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 is a challenge in biology to obtain high-resolution information and maintain the global perspective from a complex system such as the entire biliary system or central nervous system using conventional embedding and sectioning methods. This modified CLARITY method allows a laser beam to penetrate through the intact sea lamprey larval liver up to a thickness of 1 to 2 mm using low-power fluorescent objective (4x) in a regular laser scanning confocal microscope. Deeper penetration can be achieved using a more advanced multi-photon confocal microscope. Three-dimensional images can be reconstructed using 3D reconstruction software. Achieving comparable results using conventional methods would be laborious, involving the piecemeal reconstruction of many sections, and would be limited to small tissue volumes. This new method provides valuable information from whole structural analyses of intact systems12, is less labor-intensive, more efficient and reproducible.&#160;

Another advantage of this new method is that the specimen can be reused multiple times for staining with different antibodies12. This makes the comparison between different molecular markers in the same specimen more reliable and reproducible. The stable framework generated by hydrogel allows effective removal of antibodies without fine structure damage and maintains the antigenicity for a long time12. The clearing solution denatures antibodies and disrupts binding, therefore acting as an antibody-stripping solution12. Several rounds of staining have been performed in the sea lamprey liver samples and the structure and staining remained optimal.

There are two major modifications in this protocol from the original &#8220;CLARITY&#8221; protocol12. (1) Free radical generating reagents (ammonium persulfate and TEMED) were used to initiate and accelerate the polymerization of the hydrogel instead of VA044. VA044 is photosensitive and not available in the U.S. (2) No custom-built electrophoresis chamber is used. In the CLARITY method12, due to the size of the tissues (mouse brain vs. lamprey liver), electrophoretic tissue clearing is required to expedite the extraction of the plasma membrane. To avoid the initial cost for a custom-built electrophoresis chamber, we first used existing agarose gel electrophoresis devices in our laboratory. This proved to be very inefficient. However, incubation in the clearing solution with raised temperature (50 &#176;C) is very effective in clearing the plasma membrane.&#160; &#160;&#160;&#160;

Although the authors for &#8220;CLARITY&#8221; cautioned against the use of saponin in the hydrogel, it improves the transparency in the sea lamprey tissue in our procedure. One drawback for confocal imaging is that sea lamprey tissue has strong auto-fluorescence. The choice of fluorophores must be tested by comparing the images before and after immunofluorescent staining.

During sea lamprey metamorphosis, the regression of the gall bladder epithelium starts between metamorphic stages 2 and 3. By metamorphic stage 4, the gall bladder disappears entirely from sea lamprey liver14. Bile duct degeneration is most dramatic between metamorphic stages 3 and 415. This modified method is a powerful tool to study the cellular and molecular mechanisms of the degeneration of the entire biliary system, which may provide insights for human biliary atresia.

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 (Figure 2) and the 3-dimensional structure was reconstructed (Figure 3). Laser scanning confocal microscopy was used to perform optical section through the intact liver since most of the biliary system is embedded inside the liver except the junction where the bile duct enters the intestine. The degenerative process occurs in metamorphic stages 2-4, starting from peripheral small bile ductules toward the central large intrahepatic common bile duct (Figure 2), consistent with earlier studies10. &#160;
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig1highres.jpg" width="500" /> 
Figure 1.&#160;Gall bladder degeneration during sea lamprey metamorphosis. Sea lamprey lose the gall bladder (black arrows) during metamorphosis. The specimens were taken from animals with external morphology between metamorphic stages 2 and 3; i.e. round eye without (stage 2) or with (stage 3) sharp differentiation of iris and pupil, prominent papilla-like projections on the inner dorsal surface of the oral hood (both stages), thickened lips of oral hood (stage 2) forming a rectangular entrance into the oral cavity (stage 3), or &#34;pug-like&#34; snout (stage 3)3. Note that the gall bladder also changed color indicating differences in bile production. I: intestine. L: liver. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51648/51648fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig2highres.jpg" width="500" /> 
Figure 2.&#160;Confocal images showing the biliary tree in sea lamprey liver during metamorphosis. The specimens were taken from animals with external morphology between metamorphic stages 2 and 3. Top panels are taken from the anterior, and the lower panels from the posterior end. Images of liver cell marker cytokeratin 19 (1:100 mouse-anti-CK19; stained with 2 &#181;g/ml Alexa Fluor 350-goat-anti-mouse IgG; blue) and anti-apoptotic marker Bcl2 (1:100 rabbit-anti-Bcl2; stained with 2 &#181;g/ml Alexa Fluor 488-donkey-anti-rabbit IgG; green) are superimposed to generate the merged images. White arrows indicate the biliary tree. Black arrows indicate the dark dendritic looking apoptotic cells and surrounding canaliculi and ductules in the liver. I: intestine. L: liver. Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51648/51648fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig3highres.jpg" width="500" /> 
Figure 3.&#160;3D images reconstructed from merged confocal z-series. 3D images were reconstructed from the confocal z-series of three livers between metamorphic stages 2 and 3. Optical sections of merged images of liver cell marker cytokeratin 19 (1:100 mouse-anti-CK19; stained with 2 &#181;g/ml Alexa Fluor 350-goat-anti-mouse IgG; blue) and anti-apoptotic marker Bcl2 (1:100 rabbit-anti-Bcl2; stained with 2 &#181;g/ml Alexa Fluor 488-donkey-anti-rabbit IgG; green) are reconstructed with the manufacturer&#8217;s software to generate the 3D images. Top panels are taken from the anterior, and the lower panels from the posterior end. White arrows indicate the biliary tree. Black arrows indicate the dark dendritic looking apoptotic cells and surrounding canaliculi and ductules in the liver. I: intestine. L: liver. Scale bar: 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51648/51648fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51648/51648fig4highres.jpg" width="500" /> 
Figure 4.&#160;A confocal optical section showing bile ducts with bile droplets in the lumen. Merged image from 3 fluorescent channels; blue: mouse-anti-c-Myc (1:100)/2 &#181;g/ml Alexa Fluor 350-goat-anti-mouse IgG; green: 2 &#181;g/ml Nissl green; Red: rabbit-anti-TGF&#946;RII (1:100)/2 &#181;g/ml Alexa Fluor 594-donkey-anti-rabbit. Scale bar: 500 &#956;m. Bile droplets are indicated by black arrows. The liver specimen was taken between metamorphic stage 2 and 3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51648/51648fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

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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

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Developmental Biology

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 ...

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and identification of an active pheromone compound. This method has resulted in the successful characterization of the chemical signals that function as pheromones in a wide range of animal species. Sea lampreys rely on olfaction to detect pheromones that mediate behavioral or physiological responses. We use this knowledge of fish biology to posit functions of putative pheromones and to guide the isolation and identification of active pheromone components. Chromatography is used to extract, concentrate, and separate compounds from the conditioned water. Electro-olfactogram (EOG) recordings are conducted to determine which fractions elicit olfactory responses. Two-choice maze behavioral assays are then used to determine if any of the odorous fractions are also behaviorally active and induce a preference. Spectrometric and spectroscopic methods provide the molecular weight and structural information to assist with the structure elucidation. The bioactivity of the pure compounds is confirmed with EOG and behavioral assays. The behavioral responses observed in the maze should ultimately be validated in a field setting to confirm their function in a natural stream setting. These bioassays play a dual role to 1) guide the fractionation process and 2) confirm and further define the bioactivity of isolated components. Here, we report the representative results of a sea lamprey pheromone identification that exemplify the utility of the bioassay-guided fractionation approach. The identification of sea lamprey pheromones is particularly important because a modulation of its pheromone communication system is among the options considered to control the invasive sea lamprey in the Laurentian Great Lakes. This method can be readily adapted to characterize the chemical communication in a broad array of taxa and shed light on waterborne chemical ecology.

Here, we present a protocol to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromone compounds of sea lampreys.

Bioassay-guided fractionation is an iterative approach that uses the results of physiological and behavioral bioassays to guide the isolation and ...

Biochemistry

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

Determining Bile Duct Density in the Mouse Liver

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques are used, including serum chemistry, histological analysis, and immunostaining for specific markers. Although these techniques can provide important information about the biliary system, they often do not present a full picture of bile duct (BD) developmental defects across the whole liver. This is in part due to the robust ability of the mouse liver to drain the bile even in animals with significant impairment in biliary development. Here we present a simple method to calculate the average number of BDs associated with each portal vein (PV) in sections covering all lobes of mutant/transgenic mice. In this method, livers are mounted and sectioned in a stereotypic manner to facilitate comparison among various genotypes and experimental conditions. BDs are identified via light microscopy of cytokeratin-stained cholangiocytes, and then counted and divided by the total number of PVs present in liver section. As an example, we show how this method can clearly distinguish between wild-type mice and a mouse model of Alagille syndrome. The method presented here cannot substitute for techniques that visualize the three-dimensional structure of the biliary tree. However, it offers an easy and direct way to quantitatively assess BD development and the degree of ductular reaction formation in mice.

We present a rather simple and sensitive method for accurate quantification of bile duct density in the mouse liver. This method can aid in determining the effects of genetic and environmental modifiers and the effectiveness of potential therapies in mouse models of biliary diseases.

Mouse is broadly used as a model organism to study biliary diseases. To evaluate the development and function of the biliary system, various techniques ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

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

Summary

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Chapters in this video

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

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

The Identification of Sea Lamprey Pheromones Using Bioassay-Guided Fractionation

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

Determining Bile Duct Density in the Mouse Liver

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

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton