Cells in fish’s spinal discs repair themselves

By Marla Vacek Broadfoot

This article was originally posted on the Duke Today blog and is re-posted here in its entirety.


In this developing backbone of a zebrafish, collapsed inner cells (green) are replaced by newly fluid-filled sheath cells (red) from the outer layer. (Credit: Jennifer Bagwell, Duke University)

Duke researchers have discovered a unique repair mechanism in the developing backbone of zebrafish that could give insight into why spinal discs of longer-lived organisms like humans degenerate with age.

The repair mechanism apparently protects the fluid-filled cells of the notochord, the precursor of the spine, from mechanical stress as a young fish begins swimming. Notochord cells go on to form the gelatinous center of intervertebral discs, the flat, round cushions wedged between each vertebrae that act as shock absorbers for the spine.

The disappearance of these cells over time is associated with degenerative disc disease, a major cause of human pain and disability worldwide.

“It is not difficult to speculate that these same mechanisms of repair and regeneration are present in humans at very early stages, but are lost over time,” said Michel Bagnat, Ph.D., senior author of the study and assistant professor of cell biology at Duke University School of Medicine. “If we are going to think about techniques that foster intervertebral disc regeneration, this is the basic biology we need to understand.”

The study appears June 22, 2017, in Current Biology.

Bagnat likens the notochord to a garden hose filled with water. The hardy structure consists of a sheath of epithelial cells surrounding a collection of giant fluid-filled or “vacuolated” cells. During development, these vacuolated cells rarely pop, despite being under constant mechanical stress. Recent research has suggested that tiny pouches known as caveolae (Latin for “little caves”) that form in the plasma membrane of these cells can provide a buffer against stretching or swelling.

To see whether the caveolae protected vacuoles from bursting, his team and collaborators from Germany generated mutants of three caveolar genes in their model organism, the zebrafish. Because these small aquarium fish are transparent as embryos, the scientists could easily visualize any spinal defects triggered by the loss of caveolae.

The researchers found that when the mutant embryos hatched and started swimming, exerting pressure on their underdeveloped backbones, their vacuolated cells started to break up. While the finding confirmed their suspicions, it turned up a puzzling discovery. “In the caveolar mutants, you see these serial lesions up and down the notochord, and yet the mature spine formed normally,” said Bagnat. “That was very puzzling to us.”

To figure out how that was possible, lead authors Jamie Garcia and Jennifer Bagwell took a closer look at the notochord of mutant fish. They marked the vacuolated cells green and the surrounding epithelial sheath cells red and then filmed the fish shortly after they hatched and started swimming. First, they could see an occasional vacuolated cell break and spill its contents like a water balloon. Then, over the course of fifteen hours, a nearby epithelial sheath cell would move in, crawl over the detritus of the collapsed cell, and morph into a new vacuolated cell.

They performed a few more experiments and found that the repair response was triggered by the release of the cell contents, specifically the basic molecular building blocks known as nucleotides. The researchers then isolated live epithelial sheath cells and treated them with nucleotide analogs to show that they turned into vacuolated cells.

“These cells, which reside in the discs of both zebrafish and man, seem capable of controlling their own repair and regeneration,” said Bagnat. “Perhaps it is a continuous release of nucleotides that is important for keeping the disc in good shape.”

The study may offer insight not only into the development of back and neck pain, but also into the origins of cancer. Their data suggests that chordomas, rare and aggressive notochord cell tumors, may begin when epithelial sheath cells leave the notochord and invade the skull and other tissues.

The research was supported by National Institutes of Health (AR065439, AR065439-04S1, T32DK007568-26, and CA193256), a Capes-Humboldt Fellowship, the Max Planck Society, and a Faculty Scholar grant from the Howard Hughes Medical Institute.

CITATION:  “Sheath cell invasion and trans-differentiation repair mechanical damage caused by loss of caveolae in the zebrafish notochord,” Jamie Garcia, Jennifer Bagwell, Brian Njaine, James Norman, Daniel S. Levic, Susan Wopat, Sara E. Miller, Xiaojing Liu, Jason W. Locasale, Didier Y.R. Stainier and Michel Bagnat. Current Biology, June 22, 2017. DOI# 10.1016/j.cub.2017.05.035

 

High school visit gets at the heart of research

Earlier this spring, a group of Wake Early College of Health and Sciences (WECHS) high school students left their classrooms to spend the day learning about cardiovascular research. The students visited the labs of Dr. Ravi Karra and Dr. Doug Marchuk, and toured the Duke zebrafish core facility. The students, many with plans to attend graduate or medical school, listened to presentations explaining  basic concepts and methodologies of cardiovascular research work.

WECHS students examine a specimen while touring the Marchuk lab at Duke

This program came to fruition under the leadership of Dr. Maria Rapoza, executive director of the Duke Cardiovascular Research Center (CVRC), Dr. Sharlini Sankaran, the executive director of Duke’s Regeneration Next Initiative, and Sruthi Valluru, a WECHS senior high schooler.

Sruthi, like many of her Wake Early College classmates, has already taken college-level courses and has been granted experiential learning opportunities in the field of medicine through WakeMed Hospital. However, when her interests turned toward medical research, she ran into roadblocks.

“[Finding ways to interact with research] was such a tricky thing for me. I had to go out and email people or have connections…It’s so difficult for any high schooler (even though there are so many smart and capable students)…to penetrate the field of research when they don’t have the proper connections.”

Valluru hopes that launching a tour program at WECHS will help her peers gain first-hand exposure to medical research. After working in both a Duke-based start-up and research lab, Valluru’s career interests turned towards medical research as a platform to transform the lives of so many. She will be studying biomedical engineering at North Carolina State University beginning this fall semester.

a few of the WECHS students with CVRC Director Dr. Howard Rockman

Valluru emphasizes the collaborative nature of research. “As we bring more people to the field, we are creating a wider community that can help us move forward in discovering new things.” Exposing students to the work of research laboratories early on in their careers helps broaden the pipeline of scientsts and cultivate interest and talent in young people.

Students enjoyed walking through Duke’s winding halls of countless lab facilities and touring Duke’s beautiful medical campus. “One of the things I enjoyed the most was the bus ride home listening to the excited chatter of my fellow classmates about how much they enjoyed [the tour],” Valluru noted.

Popular culture often portrays researchers as socially awkward science nerds who work in a dimly-lit basement. The societal conceptions of research work are not aligned with the reality that researchers enjoy rich social lives and span a wide range of types of research.

Many WECHS students expressed that participating in the tour changed their perspectives on the research work. One student said, “This isn’t what I expected research to be like. I expected research to be this one person working by themselves.” At the end of each lab visit, WECHS students asked the lab members for contact information, eager to continue learning about research work at Duke.

The Duke CVRC, Regeneration Next Initiative, and WECHS team would like to thank everyone who made this program possible. The team looks forward to scaling future collaborations to a wider scale with high schools and research universities across the Triangle area.

Guest blog post by Jacqueline Xu, Duke Department of Medicine undergraduate intern.

NC Health Advocacy Day

L-R: Andrew George, Representative Marcia Morey (Durham County), Senator Terry Van Duyn (Buncombe County), Sharlini Sankaran, Dan Keeley, and Will Barclay at the NC legislative building.

As a scientist, it is easy to get caught up in the day-to-day workflow of research and lose sight of the bigger picture. We are often so focused on generating and reporting solid, exciting data that we neglect another major aspect of our job; sharing our work and its impacts with the broader community. On Tuesday May 23rd, a group of graduate students from Duke went to the North Carolina legislative building to do just that.

Dr. Sharlini Sankaran, Executive Director of Duke’s Regeneration Next Initiative, organized a group of graduate students to attend the North Carolina Hospital Associations (NCHA) “Partnering for a Healthier Tomorrow!” advocacy day at the state legislature in Raleigh. The event gave representatives from various hospital systems an opportunity to interact with state legislators about the work they do and issues affecting healthcare in the state. Andrew George, a graduate student in the McClay Lab, Will Barclay, a graduate student in the Shinohara Lab, and I joined Dr. Sankaran to share some of the great tissue regeneration-related research going on at Duke.

Our morning was busy as elected officials, legislative staff, executive branch agency officials, and staff from other hospital systems stopped by our booth to hear what Regeneration Next is all about. We talked about the focus on harnessing Duke’s strengths in fundamental research on molecular mechanisms underlying regeneration and development, then pairing that with the expertise of our engineers and clinicians. We discussed topics including spine and heart regeneration mechanisms from the Poss Lab, advances in engineering skeletal muscle from the Bursac Lab, and clinical trials of bioengineered blood vessels for patients undergoing dialysis from Duke faculty Dr. Jeffrey Lawson.

It was remarkable to hear how engaged everyone was, we got great questions like ‘what is a zebrafish and why do you use them?’ and ‘why would a bioengineered ligament be better than one from an animal model or cadaver?’.  Every person who stopped by was supportive and many had a personal story to share about a health issue experienced by friends, family, or even themselves. As a graduate student who does basic research, it really underscored how important these personal connections are to our work, even though it may be far removed from the clinic.

Communicating our research to legislators and others at NCHA advocacy day was a great and encouraging experience. Health issues affect all of us. Our visit to the legislature on Tuesday was a reminder that there is support for the work that we do in hopes it will help lead to a healthier tomorrow.

Guest blog post by Dan Keeley, graduate student in Biology

Announcing: RNI’s first student travel awards

Regeneration Next is pleased to announce travel grants to support six students. The travel award supports graduate students who present tissue regeneration-related research at relevant scientific meetings. Our first round of awardees are:

Colleen Drapek, Benfey Lab, Department of Biology. Colleen will attend the American Society of Plant Biology meeting and will present her work on “The Minimal Gene Regulatory network for Arabidopsis Root Endodermis differentiation.”

Lauren Heckelman, DeFrate lab, Department of Biomedical Engineering. Lauren attended the Orthopaedic Research Society conference and presented two projects: “In Vivo Behavior of Patellar Cartilage in Response to and Twenty-Four Hours following Running” and “In Vivo Exercise-Induced Glenohumeral Cartilage Strains.”

Jennifer Kwon, Gersbach lab, Department of Biomedical Engineering. Jennifer will attend the International Society for Stem Cell Research meeting to present her work on “Directing skeletal myogenic progenitor cell lineage specification with CRISPR/Cas-9 based transcriptional activators.”

Jason Wang, Bursac lab, Department of Biomedical Engineering. Jason will attend the EMBO conference on Muscle Wasting and will present his work on “Developing an in-vitro model for human skeletal muscle regeneration.”

Lifeng Yuan, Wang Lab, Department of Pharmacology and Cancer Biology. Lifeng will attend the International Society for Stem Cell Research meeting and will present work on “Negative regulation of mitochondrial intermembrane peptidase drives metabolic alteration and cell death blockage for initiating cellular senescence.”

Congratulations to these deserving students! The next deadline for the Regeneration Next travel grants will be September 28, 2017. Details and application instructions are here.

New RNI Postdoctoral Fellows announced!

Regeneration Next is pleased to announce the 2018 – 2020 Regeneration Next Postdoctoral Fellows. This group of outstanding scientists was chosen from a competitive pool of applicants. Now in our second year, the RNI Postdoctoral Fellows program identifies promising young researchers who will have a key role in shaping and growing the tissue regeneration community at Duke. Please join us in congratulating our four new RNI Fellows!

Valentina Cigliola, Ph.D.

Valentina Cigliola received her PhD in Biology at the University of Geneva, in Switzerland, where she worked on mechanisms of islet cell fate changes during pancreas regeneration in diabetes, under the supervision of Dr. Pedro Herrera. She also was involved in characterizing a variant form of the human connexin 36 protein altering β-cell function and survival, under the supervision of Dr. Paolo Meda. She recently started a postdoctoral fellow position in the laboratory of Dr. Kenneth Poss, in Duke’s Cell Biology department. As an RNI fellow, she aims to characterize the cellular and molecular basis of heart regeneration, whose discovery is highly relevant to human disease such as ischemic myocardial infarction and heart failure.

Brian Cosgrove, Ph.D.

Brian recently started as a postdoctoral fellow in Duke’s Department of Biomedical Engineering, where he works in Dr. Charles Gersbach’s lab. During his PhD training at the University of Pennsylvania, Brian developed new biomaterial platforms that better recapitulate the developmental mesenchymal microenvironment in order to more precisely interrogate how multiple mechanobiological signals are integrated by stem cells. As an RNI fellow in Dr. Gersbach’s lab, he hopes to further understand how mechanical cues can influence the epigenetic state of stem cells and utilize this information to better direct and maintain stem cell fate decisions in therapeutic applications.

 

Abdelhalim Loukil, Ph.D.

Halim recently started a postdoctoral fellowship in Dr. Sarah Goetz‘s laboratory in the Department of Pharmacology and Cancer Biology at Duke’s School of Medicine. He has focused his efforts on studying the biogenesis of a fascinating tiny organelle called “primary cilium.” He recently published exciting results on the critical role of the daughter centriole in cilia formation. The primary cilium acts as a specialized organelle that regulates several developmental signaling pathways. Disrupting cilia structure or function causes numerous hereditary human diseases commonly called ciliopathies. These disorders are generally accompanied with a range of neurological impairments and neurodegeneration. With the support of the Regeneration Next Fellowship, he will investigate the in vivo roles of primary cilia in neural function. He aims to dissect the molecular mechanisms by which cilia mediate neuronal function and homeostasis in adult brain.

WenXiu Ning, Ph.D.

Wenxiu completed her doctoral work at the Chinese Academy of Sciences. Her previous work uncovered a novel crosstalk between microtubule and F-actin which contributes to wound healing of epithelial cells. To extend her studies from cultured cells to understand how tissues are made, she has joined Dr. Terry Lechler‘s lab in Duke’s Department of Dermatology as a postdoctoral fellow. With the support of RNI fellowship and resources in the Lechler Lab, she plans to determine the functions of intermediate cells and their proliferation in epidermal development and to uncover cell intrinsic and extrinsic pathways that promote intermediate cell proliferation in order to better understand how the skin stratifies and how epidermal cells influence and communicate with each other to achieve homeostasis and wound healing.

The next call for applications for Regeneration Next Postdoctoral Fellowships will be released in September 2017. Read about last year’s inaugural cohort of Postdoctoral Fellows here.

Durham Students Give Themselves a Hand Up

(This post originally appeared on the Duke Research Blog on April 7, 2017 and is reposted here in its entirety).

Picture this: a group of young middle schoolers are gathered trying to get a “hand” they’ve built out of drinking straws, thread and clay to grasp a small container. What could such a scene possibly have to do with encouraging kids to stay in school and pursue science? It turns out, quite a lot!

Angelo Moreno (right), a graduate student in molecular genetics and microbiology, helps students with their soda straw hand.

This scene was part of an event designed just for boys from Durham schools that took place one March evening at the Durham Marriott and Convention Center. It was hosted by Made in Durham, a local non-profit focused on helping Durham’s young people graduate from high school, go to college, and ultimately be prepared for their careers, and My Brother’s Keeper Durham, the local branch of former President Obama’s mentoring initiative for young men of color.

The first evening of a convention centered on building equity in education and was geared toward career exploration. Each of the boys got to choose from a series of workshops that highlighted careers in science, technology, engineering, art, and mathematics — also known as STEAM. The workshops ranged from architectural design to building body parts, which was where they learned to build the artificial hands.

Sharlini Sankaran, the executive director of Duke’s Regeneration Next Initiative, who heard about my outreach activities from earlier this year, contacted me, and together we drummed up a group of scientists for the event.

With the help of Victor Ruthig in Cell Biology, Angelo Moreno in Molecular Genetics and Microbiology, Ashley Williams in Biomedical Engineering, and Devon Lewis, an undergraduate in the Biology program, we dove into the world of prosthetics and tissue engineering with the young men who came to our workshop.

Angelo Moreno (right), a graduate student in molecular genetics and microbiology, helps students with their soda straw hand.

Biology student Devon Lewis (top) works with several of the students.

After some discussion on what it takes to build an artificial body part, we let the boys try their hand at building their own. We asked them what the different parts of the hand were that allowed us to bend them and move them in certain ways, and from there, they developed ideas for how to turn our household materials into fully functioning hands. We used string as tendons and straws as finger bones, cutting notches where we wanted to create joints.

There was a lot of laughter in the room, but also a lot of collaboration between the different groups of kids. When one team figured out how to make a multi-jointed finger, they would share that knowledge with other groups. Similar knowledge sharing happened when one group figured out how to use the clay to assemble all their fingers into a hand. Seeing these young men work together, problem solve, and be creative was amazing to watch and be a part of!

According to feedback from event organizers, “ours was the most popular session!” Sharlini said. When we reached the end of our session, the kids didn’t want to leave, and instead wanted to keep tinkering with their hands to see what they could accomplish.

The boys had a lot of fun, asked a lot of good questions, and got to pick our brains for advice on staying in school and using it to propel them towards career success. I have distilled some of the best pieces of advice from that night, since they’re good for everyone to hear:

  • Don’t be afraid to ask a lot of questions.
  • Don’t be discouraged when someone tells you no. Go for it anyways.
  • Don’t be afraid of failure.
  • And don’t think you have to fit a particular mold to succeed at something.

“I left feeling really inspired about our future generation of scientists and engineers,” Sharlini said. ”It’s good to know there are so many Duke students with the genuine and selfless desire to help others.”

It was a joy to participate in this event. We all had fun, and left having learned a lot — even the parents who came with their sons!

Outreach like this is incredibly important. Being mentors for young people with a budding interest in science can make the difference between them pursuing it further or dropping it altogether. Engaging with them to show them the passion we have for our work and that we were kids just like they are allows them to see that they can do it too.

Guest Post by Ariana Eily

5 questions with Humacyte’s Jeff Lawson

Sharlini Sankaran spoke with Dr. Jeff Lawson, who is on sabbatical from Duke University to serve as Chief Medical Officer (CMO) for Humacyte, about his work with engineered blood vessels. Humacyte’s investigative human acellular vessel recently received one of the US Food and Drug Administration (FDA)’s first Regenerative Medicine Advanced Therapy (RMAT) designations.

What excites you about your work?
What excites me is the potential to regenerate something as simple as a blood vessel; that may be a platform which we can use to regenerate a litany of organs in the future. I’m reminded that the first barrier to transplantation wasn’t the immunosuppression, but the vascularization. When Alexis Carrel won the Nobel prize it wasn’t for transplantation, but for vascular anastomosis – which lay the groundwork for him to one day do the first kidney transplant. So, I think we have to get the blood vessel right first before you can do any more complex organ, because they are all absolutely dependent on the blood vessels. In fact, the blood vessel isn’t just a tube – the vasculature functions as its own organ with its own physiology. So, to create a functioning blood vessel from a manufacturing standpoint is a very exciting opportunity and reflects a very do-able state of science in 2017. Some day, we may 3D print an organ which we can then pre-incubate with stem cells, which may then differentiate into a kidney. And I think we should try! In the 90s, when we first started doing this, we didn’t know that it would take twenty-some years to get it right and to be in Phase III clinical trials.

So that’s where Humacyte is now, right? Tell me more about your role at Humacyte and what drives you.
Yes, we are in global clinical trials right now. We just enrolled our 190th patient in our global clinical Phase II trial. It’s so exciting. The other exciting thing is, that I get to participate in something which may impact the lives of patients whom I’ve cared for. You can’t save humanity as one surgeon – you can only do one surgery at a time and there are more to be cared for. And you get to a phase in your career where you think, maybe I can impact 10,000 patients, maybe I can work on something that changes the way care is done for the better. So for me, the opportunity to change the vascular platform which surgeons can use to treat patients is very unique and very impactful. You only get one shot at this kind of chance – that is part of what drove my decision to uncouple my day job as an academic vascular surgeon for a period of time to get the clinical execution of the trial done by taking the position at Humacyte.

Describe why your research, and Humacyte’s products, are so important to human health.
What we’re doing is making the blood vessels that can be used in the initial trial. We have a number of products in clinical trials and the one that’s furthest along the regulatory path is in dialysis care. There’s a huge unmet need for patients to have blood vessels working in their arm that they can then use to get dialysis. We have also already initiated a number of Phase II clinical studies that are more arterial reconstruction based. These fall into two domains: one for patients with peripheral arterial disease, and the possibly more intriguing one is for emergency vascular trauma, when someone is injured and you have no synthetic material to reconstruct their blood vessels with today – we think we can provide a human tissue that is safe and potentially infection resistant.

We have support from the Department of Defense to develop a blood vessel that could be deployed to far forward military facilities, civilian trauma centers, and can also be used to reconstruct failing arteries from conventional atherosclerosis in an aging population. Where we started this story a long time ago, was to be a conduit for coronary heart surgery and we have every intent of having the vessels available in a smaller size. We currently make it in one size for clinical application but we have the capacity to manufacture the blood vessel in different sizes and shapes.

We’re also looking at extending the manufacturing of tubes to other organs outside the vascular system. We are looking at things like urinary conduit, and the esophagus, and the trachea. We would first like to be able to make different types of tubes that are necessary and can be replacement human tissue, and at that point we can look at transitioning to more complicated regenerative medicine. We’ve actually done some preclinical work already with urinary conduit, we know we can make the constructs for things like trachea and esophagus. It takes the same conceptual platform of taking human cells, and making the relevant shape and structure. The other questions are, do you have to have unique attributes to the matrix that’s made? If it’s placed in a different anatomic location, will it remodel and repopulate with the host cells in similar fashion? Those are questions that will be answered in the next 20 years – certainly through the rest of my career!

What has been your biggest challenge in transitioning from the university environment to chief medical officer in a startup, recognizing you’re still doing both?
I love being a physician-scientist, it’s what I’m emotionally suited to do. I never intended to become an expert in certain regulatory, or business-oriented, or administrative things. It’s interesting where your career takes you – I’m doing things that I never trained to do. The hardest thing for me, has been giving up the things I know how to do well, like operating. The operating room is such a comfortable environment for me, and I’ve been doing it for 20 years – being out of that environment has really given an appreciation for doing the things that are part of a conventional clinical practice.

One thing I’ve learned: as a CMO, you have to be committed to working as a team. As a surgeon, you are of course working with your surgical team, but a lot of the final decision-making is the surgeon’s call. In the corporate world, there are a lot of different people that you have to negotiate with as you make decisions that impact the company. So that has been a different skillset than saying “I’m the surgeon, I’m making this decision.” It’s a skillset that says: “all right we’ve got to sit down and negotiate and talk about how we are going to solve this problem.” So that’s been a transition that’s been a good one, but a challenge. Many of these decisions are made at an organizational level where the CEO, CMO, COO and a few other people all have to concur if we are going to proceed with a decision.

One of the things that’s really challenging from a company standpoint, is that we have all of this exciting stuff that’s happening and we are growing so fast. You raise enough money to do these three clinical trials, you got to jump all in: you got to finish the studies, you gotta be able to manufacture the vessels, you gotta scale up the workforce. But that means you gotta burn a lot of money over a short period of time. The analogy that often gets used is: you are building the plane as you are flying it. Even though we haven’t figured out everything yet, we have to make it work as we go. It’s an incredible pace for the entire company.

How do you do maintain the different professional roles? What advice do you have for people who are at the beginning of their careers and looking to follow a similar path?
I operate one day a month and I have clinic one day a month, but the rest of my time is primarily dedicated to Humacyte activities while we are in this clinical trial. When I spoke with Duke’s leadership about taking the Humacyte CMO position as a sabbatical, they were fully supportive. They appreciated that we had the ability to translate something from its inception as a research project, to something that could eventually help save so many lives. Duke’s leadership understands the need for faculty to have time to innovate.

In terms of advice, there are three things I would say: First, when an opportunity comes around the corner and smacks you upside the head, don’t pass it up. For example, Laura (Niklason, co-founder of Humacyte) and I met by accident in the operating room and realized our research had a lot in common. She was trying to make a vascular tube and I was looking at endothelial function, and she was looking for someone to put cells in the tube, and we realized, we have a collaboration!

Secondly, I emphasize the importance of building teams with different domains – clinicians, researchers, engineers, regulatory specialists, et cetera. The whole really is stronger than the parts. By building a complementary team, we were able to successfully compete for one grant, followed by another, and another, to where we are now. Finally, never let your ego get in the way of a successful collaboration. We have had a longitudinal collaboration for 20 years that has transcended a lot of things – the concept of keeping your collaborators and your team, really goes a long way. It isn’t always about who’s last author, or who’s main PI on a grant, it’s about what the team can achieve.

The first human engineered blood vessel transplant was performed in 2013 – watch a short video featuring Dr. Lawson, Humacyte co-founder Dr. Laura Niklason, and the patient here (contains brief footage of surgical procedure). Interested in learning more about Dr. Lawson’s work? Check out this TedX talk: Engineered human-tissue blood vessels .

Resolving heart regeneration at the molecular level

Humans are incapable of sufficiently healing heart muscle after injury. Zebrafish on the other hand have a remarkable ability to regenerate their hearts. A new paper from the Ken Poss lab (Goldman et al, Developmental Cell, Feb 27, 2017) documents the development of a map of the zebrafish genome that gets activated during regeneration. This is the first such genome map produced for a regenerating tissue. From this map, postdoctoral researcher Aaron Goldman identified the genes and enhancers that are induced during regeneration. This map will help to unlock what is so special about the fishes’ ability to regenerate.

During regeneration, the types and proportions of cells within an organ change drastically. Thus, observed changes in gene activity can mask changes that are specific to regeneration. Since muscle is the most critical cell-type, Goldman used a novel technique to map genes and their enhancers from just the heart muscle of the fish rather than the whole heart organ. From this map we can begin to reveal the factors that are controlling required genes during regeneration. Molecules that are important to regeneration in fish will help guide and support therapies to achieve a similar result in humans.

Cardiac muscle cells in an uninjured zebrafish heart. Image courtesy Aaron Goldman.

Five Questions with Louis-Jan “LJ” Pilaz

In this continuing “Five questions with…” series, Sharlini Sankaran talks with Louis-Jan “LJ” Pilaz, Regeneration Next Postdoctoral Fellow from the Silver Lab at Duke, about his recently-published paper in the journal Current Biology.

What excites you about your work?
I have always been fascinated by the brain, this incredibly complex organic machine. The developing brain contains these cool radial glial cells that give rise to our “thinking units”, our neurons. But radial glial cells do so much more. They generate specialized brain cells such as astrocytes. They form a barrier between the brain and the rest of the body during development, and they also serve as a scaffold for migrating neurons.

They can do all this thanks to their unique morphology. Radial glial cells, are absolutely gorgeous and remain full of mysteries. I love to spend time thinking about them and I’m driven by the need to know more about them. Anything impacting their functioning can have devastating impacts on brain development and eventually lead to neurodevelopmental diseases. The more we know about them, the more likely we will be able to find cures for those diseases.

Can you describe the breakthrough discovery that led to the publication of this paper?

Click image to play a live video of messenger RNAs moving along the radial glial cell’s basal process. The messenger RNA can be seen as a bright spot moving from the bottom of the image to the top.

Radial glial cells bear two long protrusions, called processes, emanating from their cell body. These processes end with structures called “endfeet” because of the way they look. One of these processes spans the whole brain radially, going from the center outwards. It is called the basal process and ends with basal endfeet tightly connected to the “roof” of the brain. In the mouse the basal process can have a length of several hundred microns, in the human several millimeters. If the basal process is not properly maintained, neurons will not properly migrate to their final destination. If the connection between radial glial endfeet and the roof of the brain is altered, neurons will migrate too far away and will end up outside the brain. Despite the importance of those structures, little is known about what is going on inside them.

In our latest paper, we show that the basal process is a highway for molecular transport. We uncovered some fascinating functions taking place in the basal endfeet: we imaged messenger RNAs being transported at high speed from the cell body to the basal endfeet within living brain tissue (see video). With the help of Ashley Lennox, a graduate student in the lab, we showed that these basal endfeet RNAs can locally produce proteins, far away from the cell body. We discovered 115 different RNAs that accumulate in the basal endfeet. Importantly about 30% of those RNAs have been implicated in neurological diseases and might play a significant role during brain development. We also show that RNA transport may be influenced by FMRP, an RNA-binding protein linked to Fragile X Syndrome, the most common cause of autism caused by a mutation of a single gene.

Why is this discovery important to the field of regenerative medicine?
Neural stem cells receive a lot of attention in the field of regeneration in the nervous system. In some areas of the adult brain, stem cells and progenitors are still proliferating and differentiating. There is hope that one day we will be able to harness those neural stem cells already in place to produce neurons that were lost after a stroke or injury. Another strategy would be to grow neural stem cells in a dish and then introduce them into damaged brains to produce new neurons. Our paper uncovers completely novel mechanisms at play in radial glial cells. This is yet another piece of the neural stem cell “puzzle” that scientists need to consider when attempting either strategy to stimulate regeneration of injured or lost neurons in the brain.

Our paper establishes radial glial cells as a model of choice to study RNA localization and local protein production in distant areas of a cell. These two mechanisms are implicated in the regeneration of axons after injury. For that reason, future studies of RNA localization in radial glial cells may yield findings critical to better understand how it plays a role in the regenerating axon.

What comes next in this research?
Scientists do not know why radial glial cells spend so much energy to transport RNAs across such long distances and what that means for normal brain function. We are now actively trying to uncover the function(s) of those localized RNAs during brain development.

Is there anything else you want to share about your work?
I feel very fortunate to be working in Debby Silver’s lab and to be part of the Duke community. All this work would not have been possible without the support of core facilities and lively discussions with other labs. Duke is an amazing place to do science!

L-J’s work was recently featured in the Duke Med School Blog. You can read more about his discoveries here.

Mouse vouchers awarded to six investigators

house_mouseRegeneration Next supports expansion of animal models at Duke for the purposes of regenerative biology research. As such, RNI has awarded six new vouchers of up to $5,000 for new transgenic or genetically modified mice that will be used for tissue regeneration and/or stem cell research. The vouchers are redeemable for provision of services at the Duke School of Medicine Transgenic and Knockout Mouse Shared Resource. Congratulations to the PIs and their trainees!

 

  • Blanche Capel: Role of Lamin B Receptor in Male Germ Cell Differentiation and Epigenetic Regulation and Identification of Lamin Associated Domains During Male Germ Cell Differentiation and Epigenetic Programming Regeneration
  • Charles Gersbach: Generation of dCas9-­KRAB transgenic mouse line for screening of genes and enhancers during regeneration
  • Dwight Koeberl: Genome editing in a humanized mouse model of Pompe Disease
  • Ruorong Yan: Modulation of heart regeneration by enhancer-delivered factors
  • Eda Yildirim: Nucleoporin 153: Regulation of gene expression during specification of hematopoietic stem cell fate and
    embryonic development

The next callout for vouchers will be posted in early 2017 with an anticipated start date of June 1.