5 Question Challenge with Victoria Deneke, Ph.D.: Synchronization in the Cell Cycle

Ahead of her publication in Cell out online today, Victoria Deneke, Ph.D. of the Di Talia lab took our 5 Question Challenge to give us more insight into her time at Duke and her recent work titled: Self-Organized Nuclear Positioning Synchronizes the Cell Cycle in Drosophila Embryos.

What excites you about your work?

One of the most extraordinary moments for me was watching the dynamics and organization that takes place in a developing organism for the first time. I am still in awe of the beauty of development and it is what keeps me motivated to understand more and more how this occurs in living systems.

Briefly describe your breakthrough discovery that led to the publication of this paper?

We have discovered in fly embryos the mechanism by which local biochemical cell cycle signals are integrated with the mechanical properties of the embryo to ensure accurate positioning of the nuclei. We have also shown that this process is necessary to maintain cell cycle synchrony throughout early development. More broadly, our work elucidates how self-organized biochemical and mechanical dynamics can arise in embryos and uncovers an important biological function of cytoplasmic flows.

These findings were made possible by a biosensor, which I was using in my previous studies (Deneke et al., 2016), measuring the activity of Cdk1 and PP1, two important proteins that regulate the cell cycle. One day we were imaging this sensor and we happened to start a movie at a very early time point in development. We immediately noticed that the cell cycle oscillations were distributed in a spatial pattern different from that expected from published studies. Specifically, we saw that oscillations were restricted to a specific location and  gradually expanded during subsequent cell cycles. That observation prompted us to ask whether there could be a functional significance to these local biochemical oscillations and led us to identify the model defined in our new paper (Deneke et al., 2019).

Why is this discovery important to the field of Regenerative Biology?

During regeneration, a damaged organ must heal in order to regain its functional size and shape. At the heart of this process are morphogenetic events that rely on the integration of biochemical and mechanical signals to coordinate growth and proliferation in space and time. Our study provides a novel quantitative model and framework for this integration and will inspire similar studies in more complex multicellular systems.

What was the most memorable experiment in your years in the Di Talia lab (whether it was successful or not)?

Figuring out how to introduce a barrier in the embryo was definitely one of the most memorable experiments in the Di Talia lab. We wanted to know whether the waves of division that we were observing could go through a barrier or not (Deneke et al., 2016). We tried all kinds of things, from using a high-power laser to ‘cauterize’ a part of the egg (think light saber cutting an embryo in half) to using optogenetics to molecularly introduce a barrier. As much as I would have liked for either of these tools to work, they didn’t. The light saber experiment ended up exploding embryos or generating a heat wave that “cooked” the egg, which was cool to watch but not useful for us. We then turned to a paper from the 70s that used what we call in the lab an “embryo guillotine”. This apparatus consists of a dull razor blade that has been mounted on a bar that you can precisely lower with a set of knobs at each end. We contacted the Duke Physics Machine shop to build this for us and we gave it a try. To our surprise, we could introduce a barrier using this method and more importantly, we found that the wave did not go through the barrier, suggesting that diffusion was necessary for the wave to spread.

What comes next for you? Or the research?

I continue to be fascinated by the amazing dynamics and emerging patterns inside living systems, especially during development. I’m intrigued to keep studying dynamical processes, now at the very earliest moments of life, in other words, right after fertilization. I’ll be starting a postdoctoral position in Dr. Andrea Pauli’s lab in September of this year, where I will be studying spatiotemporal coordination following fertilization in zebrafish embryos.

Interested in learning more about the Di Talia lab? Check them out here.  Interested in taking the Regeneration Next 5 Question Challenge about your recently published or upcoming work, email regeneration@duke.edu

Undergrad in the Lab: A look into Lily Hiser’s work in the Bursac Lab

Lily Hiser, a junior at Duke, has been working in Dr. Nenad Bursac‘s lab.  Lily is pursuing a degree in Biomedical Engineering

Lily Hiser, undergraduate researcher in the Bursac Lab

What excites you about your work?

To me, tissue engineering is the most exciting area of research in the biomedical field. It is the perfect mixture of biology and engineering, as it takes prowess in both to create a successful tissue. Thinking big picture, I can personally imagine a future where engineering tissues is an everyday part of medical research, whether this is for drug testing, implantable devices, or some other application. Being in such a cutting-edge area of research is such a privilege for me, and I am still amazed every day I go into the lab and see the muscle tissues that we have created working like a muscle should!

  1. Why did you choose to study in Dr. Nenad Bursac’s lab for your undergraduate research project?

Before matriculating into Duke, I had the privilege of doing research in high school at the Wake Forest Institute for Regenerative Medicine. I had never been exposed to that caliber of research before, and I was particularly fascinated by the area of tissue engineering. My experience there propelled me to pursue a degree in biomedical engineering, and I knew that I wanted to do research in tissue engineering while I was at Duke. I had always been impressed with Dr. Bursac’s groundbreaking work, and I knew that doing research in his lab would be an incredible environment for learning and the development of my own research skills. I chose to study in his lab because I knew it was an area of research that I was very passionate about, and I was excited by the opportunity to experience it firsthand.

  1. What has been your favorite project in the lab?

So far, my favorite project in the lab has been my work with the Duchenne muscular dystrophy (DMD) project. DMD is a genetic disorder that causes a mutation in dystrophin, a protein which is important for maintaining muscle structure. Currently, there is no curative treatment for DMD, but there are a variety of drug treatments being developed. However, the animal models which are used for DMD drug testing, particularly the murine models, do not provide realistic modeling of the severity of the disease in humans. DMD can be caused by over 4000 unique genetic mutations, each one causing the disease to present itself in varying severities. The current mouse models only possess two genetic mutations, and it takes over a year for the model to display more advanced disease symptoms. Even then, these models are much milder than what is seen in DMD patients. This project is focused on creating an in vitro, patient-specific human model of DMD, which would more accurately model the various, unique severities of individual patients. My work has been focused on increasing the functionality and accuracy of the model, as well as using the model to test prospective drug treatments.

In order to determine the efficacy of the treatments, the engineered muscles are force tested to quantify how strong the muscle is and are subjected to damaging eccentric contractions to determine how resistant the muscle is to injury. We can only perform these tests because our model is three-dimensional, and the functional data we collect provides strong evidence towards the effectiveness of prospective treatments – as we ultimately want to make muscle stronger and more resistant to injury. This project has been incredibly exciting to work on because not only are we able to produce effective human in-vitro DMD models, but we are also able to use them in real-world applications to provide support for the scientists working to cure the disease.

  1. The Bursac lab has 5 undergraduates working currently in his lab. Is there much overlap in undergrad projects or are you all working on different projects?

For the most part, each of the five undergraduates in Dr. Bursac’s lab is working on his or her own individual projects. Personally, I work very closely with another undergraduate student, Simal Soydan. We were trained together and share a lot of responsibilities on various projects. However, even though all the undergraduates do not work together, we do collaborate as far as communicating about different techniques. If someone has experience with a specific lab technique or expertise in a subject, we are always ready to collaborate and share our knowledge.

  1. What’s next for you? Your research?

As I continue to do research in Dr. Bursac’s lab, I am looking forward to continuing to grow my laboratory technique portfolio, as well as simply learning as much as I can from all of the amazing people I work with. For my research, I am looking forward to continuing to work on the DMD model, particularly with the possibility of testing some prospective gene therapies for DMD patients. Our project is also looking for ways to enhance the ability of the muscle to simulate human biology, specifically by developing methods for incorporating other biological systems, such as immune cells, into the mu

scle model. By doing this, the model will be able to demonstrate how the various DMD treatments affect not only the muscle, but the body as a whole.

Side by side comparison of engineered healthy muscle tissue (left) and DMD model muscle tissue (right)Side by side comparison of engineered healthy muscle tissue (left) and DMD model muscle tissue (right)

5 Questions Challenge with Jieun Esther Park, Ph.D. candidate: Enterocytes in the Vertebrate Gut

LREs in the zebrafish gut shown using fluorescent microscopy and IHC
Jieun Esther Park, Ph.D. candidate in the Bagnat lab took our 5 Question Challenge for her recent publication in Developmental Cell: Lysosome-Rich Enterocytes Mediate Protein Absorption in the Vertebrate Gut.  For this publication, Park collaborated with three other Regeneration Next Labs.

What drove you to become an expert on enterocytes?
I don’t know if I can call myself an expert on enterocytes by any means.
But my first project in the lab was characterizing LREs (Lysosome-Rich Enterocytes – specialized enterocytes in zebrafish and pre-weaning mouse) and that’s how I got to start my research on enterocytes. I was fascinated by the distinct characteristics of LREs and I loved studying them in the zebrafish system where I can visually see the cellular processes occurring in these cells. This is how I got to learn a lot about enterocytes over the last few years!

If you could tell the 1st year graduate student version of yourself one thing, what would it be?
Take advantage of lots of opportunities on campus as a student. There are so many certificate programs, classes, outreach programs you can participate in. Take advantage of the resources you have to build the skillsets you want and need!

What experiment or result did you find the most exciting or surprising?
I spent first two years of my graduate school trying to find the direction of my project. I made a lot of CRISPR/Cas9 genetic mutants and zebrafish tools, but I was not sure whether I would see phenotypes with my mutants and whether I will be able to carry out my project to find interesting results. But I took time to develop a very solid assay for quantifying cargo uptake in LREs and then tested one of my mutants (cubn mutant). And when I saw that cubn mutant showed defect in protein uptake, I was so excited!!

Say I’m a high school student, can you briefly describe to me your research and the impact your discovery has on Regenerative Biology?

When you eat food, proteins in your diet are broken down into smaller pieces (called peptides, amino acids) in your stomach & intestine, and then are absorbed by your intestinal cells. However, protein digestion occurs differently in babies during the pre-weaning stage. Babies with immature digestive system cannot digest proteins efficiently in their stomach or intestine. However, they instead have special intestinal cells that gulf up undigested proteins and digest them inside the cells. We named these cells Lysosome Rich Enterocytes (LREs). These cells play a very important function in absorbing and digesting proteins during the pre-weaning stage and they disappear after weaning. Our research looked at how LREs absorb proteins and showed that a defect in LREs leads to slower growth and in serious cases, decreased survival.

Our current research findings do not have direct impact on Regenerative Biology but now we have generated the tools to test how regenerative LREs are. Since LREs are intestinal cells that only exist in neonatal/developing gut in mammals, if they are more regenerative than adult enterocytes, that will lead us to understand and explore the differences in the regenerative capacity of neonate vs adult intestine and the mechanism behind it.photo of Jieun Esther Park, Bagnat Lab graduate student

What prompted you to bridge your work in zebrafish, with a collaboration in the mouse system?

Because we believed LREs are a conserved cell type in zebrafish and pre-weaning stage mammals, we decided to use the mouse system to back up our zebrafish data. We showed that LREs are indeed highly conserved in zebrafish and pre-weaning mouse and that they also share the same molecular machinery for their endocytic function.


Jieune Esther Park is a 6th year graduate student in the Pharmacology program.


Interview by Raymond Allen, DSCB graduate student – McClay Lab.
photo of Raymond Allen, DSCB graduate student om tje

 

 

 

 


If you would like to be a guest writer/social media contributor, please contact Amy Dickson.

Understanding the Source of Regenerative Ability in Animals

Dr. Alejandro Sanchez Alvarado, an Investigator at Stowers Institute, is a world leader in mechanisms of tissue regeneration.  In the mid-1990’s he initiated a novel program employing molecular tools to explore the spectacular regenerative abilities of planarian flatworms.  His groundbreaking work over the past two decades has revealed key secrets behind the neoblast stem cells that enable planarian regeneration, and has launched successful independent careers for an impressive list of investigators who once trained with him.  Please join us at this special seminar hosted by Regeneration Next and the School of Medicine.

January 31, 2019 – 12:30PM. Room 147 Nanaline Duke Building

Strong community drives support for DDX3X research

Mariah Hoye, a Regeneration Next Fellow and Postdoctoral Research associate in the laboratory of Debra Silver, shares her experience at the 4th annual DDX3X foundation meeting.

Some of the scientists at the DDX3X meeting (L to R): Bethany Johnson-Kerner, MD, PhD; Ruji Jang; Elliot Sherr, MD, PhD, Debra Silver, PhD; Mariah Hoye, PhD; Lindsey Suit.

Our lab studies brain development in mice, with a particular interest in neural stem cells. My work specifically focuses on a protein, DDX3X, which is required for neural stem cells to function properly during brain development (Lennox AL et al, 2018).

In 2014, researchers showed that mutations in DDX3X account for 1-3% of intellectual disability (ID) diagnosed in females. Now that we have established that DDX3X plays a vital role during brain development, we aim to further dissect its function in the brain and try to develop novel therapies for children with DDX3X-ID.  Specifically, I am generating a mouse model to understand how loss of DDX3X impacts the behavior of neural stem cells and neurons in the developing brain. I am also using genome-wide assays to characterize how normal and ID missense DDX3X mutants influence neural stem cells at the molecular level. Using these different approaches, we will gain insight into the consequences of loss of normal DDX3X and the consequences of ID mutant DDX3X, which may differentially influence brain development.

So, what is DDX3X-ID?

DDX3X-ID is caused by spontaneous mutations in the DDX3X gene. For the vast majority of cases, these mutations were not inherited from either parent. There are only very rare cases of inherited DDX3X-ID. Females are most commonly affected because the DDX3X gene is on the X chromosome. In addition to intellectual disability, DDX3X-ID individuals can also exhibit developmental delay, seizures, epilepsy, low muscle tone and autism. For more information, see https://ddx3x.org/

Because of our work on DDX3X, Dr. Silver and I were invited to attend the 4th annual DDX3X foundation meeting in Philadelphia a few weeks ago. The DDX3X foundation was created by the parents of two DDX3X-ID children, Beth Buccini and Liz Berger. Since the discovery of DDX3X-ID in 2014, the foundation has organized an annual meeting for families, clinicians, and scientists to share the latest research and discuss implications for children with DDX3X mutations. In the last year, the DDX3X foundation membership has grown 122% and this is only expected to continue increasing as screening tools improve and awareness of the condition continues to grow.

On the first day of the DDX3X foundation meeting, researchers and clinicians met together to engage in round table discussions and presentations of our findings. We discussed updates on the clinical presentation of DDX3X-ID, the development of new models of DDX3X-ID (including mouse models and cell-based models) and the need for a natural history study of DDX3X-ID patients. In this intimate group, we identified the best next steps to continue advancing the research.

That night, there was a welcome dinner for all of those in attendance at the DDX3X foundation meeting. Midway through the evening, families went around the room and shared their stories. It was a homecoming. Each story echoed the other- parents knowing something was wrong early on, while doctors reassured them their children would catch up, and then later, running test-after-test only to come up short with no answers. For many, finding the DDX3X foundation community was not about getting an explanation, but finally having a community that truly understands, as one family noted ‘to see other girls who look and act like your own and have the same infectious joy’.

It’s hard to describe, but I left that night feeling connected to these families and their children, and I knew I would never see my research the same way. As basic scientists, we do not often get to interact with individuals affected by the syndromes we study. This is a huge detriment because it shields us from the personal motivation and investment that many clinician scientists have towards their research, because it involves their own patients. At the same time, the DDX3X foundation meeting really reinforced the importance of basic science and our interactions with clinical scientists. Our group had previously joined forces with Dr. Elliot Sherr, MD, PhD, a child neurologist studying many different neurodevelopmental conditions. By combining his clinical data on individuals with DDX3X-ID with our cellular and mouse model data, we were able to develop a much more complete picture of the role of DDX3X during brain development. This type of collaboration is the ideal model for moving research from “bench-to-bedside”, as it ensures that us basic scientists are addressing the most clinically-relevant questions, but also the cellular mechanisms underlying disease physiology.

Thank you to the DDX3X Foundation for inviting us to the annual meeting to share our work and to meet all of the amazing families. I would also like to thank the RNI community for funding me as one of their 2018 fellows!

Regeneration Next Collaborations grow from seed funding

In 2017, Regeneration Next received an Intellectual Community Planning Grant to seed collaborative teams, a priority outlined in the Together Duke Strategic Plan. Our goals for grant outcomes included better communication amongst researchers, engineers, clinicians, and experts and ultimately more effective translation of tissue regeneration research to potential treatments in an ethical and safe manner. We focused on two strategies: building multidisciplinary research communities at Duke, and  increasing recognition of Duke’s regenerative medicine strengths.

Screenshots from three short videos that highlight interdisciplinary collaborations in the fields of heart, muscle/joint, and neural/brain regeneration.

A blog post on the Duke Interdisciplinary Studies website chronicles the activities and outcomes that resulted from the Intellectual Community Planning grant. Read about some of our early successes in building collaborative communities>

Pancreatic cells can morph to make insulin

Diabetes is characterized by persistent high blood sugar levels that occur when pancreatic insulin-producing β-cells are destroyed or are no longer able to secrete insulin. But researchers have discovered that other pancreatic cells can come to the rescue when insulin-producing cells are destroyed. In a new study published in Nature Cell Biology, a group of researchers including Duke Regeneration Next Fellow Valentina Cigliola, Ph.D, have reported that certain pancreatic cell types, the α- and δ-cells, can change their identity and reprogram into insulin-producers to make up for the insulin deficit upon β-cell loss.

Mouse pancreatic islet in which alpha-cells have been labeled with a fluorescent tag (green). Some of them have started to produce insulin (red), and the morphed cells appear as yellow (green-and-red merge). Image courtesy V. Cigliola.

Cigliola, her mentor Pedro Herrera, Ph.D., and their colleagues at the University of Geneva report on the molecular mechanisms controlling how α-cells behave after β-cell loss. By genetically and pharmacologically altering these mechanisms, Cigliola and colleagues were able to significantly increase the number of α-cells that produce insulin. They also showed that this newfound insulin-producing capacity is reversible, with α-cells switching back to their original role when physiological conditions are restored.

This study challenges the current belief that mature cell types remain stable and cannot morph into other stem cells, and has implications not only for the treatment of diabetes, but for tissue regeneration in many other organs and disease conditions.

Citation:   Valentina Cigliola, Luiza Ghila, Fabrizio Thorel, Léon van Gurp, Delphine Baronnier, Daniel Oropeza, Simone Gupta, Takeshi Miyatsuka, Hideaki Kaneto, Mark A. Magnuson, Anna B. Osipovich, Maike Sander, Christopher E. V. Wright, Melissa K. Thomas, Kenichiro Furuyama, Simona Chera, Pedro L. Herrera. Pancreatic islet-autonomous insulin and smoothened-mediated signalling modulate identity changes of glucagon α-cells. Nature Cell Biology, 2018; 20 (11): 1267 DOI: 10.1038/s41556-018-0216-y

 

Immune Cells Help Older Muscles Heal Like New

Macrophages enable regeneration of lab-grown adult muscle tissue

By Ken Kingery
This article first appeared on Duke’s Pratt School of Engineering website, and is re-posted with permission.

colorful stain of cells and muscle tissue

Macrophages (red) within an engineered skeletal muscle tissue disrupted along mature, contractile myofibers (green). Following injury, the macrophages rescue the myofibers from cell death and muscle stem cells activate and contribute to tissue regeneration.

Biomedical engineers at Duke University have found a critical component for growing self-healing muscle tissues from adult muscle—the immune system. The discovery in mice is expected to play an important role in studying degenerative muscle diseases and enhancing the survival of engineered tissue grafts in future cell therapy applications.

The results appeared online October 1 in Nature Biomedical Engineering.

In 2014, the group led by Nenad Bursac, professor of biomedical engineering at Duke, debuted the world’s first self-healing, lab-grown skeletal muscle. It contracted powerfully, integrated into mice quickly and demonstrated the ability to heal itself both inside the laboratory and inside an animal.

The milestone was achieved by taking samples of muscle from rats just two days old, removing the cells, and “planting” them into a lab-made environment perfectly tailored to help them grow. Besides a three-dimensional scaffolding and plenty of nutrients, this environment supported the formation of niches for muscle stem cells, known as satellite cells, that activate upon injury and aid the regeneration process.

For potential applications with human cells, however, muscle samples would be mostly taken from adult donors rather than newborns. Many degenerative muscle diseases do not appear until adulthood, and growing the muscle in the lab to test drug responses for these patients would benefit from the use of the patient’s own adult cells.

There’s just one problem—lab-made adult muscle tissues do not have the same regenerative potential as newborn tissue.

“I spent a year exploring methods to engineer muscle tissues from adult rat samples that would self-heal after injury,” said Mark Juhas, a former Duke doctoral student in Bursac’s lab who led both the original and new research.

“Adding various drugs and growth factors known to help muscle repair had little effect, so I started to consider adding a supporting cell population that could react to injury and stimulate muscle regeneration,” said Juhas. “That’s how I came up with macrophages, immune cells required for muscle’s ability to self-repair in our bodies.”

Macrophages are a type of white blood cell in the body’s immune system. Literally translated from Greek as “big eaters,” macrophages engulf and digest cellular debris, pathogens and anything else they don’t think should be hanging around while also secreting factors that support tissue survival and repair.

After a muscle injury, one class of macrophages shows up on the scene to clear the wreckage left behind, increase inflammation and stimulate other parts of the immune system. One of the cells they recruit is a second kind of macrophage, dubbed M2, that decreases inflammation and encourages tissue repair. While these anti-inflammatory macrophages had been used in muscle-healing therapies before, they had never been integrated into a platform aimed at growing complex muscle tissues outside of the body.

It took several additional months of work for Juhas to figure out how to incorporate macrophages into the system. But once he did, the results changed dramatically. Not only did the new muscle tissues perform better in the laboratory, they performed better when grafted into live mice.

“When we damaged the adult-derived engineered muscle with a toxin, we saw no functional recovery and muscle fibers would not build back,” said Bursac, who is a co-director of Duke’s Regeneration Next initiative. “But after we added the macrophages in the muscle, we had a wow moment. The muscle grew back over 15 days and contracted almost like it did before injury. It was really remarkable.”

The success appears to stem primarily from macrophages acting to protect damaged muscle cells from apoptosis—programmed cell death. While newborn muscle cells naturally resist the urge to throw in the towel, adult muscle cells need the macrophages to help them push through initial damage without going into cell death. These surviving muscle fibers then provide a “scaffold” for muscle stem cells to latch onto to perform their regenerative duties.

Bursac believes the discovery may lead to a new line of research for potential regenerative therapies. According to a popular theory, fetal and newborn tissues are much better at healing than adult tissues at least in part because of an initial supply of tissue-resident macrophages that are similar to M2 macrophages. As individuals age, this original macrophage supply is replaced by less regenerative and more inflammatory macrophages coming from bone marrow and blood.

“We believe that the macrophages in our engineered muscle system may behave more like the muscle-resident macrophages people are born with,” said Bursac. “We are currently working to understand if this is indeed the case. One could then envision ‘training’ macrophages to be better healers in a system like ours or augmenting them by genetic modifications and then implanting them into damaged sites in patients.”

That work is, of course, still years into the future. While this study also showed that human macrophages support the healing of lab-grown rat muscle, and separate work in Bursac’s group has grown complex human muscles containing macrophages, there is not yet a good lab or animal system to test the regenerative powers that this approach may have in humans.

“Building a platform to test these results in engineered human tissues is a clear next step,” said Bursac. “Along the same lines, we want to better understand the potential roles that macrophages within engineered muscle play in its vascularization and innervation after implantation. We hope that our approach of supplementing lab-grown muscles with immune system cells will prove to be a general strategy to augment survival and function of other lab-grown tissues in future regeneration therapies.”

This research was supported by the National Science Foundation and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR070543, AR065873).

CITATION: “Incorporation of Macrophages Into Engineered Skeletal Muscle Enables Enhanced Muscle Regeneration.” Mark Juhas, Nadia Abutaleb, Jason T. Wang, Jean Ye, Zohaib Shaikh, Chaichontat Sriworarat, Ying Qian and Nenad Bursac. Nature Biomedical Engineering, October, 2018. DOI: 10.1038/s41551-018-0290-2

Link to free view-only article

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New faculty Yarui Diao sheds light on DNA non-coding “Dark Matter”

From Regeneration Next Director, Ken Poss, Ph.D.:
On behalf of the Regeneration Next Initiative, I’m excited to welcome Yarui Diao, Ph.D., to Duke University. This month, Yarui joins us as a new Assistant Professor in the Department of Cell Biology. Yarui will also be an affiliate of Regeneration Next and hold an appointment in Orthopaedic Surgery. Regeneration Next partnered with Dean Klotman to recruit Yarui to Duke as part of an open search this past year conducted with the departments of Cell Biology and Biomedical Engineering.

Yarui followed a highly productive training in skeletal muscle regenerative biology with postdoctoral work in Bing Ren’s lab at UCSD, where he applied cutting edge gene-editing techniques to dissect genome regulatory elements at high throughput in stem cells. Here, he intends to explore the gene regulatory mechanisms that control skeletal muscle stem cell activity and is excited to form new collaborations – indeed, his work in both biology and technology spaces should be of interest to many faculty at Duke.

Regeneration Next Executive Director Sharlini Sankaran, Ph.D. spoke with Diao about his research and future plans.

Yarui Diao, Ph.D.

SS: Your research focuses on gene regulatory elements. What are they and why are they important?
YD: As the name suggests, “gene regulatory elements” refer to the non-coding DNA regions which control gene expression. When the human genome was first sequenced in 2003, scientists were surprised to find that only 2% of the human genome was made up of so-called “coding” genes. More than 98% of the sequence was non-coding. In fact, about 15 years ago these non-coding gene sequences used to be called “Junk DNA” or “Dark Matter”, and many people used to think they were non-functional.

But then came the rapid development of the field of epigenetics and next-generation sequencing technologies. These tools allowed us to learn that these non-coding regions of the genome contain extremely critical information to help the cells in our body learn when, where, and how much to turn on or off the gene expression program. These “non-coding” regulatory regions of the genome show highly cell type-specific activity. This activity is what directs cell differentiation – it is what makes our brain cells become brain cells, a muscle cell become a muscle cell, and so on. Each cell type has different morphology, different function, and different gene expression program.  You can think of gene regulatory elements as the “Menu” and “instructions” that tell the cell how develop and how to function.

SS: These elements are very important in cell development and differentiation, but they also play an important role in tissue regeneration. Can you tell us how gene regulatory elements are important in regenerative medicine?
YD: Gene regulatory elements play an important role in almost all biological processes, including tissue regeneration. Let’s take stem cells as an example. In different tissues, there are tissue-specific stem cells, and there are lineage-specific transcription factors that are tightly controlled by gene regulatory elements. These transcription factors need to recognize specific regulatory elements to “turn on” their activity. If we can get a better understand of how gene regulatory elements work, we can get a better understanding of stem cell fate, or how stem cells differentiate into different cell types.

If you get a better understanding of gene regulatory elements, it can help you understand the disease mechanisms that are involved in many degenerative disorders and also in the aging process.  We know that in aging, a lot of tissue types fail to regenerate at the same rate as in younger people, and these have to do with changes in gene regulation.

SS: How did you become interested in this area of research? Tell me a little bit more about your research.
YD: I spent my entire postdoctoral career studying gene regulation, but my interest in tissue regeneration dates back to my PhD studies. I wanted to understand how transcription factors can control stem cell fate (what types of cells the stem cells mature into). I found that the transcription factors need to bind to specific gene regulatory elements to do the job.  At that time, just 5 – 6 years ago, despite the crucial function of these regulatory elements, it was difficult to precisely identify them in the genome because there were no good tools to characterize the 98% of the DNA that consist of these non-regulatory elements.

So for my postdoctoral training, I took a departure from my PhD research of studying muscle stem cells to join Dr. Bing Ren’s lab to study gene regulation. Dr. Ren is well known for using cutting edge technology and tools for studying functions of the non-coding genome. Luckily, at that time, in 2013, there was a new technology called CRISPR-Cas9 that was being developed. This new technology gave me the ability to develop a new method and lead a project to study the functionality of these non-coding gene regulatory elements in a high throughput manner.

In my Ph.D. research I, used muscle stem cells as a model system for studying the gene regulation mechanism centered on a master transcription factor called Pax7. We know that Pax7 regulates muscle development and regeneration. Mice lacking Pax7 show severe defects in muscle stem cell activity and muscle regeneration and growth. Very interestingly, I found that loss of Pax7 causes cells to transition into adipocytes (Fat cells) instead of muscle cells.

Muscle stem cells are isolated (top) and undergo proliferation, differentiation, and fusion to form muscle fibers (bottom row, left to right). The entire process is tightly controlled by gene regulatory elements, whose dysregulation leads to muscular disorders in disease and aging. Image courtesy Yarui Diao.

SS: To clarify – muscle stem cells turned into fat cells? There may be a whole weight-loss industry that would be very interested in this!
YD: Yes. We showed that Pax7 binds to a few non-coding regulatory elements to suppress the fat cells’ fate. Without the Pax7, muscle stem cells that would have become muscle cells, matured into fat cells instead.

SS: What are the future implications of your research for human health and regenerative medicine?
YD: I plan to use muscle stem cells as a model system to understand how gene regulatory networks control muscle stem cell activity. Muscle stem cells are not as “exciting” or popular to study compared to, for example, brain or heart stem cells. The significance of muscle stem cells and their remarkable regenerative potency is often overlooked. We know it’s very hard for the brain to regenerate. It’s very hard for the heart to regenerate. But tissues like muscle and skin have remarkable regenerative ability and we should study them more.

If we look at diseases related to muscular degenerative disorders, I would say there is a very urgent need to understand the mechanisms that control muscle regeneration. For example, there are a lot of patients suffering from disorders like muscular dystrophy and muscular atrophy. Remember, muscles are not just there to support our limb movements. Muscles also control essential functions like moving the ribcage when we breathe – so if you have a defect in your muscle function, it can cause huge life-threatening problems.

There’s another type of condition called cachexia which occurs in patients with some chronic diseases like cancer. Cachexia is a muscle-wasting condition that causes a rapid loss of muscle function. The genetic and epigenetic basis of cachexia was overlooked by both scientists and physicians for many years, because it is not a direct cause of many diseases. But loss of muscle mass and function causes big trouble for patients. Cachexia is one of the provocative questions we need to address in cancer treatment. If we could find a way to manipulate muscle stem cell function, that would be very helpful to restore mobility and strength to these patients.

Also, aging is a condition that affects everyone eventually. Scientists know that in aged mice and aging people, muscle stem cells cannot be properly activated to generate new muscle cells. When injury occurs in older people, these muscle stem cells are less likely to be activated to heal the injury by new tissue formation. So, all these clinical needs are currently unmet mainly due to our limited understanding of the gene regulation mechanisms that control muscle stem cell activity.

SS: What are you looking forward to as you move to Duke?
YD: I’m very excited to establish my own research lab, as anyone would be after years of PhD and postdoctoral training! As I mentioned, I studied muscle stem cells in my PhD, and I studied gene regulatory elements in my postdoc, and I’ve had a long-standing interest in regenerative capacity and disease conditions. When I published my Cell Stem Cell paper during my PhD, I got a lot of phone calls from patients. Even though my research was very far removed from clinical treatments, there was still a lot of interest from patients. This encourages me as a scientist to do a better job of learning how translate this knowledge into future therapies to help people.

At Duke, and in North Carolina in general, there’s a very good environment to collaborate and push forward my research. There are a lot of people working to understand the role of gene regulatory elements in general, and also specifically in muscle stem cell function. For example Charlie Gersbach is a genome engineering expert who is also very interested in muscular disorders, and Ken Poss is a pioneer in heart regeneration who has uncovered tissue regeneration enhancer elements that help zebrafish hearts regenerate. I really look forward to working with these great people, trying to generate new knowledge and make an impact in science.

I’m also very excited to become a teacher. My parents are teachers in China, and my parents-in-law, my grandmother and grandfather, were all teachers. When I was a kid, I thought being a teacher is the best job in the world. I’m so happy to come to Duke to be a researcher in a great environment, but also to become a teacher to help train a new generation of future scientists.

“Fizzy-related” gene regulates organ repair after injury

Most of us take for granted that our bodies and organs have the ability to heal after an injury, but rarely stop to ponder how. We know that the healing process occurs by filling the space created by the injury. This can happen in one of two ways: by making cells bigger (a process called hypertrophy) or by dividing cells to make new ones.  For over a hundred years, scientists have sought to understand why some organs undergo hypertrophy, and if that process has any advantages over cell division.

In a new study published in the journal eLife, Duke researcher Donald Fox, Ph.D., and his team identified a gene, known as “Fizzy related,” that regulates hypertrophy in fruitfly cells after organ injury. They found that in injuries requiring many cell divisions or many rounds of hypertrophy to heal, cell divisions led to organ distortion and loss of permeability, whereas hypertrophy had no substantial effect on the repaired organ.

Intestinal cells in fruitfly larva showing repair after injury. Magenta = intestinal cells, green – cell nuclei. Image courtesy Erez Cohen, Duke graduate student and the paper’s primary author.

These findings may benefit researchers interested in therapeutic organ regeneration, because the results identify a molecular target that could potentially be used to alter an organ’s repair capacity. They also highlight a potential protective effect of hypertrophy in injured tissues. Hypertrophy has been observed in humans, following injury to the liver, heart, or kidney. While often viewed as mal-adaptive and non-regenerative, this study’s results suggest that there may be a protective effect to hypertrophic tissue repair.

Read more about this paper and its implications in the Duke Medical School Blog.

Paper Citation: Cohen E, Allen SR, Sawyer JK, and Fox DT. Fizzy-related dictates a cell cycle switch during organ repair and tissue growth responses in the Drosophila hindgut, eLife 2018;7:e38327 doi: 10.7554/eLife.38327