Generation of Naïve Blastoderm Explants from Zebrafish Embryos

Even very early embryos contain many different signaling molecules, which can make it challenging to discern the complete function of any individual embryonic signal. By isolating cells from endogenous signaling centers, these explants enable researchers to examine the role of a given signaling molecule in relative isolation. The main advantage of this technique is that we are able to recapitulate embryonic timing, cell movements, and gene expression patterns within a simplified ex-vivo system without the time or effort required to maintain stem cells in culture.

Begin by cutting the explants from the uninfected embryos first. If the explants extending culture, then the cut was made to close to the yolk. Begin by filling a pulled glass capillary needle with RNA.

Place the filled needle into a micro-manipulator and break the tip of the needle with the forceps. Calibrate injection volume using a stage micrometer with a drop of mineral oil, adjusting injection time and pressure on the pneumatic injector to achieve a bolus of the desired size. Keep the RNA needle tip submerged in the oil until ready to inject.

Load the embryos into the injection plate using a Pasteur pipette and pipette pump, and then use a gloved finger to press the eggs into the troughs gently. Inject 10 picograms ndr2 RNA into the yolk of single cell embryos until the desired number of embryos is reached or until embryos begin to divide. Use a gentle stream of egg water from a squeeze bottle to wash the embryos out of the injection plate into a labeled Petri dish.

Place the embryos into the 28.5 degrees Celsius incubator until they reached the 128 cell stage. Remove unfertilized eggs and dead embryos from the dish. Once the embryos have reached the 128 cell stage, place them into labeled glass Petri dishes, and decant as much egg water as possible from them.

Label glass crystallizing dishes with lab tape corresponding to small dish names, and fill two thirds of the way with egg water. Place these dishes next to the dissecting microscope for quick accessibility. Add one milliliter of 20 milligrams per milliliter pronase stock, thawed on ice to 15 milliliters of 3X Danieau's solution in a 50 milliliter conical tube.

Add at least five milliliters of pronase solution to each glass Petri dish containing embryos. Agitate the glass dishes in a circular motion, monitoring the progress of dechorionation consistently under a dissecting microscope. Once the chorions begin to wrinkle and one to two embryos are out of their chorions, carefully dunk the glass Petri dish containing pronase and the embryos into the corresponding glass crystallizing dish containing egg water.

Wash the dechorionation embryos three times with egg water and decant the egg water from the dish. Perform the third and final wash with 0.3X Danieau's solution. Cover the dechorionated embryos with a Petri dish lid, and return them to the incubator at 28.5 degrees Celsius until they reached the 256-cell stage.

Fill an agarose coated Petri dish with 3X Danieau's solution. Once the embryos are at the 256-cell stage, transfer them into the agarose coated plate containing 3X Danieau's solution, lining them up along the center of the dish. Use one pair of forceps, held closed to stabilize the embryo, and use the other to cut through the blastoderm.

To cut, gently squeeze the blastoderm cells with one pair of forceps, then take the stabilizing forceps and run them along the other forceps to slice approximately halfway across the blastoderm. Rotate the embryo, placing the forceps into the existing cut and then sever the remaining blastoderm orthogonal to the first cut. Keep explants in 3X Danieau's solution for at least five minutes to heal, and then transfer them to the agarose coated well of a six-well plate filled with four milliliters of explant media.

Place the explant culture plates into the 28.5 degrees Celsius incubator until the desired time point or stage is reached. To cut the chimeric explants, use a dish with agarose molded into 12 small shallow wells using one millimeter glass beads. Fill the plate with 3X Danieau's solution.

Prepare by adding 12 embryos of one genotype or condition to the left side of the plate, and 12 embryos of the other genotype are conditioned to the right side of the plate. Move one embryo of each condition to the center of the plate near one of the 12-wells. Using forceps, cut an explant from each embryo as described for single embryo explants.

Quickly press the cut edges of the two explants together within the shallow well using forceps to allow the two halves to heal together into a single explant. Continue with the remaining 12-wells within the plate. Once the explants are healed, transfer them to the agarose coated well of a six-well plate filled with four milliliters of explant media.

Repeat until the desired number of explants is achieved. Culture explants in the 28.5 degrees Celsius incubator until intact sibling embryos reached the desired stage. Control explants cut from the uninfected wild-type embryos or those injected with 50 picograms of mRNA encoding green fluorescent protein remained rounded throughout the culture period, and failed to express markers of mesoderm, endoderm, or neuroectoderm.

Explants cut from embryos injected with 10 picograms of ndr2 mRNA became highly elongated after eight to nine hours in culture. Live time-lapse imaging of these explants by differential interfering contrast microscopy revealed that the extension onsets at or around eight hours post-fertilization. Explants from embryos injected with 10 picograms of ndr2 exhibited robust expression of the mesoderm markers, tbxta, noto, tbx16, and the neuroectoderm marker sox2.

It is very important to cut the explants well above the embryonic margin. Otherwise, the explants will contain endogenous signals from the margin and not be naive. In this method, explants were used to address the role of nodal signaling in gastrulation morphogenesis.

In the future, other researchers can use this approach to test the role of additional signaling molecules, for example, in any number of developmental processes.