The aim of this procedure is to modulate the function genes in Kavas vesicle or the KV in zebrafish embryo that is responsible for left right patterning and analyze asymmetric fluid flow generated by motil clia by delivering fluorescent beads into the kv. This is accomplished by first injecting fluorescent morph oligonucleotides that repress expression of a specific gene of interest in the yolk cell of a mid blash Dilla stage embryo, so that they load into dorsal forerunner cells that give rise to the kv. Next injected embryos that have fluorescent morphos only in the yolk cell and the kv are selected for further analysis.
Then the embryos are mounted in depression slides and fluorescent beads are injected into the fluid-filled vesicle lumen of the kv. Finally, video microscopy is used to record beads flowing inside the kv. Ultimately, results can be obtained that determine whether tissue specific depletion of a candidate gene in the KV alters asymmetric fluid flow through the quantitative analysis of bead movements.
Viral demonstration of this semester is critical as stage specific molino injections and fluorescence bead injections are difficult to learn because both depend on propositioning of the embryo and the developmental timing of the experiment. To perform global morph or MO injections, collect embryos immediately after fertilization and load them into an injection plate. Use a fire polished past our pipette to place the embryos into the injection plate to immobilize the embryos.
Break the tip of an injection needle and adjust the injection settings to create an injection drop that has a volume of one nanoliter. Using a dissecting microscope, inject one nanoliter of MO into the yoke of at least 50 embryos between the one and four cell stages. For DFC targeted M mo injection collect embryos immediately after fertilization and incubate at 28.5 degrees Celsius.
When the embryos reach the 256 cell stage, quickly load them into an injection plate and inject one nanoliter of fluorescent MO into the yolk. Inject at least 100 embryos between the 256 and 1000 cell stages to perform yolk targeted injection. After incubating fertilized eggs to dome stage, inject one nanoliter of fluorescent MO in the yoke of at least 100 embryos between the dome and 30%are ply stages.
When M mo injected embryos reach 75%a ply stage. Remove unfertilized and dead embryos under a dissecting microscope for global MO injections. Choose living embryos for fluorescence evenly distributed throughout all cells.
For DFC targeted M mo injections, select embryos with fluorescence diffused throughout the yolk and concentrated at the dorsal blasted derm margin. For yolk targeted MO injections. Choose embryos in which fluorescence is evenly distributed throughout the yolk and not observed in any embryonic cells between the two to four.
So mite stages. Select DFC targeted embryos that exhibit fluorescence only in the KV cells and the yolk and discard the rest for yolk targeted injections. Select embryos with fluorescence exclusively in the yolk to analyze asymmetric flow in the KV of MO injected embryos begin by using sharp forceps to carefully coate around 20 embryos between the four to six.
So might stages transfer one embryo to a glass depression slide and remove as much water as possible. Immobilize the embryo by adding enough warm 1%low melting point aero to just cover it as the aero solidifies. Use forceps to position it so that the KV is facing up mount 10 embryos working quickly to ensure all injections are completed by the 10.
So mite stage. After diluting 0.5 micron diameter fluorescent micro beads to one to 50 in sterile water. Mix the beads thoroughly and load three microliters into a capillary needle using the same micro injector used for MO injections.
Use forceps to break the needle tip and adjust the injection settings to generate the smallest possible injection.Drop. Next, under a dissecting microscope, align the needle with the KV lumen of the first mounted embryo. Then insert the needle into the lumen and inject less than 0.5 nanoliters of beads.
Add a drop of sterile water onto the aros to avoid the embryo from drying out under an upright fluorescent microscope. Using a 20 x objective screen the injected embryos for successful delivery of beads for each selected embryo with beads inside the KV at a drop of sterile water to cover the aros. Then observe using a fluorescent compound microscope with a 63 x water dipping objective.
Using a high speed camera mounted on the microscope record a ten second movie. Use the fluorescent channel to record the beads at approximately 70 frames per second and either brightfield or differential interference contrast to record the KV lumen. Import the movies into Image J.Create maximum projections and use the software to track the movements of the individual beads.
This figure shows the distribution of fluorescent MO in successful stage specific live injected embryos. An unsuccessful MO injection in which the solution remains aggregated in the yolk cell is shown here. Fluid flow in successfully injected embryos can be measured qualitatively or quantitatively in a control embryo with normal flow beads.
Follow counterclockwise paths that can be visualized by making a maximum projection of fluorescent bead positions over time or by tracking individual beads over time. To demonstrate loss of coordinated flow embryos were injected with MO to break down row kinase two B or rock two B, which disrupts flow, and as a result, the beads move randomly in kv, the average velocity of five beads from control and rock two B mo embryos is shown here After its development. This technical paved the way for researchers in the developmental biology field to explore genes and mechanisms in establishing left right body access in zebrafish.
