Name: observing mitotic division and dynamics in a live zebrafish embryo
Uploaded: 2016-07-15T14:00:00
Description: Watch this Scientific Journal Video about Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo at JoVE.com

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 (P30CA013148). S.M.P. is supported by the Cell and Molecular Biology T32 Training Grant (5T32GM008111-28).

1. In Vitro Transcription
<ol>
	<li>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&#39; capped mRNA products from each template, according to manufacturer&#39;s protocol.</li>
	<li>Purify the capped mRNA using a purification kit. Follow manufacturer&#39;s instructions. Elute with RNase-free H2O.</li>
	<li>Determine the concentration of RNA by absorbance at 260 nm using a spectro...

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...

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-on microtubule attachment and tension forces orient the chromosomes forming a metaphase plate1. If all chromosomes are attached correctly, the spindle assembly checkpoint is satisfied, cohesin rings holding the sister chromatids together are cleaved, and microtubules shorten to pull sister chromatids to opposite poles during anaphase2,3. The final phase, telophase, involves elongation of the cell and reformation of the nuclear envelope around the two new nuclei. Cytokinesis completes the division process by separating the cytoplasm of the two new daughter cells4-6. Alteration of key mitotic pathways (i.e., spindle assembly checkpoint, centrosome duplication, sister chromatid cohesion, etc.) can result in metaphase arrest, missegregation of chromosomes, and genomic instability7-10. Ultimately, defects in pathways controlling mitosis can cause developmental disorders and cancer, necessitating visualization of mitosis and its defects in a live, vertebrate, multi-cellular organism10-16.
Zebrafish embryos serve as a great model organism for live imaging due to the transparent tissue, ease of microinjection, and fast development. Using zebrafish, the overall goal of this manuscript is to describe a method of live 5D (dimensions X, Y, Z, time, and wavelength) imaging of mitosis17 (Figure 1C). The use of mutant zebrafish defective in different mitotic pathways demonstrate the consequence of such defects. For this protocol, Aurora B and Esco2 mutants were chosen to illustrate these defects. Aurora B is a kinase that is part of the chromosome passenger complex (CPC) involved in spindle formation and microtubule attachment. It is also required for cleavage furrow formation in cytokinesis18,19. In zebrafish, Aurora B deficiency leads to defects in furrow induction, cytokinesis, and chromosome segregation20. Esco2, on the other hand, is an acetyltransferase that is essential for sister chromatid cohesion21,22. It acetylates cohesin on the SMC3 portion of the ring thus stabilizing cohesin to ensure proper chromosome segregation at the metaphase-anaphase transition23. Loss of Esco2 in zebrafish leads to chromosome missegregation, premature sister chromatid separation, genomic instability, and p53-dependent and independent apoptosis24,25. Due to the availability, auroraBhi1045, and esco2hi2865 mutant zebrafish (hereafter referred to as aurBm/m and esco2m/m, respectively) will be used to illustrate this technique25-27.
Coupling confocal microscopy with fluorescent-tagged cell machinery has enabled researchers to visualize chromatin and cell membrane dynamics during mitosis25,28,29. Fluorescent-tagged histones have historically been used to visualize chromatin. Histones are nuclear proteins composed of four different pairs (H2A, H2B, H3, and H4) that are responsible for the nucleosome structure that composes chromosomes30. While H2B is arguably the most used histone for fluorescent proteins in mouse and cell culture, use of Histone 2A, Family Z (H2A.F/Z) has proved well for use in zebrafish31,32. Concanavalin A and casein kinase 1-gamma for example, localize to the cell membrane and have previously been shown effective in visualizing the cell membrane in sea urchins and drosophila33,34. Other studies have shown that the CAAX fluorescent-tagged protein labels the cell membrane and was successful in zebrafish31. CAAX is a motif that is recognized by post-translational modifying enzymes such as farnesyltransferases and geranylgeranyltransferases. Modifications by these enzymes cause proteins to become membrane-associated, thus labeling the cell membrane35.
Due to the prior use in zebrafish, this protocol chose to use H2A.F/Z and CAAX to label chromatin and the cell membrane. Application of this method will allow the researcher to monitor mitosis at the individual cell level to observe individual chromosome dynamics, as well as simultaneously monitor multiple cell divisions that may impact tissue differentiation and development. This article will focus on imaging the dynamics of chromosome segregation during mitosis at the individual cell level. Within this manuscript, the ability to observe several mitotic divisions, calculate division time, and decipher the mitotic phenotypes will be illustrated and discussed. By using these parameters, physiologically relevant data can be collected and applied to several disease states affected by mitotic defects.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pCS2 vectors</td>
			<td style="width:175px;">Gift from K. Kwan</td>
			<td style="width:129px;"></td>
			<td style="width:460px;">For plasmid of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NotI-HF restriction enzyme</td>
			<td style="width:175px;">New England BioLabs</td>
			<td>R3189S</td>
			<td>For restriction digest of plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">mMessage SP6 kit</td>
			<td style="width:175px;">Life Technologies</td>
			<td>AM1340</td>
			<td>For in vitro transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">RNeasy Mini kit</td>
			<td style="width:175px;">Qiagen</td>
			<td style="width:129px;">74104</td>
			<td style="width:460px;">For purifying mRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">100 x 15 mm petri dishes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:129px;">FB0875712</td>
			<td>For housing embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">microinjection mold</td>
			<td style="width:175px;">homemade</td>
			<td style="width:129px;"></td>
			<td>For holding embryos during microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agarose II</td>
			<td style="width:175px;">Amresco</td>
			<td style="width:129px;">0815-25G</td>
			<td>For embedding embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tricaine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>E10521-10G</td>
			<td>For anesthetizing embryos</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Sodium Chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">S9888</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Potassium Chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">P3911</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">C8106</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Magnesium Sulfate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:129px;">M7506</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Methylene Blue Hydrate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">MB1</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">100 mm culture tube</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>50-819-812</td>
			<td>For melted agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">35 mm glass coverslip bottom culture dish&#160;</td>
			<td style="width:175px;">MatTek Corp</td>
			<td>P35G-0-20-C&#160;</td>
			<td>No. 0, 20 mm glass, For embedding embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">#5 tweezers</td>
			<td style="width:175px;">Dumont</td>
			<td style="width:129px;">72701-D</td>
			<td>For dechorionating embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">21G 1 1/2 gauge needle&#160;</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:129px;">305167</td>
			<td>For positioning embryos in agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dissecting microscope</td>
			<td style="width:175px;">Nikon AZ100</td>
			<td style="width:129px;"></td>
			<td>For screening and embedding embryos, any dissecting scope will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Confocal microscope</td>
			<td style="width:175px;">Nikon A1+</td>
			<td style="width:129px;"></td>
			<td>For time-lapse imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Confocal software</td>
			<td style="width:175px;">NIS Elements AR 4.13.00</td>
			<td style="width:129px;"></td>
			<td>For image acquisition and processing</td>
		</tr>
	</tbody>
</table>

observing mitotic division and dynamics in a live zebrafish embryo

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.

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,...

Watch this Scientific Journal Video about Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo at JoVE.com

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

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 ...

Education

Research

JoVE Core

JoVE Journal

Cell Biology

Developmental Biology

Molecular Biology

Anatomy and Physiology

Unit 1

Unit 2

Cell Division

Essential Cellular Processes

SCIENTISTS IN ACTION

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...