Hear from some of the many extraordinary graduate students and alumni of the Johns Hopkins Doctoral Program in Cell, Molecular, Developmental Biology, & Biophysics, in their own words.
Alexis Ceasrine, PhD ’18
Alexis Ceasrine, PhD ’18
Walking Down the Islets
At Johns Hopkins, Alexis Ceasrine studied metabolic disorders and how they affect males and females differently
It’s well known that many research studies either don’t report gender in subjects or only use males. But sex differences matter in terms of development, health concerns, and more.
Alexis Ceasrine, Ph.D., who received her doctorate in 2018 from Johns Hopkins’ Cell, Molecular, Developmental Biology, and Biophysics (CMDB) program, evaluated the development of metabolic disorders, such as glucose intolerance and defective insulin secretion: hallmarks of diabetes, and how males and females are susceptible due to developmental differences.
Islets in the stream
While at Hopkins, Ceasrine worked in the lab of Rejji Kuruvilla, Ph.D. Kuruvilla’s lab examines the role of the sympathetic nervous system in development; Ceasrine’s work was an offshoot of that, she says. She studied how pancreatic islets and their associated micro-vasculature co-develop with the sympathetic nervous system.
Glucose levels in the body are primarily maintained by insulin from the pancreas—or more specifically, the islets in the pancreas. Even though islets only make up about 2-10 percent of the entire pancreas, they play an enormous role: Dysfunction of islet cells causes abnormal glucose levels, and can lead to diabetes.
As Ceasrine’s work progressed, she says, she turned to the role of beta-adrenergic signaling, and how that affects the development of islet cells.
In mouse models, when Ceasrine removed beta-2 adrenergic receptors in the pancreas during development, it created hyper-vascularized islets, which disrupted how the body produces insulin. This was due to increased expression of a vascular growth factor in beta-cells.
Surprisingly, this only happened in female mice.
“I would hypothesize it’s a hormone-dependent phenomena,” Ceasrine says. This is due to the necessity of the beta-2 adrenergic receptor only during a critical neonatal window of time. This critical window coincides with a perinatal testosterone surge that only occurs in males.
Islet transplants are a fairly involved way to treat type 1 diabetes that has been resistant to other treatments. During transplant, the majority of islets are lost because they fail to connect to blood vessels in the host, explains Ceasrine. This is one of the reasons that excess islets are often required for a transplant.
“But if we can better understand how beta cells might promote vascular growth, this could influence islet transplantation,” she says.
She adds that this is an example of why it’s so important to pay attention to sex differences in biology research. Males and females have fundamental differences, from development through adulthood.
“This is a field that needs more attention,” Ceasrine urges.
Anyone have antibody?
Initially, Ceasrine says, she didn’t want to go to grad school. She wanted to work in a forensics lab. But a mentor during her time as a biology major at Hofstra University urged her to consider a Ph.D. program—if only to take on more responsibility in a forensics lab once she obtained a doctorate.
“Hopkins was only my second visit and interview to graduate programs,” she says. “I was so impressed with the people and the research being done there. I liked that it was a true umbrella program, and yet a tight-knit community. It’s graduate student-focused. After my weekend here, I canceled all my other interviews.”
As part of the program, Ceasrine underwent five rotations in different labs her first year, with Jocelyne DiRuggiero, Ph.D., studying microbial ecology in extreme environments; a yeast genetics lab with Kyle Cunningham, Ph.D., a zebrafish metabolism lab with Steven Farber, Ph.D.; a cancer cell culture lab with Young-Sam Lee, Ph.D.; and Kuruvilla’s lab.
Ceasrine enjoyed working with Kuruvilla, and thought of her as a key mentor in her time at Hopkins. “I needed guidance because I wasn’t sure what I wanted to do at first, and she was so helpful. Rejji is a huge supporter of women in science. She had my back, no matter what.”
And she also found the program in general to be a collaborative, student-focused environment. “You get to know the other students so well. There’s research going on everywhere. I had no problem [doing things like] asking the lab next door, ‘Anyone have antibody?’”
Now that she’s received her doctorate, Ceasrine has moved to North Carolina to work as a postdoc in Staci Bilbo’s lab at Duke, where she is once again studying sex differences in development, but this time in a neuro-immunology lab, researching microglia and immune cells in the brain and placenta, and how a mother’s high-fat diet may affect fetal development. Ideally, Ceasrine would one day like to be running a lab of her own as a PI.
For students considering the program, Ceasrine suggests maintaining a focus on work-life balance. “There can be a ‘work, work, work’ culture that if you’re not working 80 hours a week, you’re not working hard enough. That will cause burnout. Remember why you are doing science at all and make time for other things you enjoy.”
Also, it’s important to have resilience when it comes to research. Not everything is going to go all right, all the time. “Your papers and grants and conference submissions aren’t going to be accepted on the first go-round every time. It’s OK to feel bummed, but it’s important that you don’t let it keep you down. Remember that every successful scientist has been rejected many times,” she says.
Kiara Eldred, PhD ’19
Kiara Eldred, PhD ’19
Dreaming in Color
Kiara Eldred studies how retinal cells develop
The retina of the eye has two kinds of photoreceptors that allow us to see: rods and cones. While rods work at low levels of light and let us see in the dark, cones allow us to see in the daytime and to perceive color. Cone cells use a photosensitive protein called opsin that allows them to respond to specific colors. We have three different types of cones, each with a different type of opsin: red, green, or blue.
At Johns Hopkins, sixth-year Ph.D. student Kiara Eldred is studying how cone cells develop, and what biological cues are required for them to form. Previous research from mouse and zebrafish studies had showed that a specific hormone, thyroid hormone, plays a large part in determining which cone cells become which color, but Eldred needed to test it on human cells.
Since retinal cells form during fetal development, this wasn’t something easily viewed. Eldred had to figure out how to grow human stem cells into retinal tissue—a creation called an organoid.
That’s when she and her faculty advisor, Bob Johnston, Ph.D., turned to the Co-Director of Johns Hopkins Center for Stem Cells and Ocular Regenerative Medicine (STORM) Donald Zack, M.D., Ph.D, , and his former postdoc, Karl Wahlin.
“I am so thankful for Karl, Don Zack, and the time they spent training me how to grow organoids,” Eldred says. “This is a really awesome thing about Hopkins: People are very collaborative here. I have been struck by how inviting everyone is and how open about their science they are.”
Once Eldred could grow those organoids, she could then test if thyroid hormone was important for making blue versus red or green cones. She found that if she eliminated one of the key receptors for the thyroid hormone in the retina, thyroid hormone receptor beta, only blue cones developed: not red or green. She then added thyroid hormone to the retinas, and saw the opposite; only red and green cones were created.This was an exiting discovery, allowing the lab to understand how those different cell types developed.
But she also wanted to know how thyroid hormone was regulated in the developing retina to produce the right proportions of the three cone types. Her research showed that blue cones are generated first, with red and green cones developing later. When she looked closely at the timing of expression of enzymes that regulate thyroid hormone levels in the retina, her findings indicated that the developing retinal tissue was controlling the levels of thyroid hormone. She saw that early there was low thyroid signaling to specify blue cones, and at later time points there was high thyroid signaling to produce red and green cones.
“What we found is that the retinal tissue itself is able to regulate levels of thyroid hormone. When these cells are born, they are directed to one cone fate or another. At early stages, low levels of hormone produce blue cones. As the retina creates more active hormone, red and green cones are then formed,” she says.
This correlates with several epidemiological studies evaluating vision in premature infants: They have a higher incidence of color vision deficiencies. When a woman is pregnant, her thyroid hormone levels are very high, so when a baby is born early, it’s removed from that high-thyroid signaling environment.
“We’re not suggesting a direct treatment, but instead a mechanism for why these premature infants may have these color defects,” she adds.
The short-term goals of Eldred’s work are to ask very basic questions about how we generate different cells, she explains. The long-term implications can be as far-reaching as the ability to build different parts of the retina for transplant.
The center of the retina houses our high-acuity vision, says Eldred. Only red and green cones are in that area; the blue cones are present as you move toward its periphery. Developing an understanding of how to make different regions of the retina that have different populations of cone cells can lead to the future development of effective retinal replacement tissue.
The more immediate plans stemming from this research are to collaborate with other scientists at Hopkins to implant the retinal cell tissue into animal models, and see if it can be used to integrate and restore sight, Eldred says. This could benefit people who are colorblind, but the more urgent wish is to help people who have lost portions of their sight, especially those affected by macular degeneration.
“The macula is located in the center region of the eye, where the densest amount of cones and the and highest number of red and green cones are located. So we are very interested in being able to make correct proportions to replace these specific regions of the eye,” she says.
At Hopkins, she recently defended her thesis and published her discovery as a first author in Science.
Under the umbrella
When Eldred first applied to graduate schools, she says, she wasn’t sure what she wanted to work on. She knew she wanted to study human biological problems, but wasn’t sure of the specifics. That’s why she appreciated Hopkins’ umbrella program, Cell, Molecular, Developmental Biology and Biophysics (CMDB), which provides access to a lot of different kinds of science.
“As a student in this program, I am focused on retinal development, but exposed to all kinds of questions, different approaches, and techniques in science. This gives me a well-rounded background and knowledge of what is relevant that I can apply to my work,” she explains.
Students in Hopkins’ biology Ph.D. program undergo four two-month rotations, and then choose what laboratory within those choices that they would like to specialize in for the remainder of their time at Hopkins.
Other rotations Eldred completed included studying differences between the left and right sides of the brain using zebrafish with Marnie Halpern, Ph.D.; in neurology working to understand how the pathways of hearing and vision are connected with Hey-Kyoung Lee, Ph.D.; and studying the microbiome and the different bacteria present in the vagina and how they contribute to women’s health with Richard Cone, Ph.D.
As an undergraduate biochemistry major at the University of Washington, Eldred worked in two separate labs: A neurology lab studying how specific brain neurons contributed to learning, and another lab that was part of the National Oceanic and Atmospheric Administration (NOAA) to study when toxic algae bloom and if we can predict it based on weather patterns.
She will be returning to the University of Washington this fall as a postdoc, working with Thomas Ray to pursue more retina-based questions. One of her projects will involve working to build a system within retinal organoids to see if what she’s done at Hopkins can be applied to fighting retinoblastoma.
A long-term goal of hers is to remain in academia; she enjoys both the teaching and research component. “I’m open to that changing, but that is currently the job I’ve seen that has the most things I’d like to do,” she says.
Greg Fuller, Current Student
Greg Fuller, Current Student
What’s up, G-Bodies?
Greg Fuller studies how cells protect themselves in stressful conditions
Normally, our cells get energy from glucose that undergoes glycolysis and then cellular respiration. However, glycolysis is an oxygen-dependent process. What happens when cells don’t have that main energy-producing pathway available? And how do they adapt to these conditions?
At Johns Hopkins, fourth-year Ph.D. student Greg Fuller studies how cells adjust to stressful settings, especially low-oxygen conditions. He is building off former Ph.D. student Ting Han’s work, which identified structures called glycolytic bodies, or G-Bodies, that form in yeast cells in response to low oxygen environments.
“We thought they might do this to decrease the amount of time the metabolites have to diffuse between glycolysis enzymes. Glycolysis is a metabolic pathway, with multiple enzymes acting in a sequence. Each enzyme catalyzes a reaction, and the next enzyme in the reaction uses the product of the previous reaction. So, if you have enzymes close together, it’s more likely the metabolites will come into contact with the next enzyme in the sequence. By concentrating all these enzymes, you can increase the rate of glycolysis, allowing yourself to produce more energy in the absence of oxygen,” Fuller explains.
What bonds us
Fuller’s research has centered on the physical properties of the G-Bodies and understanding what physical mechanisms are required to get them to form. In addition, he’s studying what they’re made of. To do this, he’s been using a number of biochemical and genetic approaches along with live cell imaging, microscopy, and mass spectrometry to identify all the proteins that localize to G-Bodies.
In this process, he’s learned that many proteins in G-Bodies bind to RNA. This, he says, correlates with a currently popular idea in biology: Many different RNAs and proteins can interact. They form a phase-separated droplet within a cell—sort of like how oil and water separate when you mix them. This seems to be a way for cells to rapidly concentrate certain proteins or RNAs to react to a stimulus: Under stressful conditions, RNA and proteins will form what he calls “stress granules” throughout the cell. G-Bodies appear to have many similarities with these structures.
“Ultimately, we want to understand G-Bodies’ function and genetic properties. If you grow yeast cells in both oxygen-rich conditions and in hypoxia, [the latter] will consume glucose at a faster rate. This is consistent with the hypothesis that G-Bodies form to enhance the rate of glycolysis,” he explains.
This could be a mechanism our cells use to survive under low oxygen conditions. For example, research in a hepatocarcinoma-derived cell line shows that structures similar to G-Bodies will form. Some solid tumors will have a gradient of hypoxia, in which there is very little oxygen toward their centers. Fuller explains that cancer cells may be using a mechanism like G-Bodies to survive in hostile conditions—basically hijacking a normal mechanism to keep themselves alive, while making the environment toxic.
“[All that] is something that will have to be explored further,” he cautions. “Right now, we want to characterize these structures in yeast first. Then we can see how they’d affect mammalian cells.”
On the way here
Fuller grew up in Ann Arbor, Michigan. His father was a biologist, and encouraged him to explore science from a young age. He went to Hopkins for his bachelor’s: Initially he wanted to study neuroscience, but as time went by, he became increasingly interested in learning how proteins and RNAs interact at the molecular level. One of his research projects involved studying the progression and disease phenotypes of amyotrophic lateral sclerosis (ALS) in Drosophila.
When he graduated, he took a job in the John Kim lab at Hopkins as a technician. It’s also the lab he works in now. He enjoyed the intellectual and collaborative environment both as a technician and now as a Ph.D. student.
“John gives us a lot of freedom to try new ideas and take different approaches to answer questions. I never felt constrained to do one type of experiment. And everyone in the lab has been very collaborative. For example, we have weekly lab meetings, where one person will present their work to everyone. You get ideas from people who haven’t been hearing about what you’re doing, and that’s hugely helpful. I remember back when I had first started, I had never done a western blot to separate and identify proteins. I was able to shadow others before trying it. Now, I’ve done hundreds of them, and have shown others how to do it,” Fuller says.
Success near and far
Success would mean discovering something and publishing on it and contributing to the larger field. But what Fuller would also like to do is continue on in academia, perhaps with a lab of his own someday—and he has Hopkins to thank for giving him a foundation in his time as both an undergraduate and graduate.
Fuller says, “I think Hopkins is really helpful in that way. It’s a small umbrella program. You’re exposed to successful faculty, and they’re all role models for students. It’s an intellectual environment that’s highly collaborative. You talk to people about what they’re working on and it helps you think critically about what you are doing, as well.”
In addition to his current spot in the Kim lab, Fuller did three other rotations at Hopkins during his first year of the program. He rotated into James Taylor’s lab where he learned about computational tools, Sua Myong’s lab to learn about imaging in biophysics, and in Sarah Woodson’s lab, where he worked with RNA biochemistry and learned new tricks about working with RNA.
“People should keep an open mind, and branch out when you do rotations. You can try something you never thought you’d try. There’s a lot to take advantage of. Make the most of it once you get here,” he adds.
Sarah Hadyniak, Current Student
Sarah Hadyniak, Current Student
Sarah Hadyniak studies how cone cells in the retina develop
Colors matter to fifth-year Johns Hopkins Ph.D. student Sarah Hadyniak. And what especially matters to her is the order in which they develop—and why that’s potentially important to treat health disorders affecting vision.
Humans have trichromatic color vision, which comes from the presence of three types of cone cells in the eye’s retina. These cone cells are responsible for color perception and react to light of blue, green, and red wavelengths.
Hadyniak’s work has focused on the development and patterning of our blue, green, and red cone cells. Up until recently, we haven’t really understood why or how the different cone colors develop and in what order, she explains.
Green and red cones are very similar in everything with the exception of a protein called opsin, which is responsible for absorbing light in the eye. Hadyniak’s project has been to determine which cone cells develop in the retina first, and the factors involved in the generation of those cone cells. She is part of the Bob Johnston lab.
Because retinal development happens in utero, the lab uses retinal organoids, derived from stem cells and grown in the lab, to study this process. And what Hadyniak has found is that Vitamin A-derived retinoic acid is responsible for the generation of red cones by expression of the red opsin protein. During fetal development, blue cones develop first. After that come green cones, and finally, red.
“We are trying to visualize the generation of these cell types and to look at different transcripts and molecules using a variety of techniques,” she says. Those include RNA sequencing to examine the opsin RNA molecules that differ among cone cells, and in-situ hybridization, which allows her to view RNA molecules within the cell.
Understanding how the retina’s cone cells develop can also help and treat various diseases, Hadyniak says.
For example, cone development takes place on the X chromosome, she explains. About 10 percent of men are colorblind, but only about .03 percent of women are. The reasoning behind colorblindness has a lot to do with these DNA sequences on the X chromosome.
But beyond colorblindness, her work has the potential to help with macular degeneration: the loss of cone cells as we age. Others in the Johnston lab are collaborating to find ways to transplant retinal stem cells into animal model systems, with a future goal of using them to fight blindness caused by this and other conditions.
In the rotation
All students in the Ph.D. program undergo four two-month rotations during their first year. Hadyniak’s rotations included examining sex determination in the drosophila gonad in Mark Van Doren, Ph.D.’s lab; molecular cloning in zebrafish with Marnie Halpern at the Carnegie Institution for Science – Department of Embryology; and Xin Chen’s lab on tracking asymmetric histone segregation in C. elegans.
Johnston’s lab, Hadyniak says, has an environment that’s fun and full of energy. “Bob loves what he does, and he loves to talk and think about science with everyone. I would pick this lab all over again,” she says.
As a biology major at Boston University, she knew she wanted a Ph.D. When she began researching developmental biology programs, she liked that Hopkins offered a strong umbrella program and emphasized broad knowledge.
For example, all incoming Ph.D. students in it must complete a “Computational Boot Camp,” which is a one-week class to teach students coding languages such as Python and new ways to view and analyze data. “I had never done it before, but it’s given me the fundamentals to use what I’ve learned in more depth,” she says.
Hadyniak enjoys a virtual trade route of information throughout Hopkins. She also gets to work with the James Taylor lab at Hopkins for assistance with some of the RNA data she collects. She collaborates closely with Hopkins’ medical school for tissue samples and organoids. Also she has a solid relationship with the Neitz Lab at the University of Washington, which focuses on genetic tests and treatments for vision disorders; they have graciously provided DNA samples and red and green cone cell ratios to help further her work.
“People at Hopkins are just really friendly and really collaborative. Doors are open, literally and figuratively. People make themselves available to talk to you. And I like the feel of Baltimore, I love the community vibe, I feel like people care a lot about this city,” she adds.
She advises potential students considering any graduate biology program to find a balance, no matter where you go. While it’s crucial to find a lab and a mentor who will support your work, you’ll also need to seek out life beyond the lab.
“Find hobbies and things outside of school and science to keep you going,” she urges. “You will have highs where you’ll want to be in the lab all the time, and then you will have some lows, and you will want a support system, whether it’s friends, family, or hobbies.”
Hadyniak is currently writing her thesis, which will center on the cone receptor patterning. Ideally, she’d like to find a postdoc program once she receives her degree, and stay in academia while doing research. She also has what she describes as a side project, which is trying to map the human retina.
“No one has been able to visualize these cell types before in fixed tissue,” she explains.
Hopkins supports its students in many ways, she says. They send students to conferences, bring in speakers, and discuss other related research in the field. Also, every student must provide regular 30-minute talks about their work within the department, which provides the opportunity to improve one’s scientific presentation skills.
“The whole community is driven to publish and be around other labs doing the same thing. But it helps you in producing quality work,” she says.
Jocelyn Haversat, Current Student
Jocelyn Haversat, Current Student
Breaking Up is Hard to Do
Jocelyn Haversat studies how sperm and egg are made, and what regulates the process of their creation
The microscopic nematode C. elegans is about a millimeter at its lengthiest. It’s generally a self-fertilizing hermaphrodite (XX), with an occasional male mutation (XO). C. elegans also shares many biological characteristics with mammals, including humans.
During meiosis, a single cell splits twice, producing four cells. Each contains half the amount of genetic information. These are the sex cells, also known as gametes. The chromosomal segregation that takes place during this time ensures the correct inheritance of genomes.
However, there are times when this process goes awry: When DNA breaks and isn’t repaired correctly, this can lead to developmental disabilities.
At Johns Hopkins, fourth-year Ph.D. student Jocelyn Haversat investigates a process called crossovers, which are required for viable eggs and sperm after meiotic division.
When chromosomes divide to become sperm and egg, they physically change chromosome pieces, or arms of DNA, says Haversat. That provides genetic diversity; for example, it’s one of the many reasons why siblings aren’t identical.
The physical exchange of those DNA pieces holds the chromosomes together. It provides tension. That tension is required during chromosomal segregation. Without it, chromosomes won’t separate properly; the cells would have the wrong number of chromosomes, called aneuploidy. Aneuploidy leads to conditions such as Down syndrome or Klinefelter syndrome. Haversat and her colleagues at the Yumi Kim lab have identified a type of protein called a kinase that sits at these sites of crossover. It appears to phosphorylate target proteins, enabling successful crossover formation. Without it, crossovers will not form, and no progeny will be viable.
During meiosis, when breaks form, they need to be repaired correctly, otherwise any abnormalities will propagate. “Where the DNA breaks and repairs with a new piece of DNA has to be really tightly controlled,” she explains. “Any abnormal repair could cause the cell to lose genes or be repaired in the wrong orientation.”
This process in C. elegans also occurs in mammals such as mice and humans. Understanding how this kinase functions could potentially lead to the development of therapeutics or improvements in screening for conditions caused by aneuploidy, Haversat adds.
Traveling and teaching
Haversat is quick to admit she doesn’t have an entirely traditional biology background. She majored in animal science at the University of Massachusetts Amherst, where her research projects included epigenetic reprogramming during pre-implantation development and cellular communication in vitro oocyte development, as well as environmental toxicology. She attended Tufts Veterinary School, and worked in a veterinary hospital preparing and analyzing patient cytology samples.
“Then I decided I didn’t want to be a vet. I wanted to work in a lab, and teach,” she says. “But first, I took a year off to hike the Appalachian Trail, and then go to Alaska.”
But when she visited Hopkins, she knew she wanted to study here within 24 hours of setting foot in Baltimore, she says. She felt that it was environment that thrived on collaboration, with labs throughout the program willing to help each other out.
“I wanted to be happy for the next five years of my life. I liked that it was a smaller umbrella department. Students seemed happy, and people were supportive. It’s a wonderful community,” she adds.
Ultimately, Haversat says, she would like to become a biology professor and teach undergraduates. Hopkins has helped in that regard, offering resources on teaching at the college level, using evidence-based teaching practices. For another project during her third year in her Ph.D. program, she got to develop and pilot a genetics lab where undergraduates are taught CRISPR and they get to use it in worms. She’s also applying to the Dean’s teaching fellowship.
“I love seeing a light bulb go off, and that ‘Aha!’ moment. I love human interactions, and propagating human kindness to students who really need it. People say, ‘Oh, if you can’t do science, you teach,’ and I don’t like that. Very good scientists should be teaching,” she says.
In the lab
Dr. Kim is extremely accessible, Haversat says. Her office door is always open. She’s supportive of Haversat’s teaching interests, and an amazing biochemist. And the way she asks and answers questions, Haversat says, is like no one else in the field.
“For example, with my project, it would be easy to just find the role of the protein, knock it out, and then analyze the phenotype. Other worm labs would do that. But Yumi has taken it a step further. She said, ‘Let’s express this in vitro at the bench, and show mechanistically what this protein is capable of doing.’ We are looking at genetics, biochemistry, and cytology and fusing together a bunch of different fields to develop a compelling and convincing story,” Haversat says.
Haversat remains excited about what she’s doing and enthusiastic about her prospects, like continuing to work with students. And she really likes Baltimore and how it supports her joint interests in science and art. “Baltimore is an authentic city; it fosters an amazing environment for community engagement. There is so much character, and people have so much love for the city for a lot of really great reasons.”
Work-life balance is very important to her: In addition to hiking and travel, Haversat plays in a bluegrass band and performs in a puppet troupe.
Advice from the teacher
Haversat urges prospective students to be true to themselves when evaluating what they’re looking for. She’s happy, she says, because she’s found a place that enables her to have the opportunity for research, as well as a life outside the lab.
And she’s also got advice for current students to help them get the most out of their time here. “Before coming here, I had never taken a biophysics class, and then I needed to. My program got me a tutor, for free. Think about what you need and if you are getting it. If you aren’t getting it, do something about it. Talk to the people you need to talk to—your boss, your department, whoever. There are so many resources here.”
Aurelia Mapps, Current Student
Aurelia Mapps, Current Student
Aurelia Mapps studies satellite glia, the “babysitters” of our sympathetic nervous system
Most of us know the nervous system as the electric wiring of the body. Its cellular unit, neurons, transmit chemical signals to other neurons. But neurons also have support cells, called glial cells, or glia, from the Greek word for “glue,” suggesting they only have a role for structural support in the nervous system. However, neuroscientists have unveiled some ways that glia create a healthy neuronal environment.
At Johns Hopkins, fifth-year Ph.D. student Aurelia Mapps studies a specific type of glial cell called satellite glial cells and how they contribute to the sympathetic nervous system, which mediates your “fight or flight” or stress responses. Satellite glia wrap around the cell bodies of neurons, essentially hugging them.
Overall, glia cells are like the “babysitters” of the nervous system, Mapps explains. They monitor the environment around neurons, making sure they’re healthy, safe from harm such as environmental factors, and in contact with the other neurons they should be.
“These glia in other systems [such as mouse models] have been shown to exacerbate neurodegenerative conditions such as Alzheimer’s disease—issues we thought were specific only to neurons,” Mapps says.
When you get scared or nervous, your sympathetic nervous system comes into play: Activated by the hypothalamus, it uses nerve pathways to start bodily reactions. Your heart rate and blood pressure increase. Your pupils dilate. Your lungs contract to take in more oxygen.
But when sympathetic neurons are overactive, this can over-stimulate your organs, like your heart. This might lead to hypertension, heart disease, or possibly even death, Mapps says.
And it’s possible that hyper-functionality could have something to do with glia. “If you remove satellite glial cells, neurons no longer have the ability to tune when they should signal or not, so they stay on. For example, your heart rate would stay too high. There’s still too much input from the sympathetic nervous system,” she adds.
More than we need
While earning her undergraduate degree in biomedical science at Huntsville, Texas’ Sam Houston State University, she participated in Research Experience for Undergraduates (REU) program here at Johns Hopkins University, where she worked in Xin Chen’s lab examining the role of a repressing gene, E (z), in fruit fly sperm production using immunohistochemistry to visualize phenotypic differences.
It was her first experience with molecular biology, and Mapps wanted to return to that kind of environment. She liked that the CMDB program was an umbrella program, where she’d sample several different labs and techniques.
During her first year at Hopkins, she rotated through Marnie Halpern, Ph.D.’s lab studying genes that control left-brain/right-brain asymmetry in zebrafish, Mark Van Doren, Ph.D’s lab, researching the sex-specific development of gonads of fruit flies, and in John Kim, Ph.D.’s lab, examining the micro-RNAs in the germline of C. elegans.
Outside of studying neuron-glia interactions, Rejji Kuruvilla, Ph.D.’s lab also studies how growth factors are trafficked in neurons in order to develop and thrive. The lab also examines what neurons might do outside of signaling for physiological responses.
“If you remove sympathetic neurons in early development, your pancreas, for example, doesn’t develop correctly. This could lead to a lot of physiological effects, such as your inability to use insulin correctly,” Mapps explains.
Mapps says that Kuruvilla is a thoughtful, enthusiastic leader. “She is extremely excited about science at all times. If you ever have data to discuss, she’s more than happy to talk about it. You can talk to her about plans and it helps you stay motivated to continue,” she adds.
“There are a lot of different techniques among all these projects,” Mapps says. “So when we go to a lab meeting, you have to go with an open mind, because you’ll never know what you’ll hear.”
It is easy to establish collaborations between labs in CMDB, she says. Several years ago, Mapps recalls, she was trying to sequence satellite glial cells and she heard a researcher in a neighboring neuroscience lab was using a droplet-based sequencing to capture individual cells and sequence the transcript of that cell.
Ultimately, Mapps would like to be a principal investigator (PI) in her own lab someday. She finds beauty in teaching and being able to talk to different audiences, from specialist to layperson. The ability to communicate successfully is something that’s always important and helps you develop as a scientist, she says.
“At Hopkins, I’ve learned to collaborate [and communicate] with different people as well as think outside the box. Because CMDB is an umbrella program, you’re going to get different input for your projects. It will expand how you think about [science],” Mapps says.
Ikenna Okafor, Current Student
Ikenna Okafor, Current Student
Cut and Paste
Ikenna Okafor studies the underlying biophysics of CRISPR-Cas9
Say you’re an orchestra conductor. And the musicians are playing a piece. But it sounds…not quite right. You can’t hear it when they all play together; it’s only once you isolate and observe each of them, individually, that you can figure out what’s wrong.
At Johns Hopkins, third-year Ph.D. student Ikenna Okafor uses methods like that to study tools used for gene editing.
Okafor works in a single-molecule biophysics lab, studying gene editing technology, primarily Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9. Cas9 is a part of a larger network of CRISPR proteins and RNAs. When used with its guide RNA, it essentially becomes a programmable DNA cutting and binding protein. This is done by “reprogramming” the guide RNA to match locations in the genome researchers want to target.
Seeing is believing
Okafor’s work also focuses on how individual molecules behave within complex biological systems. He designs different assays to measure CRISPR-Cas9’s accuracy in different scenarios, and how changes to the protein and RNA affect its ability to assemble a complex or target different places in the genome.
He does this using high-powered optics, such as multicolor fluorescence, super-resolution imaging, combined force and fluorescence spectroscopy, and single-molecule pull-down that provide nanometer resolution and millisecond temporal resolution. This is beneficial because most things in the cell happen at this spatial and temporal scale, which gives researchers the ability to observe biochemical reactions in real-time.
“We are interested in studying DNA- and RNA-binding proteins because they have many roles in controlling gene expression,” says Okafor. “These proteins are implicated in many diseases, including cancers. By understanding how they behave individually, we can find spots on the protein that may be amenable for drug targeting and protein engineering.”
The CRISPR Cas-9 protein is a workhorse in terms of gene editing: It can correct any disease that arises from gene mutations, Okafor explains. For example, it can be used to fix mutations that cause hereditary forms of blindness and deafness. Mutations in the cell can also contribute to cancers: If a cell acquires too many mutations to certain genes, it can become malignant.
Outside of health, CRISPR also has implications in agriculture and food production—such as genetically editing a type of tomato to become more cold-adaptive. But that’s a long, long way away, Okafor says.
“CRISPR and Cas-9 are normally used in research to study gene function, and how this turns on and off genes. You can use it to turn off a particular gene, and see what happens,” he says. “You study the function by breaking it.”
Okafor is quick to point out that his background is not in biochemistry or biophysics. He received his undergraduate degree in neuroscience from George Mason University, because he wanted to do neuroscience research.
But after working in that area for a while, he learned from his mentors that most neuroscientists didn’t have a neuroscience bachelor’s degree. Okafor decided to broaden his background, so he ended up getting a master’s degree in biology from University of Maryland Baltimore County (UMBC), which provided a more comprehensive knowledge of developmental biology.
Growing up in Maryland, Okafor knew of Hopkins’ reputation: “Hopkins was just the best,” he says.
He came here because he liked the umbrella program aspect of CMDB, and wanted to do a rotation in each of the four specialties: cell biology, molecular, developmental, and biophysics. Even with obvious overlap between the specialties, he found this approach useful.
“I tried to explore everything and figure out what excited me the most, and what environment I felt I could thrive in. And I knew cutting-edge technology and gene editing was something I wanted to know more about, and studying it in such great detail was really interesting. I can also collaborate and reach out to anyone I had a rotation with, which I did and have been doing,” he says.
The Taekjip Ha lab, where Okafor currently works, uses sophisticated biophysical manipulation techniques with fluorescence imaging to visualize and manipulate protein, RNA, and DNA molecules. Okafor chose this lab because he felt a strong connection with Ha, as well as the other trainees in the team, the work, and the overall environment, he says.
“TJ is really smart, really excited about the science. When we show him an interesting piece of data, he’s like a little kid. And he lets you do your thing, and encourages you to try out new ideas,” he explains.
Within the lab, collaboration happens often. For example, one of the postdocs there is a chemist who is interested in protein engineering and is working on a light-activated type of protein to open up DNA; when the light gets turned on, so does the protein, and when it’s turned off, the protein does the same. To test it, the two use an “optical trap,” which traps a single piece of DNA between two beams. When you pull on the piece, the DNA acts like a spring, and they measure the force it exerts and watch DNA-binding proteins interact with the DNA. Then they discuss the results together.
Okafor says, “I love my lab. It’s like the UN: people from all over the world. Each person in the lab had a team to root for in the World Cup. It’s diverse in specialty, too: physicists, biologists, chemists, all in one group.”
When he’s through the program, Okafor sees himself on the non-academic path. He’s interested in potential roles in research and development or management at gene editing companies. “I like to make decisions and lead teams,” he explains.
He does love mentoring undergrads, too, however. He adds, “You get to see them grow, and it’s easy to see yourself grow along with them. They’re exciting. They’re passionate. They show up and they’re ready to go, and want to share the knowledge.”
He would advise anyone considering the program to be open-minded about their research and research in general. Keep an open mind. Try something new. “Reach out to people you may not think you’re interested in; they can frame science in a way you hadn’t thought about before,” he says.
Diego Rivera Gelsinger, Current Student
Diego Rivera Gelsinger, Current Student
Diego Rivera Gelsinger studies how life can live on little water
Chile’s Atacama Desert is one of the driest places on earth. It averages 15 millimeters—about half an inch—of rain a year. Some weather stations throughout the desert have never recorded rain. At 13,000 feet with cloudless skies, it also has the highest level of surface ultraviolet (UV) radiation in the world.
It is, as sixth-year Ph.D. Johns Hopkins student Diego Rivera Gelsinger describes, “The endpoint of water availability.”
So, what forms of life dwell there, and why are they important?
Water is life
At Hopkins, Gelsinger studies archaea, single-celled microorganisms that can survive in hostile conditions. In areas with weather generally ill-suited to supporting life, such as the Atacama, the only forms that make it are microbial life forms that reside inside various types of rocks.
These rocks, Gelsinger says, serve as little refuges in a big desert: Because of their pores and cracks, archaea live off of the tiny bit of the humidity in the atmosphere that gets trapped inside the rocks. The archaea found in these rocks are adapted to high-salt conditions and resistant to radiation and its resultant oxidative stress.
His work has been focusing on what these types of archaea do to regulate their oxidative stress and survive. And what he’s learned is that small, non-coding RNAs (also known as sRNAs) play a large role. Specifically, a particular sRNA named SHoxi (Small RNA in Haloferax Oxidative Stress), which regulates how much damage occurs inside the archaea, If SHOxi is removed, Gelsinger explained, the cell only has a 20 percent chance of survival.
“This is only one of the sRNAs that can be [activated] in this stress condition. This, along with other sRNAs and other non-coding RNAs, are important in other organisms as well—like our own cells. Non-coding RNAs could potentially help them become more resistant toward extreme environments like archaea. Oxidative stress is implicated in aging and cancer, so it’s important to figure out how to deal with it,” Gelsinger says.
Around the world and beyond it
This knowledge also has implications beyond our bodies and the world at large. Climate change on earth may mean we need to find ways to adapt to harsher conditions. And as we seek life—or hospitability to life—on other planets, we may need to rethink what water means for life.
Not only has Hollywood tapped the Atacama to resemble other planets, but NASA also calls upon it as well. NASA rover teams go there because it closely compares to how dry it gets on Mars. And a recent NASA rover expedition on Mars discovered hydrated salts in rocks, similar to what Gelsinger studies.
He’s been to the Atacama twice, he says, both with NASA expeditions. He says it’s the quietest place he’s ever been. “You’re eight hours from the nearest city. There’s no hum of traffic anywhere. What you do hear is the earth moving, rocks cracking. It’s truly beautiful. Clear skies. No light pollution. You can see galaxies with the naked eye,” he explains.
Life at the extremes
Gelsinger also worked with archaea as a microbiology major at San Francisco State University, where he studied the physiology and ecology of thermophilic archaea in geothermal hot springs.
He claims he’s always been drawn to life at the extremes. “When you study biology, they teach you that all of life has these same building blocks, yet you have organisms that survive in crazy conditions that we have never evolved to do. It’s a little sci-fi,” he says.
He learned about the CMDB program at Hopkins while part of a National Institutes of Health (NIH) fellowship as an undergraduate, but wasn’t interested at first, he says. He thought the program was very biomedical, which didn’t capture him. But after discussing it with his college mentors, he began to see the advantages of a rotation program. And when he visited Hopkins, he appreciated that students were the primary focus in the lab.
At Hopkins, his rotations included a yeast aging lab with David Zappulla, Ph.D., studying telomerase non-coding RNA; a peripheral nervous system lab with Rejji Kuruvilla, Ph.D., examining how brain neurons communicate with the body’s organs; a computational lab emphasizing human evolution of regulatory regions in the incorrectly named “junk DNA” with James Taylor, Ph.D.; and in Jocelyne DiRiggiero, Ph.D.’s lab, where he is currently working.
“The rotation program made me realize how much I loved all aspects of biology, and I joined Jocelyne’s lab because it was so interdisciplinary. It’s sequencing-based, which involves computational analysis, but it also allowed me to work in a controlled laboratory setting as well as in the field,” Gelsinger says. “Jocelyne is a great scientist—but also very compassionate and caring. She’s been very supportive of me networking.”
His experience in the lab has been positive as well, he says. There are so many resources. And everyone’s been interested in exploring and testing out ideas and bouncing them off each other.
This happens outside the lab, too: An avid brewer in his spare time, Gelsinger and his team in the lab finally made “Atacama Ale,” a Belgian-style creation, which is Dr. DiRuggiero’s favorite style.
Gelsinger will be defending his thesis in December. After that, he’s interested in doing a postdoc with a goal of running his own lab someday. Once he’s through here, he’d like to expand his horizons and move away from archaea and push further into the world of non-coding RNA. One option he’s considering is researching the gut microbiome, and how it affects the brain.
He’d advise students who are interested in the program that it’s a challenge—especially at the start—but worth it.
Gelsinger says, “Keep your head up the first year. It’s tough, because you’re balancing classes and research…and you can feel like you’re not good enough. We are all smart and creative here, and persistence is the biggest part of doing well in a Ph.D. program.”
James “Jay” Thierer, Current Student
James “Jay” Thierer, Current Student
Light Up Our Lives
Jay Thierer makes bad cholesterol glow
Say you eat a cheeseburger. The fat in that burger gets absorbed by your intestine, which makes particles called lipoproteins. They carry the fat out of the intestine and through the bloodstream and over to areas including the heart, brain, or muscles, where it can be used as energy.
But while lipoproteins—such as low-density lipoprotein (LDL) otherwise known as “bad” cholesterol—are circulating around the bloodstream, they can get trapped in the walls of blood vessels. Over time, these particles can build up and cause cardiovascular disease, such as heart attacks and strokes.
Although several different classes of lipoproteins exist, the types carrying Apolipoprotein-B are particularly likely to cause these kinds of health conditions. That’s what Ph.D. candidate James “Jay” Thierer studies at Johns Hopkins. He sees cholesterol blockages clearly: through translucent zebrafish.
Finding the spark
Until zebrafish are about a week old, their bodies are translucent: Researchers can see right through them, including to their eggs.
“Where some people see caviar, I see 10,000 experiments,” says Thierer.
To track cholesterol in zebrafish, Thierer and his colleagues use NanoLuc, a protein very similar to the Luciferase protein that makes fireflies light up bright yellow at night. They have genetically engineered the zebrafish so that every time the fish make bad cholesterol, they also make this glowing protein.
These light-up zebrafish get used for numerous experiments: First, to find new drugs that can lower cholesterol and prevent heart disease. And second, for genetics research: To understand the gene that makes bad cholesterol go up and down, and examine why some families have high cholesterol and why some don’t.
How it’s done
Since zebrafish aren’t exactly swallowing pills, how do Hopkins researchers assess a drug’s effectiveness on them? Thierer collaborates with Jeffrey Mumm, Ph.D at Johns Hopkins Medicine, who has an automated robotics platform called ARQiv-HTS that will take fish larvae and place it in a well with a particular drug. After two days, it will measure how much the fish is glowing. If the fish glow less, this is a drug that can potentially lower cholesterol levels.
The team is currently working through a library of existing drugs, known as the Johns Hopkins Discovery Library (JHDL), which is a grouping of about 3,000 drugs that have been cleared for use in humans. In about three months they will complete that group and move onto their next set: a 30,000-compound library.
“It’s not completely random,” Thierer says. “The drugs are biased toward somewhat smaller, relatively uncharged molecules. Every week we screen about 100 drugs, and every week we get about one that makes the fish glow less.”
Humans and zebrafish have a surprisingly similar genetic structure, and the majority of genes associated with human disease have a zebrafish counterpart. In zebrafish, the gene “pla2g12b” has been identified to lower cholesterol levels significantly. Researchers can then study humans who have a mutation on this gene and see if it also lowers or raises cholesterol.
Cardiovascular disease is the leading cause of death worldwide. And although we do have medication to address it, we need new and better ways of helping people, says Thierer. “My project is unique in how directly it relates to human diseases…using zebrafish opens up a lot of opportunities to understand why heart disease runs in families and how to lower bad cholesterol.”
Love at first sight
Thierer fondly remembers the first time he ever peered into zebrafish under a microscope. “I was so blown away,” he says. “I could watch every part of their bodies, their hearts beating, and the blood moving around. It’s a powerful system where you can physically see what’s going on.”
As a biology major at the University of Maryland, College Park, Thierer’s diverse research projects ranged from the ethylene hormone signaling pathway in algae to cholera epidemiology. He liked research because although there were absolute properties of science, you could still question and challenge through hypotheses. You could make discoveries, but remain quantitative.
His rotations in Hopkins’ CMDB program included a yeast lab with Kyle Cunningham, Ph.D.; an in vitro RNA lab with Sarah Woodson, Ph.D.; and a neurobiology lab with Marnie Halpern, Ph.D. “I think this is a good way to ensure people make an educated decision, and it allows you time to learn lots of different techniques,” he adds. After his rotations he decided to join Steven Farber, Ph.D.’s lab at the Carnegie Institute because it combined his dual interests in zebrafish and cardiovascular disease.
There was a lot that could have gone wrong with the work, he says. For example, the glowing protein could have blocked cholesterol instead of mirroring it. But it didn’t, and they’re testing drugs constantly to make progress in cardiovascular disease treatment. And while he is thrilled with the work that’s been coming out of the lab, he emphasizes that in no way could he have done it alone.
“This is not just my work. A huge number of people have been involved. It has ballooned to encompass many professors, graduate students, and postdocs. It’s a growing team,” he says.
Thierer has submitted his thesis and will receive his diploma in December. He’ll still be working at Carnegie for the next several months on this project. After that, he may continue working with zebrafish as a postdoc, or get involved with an independent research position.
Regardless, he’d advise potential students considering Hopkins to find something they’re passionate about: If you truly care about your work, he says, everything else will fall into place. Also that Hopkins’ rotations serve as a wonderful way to try out new research experiences and not limit yourself. And finally, don’t fear “imposter syndrome,” because it’s a lot more common than you think.
“I think if you care about something and work hard, nothing is going to keep you from succeeding,” Thierer says.
Annie Vemu, Current Student
Annie Vemu, Current Student
A Structured Environment
Annie Vemu studies microtubules, the skeleton of the cell
Microtubules provide the fundamental structure of a cell. They’re small, cylindrical structures that contribute to its shape. Microtubules also work like mini-highways within a cell, transporting cargo from one place to another so cells can survive and adapt to an ever-changing environment.
At Johns Hopkins, fifth-year Ph.D. student Annie Vemu looks at how microtubules get their shape, what happens when this skeleton of the cell gets pulled apart, and how it can be changed. She’s part of the Hopkins-National Institutes of Health (NIH) GPP program: Although she attends classes at Hopkins, all her research is done at the NIH.
The shape of things
All cells have different shapes. For example, a neuronal cell is long, which supports transport of information. And although all cells have microtubules, they’re not all the same. They aren’t static structures that sit around during the lifetime of a cell.
Because the cell is experiencing different environmental cues throughout its life, its microtubules are constantly growing and shrinking.
A microtubule’s basic building blocks are called alpha- and beta-tubulin heterodimers, which interact with each other to form dynamic polymers. Upon closer inspection, Vemu says, microtubules are actually quite heterogeneous. There are different tubulin isoforms that are decorated with diverse post-translational modifications. And in certain cells, such as cancer cells, when certain types of tubulin become over-expressed, the dynamics of the microtubules change, and they become resistant to chemotherapeutic agents.
Vemu says that although tubulin was discovered about 50 years ago, researchers are still trying to understand how a single tubulin isoform or single post-translational modification affects microtubule dynamics and microtubule associated protein recruitment in the cell. Research has involved developing a protocol to isolate and purify single tubulin isoforms to understand how just a single tubulin isoform could change the microtubule dynamics of cells and the whole body ecosystem.
Vemu’s Ph.D. thesis, she says, was inspired by her time evaluating microtubule severing enzymes: spastin and katanin. They are essential for the maintenance and organization of microtubule arrays found in mitotic and meiotic spindles and neurons; if their activity is disrupted, she explain, it can lead to neurodegenerative conditions such as hereditary spastic paraplegia and microcephaly.
“When you remove those enzymes, you get disregulation of organelle trafficking as well as decreased neurotransmitter release,” Vemu says.
When the enzymes break the microtubule, they pull out tubulin dimers within the microtubule; this creates potholes within the microtubule lattice, Vemu explains. But within the cell, there is so much free tubulin floating around, the potholes could be plugged quickly by the tubulin that’s in reach. Also, this new tubulin that plugs up the potholes prevents the microtubule from falling apart and facilitates the growth of new microtubules.
Also, location matters. “The prevailing thinking until recently was that tubulin addition and removal happened only at the tips of the microtubules. But what we have found is that tubulin addition occurs all along the microtubule, because of the severing enzymes’ activity on the microtubule lattice. One advantage this could provide is that instead of completely destroying the microtubule array, you could take out old tubulin and put in new tubulin to rejuvenate the microtubule lattice. This would be much less energetically costly,” she explains.
The long-term implications of this work are meaningful for neurodevelopment, Vemu says. The activity of these enzymes can help explain how cells like neurons develop. And now that we understand that this enzymatic action occurs throughout the microtubule, we can shift our focus to all over, not just the tips.
Because, Vemu says, sometimes cells need to make a lot of microtubules very fast. “Let’s say there is a neuronal injury, and the cell body or microtubule organizing center is far away from the end of an axon. To make the microtubules, you will have to make the cell body, make the microtubule and then transport it all the way to the axon. But if you have severing enzymes, you can make microtubules quickly and not have to wait for a signal from the cell body,” she says.
Background and next steps
Vemu works in Antonina Roll-Mecak, Ph.D.’s lab at the NIH. Prior to coming to Baltimore, Vemu was a biochemistry/chemistry major at the University of California San Diego, where she also received a master’s degree in chemistry.
“I really enjoyed getting to the mechanistic perspective and the basics of things,” she explains. “And then when I went to Hopkins I got the opportunity to study things like genetics and development. That gave me a different perspective on science, which I never would have gotten if I were in the bubble of doing only biochem.”
Although she has spent a lot of time looking at the mechanical side of things, she’d like to take on another layer once she’s through with the program. Perhaps something less basic and more translational, she says. Maybe integrating neurobiology. Regardless, she’s happy to do something she’s enjoying and encourages prospective students to do the same.
“Working on something you really enjoy takes 90 percent of the struggle out of it. When experiments don’t work—and they won’t sometimes, that’s just how it is—when you are doing something you are curious about, you will be resourceful enough to figure out a way. That helped me get over the bad experiment days. Even if they don’t work, they still tell you something,” she adds.
Gabby Vidaurre, Current Student
Gabby Vidaurre, Current Student
Everything Has a Place: But Why?
Gabby Vidaurre examines one region of the genome and how it’s regulated and replicated
When a stem cell divides, it creates two cells. One will remain a stem cell, and the other will differentiate into a cell type that particular stem cell is used for.
This is particularly interesting in Drosophila, the common fruit fly, because both male and female Drosophila have stem cell populations that create both ovarioles, which produce eggs, and testes, which produce sperm.
At Johns Hopkins, third-year Ph.D. student Gabby Vidaurre studies germline stem cells in male Drosophila—and what they might mean for human genetics.
Location, location, location
To understand stem cells, it’s also important to understand histones, proteins that wrap around double helix DNA and give it support. In male Drosophilia germline stem cells, when a stem cell divides to create a new stem cell and a differentiated cell, the stem cell keeps the old histone, and the differentiated cell inherits the new histone.
Histones help regulate how accessible DNA is: Certain modifications on histones could cause a histone to hold onto to its DNA more tightly or loosely, Vidaurre explains. The looser the DNA, the easier it is to access for transcription.
Vidaurre’s work focuses on the location of histone proteins on DNA. “Flies, mammals, and most species have a histone locus, a specific region of their DNA where histone proteins and their genes are located. The histone locus is where histone protein is transcribed, but is translated outside of the cell and then brought back in. Proteins at the histone locus are involved in replication, transcription, and pre-messenger RNA processing. All RNA undergoes processing before it is translated to protein, and histone messenger RNA is unique since it has special processing proteins. This is a special region because genes don’t normally locate in one region, all together. They are usually more dispersed,” she says.
Another thing she’s noticed is that the histone locus is very large in germ cells, in that there are many more proteins in this area that support the process of transcribing RNA into DNA. “Replication proteins, transcription proteins, and mRNA processing proteins are super-enriched [in this area],” she explains. “There are a lot of those proteins there, much more than in other cell types at the same region of DNA.”
When DNA is synthesized, Vidaurre says, the cell has to make all new histones as well. This is convenient because all the histone genes are in one area, so this is where DNA replication in stem cells may begin.
Keeping it under control
How tightly proteins wrap around DNA can be important. There are some areas that we don’t want RNA transcription to occur; for example, in genes that are potentially only used during development. This would be an area where the proteins should wrap tightly around the DNA, keeping it away from rogue transcription. But there are other situations in which copies need to be easily made, such as what Vidaurre describes as “housekeeping genes,” such as cell cytoskeleton genes, which are always needed. Since they keep cells going, a looser fit works.
Studying the germline is important, she says, because it’s where proper cell development comes from. If cell growth starts out problematic, that’s going to lead to a host of developmental difficulties and beyond. “If you don’t have proper replication regulation, you’ll get [a situation like] an over-proliferation of stem cells, which can lead to cancer,” she says.
Vidaurre splits her time between two labs: Sua Myong, Ph.D’s lab, studying single molecule techniques in vitro—e.g., studying how a single protein interacts with RNA or DNA; and Xin Chen, Ph.D’s lab, focusing on stem cells in Drosophila. She enjoys working with Drosophila because not only is it a model organism, she says, but they’re relatively simple to modify for genetic study.
“Drosophila are easy to image, and easy to work with. And what’s cool is that there are regions where it’s straightforward to genetically manipulate them to modify their genome,” she adds.
She gets a lot out of both labs, she says, though each Principal Investigator (PI) has their own style. “Xin is super-intelligent and very nice and easy to talk to, and when you need help from her, she’s definitely available. And Sua is a hands-on mentor who likes to have us present what we’ve done and discuss next steps, weekly.”
Before coming to Hopkins, Vidaurre was a biology major and chemistry minor at the College of William and Mary, in Williamsburg, Virginia. Although she was always into science, she says, even from a young age, it was her undergraduate research experiences—especially with Drosophila—that got her excited about following that path after graduation. For two summers, she researched male and female Drosophila reproductive systems, and found she liked working with genetics and flies, and this fit with her interests in gene expression and DNA/RNA transcription.
While looking at graduate schools, she wanted to stay in the general area, and applied to Hopkins as well as other schools relatively nearby. But of all her interviews, she felt best at Hopkins, she says.
“I really liked the people. I felt they were very genuine and easy to get along with. And I liked the research: Hopkins has lots of different kinds of labs and research interests, but at the same time, it’s a small community. Everyone knows about each other and about each others’ work,” she explains.
Vidaurre advises prospective students to not only research potential programs, but also immerse themselves in research itself. And be prepared for the ups and downs of trying to discover something. “It’s not going to be easy, and what you’re working with might not pan out completely. You may have to switch your focus. Things may not work out immediately how you want them to,” she says.
She’s still deciding about what she wants to do once she finishes the program—other than being a first author on a paper or two.
Vidaurre believes she made the right decision, both entering a Ph.D. program and doing it at Hopkins. She says, “I’ve had to push myself mentally, and though I’m not doing everything on my own, I have support. I think I have grown a lot here.”
Matthew Wooten, PhD ’19
Matthew Wooten, PhD ’19
Searching for our identity
Matthew Wooten studies what makes a cell a cell
How does your skin cell know it’s supposed to be a skin cell? Why does an eye cell turn into part of an eye? The DNA in our cells is identical. Their blueprints are basically the same. So what accounts for the wild variety of differences?
It’s the proteins that interact with the DNA, says sixth-year Johns Hopkins Ph.D. student Matthew Wooten. They regulate what is out in the open being read, and what other DNA is packaged away. Other research has shown in that in the eye, for example, all the information that makes a cell a liver cell is packaged away very tightly.
But it’s unclear how it works, and how it knows to put away what isn’t necessary.
When a cell is dividing to create new cells, “Instead of propagating an identity the organism doesn’t need, DNA replication creates an opportunity for a cell to forget what it was. It can now go on and change,” Wooten explains.
Live and let fly
This process appears to be the case in the germ line of Drosophila melanogaster. Drosophila stem cells divide asymmetrically, and when they split to become two daughter cells, they take on different fates. One remains a stem cell, whereas the other goes onto differentiate, maturing eventually into a fully functional sperm cell. The histone proteins, which help provide structure to a chromosome, segregate asymmetrically during this division.
This separation may lay the groundwork for establishing different cell identities: The old histones segregate toward the stem cell, and the new histones, like a blank canvas, segregate toward the cell that’s going to change.
Wooten’s research in the Xin Chen lab at Hopkins centered on answering the question of how histones are established on DNA, and if there is a set pattern to it. Conceptually it’s simple to understand—new histones are placed during replication—but research over the years had shown differing results, Wooten says.
DNA replication is an inherently asymmetrical process: When two new strands of DNA are built, the leading strand synthesizes continuously, while the lagging strand gets broken up into pieces called Okazaki fragments.
Hypothetically, these differences in strand synthesis (leading versus lagging) could have important impacts on the process of histone deposition, but a definitive pattern has remained elusive: Some researchers showed old histones segregated toward the leading strand; others showed a more a symmetric process; and then others demonstrated that the lagging strand got a majority of the old histones. In spite of numerous research efforts, a consensus pattern in histone inheritance remained unclear.
That’s where Wooten and the Chen lab came in. They used two techniques that hadn’t been used together: super-resolution microscopy and chromatin fibers to visualize how histones segregate. They broke tissue from the Drosophila germ line down into single cells, put them in a buffer that popped them to release their contents, and flowed the contents of the popped cells over a glass slide. Wooten and colleagues then used super-resolution microscopy to directly visualize histone inheritance during ongoing replication.
This showed that in the Drosophila male germline, histones segregated asymmetrically, with the old histones going to the leading strand and the new histones with the lagging strand.
Given previous work demonstrating the importance that histone proteins play in regulating how genes are transcribed, these asymmetries in histone inheritance could impact how genes are differentially transcribed on the two new strands of DNA, and therefore how cell identity is specified once those strands are segregated to different cells during mitosis. “It’s more than just a basic copying mechanism. It suggests that the way in which the DNA is made is meaningful,” explains Wooten.
Problems with histone regulation during development have been shown to lead to major issues with tissue formation and homeostasis. Given the importance that cell fate specification has in fields such as regenerative medicine and cancer research, it is possible that understanding how histone segregation contributes to cell fate specification could help further future research efforts in each of these respective fields.
Riddle me this
Wooten says he came to Hopkins knowing he wanted to study biology, but was unsure of the specialty within the discipline. When he met with Chen, he became interested in how a cell determines its sense of self: All cells have the same blueprints, but behave very differently.
“It was the coolest type of riddle,” Wooten says.
As a biology major at the University of Maryland, one of his research projects included learning about toxic algae blooms in the Chesapeake Bay, and how to mitigate them. He also worked at Johns Hopkins Hospital as a research tech in electrophysiology, learning about neural mechanisms of itch and pain.
Before coming to Hopkins, Wooten says, he wanted to study neuroscience. But when he interviewed at neuroscience programs, it seemed less and less like that was the right fit. All that changed when he interviewed at Johns Hopkins. “I felt very comfortable at Hopkins. There was a sense of community, with students and faculty. The professors cared about your success—even those who weren’t in your lab.”
In his lab
Dr. Xin Chen has been a fantastic mentor who is wonderful to work with, says Wooten. And he appreciates the collaboration opportunities throughout Hopkins, as well as the Carnegie Institute right next door. He cites Joe Gall, Ph.D., who is 91 and still working at the bench, as one of his inspirations.
“He was a mentor to scientists who were some of the early pioneers studying chromatin and the structure of the nucleus. He has been so helpful, and I would never had been able to do any of this work without his and his post-doc Zehra Nizami’s advice and support,” Wooten explains. And I would definitely be remiss if I didn’t mention Jonathan Snedeker, who co-created the fiber technology with me and was there in the early days, making inroads in this protocol.”
Wooten has always wanted to be a professor, he says. And Dr. Chen has been very supportive of sending him to conferences, meetings, and helping him build his personal network, from making contacts to finding reagents.
Wooten has defended his thesis, and is planning to travel to Seattle in January 2020 to become a post-doc at Fred Hutchinson Cancer Research Center in the laboratory of Steve Henikoff. There, he plans to develop approaches to look at how proteins bind to DNA in a single-cell capacity, and what makes a stem cell a stem cell.
Although he’s almost out of the program, he has advice for those considering Hopkins or graduate school in general. Wooten says, “Every graduate students career has its ups and downs. Its important to find things outside the lab that you can go to when work is getting you down. Sometimes, your best ideas can come to you away from the bench!”
He is deeply grateful for Chen’s mentorship as well as the camaraderie he’s experienced in his lab and at Hopkins in general. “At the end of the day, the most important thing is a mentor you like and respect, and friends and colleagues who can help you along the way. Hopkins has all those things.”