title: Live imaging of mitosis in the developing mouse embryonic cortex.
abstract: Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-α-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center.
authors: Louis-Jan
authors: LJ
authors: Pilaz
authors: Department of Molecular Genetics and Microbiology, Duke University Medical Center; Departments of Neurobiology and Cell Biology, Duke Institute for Brain Sciences, Duke University Medical Center; debra.silver@duke.edu.
authors: Debra L
authors: DL
authors: Silver
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002540:Cerebral Cortex / Q000166:cytology* / Q000196:embryology*; D018274:Electroporation; D005260:Female; D007091:Image Processing, Computer-Assisted / Q000379:methods; D051379:Mice; D008822:Mice, Transgenic; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D058953:Neural Stem Cells / Q000166:cytology*; D011247:Pregnancy