny over time. Scale bars: 100 μm (wholemount images); 10 μm (sections).