conservation of somatic cell replacement during early development of mouse female germline cysts Wanbao Niu, Allan C. Spradling HHMI lab, Department of Embryology, Carnegie Institution for Science Abstract In mice fetal ovary, Wnt4-expressing somatic cells we term "escort-like cells (ELCs)" in- teract with early developing cysts of both sexes. After E12.5, Lgr5+ pre-granulosa cells ingress from the ovarian surface epithelium, and our lineage tracing showed that they replace escort-like cells in the cortical region to establish the wave 2 granulosa cell pop- ulation supporting primordial follicles. In contrast, lineage marking of ELCs at E10.5 showed that early somatic cells are not heavily replaced in the medullar ovarian region that give rise to wave 1 follicles. Reflecting their distinct somatic cellular origins, second wave follicles were ablated by diptheria toxin treatment of Lgr5-DTR-EGFP mice at E16.5, while first wave follicles developed normally and supported fertility. These findings argue that somatic cell replacement in mice and Drosophila has been evolu- tionarily conserved, and while not essential, likely aids female gamete development. Figure 1 (A) In situ hybridization (ISH) analysis shows Wnt6 (blue) and Fmr1 (red) mRNA expression in the E14.5 ovaries. (B) Cellular localization of Id1 in E14.5 ovaries. Ovaries were stained for Id1, and the oocyte marker DDX4 at E14.5 by immunofluorescence. (C) Cellular localization of Gata4 in E14.5 ovaries by immunohistochemistry. (D) Electron micrograph of E14.5 ovary showing part of a germline cyst sur- rounded by ELCs (yellow asterisks). Squamous membranes of ELCs surrounding the germ cells are indi- cated by arrowheads. Figure 2 (A) Differentiation trajectory of E12.5, E14.5, and E18.5 ovary beginning with escort-like cells and epithelial cells and splitting into two branches (Gw1, Gw2) constructed by Monocle. Cell colored by the developmental state. (B) In situ hybridization analysis shows Wnt6 (blue) and Lgr5 (red) mRNA expres- sion in the E14.5 ovary. Lgr5+ pregranulosa cells were detected in the cortical but not in the deeper sub- regions. E14.5 Ovary wnt6 fmr1 E14.5 ovary wnt6 lgr5 F’ F’ F’’ F’’ Developmental State E12.5 E14.5 E18.5 Component 2 Component 1 0 5 -5 0.0 2.5 5.0 -2.5 Gw1 Gw2 Escort-like cells Epithelial cells Epithelial cells E10.5 E19.5 E12.5E15.5 P21 Tmx Analyze CreERT2 Crossing: Axin2CreERT2/+; R26RYFP/+ YFP Stop Axin2 Rosa26 } Cortex Medulla E12.5 10 μm Cortex Medulla E19.5 30 μm 10 μm YFP DDX4 DAPI DDX4 tdTomato DAPI P1 30 μm 30 μm 30 μm 20 μm E14.5 20 μm 20 μm 20 μm 10 μm WT-control Ddx4 DAPI (P21) WT-control Lgr5DTR/+ Lgr5DTR/+ Ddx4 DAPI (P5) 30 μm 30 μm 30 μm 30 μm 30 μm 30 μm 30 μm 30 μm WT-control Lgr5DTR/+ PrimordialPrimary Antral Number of follicles (P21) *** * NS Number of follicles (P5) *** NS Primordial Primary WT-control Lgr5DTR/+ E16.5 P5 P21 DT Analyze DTR-EGFP Lgr5 promoter (chromosome 10 in) 10 μm E14.5 Gata4 20 μm E14.5 A B C D A B B A C D E F A B C D 2 μm ELCs ELCs * * * * E13.5E14.5 E16.5 E18.5 P1 Tmx Analyze CreERT2 Crossing: Lgr5CreERT2/+;R26RtdT/+ tdTomato Stop Lgr5 Rosa26 } Number of labeled ELCs Number of labeled pre- granulosa cells Figure 3 (A-B) Schematic of ELC and surface granulosa cell trac- ing strategy. (C) Lineage tracing of Axin2+ escort-like cell proge- ny for E12.5 and E19.5 demonstrates that Axin2+ escort-like cells mainly contribute to the first wave follicles. (D) Lineage tracing of Lgr5+ surface-derived pregranulosa cell progeny demonstrates that surface pregranulosa cells mainly contribute to the cortical second wave follicles. (E-F) YFP labeled ELCs and tdTomato la beled cortical cells were quantitated. Results Discussion Figure 4 (A) Experimental strategy to ablate Lgr5-expressing cells using the Lgr5-DTR-EGFP mouse model. (B) Histological analysis of the ovary from wild-type mice and Lgr5DTR/+ animals at P5 and P21. (C-D) Follicle quantification in the ovary at P5 and P21 after DT administration at E16.5. N