The overall goal of this procedure is to follow endocardial cells during atrioventricular canal and heart valve formation. This method can help to address key question in the field of cardiac development, such as to understand how endocardial cells behave in different types of mutants. Tracking cells during AVC and valve formation is challenging because of the rapid beat of the heart.
It makes it difficult to follow cells via traditional time-lapse microscopy. The main advantage of this technique is that it bypasses some of these difficulties. Demonstrating the procedure will be Renee, a postdoc in my lab.
After creating a mold according to the text protocol, pipette about 1.5 milliliters of melted 1%agarose into a 35-millimeter plastic mounting dish. Then, position the plastic mold in the liquid agarose, taking care to avoid trapping air between the mold and the agarose. Place the dish at four degrees Celsius until the agarose hardens.
Then, remove the plastic mold from the agarose. Next, add two milliliters of previously prepared mounting medium to the mounting dish, and allow 15 to 30 minutes for the solution to diffuse into the agarose. In the meantime, melt a one-milliliter aliquot of 0.7%low melting point, or LMP, agarose by placing the tube in a 70-degree Celsius heat block for about five minutes.
Pipette the LMP agarose up and down to cool it slightly. Then, add 25 microliters of eight milligram per milliliter tricaine and 40 microliters of one molar BDM solutions to the tube. Mix by pipetting up and down.
Then, transfer the tube to a 38-degree Celsius heat block to keep the LMP agarose in its liquid state. Transfer the embryos to a separate dish containing two milliliters of mounting medium. Then, when the hearts have stopped beating, transfer the embryos to the mounting dish and use forceps to arrange them in the wells so that they lie at a 25-degree angle, tails pointing toward the deeper part of the well, with the yolk facing up.
When the embryos are roughly in place, remove the medium, and embed them in approximately 200 microliters of 0.7%LMP agarose containing tricaine and BDM. Then, readjust the embryo positions, tilting them to the left or right as necessary. Press down on the walls of the indented square to allow excess agarose to overflow to the side areas of the mounting dish.
Wait approximately five minutes for the LMP agarose to set, then add mounting medium to the dish. The embryos are now ready to be photoconverted. To carry out photoconversion, on an upright confocal microscope equipped with a 405, 488, and 561-nanometer laser sources, use transmitted white light and an objective lens to locate the embryo's heart.
Using the 561-nanometer laser, check for red Kaede fluorescence. Then, use the 488-nanometer laser to visualize the endocardium. Next, on the microscope acquisition software, enter FRAP mode.
Then, select approximately 1%laser power for both the 488 and the 561-nanometer lasers. While focusing on the plane of interest, select a region of interest, or ROI. Then, using the 405-nanometer diode laser at 25%laser power, photoconvert the cells, scanning over the ROI three times.
Should insufficient Kaede be photoconverted in the ROI, scan the 405-nanometer laser over the area three times again. Return to the TCS SP8 mode on the software, and using the 561 and 488-nanometer lasers sequentially, in Bidirectional mode, acquire a Z-stack. After photoconversion, use a glass pipette to press down slightly just above the head of the embryo to break the LMP agarose.
Then, gently suck up the embryo. To wash away the BDM containing medium, eject the embryo from the glass pipette into an 85-millimeter Petri dish containing embryo medium with PTU. Transfer the embryo to a well in a six-well plate containing embryo medium with PTU.
During this step, make sure to keep note of which embryo goes into which well, as this is essential to correlate the position of photoconverted cells at later developmental stages. Now that the embryos have been removed from the BDM containing medium, their hearts should start beating again in about five minutes. Return embryos to the dark at 28.5 degrees Celsius to allow them to continue to develop normally.
At the desired stage, stop the heart and re-embed the embryos, as demonstrated earlier in this video, with the important difference that the embryos must be treated with BDM in separate, marked dishes to keep track of them. For embryos up to 62 hpf, acquire Z-stacks of the endocardium using 561 and 488 nanometers for red and green fluorescent signals, respectively. For embryos older than 62 hpf, use a multiphoton laser set to 940 nanometers to image green Kaede instead of the 488-nanometer laser.
For embryos younger than 48 hpf, it is possible to project the three-dimensional AVC onto a two-dimensional image for analysis. First, threshold the image. Then, erase points that lie outside of the heart.
To perform manual segmentation, orient the image on the left to match the image on the right. Segment the side of the heart in which the orange vector passes through, and add points in the direction of the blue vector. Next, interpolate the cross-sections, and project the color intensity onto the surface.
Finally, adjust the orientation of the AVC, and project it onto a 2D surface. An example of an embryonic heart just after photoconversion at 48 hpf is shown here. Cytoplasmic Kaede in several cells of the superior side of the AVC has been converted from green to red.
In the same embryo at 80 hpf, it's evident that the photoconverted cells have proliferated and have migrated to the inner side of the superior atrioventricular valve. An example of a 36 hpf embryo where the EdCs in the atrium and ventricle have been photoconverted is shown here. The AVC is then projected into two dimensions for easier visualization and analysis and allows one to quantify the distance between the two photoconverted regions.
While attempting this procedure, it's important to keep the time the heart is stopped to a minimum to avoid affecting normal development. With practice, photoconversion can be completed within about 30 minutes provided that everything is prepared in advance. After watching this video, you should have a good understanding of how to track endocardial cells after photoconversion in live zebrafish embryo and analyze your data sets.
