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Developmental Biology

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Published: July 15th, 2016



1Department of Pharmacology and Toxicology, University of Alabama at Birmingham

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is a critical cellular process essential for growth, differentiation, and regeneration in a living organism. Upon accurate preparation and replication of DNA in interphase, the cell is primed to divide. The first phase of mitosis, prophase, is initiated by activation of cyclin B/Cdk1. Prophase is characterized by condensation of chromatin material into chromosomes. Nuclear envelope breakdown occurs at the transition between prophase and prometaphase. In prometaphase, centrosomes, the nucleating center for spindle formation, begin to migrate to opposite poles while extending microtubules in search of kinetochore attachment. Upon attachment, conversions to end-o....

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1. In Vitro Transcription

  1. Linearize pCS2-H2A.F/Z-EGFP and/or pCS2-mCherry-CAAX vectors by NotI restriction enzyme digest31. Using an RNA in vitro transcription kit, generate 5' capped mRNA products from each template, according to manufacturer's protocol.
  2. Purify the capped mRNA using a purification kit. Follow manufacturer's instructions. Elute with RNase-free H2O.
  3. Determine the concentration of RNA by absorbance at 260 nm using a spectro.......

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Figure 2 demonstrates the ability to observe many cell divisions using a wide field view of an AB wild-type zebrafish tail. Over seven mitotic cells are imaged in a 14 min time frame (Movie 1). Within the two hr time-course, over 40 mitotic events were captured. On average, 50 dividing cells were observed in the AB and 30 dividing cells in aurBm/m embryos (Figure 2B). To account for the number of cells imaged,.......

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Use of this method allows one to infer nuclear envelope breakdown, formation of a metaphase plate by microtubule-kinetochore attachments, and segregation of sister chromatids to form two new cells in vivo and in a time-dependent manner. The ability to observe mitosis in zebrafish is advantageous over fixed samples and cell culture systems because the cells are being imaged in the natural physiology, the tissue is transparent which allows for fluorescent proteins to be used, they develop relatively fast, and time.......

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We thank Kristen Kwan for the pCS2-H2A.F/Z-EGFP and pCS2-mCherry-CAAX vectors. We thank Chris Rodesch for tutoring us in live imaging in zebrafish. We thank Shawn Williams, Erik Malarkey and Brad Yoder for assistance in confocal imaging at UAB and the High Resolution Imaging Facility at UAB. The High Resolution Imaging Facility is supported by the UAB Comprehensive Cancer Center Support Grant (P30CA013148) and the Rheumatic Disease Core Center (P30 AR048311). J.M.P. is supported by the National Institute of Neurological Disease and Stroke (NIH R21 NS092105), and pilot grants from American Cancer Society (ACS IRG-60-001-53-IRG) and the UAB Comprehensive Cancer Center (....

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Name Company Catalog Number Comments
pCS2 vectors Gift from K. Kwan For plasmid of interest
NotI-HF restriction enzyme New England BioLabs R3189S For restriction digest of plasmid
mMessage SP6 kit Life Technologies AM1340 For in vitro transcription
RNeasy Mini kit Qiagen 74104 For purifying mRNA
100 x 15 mm petri dishes Fisher Scientific FB0875712 For housing embryos
microinjection mold homemade For holding embryos during microinjection
Agarose II Amresco 0815-25G For embedding embryos
Tricaine Sigma-Aldrich E10521-10G For anesthetizing embryos
Sodium Chloride Sigma-Aldrich S9888 For embryo water (E3 Blue), dissolved in UltraPure H2O
Potassium Chloride Sigma-Aldrich P3911 For embryo water (E3 Blue), dissolved in UltraPure H2O
Calcium Chloride Dihydrate Sigma-Aldrich C8106 For embryo water (E3 Blue), dissolved in UltraPure H2O
Magnesium Sulfate Fisher Scientific M7506 For embryo water (E3 Blue), dissolved in UltraPure H2O
Methylene Blue Hydrate Sigma-Aldrich MB1 For embryo water (E3 Blue), dissolved in UltraPure H2O
100 mm culture tube Fisher Scientific 50-819-812 For melted agar
35 mm glass coverslip bottom culture dish  MatTek Corp P35G-0-20-C  No. 0, 20 mm glass, For embedding embryos
#5 tweezers Dumont 72701-D For dechorionating embryos
21G 1 1/2 gauge needle  Becton Dickinson 305167 For positioning embryos in agar
Dissecting microscope Nikon AZ100 For screening and embedding embryos, any dissecting scope will do
Confocal microscope Nikon A1+ For time-lapse imaging
Confocal software NIS Elements AR 4.13.00 For image acquisition and processing

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