Still Learning

Today was focused on cleaning plates after breaking down the crosses we set up yesterday. To 
clean 
the 
dish, 
I had to place the 
embryos under 
a 
dissecting
 microscope
 and
 removed 
unfertilized
 or 
damaged
 eggs.

 The most challenging part of cleaning is knowing when the embryo is fertilized or unfertilized - unfertilized
 eggs 
will 
not 
undergo
 the 
first 
mitotic
 division.



The first embryo from the left shows an embryo that is activated at the one-cell stage and ready to be fertilized, but many cells become stuck at this stage and are therefore infertile. The second embryo is fertilized and the cell has multiplied into thousands of tiny cells. The third embryo is at 50% epiboly in the gastrula period, meaning it has formed an epiblast and hypoblast after about 5.5 hours. The second to last embryo is at the bud stage where it has formed an embryonic axis. Finally, the last embryo is characteristic of an 18-somite, where neuromeres develop and a tail appears.

Getting My Bait

My second week at NIH has begun and I am starting to feel much more comfortable with the environment and projects I've been confronting. My wonderful Monday morning and afternoon were focused on sexing and crossing 92 zebrafish pairs of F2 generations. The first skill I had to master was how to distinguish males from females - not an easy task.

                                                                                                       
The picture above shows a female fish at the top and a male zebrafish below it. The main characteristics to look for in each sex are the following:

Females:

• Have a larger stomach (usually because of the eggs they carry)
• Have clear tails with yellow tips
• Have very yellow dorsal fins
• Are generally larger than male fish   

Males:

• Are usually torpedo shaped
• Retain a pinkish color
• Have clear dorsal fins
• Have very yellow tails 

The process of sexing seems easy when the differences are described so clearly, but in reality, many males and females are difficult to distinguish - especially if a male fish is full and looks like it is carrying eggs. Nevertheless, after sexing the fish, it was time to cross all 92 pairs.
           
Crossing is done by placing one male (sometimes two) and one female fish in a crossing tank like the one shown above. The male fish then swims on the side or behind the female, stimulating her to release eggs while the male externally fertilized them. Spawning usually ends 10-30 minutes from the time it starts and will ideally leave eggs at the bottom of the tank to be collected!

                                                                                                                                                         

Assembling the Fishing Rod

This blog is to reflect on my experience at NIH as an undergraduate intern in the Eunice Kennedy Shriver National Institute of Child Health and Human Development Summer Student Intramural Training Award Program. I hope this blog also serves to encourage other undergraduate students to participate in research, even if it seems like a very intimidating career at the undergraduate level. If you know me as a person, you will know I have always been torn between pursuing biological research and developing my skills as an English major. Therefore, I found this blog to be the perfect matrimony between my two interests. 


So now that the formality before informality has been established, I can continue on to tell my followers (if I end up having any) about my first week at the NIH campus in Bethesda,MD. The first day felt like the longest day of my life - probably because waking up at 7 am seems like the most ungodly thing after two years of scheduling classes after 11 am at the University of Maryland, CP. But normal people wake up early right? Anyways, I was introduced to the campus, got my official badge with the most unattractive photograph of my life and was sent to the lab of Dr. Brant Weinstein, Section onVertebrate Organogenesis / Program in Genomics of Differentiation Program (PGD). Yeah, who knows what the latter part of that sentence even means...hopefully you will notice my growth and development throughout this blog so I'll be able to tell you in the future (far future). 


If anyone has taken a college bio class, imagine sitting in the class for 6 hours. That's what the first day was like, but trust me I needed it. The thing about labs is that they are very specific to the subject being researched - so despite having taken biology classes and having done previous research at UMD, I was totally lost on what was going on in Dr. Weinstein's lab. This makes me wonder if it would have been more productive to do research before taking the classes. To be honest, I can't recall even 15% of what I learned in a classroom, but I don't find it too difficult to remember what I did in a lab. This hands-on learning may simply be my cup of tea, but it also puts classroom biology in a much clearer perspective. Explaining it to you now will help me to understand it more as well.


So here's the gist: 
Our lab uses zebrafish, a tropical freshwater fish that is an important model for vertebrate development because of its fully sequenced genome, to study developmental angiogenesis in the vertebrate vascular system. It's a pretty complicated field of many different projects and hopefully I'll be starting my own individual project soon! But let me break it down for you even further.
Let's start with the zebrafish - here's a nice little picture taken from wikipedia.

I definitely want to post some first-hand pictures from my own procedures at NIH, but I'm going to have to ask Dr. Weinstein first if I'm allowed to do so. So, until then, you will have to be satisfied by wiki. Regardless, that's what a zebrafish looks like and I get to work in a lab that has a facility with over 19,000 tanks of these little fishes! Basically, the work I do involves crossing different families of these fish and collecting the embryos of the fish so that we can observe them for mutations or expressions of genes we are focusing on. There is so much more involved in our research than this general overview, but I will get into specifics as I continue to post!


Cool things I've gotten to do so far - 

• Looking for swim bladders aka gas filled organs in the dorsal part of a fish which allow them to control their buoyancy. The little silver circle in the middle is a swim bladder.                      What's cool is that swim bladders are evolutionary very similar to lungs. Embryos form an outpocketing of pharynx or esophagus that becomes one or a pair of sacs (swim bladders or lungs) filled with gases derived directly or indirectly from the atmosphere. So yeah you breathe like a fish.
• Injecting embryos with mRNA. I line up fertilized embryos in their one-cell stage in an agarose gel plate (as shown below) and inject mRNA into the blastomere. This allows for gene expression of the gene that the mRNA encodes.                                                                                                                                                                                                                                                              Injecting embryos is a VERY difficult process for a beginning intern like me. The needle used to inject the embryos is extremely thin and breaks quite easily, which can become very frustrating. Also, getting through the chorion, or the outermost membrane of the embryo, can be tricky - and getting into the blastomere is even more difficult! Because it is best to inject into the cell, rather than the yolk, the embryos need to be oriented very carefully on the gel and this take A LOT of time. However, mRNA can also be injected into the yolk so if I have trouble with the blastomere, not all is lost.
• Dechorionating cells. Dechorionation involves removing the outermost membrane surrounding the developing embryo. I do this by grabbing both sides of the chorion with two foreceps and gently pulling the chorion apart without disrupting the embryo itself. This way the embryo can be observed more accurately under the microscope!