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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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+of+tuberous+sclerosis+complex+reveals+cell-autonomous+and+non-cell-autonomous+functions+of+mutant+tuberin.">Zebrafish model of tuberous sclerosis complex reveals cell-autonomous and non-cell-autonomous functions of mutant tuberin.</a> Disease Models &amp; Mechanisms. 4 (2), 255-267 (2011).</li><li>Delous, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox9b+is+a+key+regulator+of+pancreaticobiliary+ductal+system+development.">Sox9b is a key regulator of pancreaticobiliary ductal system development.</a> PLoS Genetics. 8 (6), 1002754 (2012).</li><li>Shin, D., Lee, Y., Poss, K. D., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restriction+of+hepatic+competence+by+Fgf+signaling.">Restriction of hepatic competence by Fgf signaling.</a> Development. 138 (7), 1339-1348 (2011).</li><li>Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+helix-loop-helix+transcription+factor,+heart+and+neural+crest+derivatives+expressed+transcript+2,+marks+hepatic+stellate+cells+in+zebrafish:+analysis+of+stellate+cell+entry+into+the+developing+liver.">The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.</a> Hepatology. 56 (5), 1958-1970 (2012).</li></ol>

The authors have nothing to disclose.

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 ...

This method provides a way to quantify liver size in larval zebrafish. It also allows for assessment of the effects of genetic and pharmacologic manipulation on liver growth and development. This technique provides a rapid and precise way to visualize and measure the liver as well as the gut and pancreas.In addition to quantifying liver size, this dissection method can be helpful for preparing the liver, gut, and pancreas for in situ hybridization or confocal imaging. Before attempting deskinning on experimental larvae, make sure you've spent ample time practicing on non-experimental samples and have a significant success rate. It is critical to visualize proper orientation and movement of zebrafish larvae and forceps in order to remove skin without disturbing surrounding organs.At three to seven days post-fertilization, after euthanizing larvae, use a glass pipette and pipette pump to collect up to 15 larvae in a two milliliter tube. Wash larvae twice with one milliliter of cold 1X PBS on ice. Remove as much PBS as possible using a glass pipette and pipette pump and add one milliliter of cold four percent PFA in PBS.Incubate at four degrees Celsius at least overnight with gentle rocking. Using a glass pipette and pipette pump, remove the PFA from the tube and rinse three times with one milliliter of cold PBS. Rock for five minutes in between rinses.Pipette several larvae in PBS into one well of nine-well round-bottom glass dish. Under a microscope, remove the skin surrounding the liver using fine forceps to hold one larva on its back, gripping on either side of the head as gently as possible. Then use very fine forceps in the other hand to grab the skin just overlying the heart.Pull skin down diagonally towards the tail of the fish in the bottom of the dish on the left or right side of the fish. Repeat for the other side. Continue grabbing flaps of skin and pulling down until all of the skin and melanophores overlying or near the liver have been removed.If yolk is present for five to six days post-fertilization larvae, lift the yolk off in one piece by holding the fish with fine forceps on its back and using the very fine forceps to gently prod the yolk. For three to four DPF larvae, hold the fish with fine forceps on its back and use the very fine forceps to stroke the yolk starting from the ventral side. Scrape the yolk off in pieces.Place the dissected larvae into fresh, cold PBS in a two milliliter tube using a glass pipette and pipette pump. To mount the larvae, pour a few milliliters of three percent methylcellulose onto the lid of clean, plastic Petri dish. Use a glass pipette and pipette pump to add larvae to the methylcellulose, adding as little PBS with the larvae as possible.Under a dissecting microscope at low magnification, use fine forceps to orient the fish so they are laying on their right side facing left. Confirm that the fish to be photographed is aligned perfectly with one eye directly on top of the other eye. If the fish's tail is bent, remove the tail by pinching it forcefully with forceps to remove it so that the fish lays flat.Zoom in to high magnification and focus on the liver, making sure that the liver's outline is clearly visible. Press the Capture Image button to snap a picture and save the file. Repeat for all fish, making sure the magnification is the same for each picture.Take a picture of a micrometer using the same magnification. To use the program R to blind all liver pictures to avoid potential investigator bias and promote scientific rigor, rename files randomly and create a randomization file containing a list of the original file names and corresponding blinded file names. Open randomized files in order, starting with file one.Choose the Freehand Selections tool and outline each liver. Press Control-M to measure the area of each liver. For any livers that cannot be accurately measured because they have residual skin, yolk, are out of focus, or are not intact, insert a placeholder measurement.Save the measurements in a text file. To unblind and analyze data, open the measurements file and randomization file in a spreadsheet program. Insert a new column in the measurements file and add the original file names for the blinded files using the Randomization file.Save this file as the Unblinded Measurements file. Sort the data by the original file name. Exclude any liver measurements for which pictures weren't adequate.If necessary, convert measurement values into the desired scale in square millimeters. Open the Scale bar in the image processing software. Use the Straight Line tool to measure one millimeter on the scale bar.The image processing software then measures in the same units as the livers, giving a conversion factor. Use the conversion factor to convert measurements in the Unblinded Measurements file. Determine the mean and standard deviation and calculate P-values.At six days post-fertilization, non-transgenic zebrafish larvae showed natural position and shape of liver overlying the gut. Transgenic zebrafish have significantly increased liver size as compared to their non-transgenic siblings. Some examples of inadequate images and micrometer are shown here.Larva with skin covering the liver, larva with yolk obscuring the liver, larva with parts of the liver pinched off, and larva with liver dislocated and falling off, larva with missing liver, larva with liver outline that is difficult to identify, and larva with improper positioning. When attempting this procedure, it is important to use small and controlled movements to prevent damage to the liver. In addition to bright-field microscopy, we can also use larvae dissected in this way, for confocal microscopy, to visualize fluorescent reporter proteins or immuno-fluorescent staining, and for in situ hybridization.The formaldehyde or PFA is an irritant and suspected carcinogen. Gloves should be worn when handling PFA and concentrated solutions should be handled in a chemical fume hood.

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Research

JoVE Journal

Developmental Biology

6.4K visualizações.  University of Utah School of Medicine. Este método fornece uma maneira de quantificar o tamanho do fígado em zebrafish larval. Também permite avaliar os efeitos da manipulação genética e farmacológica no crescimento e desenvolvimento do fígado. Esta técnica fornece uma maneira rápida e precisa de visualizar e medir o fígado, bem como o intestino e pâncreas.Além de quantificar o tamanho do fígado, este método de dissecção pode ser útil para preparar o fígado, intestino e pâncreas para hibridização in si...