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

Cell survival pathway linked to regeneration in aged skeletal muscle

Skeletal muscle stem cells (MuSCs) play a vital role in the repair and regenerative functions of our muscles after injury. As those of us over forty may know firsthand, this function declines with age and contributes to impaired muscle regeneration in older individuals. Despite what you may see in internet ads, there are currently no stem cell injection treatments that can reverse this decline in muscle regeneration function. However, thanks to a new report from Duke University’s Department of Medicine (Hematology), scientists are a step closer to identifying treatments that target molecular pathways related to cell survival. They hope that age- or disease-related muscular degeneration can be slowed or even reversed by identifying and restoring function of these molecular pathways.

Top to bottom muscle stem cells in young, middle-aged, old and geriatric mice. Blue is nuclei, red is cytoplasm and green shows autophagy.

Top to bottom: muscle stem cells in young, middle-aged, old and geriatric mice. Blue is cell nucleus, red is cytoplasm and green shows autophagy. Image courtesy James White

In this recently published report, Duke researcher James White and colleagues identified a molecular pathway that regulates several natural phases of the MuSC cell cycle. This pathway, the AMPK/p27Kip1 pathway, regulates key processes including autophagy (cellular recycling to turn old proteins and other substances into energy and materials for new structures) and apoptosis (cell death). Aging cells tend to exhibit less autophagy and more apoptosis, which ultimately leads to reduced regenerative and repair capacity in older adults.

In the paper, published in the journal Stem Cell Reports, White and colleagues found that the balance between the processes of autophagy and apoptosis is crucial in determining whether an MuSC survives and can assist with muscle repair. They found that MuSCs in aging mice exhibit less autophagy and more apoptosis, and are more susceptible to cell death than the MuSCs of younger mice.

White and colleagues determined that aging MuSCs have dysfunctional signaling of the AMPK/p27Kip1 molecular pathway. When they activated the AMPK/p27Kip1 molecular pathway in old MuSC, they found increased cell proliferation. In addition, restoring AMPK/p27Kip1 signaling increased the survival rate of transplanted aging MuSCs. White and colleagues conclude that this pathway could be an important potential therapeutic target for improving muscle regeneration in older individuals.

Citation: James P. White, Andrew N. Billin, et al, The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells, Stem Cell Reports, 2018,
https://doi.org/10.1016/j.stemcr.2018.06.014.

Astrocyte-to-neuron signaling is key to building brain connectivity

Duke researcher Cagla Eroglu, PhD and her colleagues have identified a key protein receptor in the brain which regulates development of important connections in the brain. A new research paper published in the Journal of Cell Biology explains how this molecule plays a pivotal part in brain development. Chris Risher, faculty member at Marshall University and a former Postdoctoral Fellow in the Eroglu lab, was the paper’s primary author. Risher says, “this work provides new insight into the development of aberrant synaptic circuitry in conditions like autism, epilepsy, and other neurological conditions.”

An epifluorescence microscopy image of a rat neuron. Bright spots reveal synapses (connection sites). Image courtesy Chris Risher.

The brain is a highly complex organ that enables us to think, remember, move, and perform simple to complicated tasks. These tasks use brain circuits that are made up of connections between cells called neurons. Neurons contact each other at sites known as synapses, and the human brain is estimated to contain trillions of these connections. The way that synapses form in the brain, and how faulty connectivity may lead to brain dysfunction, are still largely unanswered questions in neurobiology.

In the last decade and a half, research has pointed to a non-neuronal basis for regulating synaptic connectivity. Star-shaped “connector” cells known as astrocytes, which far outnumber neurons in the brain, secrete factors that modulate the timing and extent of synapse formation. One of these factors, an extracellular protein called thrombospondin (TSP), was previously shown to promote synapse formation via a receptor, α2δ-1. But the mechanism of synapse formation was not yet known.

Risher used a technique called three-dimensional electron microscopy to study synapse formation in mice brains. In this paper, they show that that α2δ-1 is required for the structural maturation of synapses. They determined that a molecule produced by α2δ-1 stimulates structural maturation of neurons. Mice neurons that lacked α2δ-1 or the molecule it produces remained in an immature state. When they restored the receptor and its functions, the synaptic deficiencies were reversed.

This paper lays the groundwork for future therapies and treatments targeting conditions that are caused by deficient astrocyte-to-neuron signaling. One such drug, the FDA-approved drug gabapentin (Neurontin) is used to treat a variety of neurological conditions and works by targeting TSP and α2δ-1. In humans, defects in TSP, α2δ-1 and their signaling partners have been implicated in autism, epilepsy, and other neurological conditions.

Citation: Thrombospondin receptor α2δ-1 promotes synaptogenesis and spinogenesis via postsynaptic Rac1. Risher, Kim, et.al., Journal of Cell Biology DOI: 10.1083/jcb.201802057 | Published July 27, 2018

Summer Fellow Jack Chang: Thank somebody today

A guest blog by Jack Chang
Mr. Chang is a Regeneration Next Summer Research Fellow in the Karra lab at Duke’s Division of Cardiology. In this blog post, he shares his research experience and reflects on how success depends on more than just individual effort.

My name is Jack Chang and I’m one of the RNI Summer Fellows this year. I’m a rising senior studying biochemistry at the University of Texas at Austin and plan on attending medical school to become a physician. This summer, I’m working in the Karra Lab studying mechanisms of cardiac tissue regeneration in zebrafish larvae, and it’s been an incredible experience. My project involves analyzing the interaction between TGF-b and vegfaa signaling on cardiomyocyte proliferation. To accomplish this, I’m using a confocal microscope to examine the hearts and the software Fiji to quantify cardiomyocytes. By the end, I expect to improve my understanding of the mechanisms of cardiac tissue regeneration in zebrafish and how this can be applied to human hearts.

In lab, I’ve grown tremendously as a scientist. I’ve been able to refine my laboratory research skills, increase my knowledge of the cardiovascular system, and learn how to better assess research. Every day, I learn something new, which is exciting. Being at Duke has been amazing as well. I love interacting with all the bright people I meet and hearing about the amazing accomplishments that have happened and are happening here in Durham.

Chang (Left,white shirt) with Ravi Karra (left, blue shirt), mentor Diana Chong (right, blue shirt) lab members at a celebratory lunch. Photo courtesy Paige DeBenedittis, lab manager (front, purple shirt).

With these highs, however, also come lows. Research can be frustrating and draining. When experiments don’t work, it’s discouraging. Before this program, I had an idealized perspective of research. I expected experiments to run accordingly and for data to be conclusive. But I’ve learned the grinding nature of science and how often finishing an experiment leaves one with more questions than answers. Adjusting to working full-time took time as well. Coming from undergrad’s easy pace, I often came home both physically and mentally exhausted. Being away from friends and family back home is hard as well. I write this to show that there are two sides to every coin and acknowledge that we all go through trials, even when it appears we’re having the time of our lives. So, if you’re feeling down, you’re not alone. Talk to someone about it. Odds are they’re able to empathize with you.

This past week, I got this fortune from a Chinese restaurant. It read:
“People make plans; fate makes the plan successful.”
Agree or disagree?

Initially, I couldn’t disagree more. In fact, when I first read it, I was appalled by this proverb. I posted on social media asking my friends their thoughts and the majority dissented as well. But one friend, whom I respect tremendously, agreed with it. I was confused as to how someone so wise would concur with such a statement. But that got me rethinking if there actually was any truth to it.

If not fate, what makes plans successful? Hard work? Talent? Fortunate circumstances? I think most people would agree that hard work brings success. But I don’t think anyone could argue all of their success in life has come from their own merit. To be fair, hard work does favor success. But how about the times when we got lucky? Or things just seemed to fall into place? For example, me being accepted into this program and coming here wasn’t all because of my own doing. Sure, I earned good grades and sought out research opportunities back home. But I’m fortunate to have friends who encourage me and great role models from my family and professors. Success isn’t dependent on just oneself. It involves factors like privilege, the actions or non-actions of other people, and even some luck.

This summer, I learned how research wasn’t an exception to this idea. To get research done, there was collaboration within a lab, as the PI, lab manager, postdocs, and even undergrads discussed ideas. Information was also shared between labs. Whether it was a joint lab meeting between two groups at Duke or reading a paper written by a lab in China, every day I saw how interwoven science is. PI’s are even dependent on grants to fund their research. With many publications and awards, it’s easy to become arrogant. But research is a team sport, with one piece of evidence being the many authors on a paper. It certainly takes more than an individual effort to account for success.

Being at Duke has been formative not just for my research skills, but also my appreciation for the people around me. Some of the skills I’ve learned include using a confocal microscope and sectioning with a cryostat. But without the guidance of the people in my lab, I would not be as fluent in these techniques, and I’m grateful for the mentors who’ve dedicated their time to training me. Above all, this experience has helped me appreciate how special every day is. I’m lucky to be at a great institution doing research and to work with people who help me be the best I can. Life isn’t just about me, but rather the people who have shaped me to the person I’ve become.

So, thank someone today. It can be your boss, co-workers, or even the cashier at the cafeteria. Show your appreciation for them and acknowledge the role they’ve played in your success. Reflect on the good has happened to you that has helped you reach the success you have today and strive to be that good to someone else. And while there is an “i” in science, you can’t spell it without the other letters.

Stay humble, be grateful, and appreciate the awesome life we all live.

Jack is always down to hear what other people are doing with their time: jack.chang@duke.edu.

Society for Developmental Biology Meeting: Recap Days 3 & 4

Ben Cox, graduate student in the Ken Poss lab, is sharing his experience at the Society for Developmental Biology’s 77th annual meeting in Portland, OR. Here’s his recap of the third and fourth day. Read his recaps of Day 1 and Day 2 .

Day 3:

Boundary between large and small intestine in the fruitfly. Image credit: Jessica Sawyer, Duke University

Day 3 of SDB featured the session that was likely of greatest interest to scientists in Regeneration Next labs. Lucy O’Brien of Stanford chaired the session titled Development Redux: Stem Cells in Regeneration and Homeostasis, and talked about her lab’s work on Drosophila (fruitfly) intestinal stem cells.

Additional highlights included Karen Liu of King’s College London, who studies bone healing in mammals. The work she presented focused primarily on neural crest-derived frontal bone, which is more osteogenic than parietal or mesodermal osteoblasts and heals faster after postnatal wounding. Her lab is working to identify the basis for this differential healing ability, including the role of Sostdc1 as a dual Wnt/Bmp signaling inhibitor.

Kacy Gordon of Dave Sherwood’s lab at Duke presented work on the C. elegans gonad distal tip cell, which the Sherwood lab uses as a stem cell niche model. She studied the interaction of the distal tip cell  and sheath cell Sh1 and found that the interface between the two cells defined a region of germ cell proliferation dependent on actin.

She was followed by Rohan Khadilkar of the University of British Columbia, who studies cell junctions in the lymph gland, a hematopoetic organ, in Drosophila. Through live imaging and RNAi of the extracellular matrix component integrin, his lab showed that the ECM is essential for maintenance of prohemocytes, which will eventually form the hemocytes necessary for immune response to pathogens.

Finally, Regeneration Next director Ken Poss presented work on Vitamin D signaling in cardiomyocyte development, homeostasis, and regeneration. After testing many molecules in a screen using the FUCCI protein system, which consists of fluorescent reporters of cell cycle activation, the Poss lab identified the Vitamin D analog alfacalcidol as a regulator of cardiomyocyte proliferation. Creation of multiple transgenic reporters and signaling mutants confirmed the role of Vitamin D signaling in cardiomyocyte proliferation in all stages of growth and that this effect is dependent on Erbb2 signaling. Further, they showed Vitamin D has broad effects on proliferation and tissue and animal size, not just in cardiomyocytes.

Day 4: The final day was most noteworthy for the award lectures but featured many other talks of interest early in the day. In the Cellular Patterns and Polarity session, Sally Horne-Badovinac from UChicago presented work from her lab, which studies the collective migration of epithelial cells using the Drosophila egg chamber as a model. The egg chamber rotates as it develops as a result of collective migration of follicle cells across the basement membrane. Work from her lab showed that the genes Fat2 and Lar are necessary to specify the leading and trailing edges of these migrating cells. She also showed work on the gene semaphorin-5c, part of a family of genes best known for its role in axon guidance during development. Horne-Badovinac showed that flies with mutations in this gene have a round egg phenotype, and that the gene normally colocalizes with Lar. Her lab has hypothesized that Semaphorin-5c signaling blocks protrusions at the backs of cells, allowing for normal collective migration to occur.

Later, in the Development and the Nucleus session, Rebecca Resnick of Stephen Tapscott’s lab at Fred Hutchison Cancer Research Center investigated the idea of epigenetic memory in development and disease. She showed that the histone variants H3.X and H3.Y, which promote relaxed chromatin and accumulate at the transcription start site of highly expressed genes, are targets of the gene DUX4, a homeobox gene linked to facioscapulohumeral muscular dystrophy. She showed that a burst of this gene during development creates epigenetic memory of the gene expression program through the H3.X and H3.Y histone variants.

Smed.jpg

The planarian schmidtea mediterranea, which can regenerate its entire body plan after injury. Wikimedia Commons Image by A. Alvarado Sanchez

The final full session of the conference featured awards lectures. Christian Petersen of Northwestern received the Elizabeth D. Hay New Investigator Award and focused more of his talk on recent and ongoing research compared to the researchers who spoke later, perhaps a result of his relative newness to being an investigator. Petersen works in the planarian Schmidtea mediterranea studying regeneration, and trained under Peter Reddien. This planarian can regenerate its entire body plan after severe injury, and it had previously been shown that inhibition of Wnt signaling was necessary to specify regeneration of the head. Thus, when Wnt signaling is inhibited, regeneration results in the formation of two heads. Several years ago, Petersen showed that the gene Zic-1 is required for anterior pole formation and that its inhibition leads to the formation of two tails. Another gene expressed in the anterior during regeneration, Notum, is downstream of Zic-1 but upstream of Wnt signaling. When Notum is knocked down by RNAi, an uninjured planarian will grow a new pair of eyes while keeping the old. However, only the new pair of eyes can regenerate after acute injury. Studies such as these highlight the role of S. mediterranea in understanding basic tenets of positional control genes and how regeneration can make use of information available via pre-existing tissue.

The other award winners were Robb Krumlauf of Stowers, winner of the Edwin G. Conklin Medal, Eric Wieschaus of Princeton, winner of the Society for Developmental Biology Lifetime Achievement Award, and Drew Noden of Cornell College of Veterinary Medicine, winner of the Viktor Hamburger Outstanding Educator Award. These highly accomplished scientists spoke both on their own work and the history of the field, placing the advances they and their immediate colleagues made into the context of multiple centuries of the study of development. It was a fitting end to a conference that featured a spectrum of research ranging from current undergraduates to a Nobel Prize winner in Dr. Wieschaus.