| Autor: |
Khaliullin RN; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego; Recursion Pharmaceuticals., Hendel JM; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego., Gerson-Gurwitz A; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego., Wang S; Ludwig Institute for Cancer Research, San Diego; Biomedical Sciences Graduate Program, University of California, San Diego; Cell Biology Section, National Institute of Dental and Craniofacial Research, National Institutes of Health., Ochoa SD; Ludwig Institute for Cancer Research, San Diego; Department of Biology, San Diego State University., Zhao Z; Ludwig Institute for Cancer Research, San Diego; Developmental and Stem Cell Biology Graduate Program, University of California, San Francisco., Desai A; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego., Oegema K; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego; koegema@ucsd.edu., Green RA; Ludwig Institute for Cancer Research, San Diego; Department of Cellular and Molecular Medicine, University of California, San Diego; regreen@ucsd.edu. |
| Abstrakt: |
C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80-100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development. |
