Maggie DeBell, Yan Zhou, and Evan Czulada
At one point in our lives, we were all embryos growing inside of our mother’s womb. Eventually, this little ball of life became full-sized human beings. Our lab studies how embryos grow and develop over time. We observe this interesting part of biology through the lens of our organismal model, the salamander. Salamander embryos develop outside of their parents’ bodies, which allows us to study them in great detail without being invasive to the parent. Furthermore, these amphibians are abundant in this region, providing us with easy access to them and their embryos.
Yet not all salamanders were created equal. Ambystoma maculatum, also known as the spotted salamander, is unique among all vertebrates. Found in this salamander’s egg capsules and tissues is a type of algae called Oophila amblystomatis. This endosymbiotic relationship–the entrance of one organism into the tissues and cells of another–is the only occurrence of its kind of all vertebrates. Learning more about this relationship allows us to understand, evolutionarily, how this type of symbiosis can impact the members of two different species. Moreover, connections can be made between the route of algal entry and a similar mechanism of pathogen invasion into human cells. This natural phenomenon has led to multiple experiments being conducted to further our knowledge on this subject matter, from several types of imaging to studying the organisms inhabiting the egg capsules of the embryos.
Whole Mount Immunochemistry: Antibody Staining MicroCT Imaging
To observe how this relationship affects the limb and organ development of Ambystoma maculatum, I have been working to establish a protocol of antibody staining these embryos for later use in a microCT machine. Antibodies are naturally produced via the immune system to protect the host against invasion, but they can also be used to bind to a specific antigen–a very specific protein, pathogen, or other part of the cell–and illuminate said antigen when positioned under a microscope. Using this technique, it is possible to show the expression of proteins vital to the development of the salamander embryos.
The specific protein that my antibodies target is called type II collagen. Type II collagen is an integral protein in the formation of cartilage, which primarily composes the skeleton of developing embryos. (Later, this cartilaginous skeleton becomes bone as the embryos become adults.) Therefore, using this technique, I am able to see where collagen-II is most abundant in the salamander embryo. Moreover, caspase 3, a marker for cell apoptosis (programmed cell death), is another antibody added to the embryos that shows where the salamander cells have terminated their use of the collagen-II protein.
Once the right protocol is finally developed, I can then proceed with other chemical treatments to permit imaging through a microCT machine. This type of imaging allows me to analyze the developing limb patterns of the young salamander in a three-dimensional context. The issues I have had with the protocol are based primarily on antibodies that are not properly binding to their selected antigens. Having obtained new antibodies, I hope to have a working mechanism for this kind of imaging in the near future.
To enhance our understanding of this symbiotic relationship, we will be collecting data on the placement and quantity of algae within salamander embryos. This can be done using episcopic imaging. The embryos are embedded in a material called JB-4, which allows us to preserve the autofluorescence of the algae. These blocks are sliced on a microtome, taking a photo after each cut. Eventually, these images will be put together to form 3-D reconstructions of the embryos, showing us exactly where the algae is.
To get a clear image of the embryos as they are being cut on the microtome, we took apart an old microscope and mounted it sideways. The apparatus holding it up allows us to focus the image.
We are currently working through issues with magnification, alignment, and knife angle optimization. There will be other details to work through, but we hope to start collecting data soon!
There is a multitude of bacteria that inhabit the egg capsule of a salamander embryo. After identifying these specific bacteria using 16s rRNA gene sequencing, we can further study these microorganisms to determine how they inhibit a type of mold that is known to kill salamander embryos.
To accomplish this feat, the intracapsular fluid surrounding the embryo was first extracted and swabbed on plates and incubated for bacteria growth. Next, those bacteria can be isolated morphologically through separation into different Petri dishes. DNA was then extracted from the bacteria and amplified through a technique called PCR using a 16s primer. PCR is a method used to find specific genes or pieces of DNA and make many more copies of it for evaluation. Through analysis of the gel and its subsequent purification, the product can be sent away for Sanger sequencing (a way to find out exactly what each bacterium is based upon their DNA). This allows us to make determinations about these types of bacteria and permit further investigation.