This protocol allows the labeling of individual ependymoglial cells in the zebrafish telencephalon to enable their long-term monitoring, as well as the manipulation of specific pathways in a cell-autonomous manner. The main advantage of this technique is that it allows single cell labeling in fast and efficient way, as well as gene editing and signaling pathway manipulation in cell-autonomous manner. This method allows the investigation of basic cell biology questions, for example, about cell delamination, the maintenance of epithelial homeostasis, and the symmetry of cell division.
Begin by using a needle-pulling apparatus to prepare glass capillaries. Use microloader tips to fill a prepared glass capillary with ten microliters of plasmid solution. Then press menu change capillary"on the injection device to allow the needle to be loaded into the needle holder.
Then switch the injection device from change capillary to inject mode. Then, using a stereomicroscope with a four times magnification, and Finden forceps, cut only the tip of the capillary. Then apply pressure to the foot pedal to confirm that the plasmid solutions runs easily out of the needle without hindrance.
When the needle is ready, transfer a zebrafish from the husbandry tank to a container of anesthetic solution. And wait a few minutes until the movement of the gills subsides. Press the sedated fish dorsal side up onto a pre-wetted sponge under the stereomicroscope, and use a stainless steel dissecting microknife with a 40 millimeter cutting edge and a 0.5 millimeter thickness to carefully create a small hole in the fish skull at the posterior side of the telencephalon, just next to the border of the optic tectum.
Tilt the fish as necessary and orient the tip of the glass capillary towards the skull in the correct angle to facilitate penetration of the capillary tip through the hole. Then, very carefully insert the tip of the capillary into the hole through the dorsal ependymal cell layer, until it reaches the telencephalic ventricle, and apply pressure to the foot pedal for about 10 seconds to deliver approximately 1 microliter of the plasmid solution. The success of the delivery can be confirmed by observing the spreading of the green liquid throughout the ventricle.
Cover the fish telencephalon with a small amount of ultrasound gel. For electroporation of the plasmid injected animal, remove the sponge from the injection set-up and immerse the inner side of the electrotips into ultrasound gel. Position the fish head between the electrodes, with the positive electrode at the ventral side of the head and the negative electrode on the dorsal side.
Then, while still holding the fish's body in the sponge, press the electrodes gently and precisely against the telencephalon and administer the current with the foot pedal, holding the electrodes in place until all five pulses are finished. If the electroporation is successful, plasmid labeled single ependymoglial cells can be observed among the non-plasmid labeled ependymoglial cells. Depending on the efficiency of the electroporation process, a higher or lower number of ependymoglial cells may be labeled.
Nevertheless, the demonstrated protocol yields a higher number of labeled cells than previously published protocols. Note that the highest density of labeled cells tends to emerge mostly at the inner ventricular side of both hemispheres due to the way in which the injected plasmid liquid distributes between the hemispheres. In this video, one hemisphere of the zebrafish telencephalon is presented in 3D, where the ependymoglial cells with radio-processes, are in magenta when observed from the side.
Through co-electroporation with another plasmid that labels nuclei, cell division of ependymoglial cells can be observed. Unsuccessful electroporation results in very low number or no labeled ependymoglial cells. Dorsal ependymal cells, however, are the most likely to be labeled.
Their soma is larger in cuboid and they do not possess radio-elongated processes as evident from a side view of the ependymoglial cell layer. tdTomato-mem labeled cells are most likely ependymal cells, which are located above the layer of ependymoglia. In contrast, the introduction of a tdTomato-mem expressing plasmid to individual ependmyoglial cells results in tdTomato-mem expression in addition to the initial cell labeling.
The most important things to remember are to cut only the tip of the capillary and to penetrate the ependymal layer while injecting so that the liquid spreads throughout the ventricle. This technique enables the long-term monitoring of single ependymoglial cells through in vivo imaging, and therefore establishing their role in brain regeneration as well as their vast in vivo genetic link.
