Adventures in Dr. Kerney’s Lab

 The Kerney Lab – Summer 2015

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The Story of the Salamander: Studying Symbiosis Between Salamander and Algae

by Huanjia Zhang and Elizabeth Hill

The Story of the Salamander: Studying Symbiosis Between Salamander and Algae

by Huanjia Zhang and Elizabeth Hill
The symbiosis between yellow-spotted salamander embryos (Ambystoma maculatum) and the green alga (Oophila amblystomitis) is a well-known example of a classic mutualism. Developing salamander embryos have masses of algae cells that live inside their egg capsules while they’re developing.  The algae produces oxygen within the egg capsule through photosynthesis while the salamander embryos gives off nitrogenous waste to the potential benefit of the algae.

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Salamander embryos with symbiotic algae

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The Northwestern salamander – Ambystoma gracile

But wait! It gets better! The yellow-spotted salamander embryo exhibits a particulary interesting relationship with their algal symbioant. This symbiotic relationship is incredibly rare in vertebrates because the algal cells not only surround the salamander embryos but also enter the embryonic tissues and cells.  This is perhaps the only known intracellular symbiosis in vertebrates.

Elizabeth Hill:

My role in furthering this research is to find an efficient way to detect the invasion of algal cells in salamander embryos. This summer I have worked on the fluorescent microscope to get good picture of what’s going on inside the salamander.  Because the algal contains chlorophyll it lights up when exposed to green fluorescent light, causing the salamanders to light up like the night sky.

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Salamander embryo under fluorescent light, the bright spots are algal cells.

Once the prescence of algae was confirmed for a given embryo, I used a histological stain called Gomori’s Silver Methenamine that usually stains fungal cells in pathology.  The goal of using this stain was to stain the cell walls and starch granules of the algal cells while leaving the salamander cells unstained.  The GMS stain works by oxidizing the ends of complex carbohydrates to aldehydes and then binding to silver ions, turning complex carbohydrates black.  After many trials of the GMS stain, there has been some success, but I still have some problems with background staining of other carbohydrates (especially glycogen) in the salamander embryos.  I am currently working on a way to optimize GMS to only stain algal cells.

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Elizabeth is sectioning to prepare the slides for staining

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GMS stain of salamander tissue, possible algae are brown.

Huanjia Zhang:

While Elizabeth has been conducting histological stains and qualitatively observing  algae under a fluorescent microscope, I have been using qPCR  to quantify and analyze the amount of algae cells within salamander embryos.
So what on earth is qPCR? qPCR stands for “quantitative PCR,” which is also occasionally called “real time PCR.” It is a commonly used technique to amplify and simultaneously detect or quantify a targeted DNA molecule. qPCR is very similar to traditional PCR. The major difference being that in qPCR the amount of PCR product is measured after each round of amplification whereas, in  traditional PCR, the amount of PCR product is measured only at the end point of amplification.

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iQTM5 real-time PCR detection systems

The concept of qPCR is simple: amplification products are measured as they are produced using a fluorescent label. During amplification, a fluorescent dye binds to the accumulating DNA molecules, and fluorescence values are recorded during each cycle of the amplification process. The fluorescence signal is directly proportional to the starting DNA concentration, and the log-linear correlation between PCR product and fluorescence intensity is used to calculate the amount of template present at the beginning of the reaction.

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As the qPCR reaction progresses, double-stranded products are generated. The flourescent dye intercalcates into these products and begins to fluoresce.

So far, I have used  qPCR to quantify fourteen samples of A. maculatum embroyos of different developmental stages. In addition to A. maculatum embryos, I have also conducted qPCR for eight samples of A. gracile embryos, which is a related species to A.maculatum that lives on the west coast of the United Sates. So, far the qPCR results of A. maculatum embryos has confirmed that there are algae cells inside the tissue. More interestingly, the qPCR results of A. gracile also showed that there are potentially algae inside these embryos, which has not been observed under the flourescent microscope. The next step for me to do is to analyze this data and try to find if there is a relationship between the amount of algae cells in the embryos at different developmental stages.

Bone Development in Xenopus tropicalis

by Michaelyn Cornish

Hello all! This summer I had the opportunity to work in Dr. Kerney’s research lab. It is hard to believe that the summer research is coming to a close already, but I can assure you that this short time in the lab was well spent. I can’t explain everything that I’ve learned so far, but I will give you a glimpse of the exciting research I have been doing! This summer I worked to continue a project started by a former biology student that involves amplifying and piecing together various genomic regions of Xenopus tropicalis, an aquatic frog from Western Africa.

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Aren’t these Xenopus tropicalis so cute?

Dr. Kerney and I were specifically interested in looking at skeletal development within these frogs. There are two distinct stages of bone formation in these organisms. The first, pre-hatching, involves cartilage formation, which is followed by a metamorphosis stage that involves both new cartilage and bone formation. Cranial cartilage and bone in these organisms originate from a population of cranial neural crest cells (CNCC). However the location of pre-metamorphic (tadpole) bone-forming neural crest cells remains a mystery.

Transcription factors, proteins that regulate gene expression, control these CNCC and ultimately play a large role their differentiation into osteoblast precursor cells that eventuall form skeleton. We are studying a transcription factor that controls bone development in X. tropicalis. The ultimate purpose of our research is to determine where exactly the bone-forming CNCC population in tadpoles originate, and when these cells differentiate into osteoblasts. The cells can eventually be traced by designing a plasmid which can be inserted into developing X. tropicalis embryo. The plasmid will contain the transcription factor’s promoter region, several other intronic regions that contain enhancer sites, and fluorescent marker protein. After all regions are amplified, work can be done to fuse the regions together, cloned them into a vector, and eventually inject the newly formed plasmid into X. tropicalis embryos in order to determine the location of the bone-forming CNCC.

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The PCR8 TOPO TA Cloning kit as well as the PCR machine that I used  are  shown above.

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Here is another picture showing one of my MANY gels that I ran this summer used to verify the PCR products.

https://tools.lifetechnologies.com/content/sfs/vectors/pcr8gwtopo_mcs.pdf

Here is a link to a  picture depicting where our PCR product will be inserted. You can see that it is fused into the vector at the 3’ T overhangs and is also flanked by EcoRI cutting sites.

But Wait! We have done more than science this Summer!

In addition to all the cool science experiments that we conducted, we all spent quality time outside of the bio lab together. Here are some photos that capture  some of the cherished memories made and the good times we had this summer!

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We had fun at the National Maker Faire in DC.

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Here is Dr. Kerney showing his artistic side burning wood using the sun and a lens!

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Huanjia’s Gettysburg G!

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Elizabeth having her own wood burning session

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Michaelyn even became an astronaut for the day!

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Our dangerous dry ice crisis.

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Fun at Mr. G’s

Our Awesome Lab Family

Dr. Kerney

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Elizabeth Hill, Rising junior biology major

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Michaelyn Cornish, Rising junior biology major.

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Huanjia Zhang,Rising junior biology major.

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