Summary
In this Q&A, Scientific Editor Emily Marcinkevicius talks to lead authors Nelson Hall and Uri Weill and co-corresponding authors Bo Wang and Jochen Rink about collaborating on their paper “Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection” and the scientific wonders of the flatworm.
Main text
Planarian flatworms have yielded foundational insights into developmental biology and regeneration. They have the potential to yield further insights into stem cell biology, cancer development, nervous system plasticity, and the link between genotypic and phenotypic variation; however, the potential of this “natural laboratory” has not been fully harnessed due to a comparative lack of standard molecular biological methods. To address this limitation, Hall and Weill et al. developed techniques for design and somatic transfection of exogenous mRNAs into isolated planarian cells as well as whole organisms. They made tractable luminescence gene-expression reporters that can be used to assess post-transcriptional gene-regulatory mechanisms. These findings lay the methodological groundwork for future lines of inquiry using this powerful model organism.
First please tell us a little bit about yourself. Where are you from originally, and where did you study previously? How did your scientific interests develop?
Nelson Hall: I was born in Dallas, Texas, but I grew up in Charlotte, North Carolina. North Carolina is a great state to get an appreciation for nature since you have the mountains to the west and the beaches to the east. I think most of my interest in the life sciences came from observing wildlife while hiking, kayaking, and fishing with my dad in those mountains and beaches. Ultimately, that all led to me pursuing a degree in bioengineering at MIT and now a PhD in the same field at Stanford.
Uri Weill: I was born in Tel Aviv, Israel, and grew up in a town called Givatayim next to Tel Aviv. I did my military service as a professional defensive diver at the Underwater Missions Unit of the Israeli Navy. During my service, I fell in love with the sea and the life in it, and so I decided to do my BSc in marine biotechnology at the Ruppin Academic Center, Michmoret, Israel. Thereafter, I completed my MSc and PhD degrees at the Weizmann Institute of Science in Rehovot, Israel, where I studied yeast cell biology. Next, I wanted to return to my passion of investigating aquatic organisms and joined the lab of Dr. Jochen Rink at the Max Planck Institute of Multidisciplinary Sciences in Göttingen, Germany.
Bo Wang: I was trained as a physicist, and my PhD thesis is on Brownian motion. When I took a developmental biology course taught by Phil Newmark, I was blown away by the beauty of development, during which individual cells are programmed in space and time to coordinate with each other and form a new organism. A few months later, I started working in Phil’s lab as a postdoc and was converted to a developmental biologist. When I was establishing my lab at Stanford, I spent a few months at Kavli Institute for Theoretical Physics at the University of California, Santa Barbara, as a part of the Evolutionary Cell Biology program organized by Rob Phillips, Michael Lynch, and Shelley Sazer. I remember being told by Rob at a lunch that I should keep my physics accent when approaching biological problems. This was important advice, and our current research has a consistent emphasis on developing new methods and applying quantitative engineering mindset to tackle the intrinsic complexity of biology.
Please tell us about working on the project together. What motivated you to develop the project?
N.H.: I had previously worked in a mammalian synthetic biology lab, and I was interested in applying those tools and techniques to a whole organism. When I heard about planarians and their regenerative capacity, I immediately imagined implementing a lineage tracing system to watch stem cells work to replace amputated tissue. The idea of using the tools of synthetic biology to reprogram and readout the processes of regeneration and development sounded so exciting. Of course, Bo informed me that transgenic tools were not available in planarians, and I took that as a challenge to see what progress could be made in that space.
U.W.: In our lab, we realized that using transgenic tools in planarian research could significantly advance every project we were working on, so we decided to dedicate our recourses to try to solve the problem.
How did you end up deciding to make this a collaboration between the two groups?
N.H.: The collaboration began organically through simply sharing our progress and data thanks to the close relationship between Bo and Jochen. The challenge of transgenesis in planarians is something that I don’t think a single person could solve entirely, so I was happy to have two labs on the job. Not long after I sent a version of the luminescent reporter to Jochen’s lab, Uri began using it to screen for other transfection reagents to perform in vitro transfections and found Viromer, which was a real game-changer and enabled many experiments we currently have in the manuscript. It was clear very early on that collaborations are essential for tackling long-standing challenges in tool development.
What strengths did each group bring to the project?
N.H.: Jochen’s lab is very skilled in the manipulation of planarians, especially microinjection into intact animals, which is critical for in vivo transfections. They also optimized the Western blotting method in planarians, which was instrumental in addressing certain reviewer concerns. Our two labs are also different in their members and culture. The fact that we approach problems differently is a strength that allows us to more rapidly traverse the wide parameter spaces that tool development presents.
U.W.: Coming from the field of bioengineering, Bo and Nelson had unique and advanced views and skills in molecular cloning and reporter design, which were essential to address planarian cell transfection. At our lab, we built on their approaches to screen for additional conditions, reagents, and parameters. This back-and-forth work between the labs has continued throughout the project and significantly advanced it.
What other group members, collaborators, or resources were instrumental in this project?
N.H.: While brainstorming transfection methods early in the project, I came across nanostraws, small hollow needles that can deliver genetic material into cells, and to my surprise, they were being developed by Nick Melosh’s group just a short walk away. Not too long after that, Sergio and I were attempting to deliver mRNA into planarian cells, which led to our first result indicating that mRNA transfection was feasible. Our readout at that point was exclusively luminescence via plate reader, but Hongquan Li helped us build a low-cost luminescence microscope, which enabled much of the imaging data seen in the paper.
U.W.: One of the main challenges we addressed was verifying exogenous reporter expression with independent lines of evidence. Dr. Leonard Drees, a postdoc in our lab, leveraged his prior experience in fly molecular biology to detect nanoluciferase protein via western blotting from tissue lysates of transfected animals. Leo also worked side by side with me to inject many planarians to test live transfection parameters. In addition, Dr. Tobias Boothe, the imaging specialist in our lab, set up an Olympus LV200 luminescence imaging system and was instrumental in getting the imaging of transfected animals going.
How was your experience with the peer-review process, and how did it strengthen your work?
N.H.: To be honest, it was very challenging, but comparing our initial submission with the final result, it’s a much more thorough exploration of the technique than it began as. With tool development, it can be tempting to stop once you demonstrate that the tool works as intended; I think the reviewers pushed us to not only show that the tool functions, but also show how the tool might be implemented to probe interesting biological questions.
U.W.: I think that one of the challenging aspects of our review process was trying to incorporate and address the comments and requests from five different reviewers.
There was ... unprecedented reviewer interest in this paper! Are there research questions or communities that you think could benefit from adopting planarians as a model to help inform their studies?
N.H.: I’ve always found planarian’s lack of obvious cancer-like diseases so bizarre, especially considering that they harbor a large quantity of proliferative stem cells throughout their indefinite lifespan. By what mechanisms do they keep these cells in line? I hope that as planarians develop as a model with increasingly robust genetic tools, we might be able to make luminescent stem cell reporters that allow us to watch clonal dynamics within a worm over time. Cancer biology is understandably very mammalian-centric, so maybe these tools might make planarians an enticing alternative system.
U.W.: Our lab is fascinated by the wide spectrum of species-specific regeneration abilities among “wild” planarian species, and along with this comes a diversity of cellular and tissue parameters during regeneration and normal growth. We hope that more and more planarian species will be adopted for molecular and genetic research to understand the mechanisms that control this observed diversity. We have already shown, as a proof of principle, that it is possible to detect luminescent signal from the transfected cells of an additional planarian species—Schmidtea polychroa, for example. We hope that the methodologies we developed could be adapted and used in many additional planarian species.
B.W.: Another fascinating aspect of planarian biology is that the brain constantly reforms through rapid turnover of neurons, and the total number of neurons within the brain can fluctuate across two orders of magnitude (∼1,000–100,000) depending on the availability of food. How are neural circuits maintained in such dynamic brains? How is memory retained during and after regeneration? Labeling and manipulating neurons in vivo could allow us to begin to approach these important questions.
On a scale of 1 to mouse, how difficult is it to work with planarians on a daily basis?
N.H.: Planarians are refreshingly simple to maintain. They’re kept in simple Tupperware containers filled with fresh water, and they’re fed macerated calf liver once per week—that’s it. I’ve been able to maintain planarians in my own home, so I think that speaks to the ease with which they can be maintained. This also makes them a wonderful system to introduce elementary school students to experimental biology since many classic regeneration experiments can be performed in even a simple classroom setting.
U.W.: Planarians are easy to maintain and work with. The challenge is in adopting existing methods from other systems to study planarians. Things like extensive mucus on body surface, large number of lipid droplets in tissues, inaccessible zygotes, and a lack of transgenesis complicate the process of developing new molecular and genetic tools, but our labs and others in our field are now beginning to overcome these hurdles, which makes it a very exciting time to work with planarians.
Is there anything about planarians that you find especially surprising or interesting as you work with them?
U.W.: When collecting planarians in the wild, you sometimes have to play detective in order to track them down, getting information from other researchers and planarian enthusiasts, arriving at the correct time of the year, and looking very carefully under rocks and vegetation.
N.H.: Sometimes you’ll come across isolated bodies of water and find planarians and wonder, “How did these get here?” I like to imagine some adventurous planarian long ago hitched a ride on a water bird’s foot, and I’m seeing its many offspring however many centuries later.
B.H.: We often assume biology always functions as how it should. However, we sometimes observe that planarians can make mistakes in their regeneration resulting in odd shapes and forms, which, however, are typically corrected during the next regeneration. The amount of plasticity and robustness built in the system is astonishing to me.
It’s that time of year again when new students are embarking on their graduate careers. Nelson and Uri, do you have any advice for more junior trainees who are just starting out?
N.H.: As difficult as it might be, avoid comparing yourself to your peers. Some members of your cohort will graduate early, other later, some with one paper, others with multiple. A healthier attitude is to look at your peers and attempt to learn and imitate the positive qualities you see in them. The PhD is a very personal journey, so focus on yourself and your own growth.
U.W.: Try to form collaborations with people in your group, and even more importantly, beyond your group and institute. This will open you up to new ideas, methodologies, and viewpoints.
What’s next for each of you in your careers?
N.H.: I’ll be looking for opportunities that allow me to continue exploring genome engineering challenges, either in industry or academia. I’m also focusing a lot more on another flatworm, Macrostomum lignano, which looks to be a very promising system for organismal synthetic biology.
U.W.: I’m currently working on another project in collaboration with Bo’s lab that utilizes planarian chimeras as a system to experimentally study the consequences of genetic heterogeneity. I hope that in the near future, I will be able to establish my own lab to study tissue homeostasis mechanisms with planarians.
Jochen and Bo, what are some of the major new/next directions your respective research groups will be moving in over the coming years?
Jochen Rink: Our paper demonstrates the expression of an exogenous reporter in planarian cells and intact animals. Finally! What lies ahead is a lot more method optimization, e.g., the development of plasmid vectors, Cas9-mediated integration, and cell type-specific drivers. One interesting question en route is whether endogenous defense mechanisms might limit planarian transgene expression, perhaps geared toward the defense of genome integrity within the adult pluripotent stem cells (so-called neoblasts). Beyond, the promise of reporter expression in specific cell types entails a whole new mechanistic dimension in planarian research. We continue to pursue our long-term goal of visualizing and quantifying cell dynamics during planarian regeneration, and we are excited about the prospects of genetic lineage tracing within the pluripotent stem cell system. In short, an exciting time to be in the field!
B.W.: A new direction we are undertaking is to understand why planarian transgenesis has been so challenging. Given that we have overcome the barrier (whatever it was) to express a reporter in planarian cells, it has become possible to identify the nature of that barrier, either physical, molecular, or cellular, which prevents exogenous genetic materials from modifying the cell. Animals, like planarians lacking adaptive immunity, may have special mechanisms to defend against viruses and other environmental sources that want to have access to the machinery in their cytoplasm and even their genomes.
A few questions about methods research: what do you think are some of the major questions that are going to drive scientific progress in the next 10 years, and what types of approaches are these questions going to require?
N.H. and B.W.: Can we engineering biological systems at the organismal level? Much of synthetic biology follows a “sense and respond” paradigm—CAR-T cells for example, sense and respond to cancer cells through a chimeric antigen receptor. However, can we zoom out further and engineer novel cell types, organ structures, and even organismal behaviors? We think that engineering such novelties will require advances in DNA synthesis and cloning to facilitate the construction of larger circuits, novel gene delivery, and editing strategies to enable the integration of these larger synthetic gene networks at defined genomic loci and an ability to readout the state of the synthetic network with tools like single-cell sequencing.
U.W.: What can we learn from the natural genetic diversity of organisms in the wild? While this question has been leading research for many years, I think it is still very relevant now and in the future, especially with new molecular technologies arising all the time. I think it is important to develop additional tools for “non-model” organisms such as those that we show in this article.
If you could wish a new method or technology into existence, what would it allow you to do?
N.H.: My dream technology would be a benchtop DNA printer that could take an arbitrarily long or complicated sequence and synthesize it on the spot. While the cost of sequencing continues to plumet, DNA synthesis remains costly, and limits on gene fragment length results in many hours dedicated to cloning. Sometimes I want a planarian promoter sequence, or a codon optimized Cas9 sequence, or a wholly novel fusion protein; with benchtop DNA synthesis, I can spend more time iterating on construct designs and less time frustratedly optimizing PCR and cloning conditions without bankrupting the lab.
U.W.: My biggest wish is to be able to image proteins in live cells without the need for exogenous reagents or genetic manipulations. Every perturbation we make to our living systems in order to investigate them might be affecting the results we get. While tagging a gene with a reporter is a very powerful and helpful tool that I have used thousands of times during my academic career, I hope someday we will not need to rely on it as much.
Richard Nelson Hall
Graduate student, Department of Biological Engineering, Stanford University, USA. Appearing holding a gumboot chiton.
Uri Weill
Postdoctoral fellow, Department of Tissue Dynamics and Regeneration, Max Planck Institute of Multidisciplinary Sciences, Germany
Bo Wang
Assistant professor, Department of Bioengineering, Stanford University, USA
Jochen Rink
Director, Department Tissue Dynamics and Regeneration, Max Planck Institute for Multidisciplinary Sciences, Germany
Published: October 24, 2022