Name: quantifying liver size in larval zebrafish using brightfield microscopy
Uploaded: 2020-02-02T13:00:00
Description: Watch this Scientific Journal Video about Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy at JoVE.com

We would like to acknowledge Maurine Hobbs and the Centralized Zebrafish Animal Resource (CZAR) at the University of Utah for providing zebrafish husbandry, laboratory space, and equipment to carry out portions of this research. Expansion of the CZAR is supported in part by NIH grant # 1G20OD018369-01. We would also like to thank Rodney Stewart, Chloe Lim, Lance Graham, Cody James, Garrett Nickum, and the Huntsman Cancer Institute (HCI) Zebrafish Facility for zebrafish care. We would like to thank Kenneth Kompass for help with R programming. This work was funded in part by grants from the Huntsman Cancer Foundation (in conjunction with grant P30 CA042014 awarded to Huntsman Cancer Institute) (KJE) and NIH/NCI R01CA222570 (KJE).

Animal studies are carried out following procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Utah.
1. Fixing Larvae
<ol>
	<li>At 3&#8211;7 days post fertilization (dpf), euthanize larvae with tricaine methanesulfonate (0.03%) and collect up to 15 larvae in a 2 mL tube using a glass pipette and pipette pump.</li>
	<li>Wash larvae twice with 1 mL of cold (4 &#176;C) 1x phosphate-buffered saline (PBS) on ice. For each wash, remove as much liquid a...

<ol>
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Quantification of liver size is crucial in studies aimed at understanding liver development, regeneration, and oncogenesis. The protocol described here is a relatively quick, easy, and cheap technique for liver size quantification in larval zebrafish. Exercising appropriate caution while performing certain aspects of the protocol can aid in increased accuracy of results and decreased frustration.
Proper fixation of the larvae is crucial towards getting well-preserved biological samples and pre...

Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver1 and the third leading cause of cancer-related mortality2. To better understand mechanisms of hepatocarcinogenesis and identify potential HCC therapeutics, we and others have developed transgenic zebrafish in which hepatocyte-specific expression of oncogenes such as &#946;-catenin3,4, Kras(V12)5,6, Myc7, or Yap18 leads to HCC in adult animals. In these zebrafish, liver enlargement is noted as early as 6 days post fertilization (dpf), providing a facile platform for testing the effects of drugs and genetic alterations on oncogene-driven liver overgrowth. Accurate and precise measurement of larval liver size is essential for determining the effects of these manipulations.
Liver size and shape can be assessed semi-quantitatively in fixed zebrafish larvae by CY3-SA labeling9 or in live zebrafish larvae using hepatocyte-specific fluorescent reporters and fluorescence dissecting microscopy5,6. The latter method is relatively quick, and its lack of precision can be addressed by photographing and measuring the area of each liver using image processing software7,10. However, it can be technically challenging to uniformly position all live larvae in an experiment such that two-dimensional liver area is an accurate representation of liver size. A similar technique for quantifying liver size involves using light sheet fluorescence microscopy to quantify larval liver volume8, which may be more accurate for detecting size differences when the liver is expanded non-uniformly in different dimensions. Fluorescence-activated cell sorting (FACS) can be used to count the number of fluorescently labeled hepatocytes and other liver cell types in larval livers8,11. In this method, larval livers are pooled and dissociated, so information about individual liver size and shape is lost. In combination with another liver size determination method, FACS enables differentiation between increased cell number (hyperplasia) and increased cell size (hypertrophy). All of these methods employ expensive fluorescence technology (microscope or cell sorter) and, except for CY3-SA labeling, require labeling of hepatocytes with a fluorescent reporter.
Here we describe in detail a method for quantifying zebrafish larval liver area using bright-field microscopy and image processing software3,12,13,14. This protocol enables precise quantification of the area of individual livers in situ without the use of fluorescence microscopy. While analyzing liver size, we blind the image identity to reduce investigator bias and improve scientific rigor15.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Camera for dissecting microscope</td>
			<td>Leica, for example</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica, for example</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fine (Dumont #5) forceps</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pipets</td>
			<td>VWR</td>
			<td>14672-608</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Image J/FIJI</td>
			<td></td>
			<td>ImageJ/FIJI can be dowloaded for free: <a href="https://imagej.net/Welcome" target="_blank">https://imagej.net/Welcome</a></td>
		</tr>
		<tr>
			<td>Methyl cellulose</td>
			<td>Sigma</td>
			<td>M0387</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>Various suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette pump</td>
			<td>VWR</td>
			<td>53502-233</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dishes</td>
			<td>USA Scientific Inc</td>
			<td>2906</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrex 9-well round-bottom glass dish</td>
			<td>VWR</td>
			<td>89090-482</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for blinding files</td>
			<td>R project</td>
			<td></td>
			<td>R can be downloaded for free: <a href="https://www.r-project.org/" target="_blank">https://www.r-project.org/</a></td>
		</tr>
		<tr>
			<td>Scientific graphing and statistics software</td>
			<td>GraphPad Prism</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spreadsheet program</td>
			<td>Microsoft Excel</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine methanesulfonate (Tricaine-S)</td>
			<td>Western Chemical</td>
			<td>200-226</td>
			<td></td>
		</tr>
		<tr>
			<td>Very fine (Dumont #55) forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying liver size in larval zebrafish using brightfield microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Transgenic zebrafish expressing hepatocyte-specific activated &#946;-catenin (Tg(fabp10a:pt-&#946;-cat) zebrafish)3 and non-transgenic control siblings were euthanized at 6 dpf and liver area was quantified using brightfield microscopy and image processing software. Transgenic zebrafish have significantly increased liver size (0.0006 cm2) as compared to their non-transgenic siblings (0.0004 cm2, p &#60; 0.0001; Figure 1...

Watch this Scientific Journal Video about Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy at JoVE.com

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

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