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

The overall goal of this procedure is to induce hepatocyte ablation in zebrafish for the analysis of biliary driven liver regeneration. This is accomplished by first exposing transgenic larva that express nitro reductase in their hepatocytes to metrona or MTZ. In the second step, the level of hepatocyte ablation is assessed based on the relative size of the liver.

After MTZ treatment in the final step, bully ablated larvae with a tiny liver are collected ultimately the size of the liver and the expression of liver markers in the regenerating liver are measured by epi fluorescence and confocal microscopy respectively. The main advantage of this technique over existing methods like rodent oval cell activation models is that with this technique, the regenerative hepatocytes are mainly derived from biliary epithelial cells. This method can help answer key questions in liver regeneration field, such as how do biliary or liver progenator cell contribute to the liver Regeneration Generally individual to new to this method will struggle because the liver size in of lead and degenerating lab is variable.

To conduct timed matings, set up adult male and female hemizygous or homozygous fish overnight with a divider between them. Remove the divider the following morning when mating is desired, using a fine plastic mesh strainer to harvest the embryos, turn the strainer upside down and use a squeeze bottle to rinse the mesh surface with a fine stream of egg water. Then transfer up to 100 embryos into a 100 millimeter Petri dish containing 25 milliliters of egg water, and place the dish at 28 degrees Celsius to inhibit pigmentation.

Add PTU to the culture up to 10 hours. Post fertilization at 80 hours. Post personalization, anesthetize the larvae with trica and use an epi fluorescence microscope to collect the CFP positive larvae, keeping only the animals with similarly sized livers.

Then replace the trica egg water with fresh egg water supplemented with PTU and return the CFP positive zebrafish larvae to the 28 degrees Celsius incubator. To treat the zebrafish larvae with MTZ first, divide the appropriate number of larvae into two different culture vessels. Keep the experimental larvae group in a solution of freshly prepared MTZ and the control group in egg water.

Supplemented with DMSO. Cover the experimental group with aluminum foil to prevent photo inactivation of the MTZ and incubate both groups of zebrafish larvae at 28 degrees Celsius at the end of the ablation period. Remove the MTZ solution from the plates and wash the MTZ treated larvae two to three times with 25 milliliters of egg water and swirling discarding the egg water After each wash.

After the last wash, immobilize the larvae with half the concentration of trica previously used to avoid heart edema and death in the MTZ treated larvae. Then use the CFP filter on an epi fluorescence microscope to assess the hepatocyte ablation levels based on the relative liver size of the MTZ treated larvae. Consider the zebra fish with large livers representing zero to 5%of the larvae to be non ablated.

Those with medium-sized livers representing 10 to 20%of the larvae to be partially ablated and those with very small livers representing 80 to 90%of the larvae to be fully ablated. After classifying all of the animals, discover the larvae with non ablated or partially ablated livers, keeping only the zebra fish with tiny livers indicating a severe hepatocyte ablation for liver analysis. At the regeneration zero hour time point, transfer the larvae into a 1.5 milliliter micro centrifuge tube.

Immediately after the MTZ wash out and CFP sourcing, replace the egg water with 3%formaldehyde MPEM buffer for an overnight fixation on a rotor of four degrees Celsius. The next morning, replace the fixation solution with one milliliter of P-B-S-D-T and rotate the larvae for an additional five minutes. Next, all the fixed larvae into a 100 millimeter Petri dish containing fresh P-B-S-D-T under a dissecting microscope and use forceps to manually remove the yolk and pectoral fins when all of the larvae have been clipped.

Transfer up to 20 animals into a 1.5 milliliter tube and replace the P-B-S-D-T with one milliliter of blocking solution. After two hours on the rotator at room temperature, replace the blocking solution with 100 microliters of the primary antibody cocktail for an overnight incubation at four degrees Celsius. With rocking the next day, remove the primary antibody solution and wash the zebra fish with P-B-S-D-T for five 10 minute washes.

After the last wash, add 100 microliters of the secondary antibody for a two hour incubation at room temperature with rocking. Finally, wash the larvae five times in P-B-S-D-T as just demonstrated. Orient the larvae laterally on a microscope slide and add a drop of mounting media.

Then carefully cover each zebra fish with a cover glass and seal the cover glass with nail polish. MTZ treatment for 36 hours from 3.5 to five days post fertilization dramatically reduces the liver size. After the MTZ washout a strong CFP and red expression reappears within 30 hours indeed as assessed by the expression of LCAM in the membrane of the biliary epithelial cells.

The intrahepatic biliary network initially collapses, but is then reestablished a 54 hours post washout indicating a rapid liver regeneration and recovery. In these images regenerating hepatocytes transgenic for a nuclear red fluorescent protein expressed exclusively in the biliary epithelial cells of the liver are shown most of these H two BM cherry positive cells in the regenerating liver express HNF four alpha at regeneration hour zero at regeneration hour four C eight. The MTZ treated CFP positive hepatocytes still retain their H two BM cherry expression indicating the conversion of the biliary epithelial cells to hepatocytes upon severe hepatocyte loss.

While attempting this procedure, it is important to remember to select fully ablated after the MTT treatment for the subsequent liver regeneration on lysis Action after its development. This technique paved the way for researchers in the field of liver regeneration to explore the mechanisms underlying liver progenitor, cell driven liver regeneration. After watching this video, you should have a good understanding of how to use this geography cyte specific operation model for bilio driven liver degeneration studies.