Understanding the Developing Brain:Neurogenesis and Corticogenesis

The first few weeks in the lab I find are the most difficult. You’re in a new place, and you're doing complicated science that you likely have no experience with. To make it even more intimidating, most members in the lab are skilled, intelligent, and extremely knowledgeable of not only the lab’s research but a plethora of subjects. In my lab, we study mitosis and differentiation of neural stem cells. Our research roughly falls into the fields of molecular genetics, microbiology, developmental and stem cell biology, and of course, neuroscience. In other words, my lab is extremely interdisciplinary.

But as I spend more and more time here I am familiarizing myself with the techniques, tools, and basics of working in a neural stem cell lab. If I wanted to sum up my research in one statement I would tell you that I am doing an anatomical study of the basal process of neural stem cells during mammalian corticogenesis. But to someone who has no background knowledge of neuroscience and molecular biology this description has very little meaning. So I’m going to start with corticogenesis.

Corticogenesis is the formation of the cerebral cortex, or the outer lining of the cerebrum. The cerebral cortex, which is also commonly referred to as the neocortex in mammals, is made of six layers which vary in neuron concentration and function. The development of this complex structure, also known as arealization, is consequently a highly specific process whose formation is largely reliant on spatio-temporal timing.

During corticogensis, neural stem cells (NSCs) also known as radial glial cells (RGCs) go through a process known as neurogenesis in which mature “adult” neurons are generated. The following figure displays a simplified version of this process:

However, as shown in the figure, this pathway is not always direct. NSCs can directly differentiation into mature neurons, or they can first differentiate into basal progenitors (BPs), also known as intermediate progenitor cells (IPCs), and then later, following another round of mitosis, differentiate into mature neurons. If NSCs do not differentiate following mitosis, this is known as proliferation. Both daughter cells will be NSCs.

The following diagram displays the direct and indirect forms of neurogenesis. It is important to note that when NSCs differentiate following mitosis, one of the daughter cells remains a NSC while the other may be either a basal progenitor or a neuron. Never do both daughter cells differentiate, except when an IPC undergoes mitosis to form two mature neurons.

My research focuses on understanding neurogenesis and the several factors that contribute to neuron differentiation. One of these factors, regulation of the cell cycle, specifically mitosis, is critical for cortical development. After all, it is the process of mitosis which produces mature neurons. Understanding the mechanisms underlying the division of these stem cells, or radial glial cells is crucial. These NSCs are long neuroepithelial cells which stretch all the way from the ventricle zone (VZ) that borders the ventricles of mammalian brains, to the pial surface of the brain. To reach such great lengths, these cells have a long epithelial structure known as a basal process. At the end of the basal process are structures known as “basal endfeet.”

It has been found that cell polarity places a crucial role in neurogenesis. There is strong evidence that supports that daughter cell inheritance of the basal or apical process following mitosis plays a crucial role in the determining of cell fate. This summer I am trying to understand  the importance of the basal process in neurogenesis. Using techniques such as immunohistochemistry, live imaging, and in utero electroporation (thanks to the assistance of my secondary mentor Louis-Jan!) on brain slices I am trying to identify some of the factors that influence cell fate and the mechanism by which they accomplish this.

We believe that RNAs localized at the basal process may play a critical role in neurogenesis. Using immunoflorescent antibodies we can label the nuclei, cell body, basal processes, and specific proteins within cells. Below is an example of some of the immunofluorescence we do here in the lab.

mCherry is a fluorophore that we use to label the cell body and basal process. HOECHST labels the nuclei, and FMRP targets mRNAs. FMR1 is the human gene that codes for the protein FMRP, which is critical for cortical development. Mutations in the FMR1 gene can lead to a variety of mental and physical disorders such as mental retardation, autism, fragile x syndrome, and parkinsons. It is just one example of the many proteins essential for cortical development. Hopefully I can contribute to our knowledge of neurogenesis so that we may better understand the mechanisms of brain development.

Learning from the Master

This summer I’ll be spending my time studying brain development in the Silver Lab. The research I’ve been doing here these past two weeks is groundbreaking. It’s been a dream of mine to work in a neuroscience laboratory for some time now. I am specifically interested however in developmental biology. With its cutting-edge neural stem cell research, the Silver Lab manages to combine two of my greatest interests, developmental biology and neuroscience.

Having just started out here, I have a lot to learn. Though I have worked in other labs previously, I find that laboratories vary with surprising specificity. Although some of skills from working in my other lab have helped me greatly, there are many techniques I have yet to master. I have tried, several times to isolate completely intact brains from developing mouse embryos, and have yet to master the art. Of course sharpening my skills and developing expertise in research requires time and often mentorship. I have the pleasure of working in a welcoming and encouraging lab. What better way to learn the ways of research than to interview a master?

I’d like to introduce my primary mentor, Dr. Debra Silver an accomplished scientist and the principal investigator of my lab. In the following interview Dr. Silver or Debby for short, reveals to me her journey and experiences in science.

Here is a photo of  my secondary mentor Louis-jan (LJ) and me in the lab!

Where are you from?

I grew up in Massachusetts in a small town called Southboro, which is 40 minutes west of Boston.

Were you always interested in science? 

Yes. I think I realized I loved science in high school. I wasn’t one of those people who grew up doing science experiments here and there. I took my first biology and chemistry courses in high school, and I went home and realized I liked doing my chemistry homework every day, well, that’s when I realized I liked science.
 

Where did they go to school and why did u go there? What was your major?

I went to Tufts in Massachusetts. I first became interested in Tufts because my father went there. I decided to go there because I loved their campus and I knew that they had a pretty strong program in biology and I wanted to be a biology major. I also wanted to be closer to Boston.

How long have you know that you wanted to pursue a career in science and specifically neuroscience?

When I was in college I started doing research for three summers. After I graduated I decided that I wanted to do more research and so I went back to the same lab that I did summer research in. I ended up staying there for four years. I originally had planned on going there for two years, to save enough money to travel around the world. I got really into my project, and my boss at the time treated me more like a graduate student than a technician. I was a lab technician at the time. I got so into it that I ended up staying for four years and I got a couple papers out. That convinced me that I wanted to go to graduate school. I applied after I had been in the lab for two years and I deferred for a year. I had one first author paper and two coauthors as a lab technician. That was how I got excited. I got interested in Neuroscience towards the end of graduate school. In graduate school I had been working on basic science questions on how cells migrate in drosophila. I decided that I wanted to move to a system that had more relevance to humans, so that was mice. I was particularly interested in the nervous system because it’s so fundamental for life.

What do you like best about being a researcher?

I love the thrill of discovering something that no one else has discovered before and I love the opportunity to peer into biology and learn about something that’s been perfected by a cell and chip away at our understanding of it. I like the creativity involved in coming up with new experiments and the thrill of finding something new.

Any disasters in the lab or embarrassing moments?

When I was a summer student I had spent a week making a radioactive probe. I lost sight of time when I was boiling it and completely melted it all over the heat block. I also found out the hard way that xylene has to be stored in glass and not plastic. It melts plastic. I did that experiment inadvertently and it melted a whole thing of plastic.
 

Have you had experience in a non-academic environment?

Well I did my postdoc in a government lab. That was a very different experience. I’ve learned that I love academics. I think it’s a huge part of being a scientist to teach other people what we know. I think the science can go in really exciting directions when people who are working on the problem have no bias as to what they think is going to happen. The government lab was a very exciting place to work. The world is your oyster as a scientist, you have so many resources.

What do you like to do outside of science?

I have two kids so most of my free time goes towards them. I love mountain biking. I like photography. I like mostly just spending time with my family, playing soccer with my kids. I like yoga too.

How did you end up at Duke?

When you are looking for a job you apply to a bunch of places that have labs. I ended up deciding on Duke because it was a great place that combined my interested in neurobiology, developmental biology, and RNA biology and there are a lot of people doing that here. I have pretty broad interests.

How has your experience been teaching?

I really like teaching. For a long time I thought I wanted a position that was more teaching and less research. I wish teaching was more appreciated. I love teaching and having people in my lab. I guess that’s more mentoring. I love when people take a class out of interest and not a requirement. I’ve always tried to teach. Actually when I was a graduate student I tried teaching students in high school in Baltimore.

What is your greatest finding?

I don’t know what my greatest finding is. But my most exciting finding was in graduate school. Hopefully everyone has that day when they find something really exciting in the lab. For me, that was in graduate school. I had generated mutations in flies in the components of the JAT/STAT signaling pathway. I had discovered that this pathway was required for cell migration in fly ovaries. My most exciting experiment in graduate school was the day that I overexpressed components of this pathway in post-mitotic cells. This caused a huge increase in the number of cells that migrated. This showed that JAK/STAT was not only required for migration but was also sufficient to tell a cell to migrate. I was third year graduate student and it was a very unique finding. That was the first really big finding of my career.

What are you looking forward to most?

Having our lab really make an impact on the field of stem cells and neural development, cortical development, and all of the members of our having the chance to taste success and make impacts on the field. I would also like to potentially identify things that have an important influence on neurodevelopmental diseases.
 

What would you change about doing science?

I wish the public appreciated the time it takes to do science and the importance of basic science towards finding cures for disease. I wish it was more appreciated.

What lessons do you have or advice for someone pursuing a career in science?

I think find out what you are passionate about and work on it. I think it’s really helpful to find a mentor, someone you can look too as a role model, and be patient because science requires a lot of patience. But if you put in the patience it will reap awards back at you. Any career, science or otherwise, is just that you are passionate about it. Graduate school is a lot of work, be passionate about it. If you are looking for opportunities in labs, look for places that are nurturing and helping you develop as a scientist as opposed to just treating you as a person who is there. You have to be willing to put in the hard work. I think it’s important to always maintain enthusiasm.

Build Your Own Door

Let me introduce myself. My name is Danielle Scarano. I'm an 18 year old rising sophomore at Duke University. I'm currently working in a neural stem cell lab  If you were to ask me who I am there are many different replies I could give. I’m an illustrator of imagination, and a master in the art of sarcasm. I’m a onetime skier, an expert taco maker, and an theoretical physics documentaries addict. I’m an atrocious singer, and an incompetent swimmer. I stand at a mere five foot one, have green eyes, and a terrible fear of spiders. I am a gold medalist in the International Sustainable World Project Olympiad, a two-time All County Artist, a Simons Research Fellow, a Siemens Semifinalist and a finalist in the Hong Kong International Science fair. I could tell you that science has always been my passion, and that I could recite the planets backwards and forwards at age three.  I could tell you many things, but alone these things do little to define me. So come along with me on my journey, as I try to figure out who I am and what I wish to pursue. 

There’s a little quote that I’m quite fond of by William J. H. Boetcker. It goes, “The difficulties and struggles of today are but the price we must pay for the accomplishments and victories of tomorrow.” In order to effectively articulate my expectations for this summer, I think it’s best to tell you a little bit about how I see things. Everyone looks at the world from a different perspective. Everyone has faced troubles, terrors and triumphs. I would say that all the Howard Hughes Research Fellows have something to be proud of. We are at one of the best institutions on the planet, especially when it comes to cutting-edge research, and all of us hit the ground running. Duke is a realm of endless opportunity, and we took it. I think that alone is exceptionally commendable. There are those who see opportunity and are too fearful to go for the gold, and there are those who take it. Life is too short. We must be bold in our pursuits. And even if opportunity doesn’t present itself to us, we shan’t let that hinder our progress. Persistence is something of incredible value. It is what distinguishes those who reach for the stars from those that actually grasp them.

As Milton Berle once stated, “If opportunity doesn't knock, build a door.” This is the philosophy I follow, or at least try my best to. To demonstrate this best I’d like to quote something from my Howard Hughes Application, “In science it is inevitable that we will sometimes fail. As we test our hypotheses and attempt to answer questions we are often wrong. Yet if our drive to find answers is crushed by a mere failed attempt than science could never be successful. If the great minds of the past had given up after their initial hypotheses failed, then science would never have progressed to level it is at today.” That is why inventors, businessmen, and most of all scientists must build doors. They must formulate alternative solutions, deduce answers to questions, and frequently oppose a widely accepted idea. They must enlighten and challenge.

So I say, today’s struggles are tomorrow’s successes. As scientists this summer there will be times that we fail, times that we mess up, when our experiments don’t go as we anticipate. Although I have already worked in several labs, I expect that I will have some failures. Even the best scientists do. Some of the most interesting and revolutionary discoveries have resulted from mishaps. Perhaps then it is wrong to describe these mistakes as “failures.” It is probably more accurate to label them as discoveries.

As Thomas Edison once said, “I will not say I failed 1000 times, I will say that I discovered there are 1000 ways to cause a failure.” This summer I expect to grow as a scientist. I am now at a point in my life where I must decide which direction I want my life to go. Although I have known since I was young that it was in science, it is only through the anticipated practice, persistence, and discovery that my time as Howard Hughes Research Fellow will provide that I will be able to develop a clearer understanding of who I am and what doors I wish to create.

Me working in the lab!

Hopefully my corny motivational speech has inspired you to be bold in your own pursuits.

Sincerely Yours,

Danielle