분석

The overall goal of this procedure is to analyze the function of a candidate gene in controlling in vivo cell migration using cell transplantation combined with live imaging in the Zebrafish embryo. This method can help address key questions in the cell migration field such as determining the role and importance of new candidate genes in in vivo cell migration. The main advantage of this technique is that it creates mosaic embryos which allows good imaging and cell adenomyosis.

To prepare cell transplantation needles use a micro pipette puller to pull a glass capilary. Then, with a microforge, cut the tip of the needle at the point where the inside diameter is 35 micrometers. Using a microgrinder bevel the cut end of the capillary at an angle of 45 degrees.

To eliminate glass residues insert the needle into a needle holder mounted on a syringe then use 2%hydrofluoric acid to rinse the inside of the needle three times by drawing the solution in and out. Follow by rinsing the needle with acetone three times. To prepare dishes for injection or cell transplantation prepare 1%agarose by heating 50 milliliters of embryo medium containing 0.5 grams of agarose in a microwave for one minute at full power.

Cool the agarose gel to about 60 degrees Celsius and pour the entire volume into a 90 millimeter petri dish. Insert a mold to create lines for injection or to create wells for cell transplantation. When the agarose gel is set use forceps to remove the mold.

To inject donor embryos, under a stereomicroscope use forceps to gently squeeze collected transgenic embryos into the lines of an injection dish filled with embryo medium. With forceps orient the embryos with the cells toward the top then incubate the dish at 28 degrees Celsius until the embryos have reach the four cell stage. Next, load an injection needle with two microliters of a previously prepared injection solution containing the actin-binding domain of the yeast actin-binding protein 140 bound to mcherry.

Then insert the needle into a needle holder attached to a micromanipulator and connected to an air transjector. Open the needle by using fine forceps to gently touch the needle tip or by pinching it's extremity. Then insert the needle into one of the four cells and inject the solution to fill 70%of the cell volume.

Inject 30 embryos in a similar manner. When the embryos have reached sphere stage use a plastic pasture pipette to transfer host transgenic goosecoid GFP embryos and previously injected donor embryos into seperate 35 millimeter petri dishes coated with one millimeter agarose in embryo medium and filled with Pen/Strep embryo medium. Using fine tweezers manually extract the embryos from their chorions.

Then place the embryos into the incubator at 28 degrees Celsius. When the embryos have reached shield stage under a flourescent microscope select donor embryos expressing ABP140 mcherry within the green shield. Using a fire polished pasture pipette transfer the host embryos into a row of wells in the cell transplantation dish filled with Pen/Strep EM.Then place the donor embryos in the neighboring row.

Next with an eyelash carefully orient the embryos with the shield up. Then using a needle holder attached to a syringe rinse the tip of the needle by drawing in and expelling out 70%ethanol. Dry the needle by drawing air into it.

When the needle is dry insert it into the needle holder connected to the Hamilton syringe with the bevel facing upwards. To perform shield to shield transplantation enter the shield of a donor embryo with the transplantation needle and draw up about 10 to 20 cells labeled with ABP140 mcherry. Carefully avoid drawing yolk.

Transplant the cells into the shield of the host embryo and repeat the cell transfer until all host embryos have been transplanted. When transplantation is complete use a fire polished pasture pipette to remove any damaged embryos. Incubate the transplanted embryos at 28 degrees Celsius for about 30 minutes to allow them to recover.

To transplant single prechordal plate pregenitor cells after dechorionating embryos as demonstrated earlier use a fire polished pasture pipette to transfer three donor embryos into a cell transplantation dish filled with Pen/Strep calcium free ringers. With fine tweezers for each embryo dissect an explant containing the injected ABP140 mcherry labeled cells and rapidly discard the rest of the embryos. Using an eyelash gently stir the explants until the cells dissociate.

Then draw up a single, isolated cell in the transplantation needle and transplant it into the shield of a host embryo as demonstrated earlier in the video. For optimal control of the aspiration and transplantation keep the air/water separation at about 20 centimeters from the end of the needle. To mount the embryos fill a glass bottom vial with one millimeter of hot 0.2%agarose in EM.Place the vial at 42 degrees Celsius in a heat block.

Transfer a selected embryo into the agarose solution and discard the excess embryo medium from the pipette. Then draw the embryo into the pipette with agarose and transfer it to a previously prepared imaging chamber along with a drop of agarose. Before the agarose is set use an eyelash to orient the embryo by placing the perspective prechordal plate upwards for imaging with an upright microscope, or on the glass bottom for imaging with an inverted microscope.

There will be about 40 seconds to orient the embryo before the agarose gel sets depending on the room temperature. Orient the embryo rapidly by manipulating the blastoderm rather than the yolk which is very fragile. After all embryos are mounted and the agarose is set use Pen/Strep EM to fill the chamber to prevent the embryos from drying.

Carry out live cell imaging and cell dynamics experiements according to the text protocol. The technique demonstrated in this video was used to analyze the role of Sin1, one of the core components of TOR complex two in controlling in vivo cell migration. This movie shows the migration of transplanted prechordal plate pregenitor cells.

Actin labeling allows visualize of actin rich cytoplasmic protrusions. Through analysis of frequency and orientation it was observed that wild type cells frequently produce large cytoplasmic protrusions, oriented in the direction of the animal pole, and of migration. As seen in this figure, loss of function of Sin1 leads to a drastic reduction in the number of protrusions and randomization of the remaining protrusions demonstrating the importance of Sin1 in controlling actin rich protrusion formation.

Interestingly the Sin1 phenotype can be rescued by expression of a constitutively active form of Rac1 strongly suggesting that TOR C2 controls actin dynamics and cell protrusion formation through Rac1. This technique was also used to analyze the role of arpin, an inhibitor of the ARP2/3 complex in cell migration. As shown here, loss of function of arpin leads to an increase in protrusion frequency, due either to more frequent protrusion formation, or an increase in protrusion stability.

Measuring protrusion lifetime in the absence of arpin revealed that the temporal persistence of protrusions doubled indicating an increase in stability. Once mastered the transplantation of about 30 embryos can be done in 45 minutes. While attempting this procedure it's important to keep an eye on the development of the embryos as all the steps are critically timed.

Following this procedure, other methods like live confocal microscopy can be performed to provide better imaging and address questions regarding the sub cell organization of cytoscotic elements, polarity markers, addition proteins, or any other cell constituent. After watching this video you should have a good understanding of how to use transplants of prechordal plate pregenitors to analyze the role of any candidate gene in in vivo cell migration.