Oocytes develop in the germline cyst, a cellular organization in which germ cells are tightly interconnected and surrounded by somatic cells. The cyst produces oocytes for follicle formation and is a hub for essential processes in meiosis and oocyte differentiation. However, the formation and organization of the cyst, and their contribution to oocyte production in vertebrates remain unclear. Here, we provide tools for three-dimensional and functional in vivo analyses of the germline cyst in the zebrafish ovary. We describe the use of serial block-face scanning electron microscopy (SBF-SEM) to resolve the three-dimensional architecture of cells and organelles in the cyst at ultrastructural resolution. We present a deep learning-based pipeline for high-throughput quantitative analysis of three-dimensional confocal datasets of cysts in vivo. We provide a method for laser ablation of cellular components for manipulating cyst cells in ovaries. These methods will facilitate the investigation of the cyst cellular organization, expand the toolkit for the study of the zebrafish ovary, and advance our understanding of female developmental reproduction. They could also be further applied to the investigation of other developmental systems.
Bibliographical noteFunding Information:
We thank A. Beckett, from the Biomedical EM unit at the University of Liverpool, for her help in processing our samples for SBF-SEM imaging. We also thank Z. Manevich, from the Faculty of Medicine Microscopy Core at the Hebrew University, and Y. Addadi, from the core facility at the Weizmann Institute, for their support of our multi-photon microscopy operation in our laser ablation experiments. This work was supported by the Israel Science Foundation (3291/19 and 558/19 to Y.E.) and a Hebrew University of Jerusalem (HUJI) International PhD Talent Scholarship (V.K.). Open Access funding provided by Hebrew University of Jerusalem. Deposited in PMC for immediate release.
This work was supported by the Israel Science Foundation (3291/19 and 558/19 to Y.E.) and a Hebrew University of Jerusalem (HUJI) International PhD Talent Scholarship (V.K.). Open Access funding provided by Hebrew University of Jerusalem. Deposited in PMC for immediate release.
© 2023. Published by The Company of Biologists Ltd.
- 3D deep-learning data
- Germ cell development and morphogenesis
- Germline cyst
- Live manipulation by laser-ablation
- Microscopy image analysis
- Zebrafish ovary