Name: a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)
Uploaded: 2014-06-06T14:15:00.000+00:00
Duration: 7 min 3 s
Description: Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

The authors acknowledge the contribution of Hammond Bay Biological Station, Great Lakes Science Center, U.S. Geological Survey. We also thank Dr. Melinda Frame at the Center for Advanced Microscopy at Michigan State University for her technical support in laser scanning confocal microscopy. This study is supported by grants from the Great Lakes Fishery Commission to YWCD and WML.

1. Solution Preparation
<ol>
	<li>Make 1 L 10x 0.1 M phosphate buffer saline (PBS, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic), 115 g sodium phosphate (dibasic), and 87.66 g NaCl. Dissolve in about 800 ml distilled H2O, adjust pH, and bring the volume up to 1 L with distilled H2O.
	<ol>
		<li>Make 1 L 0.1 M phosphate buffer saline (pH 7.4): Take 100 ml 10x PBS, and add 900 ml distilled H2O.</li>
		<li>Make 1 L 0.1 M phosphate buffer saline (pH 7.4) with 0.1% Triton X-100: Take 100 ml 10x PBS, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H2O.</li>
	</ol>
	</li>
	<li>Make 1 L 10x 0.1M phosphate buffer (PB, pH 7.4): Weigh 26.2 g sodium phosphate (monobasic) and 115 g sodium phosphate (dibasic). Dissolve in about 800 ml distilled H2O, adjust pH, and bring the volume up to 1 L with distilled H2O.
	<ol>
		<li>Make 1 L 0.1 M phosphate buffer (pH 7.4): Take 100 ml 10x PB, and add 900 ml distilled H2O.</li>
		<li>Make 1 L 0.1 M phosphate buffer (pH 7.4) with 0.1% Triton X-100: Take 100 ml 10x PB, add 1 ml Triton X-100, and bring the volume up to 1 L with distilled H2O.</li>
	</ol>
	</li>
	<li>Make 400 ml hydrogel monomer solution (4% acrylamide, 0.25% bis-acrylamide, 4% paraformaldehyde, 0.0075% ammonium persulfate, 0.0005% saponin in 0.1 M PBS): Weigh 0.3 g ammonium persulfate and 0.2 g saponin. Add 210 ml distilled H2O, 40 ml 40% acrylamide, 10 ml 2% bis-acrylamide, 40 ml 10x PBS (pH 7.4), 100 ml 16% paraformaldehyde, and mix well.</li>
	<li>Make 4 L clearing solution (4% SDS in 0.2 M boric acid): Weigh 49.464 g boric acid and 160 g SDS. Dissolve in about 3.5 L distilled H2O, adjust pH to 8.5 with NaOH, and bring the volume up to 4 L.</li>
	<li>Make 1 L 80% glycerol solution: Measure 800 ml glycerol, add 200 ml distilled H2O, and mix well.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Prepare hydrogel monomer solution and aliquot 10 ml to each 15 ml centrifuge tube.</li>
	<li>Dissect the whole tissue and fix in the hydrogel for 2 days at 4 &#176;C. Make sure that the hydrogel solution is at least 10x the volume of the tissue.</li>
	<li>Bring the hydrogel fixed tissue (in the 15 ml centrifuge tube) to a fume hood.</li>
	<li>Polymerize the hydrogel fixed tissue by adding 5 &#181;l TEMED, mix well, and let sit in the fume hood at room temperature for 2-3 hr.</li>
	<li>Remove the excess gel thoroughly. Note: Extra gel will hinder the clearing process and the antibody penetration during staining.</li>
	<li>Incubate the fixed tissue in 10 ml clearing solution in an incubator shaker set at 70 rpm and 50 &#176;C for several days until the tissue becomes transparent. Change clearing solution at least once per day and remove the excess gel from the tissue when changing the clearing solution.</li>
	<li>Rinse the tissue in 10 ml 0.1 M PB with 0.1% Triton X-100 at 70 rpm and 37 &#176;C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.</li>
	<li>Incubate the clarified tissue in the primary antibody solution in PB with 0.1% Triton X-100 at 70 rpm and 37 &#176;C for 2 days. Note: Make enough primary antibody solution to submerge the whole tissue.</li>
	<li>Rinse the tissue in 10 ml PB with 0.1% Triton X-100 at 70 rpm and 37 &#176;C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.</li>
	<li>Perform the following steps in a dark room or under dim light. Cover the samples with foil to prevent photo bleaching.</li>
	<li>Incubate the clarified tissue with the fluorescent secondary antibody solution in PB with 0.1% Triton X-100 and the blocking serum at 70 rpm and 37 &#176;C for 1 day.</li>
	<li>Rinse the tissue in 10 ml PB with 0.1% Triton X-100 at 70 rpm and 37 &#176;C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.</li>
	<li>Rinse the tissue in 10 ml PBS at 70 rpm and 37 &#176;C overnight. Repeat this step 3 times and remove the excess gel from the tissue when changing the buffer solution.</li>
	<li>Incubate the clarified tissue in 80% glycerol at 70 rpm and 37 &#176;C overnight.</li>
	<li>Store the fluorescent stained tissue at 4 &#176;C prior to confocal microscopy.</li>
</ol>

<ol>
	<li>Applegate, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+history+of+the+sea+lamprey+(Petromyzon+marinus)+in+Michigan.">Natural history of the sea lamprey (Petromyzon marinus) in Michigan.</a> US Fish and Wildlife Service Special Science Report on Fishery Service. (55), (1950).</li><li>Hardisty, M. W., Potter, I. C., Hardisty, M. W., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+general+biology+of+adult+lampreys.">The general biology of adult lampreys.</a> The biology of lampreys. 1, 127-206 (1971).</li><li>Youson, J. H., Potter, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+description+of+the+stages+in+the+metamorphosis+of+the+anadromous+sea+lamprey,+Petromyzon+marinus+L.">A description of the stages in the metamorphosis of the anadromous sea lamprey, Petromyzon marinus L.</a> Can. J. Zool. 57, 1808-1817 (1979).</li><li>Boomer, L. A., 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=Cholangiocyte+apoptosis+is+an+early+event+during+induced+metamorphosis+in+the+sea+lamprey,+Petromyzon+marinus+L.">Cholangiocyte apoptosis is an early event during induced metamorphosis in the sea lamprey, Petromyzon marinus L.</a> J. Pediatr. Surg. 45, 114-120 (2010).</li><li>Kasai, M., Suzuki, H., Ohashi, E., Ohi, R., Chiba, T., Okamoto, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+and+results+of+operative+management+of+biliary+atresia.">Technique and results of operative management of biliary atresia.</a> World J. Surg. 2, 571-580 (1978).</li><li>Suzuki, T., Hashimoto, T., Kondo, S., Sato, Y., Hussein, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+patients&#8217;+outcome+post-Kasai+operation:+a+19-year+experience+with+modification+of+the+hepatic+portoenterostomy+and+applying+a+novel+steroid+therapy+regimen.">Evaluating patients&#8217; outcome post-Kasai operation: a 19-year experience with modification of the hepatic portoenterostomy and applying a novel steroid therapy regimen.</a> Pediatr. Surg. Int. 26, 825-830 (2010).</li><li>Hartley, J. L., Davenport, M., Kelly, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia.">Biliary atresia.</a> Lancet. 374, 1704-1713 (2009).</li><li>Morecki, R., Glaser, J. H., Cho, S., Balistreri, W. F., Horwitz, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia+and+reovirus+type+3+infection.">Biliary atresia and reovirus type 3 infection.</a> New Engl. J. Med. 307, 481-484 (1982).</li><li>Shimadera, S., Iwai, N., Deguchi, E., Kimura, O., Fumino, S., Yokoyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inv+mouse+as+an+experimental+model+of+biliary+atresia.">The inv mouse as an experimental model of biliary atresia.</a> J. Pediatr. Surg. 42, 1555-1560 (2007).</li><li>Sidon, E. W., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+changes+in+the+liver+of+the+sea+lamprey,+Petromyzon+marinus+L.,+during+metamorphosis:+I.+Atresia+of+the+bile+ducts.">Morphological changes in the liver of the sea lamprey, Petromyzon marinus L., during metamorphosis: I. Atresia of the bile ducts.</a> J. Morphol. 177, 109-124 (1983).</li><li>Yeh, C. -. Y., Chung-Davidson, Y. -. W., Wang, H., Li, K., Li, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+synthesis+and+secretion+of+bile+salts+as+an+adaptation+to+developmental+biliary+atresia+in+the+sea+lamprey.">Intestinal synthesis and secretion of bile salts as an adaptation to developmental biliary atresia in the sea lamprey.</a> Proc. Nat. Acad. Sci. U.S.A. 109, 11419-11424 (2012).</li><li>Chung, K., 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=Structural+and+molecular+interrogation+of+intact+biological+systems.">Structural and molecular interrogation of intact biological systems.</a> Nature. 497, 332-337 (2013).</li><li>Alarc&#243;n, V. B., Filosa, M. F., Youson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratins+in+the+liver+of+the+sea+lamprey+(Petromyzon+marinus)+before+and+after+metamorphosis.">Cytokeratins in the liver of the sea lamprey (Petromyzon marinus) before and after metamorphosis.</a> Cell Tissue Res. 287, 365-374 (1997).</li><li>Youson, J. H., Ogilvie, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructural+features+of+degeneration+of+the+gallbladder+during+lamprey+biliary+atresia.">Ultrastructural features of degeneration of the gallbladder during lamprey biliary atresia.</a> Tissue and Cell. 22, 477-492 (1990).</li><li>Morii, 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=Onset+of+apoptosis+in+the+cystic+duct+during+metamorphosis+of+a+Japanese+lamprey,+Lethenteron+reissneri.">Onset of apoptosis in the cystic duct during metamorphosis of a Japanese lamprey, Lethenteron reissneri.</a> Anat. Rec. 293, 1155-1166 (2010).</li></ol>

The authors have nothing to disclose.

This protocol is modified from a new method called &#8220;CLARITY&#8221;12, which crosslinks intact tissue with polyacrylamide to form a nanoporous hydrogel, and then strips away the plasma membrane of the tissue to achieve optical transparency and macromolecular permeability. &#8220;CLARITY&#8221; allows intact-tissue imaging of long-range projection and local circuit wiring in the nervous system. This new method can be used to visualize the entire biliary system in sea lamprey liver during metamorphosis. It ...

Sea lamprey develop through three distinct life stages1,2. Larval sea lamprey (L) spend most time in burrows as benthic filter feeders. After going through seven metamorphic stages of dramatic changes in external morphology and reorganization in internal organs3, the resulting juveniles (JV) enter a parasitic stage during which they feed on blood and tissue fluids from host fish, increasing the body mass more than 100 times. After 1.0 to 1.5 years feeding on the host fish in the ocean or large lakes, adults cease feeding during the early spring and migrate into rivers to spawn and then die1,2.

During metamorphosis, the sea lamprey liver loses the gall bladder and the entire biliary tree, an evolutionary mutant phenotype that mimics the human infant disease biliary atresia. Infant biliary atresia is a rare pediatric liver disease with severe medical complications4,5,6,7,8,9,10, however the pathogenesis and etiology of biliary atresia are largely unknown4. Patients with biliary atresia die within two years after birth unless surgical intervention (Kasai procedure) is performed5. Subsequently, these patients require extensive clinical management and often liver transplantation&#160;6. Many theories of biliary atresia etiopathogenesis have been proposed, such as viral infection, congenital malformation, autoimmune disease, and toxic insult. However, the contribution of each to the development of biliary atresia remains inconclusive7,8,9,10.

Unlike infants that suffer pathological biliary atresia, sea lamprey undergo developmentally programmed biliary atresia without extensive necroinflammation, fibrosis or cirrhosis10. The animals may suffer transient cholestasis during this process10, but adapt to this developmental condition via de novo synthesis and secretion of bile salts in the intestine after developmental biliary atresia, in addition to known mechanisms such as reduction of bile salt synthesis in liver11. This developmental process in sea lamprey provides the only known opportunity to examine the progression of biliary atresia.

A newly-developed method called &#8220;CLARITY&#8221; enables high-resolution imaging in complex mammalian nervous systems by transforming intact tissue into an optically transparent nanoporous hydrogel12. Using sea lamprey liver and a modified CLARITY protocol, intact-tissue imaging of biliary degeneration can be documented throughout liver metamorphosis.

<table><tbody><tr><td>40% acrylamide&amp;nbsp;</td><td>Bio-Rad</td><td>161-0140</td><td></td></tr><tr><td>2% bis-acrylamide&amp;nbsp;</td><td>Bio-Rad</td><td>161-0142</td><td></td></tr><tr><td>TEMED</td><td>Bio-Rad</td><td>161-0800</td><td></td></tr><tr><td>ammonium persulfate&amp;nbsp;</td><td>Sigma</td><td>A3678-25G</td><td></td></tr><tr><td>boric acid</td><td>Sigma</td><td>B7901-1KG</td><td></td></tr><tr><td>saponin&amp;nbsp;</td><td>Sigma</td><td>47036</td><td></td></tr><tr><td>sodium dodecyl sulfate&amp;nbsp;</td><td>Sigma</td><td>L337-500G</td><td></td></tr><tr><td>sodium phosphate (monobasic)</td><td>Sigma</td><td>04269-1KG</td><td></td></tr><tr><td>sodium phosphate (dibasic)</td><td>Sigma</td><td>S5136-1KG</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100-500ML</td><td></td></tr><tr><td>glycerol&amp;nbsp;</td><td>Sigma</td><td>G9012-500ML</td><td></td></tr><tr><td>16% paraformaldehyde&amp;nbsp;</td><td>Electron Microscopy Sciences</td><td>15710-S</td><td></td></tr><tr><td>NaOH pellets&amp;nbsp;</td><td>EMD</td><td>SX0590-3</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Any brand</td><td></td><td></td></tr><tr><td>dissecting tools&amp;nbsp;</td><td>Any brand</td><td></td><td></td></tr></tbody></table>

a new clarification method to visualize biliary degeneration during liver metamorphosis in sea lamprey (petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian countries, with a reported frequency of 1 in 5,000 in Taiwan. Although much is known about the management of biliary atresia, its pathogenesis is still elusive. The sea lamprey (Petromyzon marinus) provides a unique opportunity to examine the mechanism and progression of biliary degeneration. Sea lamprey develop through three distinct life stages: larval, parasitic, and adult. During the transition from larvae to parasitic juvenile, sea lamprey undergo metamorphosis with dramatic reorganization and remodeling in external morphology and internal organs. In the liver, the entire biliary system is lost, including the gall bladder and the biliary tree. A newly-developed method called &#8220;CLARITY&#8221; was modified to clarify the entire liver and the junction with the intestine in metamorphic sea lamprey. The process of biliary degeneration was visualized and discerned during sea lamprey metamorphosis by using laser scanning confocal microscopy. This method provides a powerful tool to study biliary atresia in a unique animal model.

Several important developmental events occur in the hepatobiliary system during sea lamprey metamorphosis. The bile duct and the gall bladder undergo apoptosis and degenerate (Figure 1). Combining the modified clarification method and staining with liver cell marker cytokeratin 19 (CK19, present in both cholangiocytes and hepatocytes before and after metamorphosis13) and anti-apoptotic marker Bcl2 using confocal microscopy, the entire biliary system was captured along the Z-axis (Figur...

Watch this Scientific Journal Video about A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus at JoVE.com

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Biliary atresia is a rare disease of infancy, with an estimated 1 in 15,000 frequency in the southeast United States, but more common in East Asian ...

a-new-clarification-method-to-visualize-biliary-degeneration-during

Research

JoVE Journal

Biology

13.7K Views.  Michigan State University. Sea lamprey lose the gall bladder and bile ducts during metamorphosis, a process similar to human biliary atresia. A new fixation and clarification method (CLARITY) was modified to visualize the entire biliary tree using laser scanning confocal microscopy. This method provides a powerful tool to study biliary degeneration.

Video: A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey Petromyzon marinus

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Developmental Biology

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Cancer Research

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Neuroscience

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.

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 ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the three-dimensional (3D) architecture of the organ at the whole-organ level. Liver architecture is characterized by multiple branching lumenized structures, which can be filled with resin, including vascular and biliary trees, establishing a highly stereotyped pattern in the otherwise hepatocyte-rich parenchyma. This protocol describes the pipeline for performing double resin casting micro-computed tomography, or &#34;DUCT&#34;. DUCT entails injecting the portal vein and common bile duct with two different radiopaque synthetic resins, followed by tissue fixation. Quality control by clearing one lobe, or the entire liver, with an optical clearing agent, allows for pre-screening of suitably injected samples. In the second part of the DUCT pipeline, a lobe or the whole liver can be used for micro-computed tomography (microCT) scanning, (semi-)automated segmentation, and 3D rendering of the portal venous and biliary networks. MicroCT results in 3D coordinate data for the two resins allowing for qualitative as well as quantitative analysis of the two systems and their spatial relationship. DUCT can be applied to postnatal and adult mouse liver and can be further extended to other tubular networks, for example, vascular networks and airways in the lungs.

Double resin casting micro-computed tomography, or DUCT, enables visualization, digitalization, and segmentation of two tubular systems simultaneously to facilitate 3D analysis of organ architecture. DUCT combines ex vivo injection of two radiopaque resins followed by micro-computed tomography scanning and segmentation of the tomographic data.

The liver is the biggest internal organ in humans and mice, and high auto-fluorescence presents a significant challenge for assessing the ...

Medicine

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

Summary

Explore More Videos

Chapters in this video

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

DUCT: Double Resin Casting followed by Micro-Computed Tomography for 3D Liver Analysis

Partial Hepatectomy in Adult Zebrafish