This demonstration shows the technique for removing the germline precursor cell from early embryos in the pod. Crustacean parli sys parli is an emerging model organism with expansive potential for use in evolutionary and developmental research. Most model arthropods are insects and the most extensively studied of these is the fruit fly Glip Melanogaster insects are nested within the sub phylum, panc cresta and studying other crustaceans outside of the insects provides a broader view of the evolutionary history of the developmental traits and molecular mechanisms that have been well studied in drosophila.
However, very few crustacean have been well-established for experimental laboratory analysis of development. Parelli is a highly tractable laboratory model system and thus represents a valuable addition to the inventory of model organisms. As a laboratory animal, parli is very easy to maintain and work with.
It can survive well in large cultures of artificial seawater, and it is easy to distinguish between males and females based on clear morphological differences. Most notably, the large hooked anterior appendages that males use to grasp the females during mating for developmental work. Parelli has several very appealing features, couples mate all year round, so it is possible to gather embryos at any time.
Females lay embryos into the brood pouch between the first several pairs of legs, and it is possible to gather the embryos very early in development without killing the female or damaging the embryos. The embryos survive in filtered artificial seawater through hatching, and there is a detailed staging table that allows accurate identification of the progress through development. Shown here is a selection of stages of paral development through hatching by the eight cell stage of development.
Each of the cells in the paral embryo is specifically faded to one of the three germ layers or the primordial germ cells. It is the specification of the germ cell population that is a particular interest in this demonstration. The cell name G for germline is known to be the unique germline precursor in parli lineage tracing experiments show that the descendants of G become all of the germ cells and no descendants are part of any other germ layers.
Additionally, experiments have been performed removing G from the embryo without harming the rest of the cells. In these experiments embryos that have G removed lacked germ cells through the rest of embryogenesis as indicated by the absence of cells expressing the protein vasa, which is a germline marker in model organisms across metazoans. We will now demonstrate how to gather parli embryos and manually ablate G from an eight cell embryo.
This video demonstrates removing G, but can be applied to any cell at the eight cell stage. For this part of the procedure, we will need a pasture pipette with the end widened a second unaltered pasture pipette filtered artificial sea water, a large container for housing couples, small Petri dishes, a glass well plate forceps, and a dissecting tool consisting of a thin curved wire in the end of a pasture. Pipette gloves are not used in this demonstration, but remember to add protective equipment in compliance with risk management at your institution to make the widened pasture pipette first score around the pipette with a diamond pen and then carefully break off the tip.
Hold over a flame for several seconds to smooth the edges. The opening should be slightly narrower than the maximum width of the pipette. To make the wire dissecting tool insert a tungsten wire into the end of a glass pipette and hold over a flame to melt the glass.
Fixing the wire in place use forceps to gently curve the end of the wire for easiest manipulation with the tool. The hooked end should be slightly over half a centimeter in length. Collect couples using the widened pasture pipette and transfer them to a separate container of artificial seawater.
Leave the couples at room temperature overnight. The next morning infuse filtered artificial seawater with carbon dioxide. Gather separated females and anesthetize them in this water.
Transfer a single female to a glass well plate in filtered artificial seawater with carbon dioxide viewing under a dissecting microscope, grasp the coxal plates along one side of the body with forceps. Embryos are visible in the brood pouch. Insert the wire dissecting tool into the posterior end of the brooded pouch and gently lift the wire towards the anterior to separate the brood pouch covering, opening the pouch and exposing the embryos.
Use a pasture pipette to flush water through the opened brood pouch. Pushing out the embryos. Transfer the female to filtered artificial sea seawater without carbon dioxide to allow recovery before reintroducing her to the main culture.
Transfer embryos to a Petri dish with filtered artificial seawater and view under the microscope to stage them. Since it is often difficult to determine the stage of the embryos before removing them from the female, we often need to gather embryos, many females in order to have sufficient embryos of the correct stage. Often the easiest way of getting eight cell embryos is collecting embryos of earlier stages and waiting for them to mature to the correct stage.
In addition to the embryos we have already gathered for the ablation procedure, we will need a pasture pipette forceps filtered artificial sea water, a Petri dish with a layer of cigar, a needle made from a pulled glass capillary, a mouth pipette with a small opening, clean Petri dishes and filtered artificial sea water with amphotericin and penicillin streptomycin for the tip of the mouth. Pipette pull a long tipped past your pipette over a flame and break off the tip of the small end inserted into the end of the mouth.Pipette. This tip is the perfect size for the procedure.
The glass needle, which will be used to puncture G during the ablation procedure must have a fine point, but be sturdy enough not to break when pushed against the corion. Shown here is one common shape used in our lab. For the ablation, we must first identify which cell is G, the embryo on the left shows G outlined in black, and in the following section, we will learn how to distinguish G from the other micro mirrors.
The embryo on the right shows what you should expect as the outcome of your successful ablation. In this embryo, G has been ablated as indicated by the black circle, and only three micro meres remain using forceps. Gently roll the embryo to view and compare the cells.
G can be identified as the smallest microme sitting on top of the smallest macro mere MAV can be identified as the smallest macro mere by rolling the embryo over. Additionally, G does not touch the cell EN, which is the microme across from it. As the cell's el and er share a border between G and EN for this embryo, we can see that the smallest microme is located on the left and we can see that it is not touching the microme across from it.
By rolling the embryo over, we can confirm that this micro mirror is sitting on the smallest macro mirror. Now located at the bottom left of the embryo, we can confidently call it G and move on to ablating. In brief.
For this procedure, we will stabilize the embryo with forceps while we puncture G with the pulled glass needle. We will then apply slight pressure to the embryo with the forceps causing the contents of G to leave the embryo through the hole we made. We use the mouth pipette to remove the contents of G after they leave the embryo so that we can see when G has been completely removed.
Now to go through that in detail, fill the silk guard plate with a shallow layer of filtered artificial seawater. Transfer an eight cell embryo to the plate using a past your pipette with forceps. In the left hand, orient the embryo with G to the right and gently grasp the embryo to stabilize it using a pulled glass needle, gently punctured G, do not insert the needle too far to prevent pushing the needle into the macro mirrors below G, switch the needle in your right hand for a mouth pipette with a pulled past pipette tip.
Hold the tip of the pipette close to the hole in G and apply very gentle suction. Gently squeeze the embryo with the forceps as the contents of G is pushed out of the corion. Pull it into the mouth pipette to allow an unobstructed view of the embryo.
Carefully watch the embryo. As G is removed, the border of G will recede towards the hole in the corion, making the intersection of the underlying cells visible. Continue applying pressure until all of G has been removed and use the forceps to roll the embryo and visually confirm that all of G is gone.
Using a pasture pipette, transfer the embryo to filtered artificial sea water plus amphotericin and pen strep. Change half the water on the embryo daily to prevent infection. Here is an embryo before and after ablation.
As you can see, there's an empty space where G was before. When done delicately and when the embryos are well cared for after the procedure, a large percent survive to hatching. In our lab, the average survival rate is just over 50%
