C psn

Ovaries were dissected within PBS under stereomicroscope (Nikon C-PSN, Tokyo, Japan) and imaged under 40× microscope (Nikon SMZ18, Tokyo, Japan) with the camera (Nikon DS-Ri2, Tokyo, Japan). Ovarioles were separated from whole ovaries within DAPI-containing medium on slides. Egg chambers of whole ovaries were imaged with microscopes (ZESS Axio-Observer, Olympus-FV1000) to obtain both DAPI and bright field. Egg chamber size was measured by Image J, and it was used as a feature in stage identification [34 (link)]. Stages 1–7 were identified by their overall size, in combination with manual verification; stages 8 and 9 were identified according to the presence or absence of the nuclei surrounding the anterior nurse cells and stages 10–14 were identified based on morphology.

Mice were housed in a specific-pathogen-free animal laboratory (humidity 60–65%; temperature 22 °C) in 12 h light–dark cycles. All animal housing and experiments conducted were in accordance with the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Institutional Animal Care and Use Committee Guidelines (KRIBB-AEC-18090).
The mouse embryonic tissues were harvested for the day of vaginal plug was dated as embryonic day (E) 0.5. The dissection of spinal cords from embryos was performed as previously described [3 (link), 4 (link)]. Briefly, the pregnant mouse (E 10.5–18.5) was anesthetized using carbon dioxide (CO₂) gas as a euthanasia agent, dissected the lower abdomen, and the uteri containing embryos were placed in a 100-mm Petri dish containing ice-cold sterile HBSS. The extraembryonic membranes overlying the embryos were removed from cranial to caudal and separated from the forebrain to the spinal cord using a dissection microscope (Nikon C-PSN, Tokyo, Japan). The isolated tissues were washed in ice-cold PBS and analyzed using RT-PCR or western blotting.