The overall goal of this experimental protocol is to feed larval Zebrafish a lipid rich meal that can be spiked with a fluorescent lipid analog to mount the larvae to visual lipid uptake dynamics and to quantify dietary lipid intake. This method can help answer key questions in the fields of metabolism and gastrointestinal biology, such as how dietary lipids are metabolized and how food intake is regulated. The main advantage of this technique is that fluorescent lipid analogs can efficiently be delivered in dietary liposomes.
The optical clarity of the larval Zebrafish enables microscopy to reveal subcellular lipid processing. To begin, suspend one milligram of powdered fluorescent lipid analog in two milliliters of 100%chloroform for a final concentration of 0.5 micrograms per microliter. Aliquot the analog into amber glass tubes with screw caps compatible with the solvent that contain a Teflon gasket.
Store these stocks in the freezer. Add 10.4 microliters of the 0.5 microgram per microliter fluorescent lipid analog stock to a 1.5 milliliter tube and dry under a stream of nitrogen, taking care not to blow the lipid out of the tube. Immediately resuspend the dried lipid in five microliters of 100%ethanol by pipetting a stream down the sides of the tube.
Then, add 95 microliters of embryo medium and mix thoroughly by pipetting up and down until the mixture appears homogeneous. Protect the tube from light and store it on ice until needed. To make a fluorescent cholesterol liposome solution, add enough fluorescent cholesterol analog stock into a 1.5 milliliter tube to make a final concentration of 2.5 micrograms per milliliter when it is added to the egg yolk emulsion.
Dry the cholesterol stock under a stream of nitrogen, as before. Immediately resuspend the fluorescent cholesterol analog in 15 microliters of room temperature 100%ethanol by pipetting a stream down the side of the tube. And mix with 85 microliters of 1%fatty acid free BSA and water.
It is essential to completely solubilize the fluorescent lipid analog in ethanol and embryo media prior to adding it to the egg yolk emulsion to ensure homogeneous distribution of the lipid in the feed and even availability to all larvae. Protect the tube from light with foil and store it on ice. To make the feeding solution, add 19 milliliters of embryo medium to a 50 milliliter conical tube, and then add a one milliliter egg yolk aliquot.
Vortex the mixture for one to two minutes to make a homogeneous emulsion. Pour the mix through a fine strainer to remove protein conglomerates and collect it into a fresh 50 milliliter conical tube. Next, pulse sonicate the egg mix five times, one second on, one second off, with a 1/4 inch tapered microtip at six watts.
Repeat the sonication program four additional times. This creates the liposomes, which should now be continuously rocked until use to maintain homogeneity. Add the appropriate amount of fluorescent lipid analogs to five milliliters of the liposome mixture immediately after sonication.
Vortex at high speed for 30 seconds to incorporate the fluorescent lipid analogs into the liposomes. Protect the mixture from light by wrapping the tube in foil, and return it to the rocker. Add fifty larvae to a feeding dish, remove the embryo medium, and add 5 milliliters of feed.
To encourage food intake, gently rock the larvae in an incubated rocker and shield from light if fluorescent lipid analogs are in use. At the end of the feeding period, wash the larvae in five milliliters of embryo medium three times. To immobilize the larvae, place a metal block on ice, overlay a tissue to prevent excessive cooling, and then place the larvae in the dish of embryo medium on top to cool.
Observe the larvae at 10x under a dissecting microscope, and determine which of them have consumed the liposome feed. Larvae that have consumed liposomes will have a darkened intestine. It is critical to determine whether a larvae have fed by checking for a darkened intestine.
Typically, one to five percent of the larvae will not eat, and could confound the experimental results if not identified and removed. For long-term, high magnification imaging with an inverted objective, first cool the larvae on a metal block on ice. Transfer a larva to a glass bottom dish, and remove the embryo medium by wicking with a tissue.
Place a drop of 42 degree Celsius low melt 1.2%agarose on top of the larva, and immediately position with a poker. Remove the dish from the cold block and allow the agarose to sit for two to three minutes. Finally, add a sufficient volume of fresh embryo medium to cover the agarose and prevent desiccation.
Specimens are now ready for imaging. At the end of a liposome feed, pool a minimum of 10 washed larvae in a 1.5 milliliter tube. Remove the embryo medium and then snap freeze the larvae.
Repeat in a separate tube with 10 unfed larvae as a control. Keeping the sample on ice, add 100 microliters of homogenization buffer to each tube, and then homogenize with a microtip sonicator. Transfer the tissue to a 13 milliliter culture tube.
Add 375 microliters of one to two chloroform to methanol to each homogenization buffer slurry. Vortex for 30 to 60 seconds, and then incubate for 10 minutes at room temperature. Add a further 125 microliters of chloroform, and vortex for 30 seconds.
Next, add 125 microliters of 200 millimolar TRIS pH 7.5, vortex for 30 seconds, and then centrifuge at 2000 times g for five minutes protected from light. Remove samples from the centrifuge carefully to avoid disturbing the separate phases. Using a clean glass pipette, collect the lower organic phase and transfer it into a clean 13 milliliter glass tube.
Carefully avoid transferring any of the upper aqueous or middle larval debris phases and discard them. Dry the organic phase under vacuum at 0.12 atmospheric pressure, while protecting it from light, taking care to stop once the liquid has evaporated. Resuspend a single sample in 10 microliters of two to one chloroform to methanol and spot the entire sample volume onto a channeled, thin-layer chromatography plate.
Repeat with the remaining samples, spotting in evenly spaced intervals. Finally, scan the plate with a fluorescent plate reader. Examination of larvae fed on a rocker under experimental conditions shows darkened intestines, typical of fish that have consumed the egg yolk emulsion.
Unfed larvae usually have clear intestines, so those that have not consumed the diet formula during the feeding period are easily distinguished. A larva fed with a fluorescent lipid analog visualizes the transport and accumulation of this dietary lipid. Here, the fluorescent signal is seen throughout the digestive organs after eight hours of feeding.
Various lipid analogs are incorporated in, and thus visualize, unique sets of cellular structures since they are differentially metabolized into complex lipids. Here, Fluorescent-C16 and C12 analogs label lipid droplets. Fluorescent-C5 labels hepatic and pancreatic ducts, lipid droplets, cellular membranes, and arterial networks.
And Fluorescent-C2 analog labels hepatic and pancreatic ducts and cellular membranes. Larval food intake assays were used to study the feeding of wild-type versus transgenic larvae overexpressing Apolipoprotein A4B1, and showed that over four hour feedings the transgenic larvae ate less than the wild-type. Breedings were normalized used paired, unfed control larvae.
Quantification of the fluorescence of wild-type and transgenic larvae, which overexpress Apo A4 following feeding, show significantly reduced ingestion by the transgenic larvae when compared to the wild-type. Once mastered, the egg yolk fluorescent lipid analog feed can be prepared in about 15 minutes. Food intake can be quantified from 10 to 12 pooled samples in approximately four hours.
While attempting this procedure, it's important to remember to screen larvae for developmental defects that may impair food intake, such as a malformed jaw, an underdeveloped intestine, or decreased mobility. Methods like pharmaceutical treatments or genome engineering can be incorporated into this experimental protocol in order to answer additional questions, like how specific proteins and cell signaling pathways regulate food intake and lipid metabolism. After it's development, this technique paved the way for researchers in the fields of gastrointestinal physiology and cell biology to visualize dietary lipid transport and accumulation in larval Zebrafish.
After watching this video, you should have a good understanding of how to prepare a lipid rich larval Zebrafish feed, screen larvae for food intake, mount larvae for short and long-term imaging, and quantify food intake. Don't forget that working with a sonicator can be hazardous. Ear protection should be worn while performing this procedure.
