New research conducted at the stem cell centre, DanStem, at the University of Copenhagen shows that insulin is a key determinant of embryonic stem cell potency in mammals. When large amounts of Insulin are around, stem cells retain their ability to make all the cell types in the body. However, too little insulin leads to embryonic stem cells being transformed into a new type of stem cell, one that can make tissues that support foetal development and helps make the different internal organs. As embryonic stem cells come from embryos around the time they implant into the mother, this study suggests that maternal insulin and diet maybe be important for the earliest stages of pregnancy. This study also points to new ways that stem cells can be made and differentiated to help treat degenerative diseases.
Insulin was found to be a new factor that is important for the identity of pluripotent stem cells, cells able to make all the cells in the body. A new study conducted at the Novo Nordisk Foundation Center for Stem Cell Biology, DanStem, at the University of Copenhagen reveals that insulin acts in unexpected ways to stabalise stem cells in the laboratory and on embryos cultured in a dish.
“As this type of stem cell is like the cells of the early embryo, it suggests that insulin could also be important for human development implying maternal diet and insulin levels could effect the earliest stages of a healthy pregnancy.
Professor Joshua Brickman
‘We were exploring how stem cells respond to signals produced by other cells, instructions that tell them to develop into cells that are specialized for organ and intestine formation, the endoderm. But then, when we added these factors to the food (or media) normally used to grow stem cells in, we were surprised that these signals could instruct stem cells to stay as stem cells. By comparing different medias we discovered a key difference, Insulin. With Insulin, they stay as stem cells, but with out it they make a special type of endoderm. As this type of stem cell is like the cells of the early embryo, it suggests that insulin could also be important for human development implying maternal diet and insulin levels could effect the earliest stages of a healthy pregnancy, says head of the study Professor Joshua Brickman from DanStem. At the same time, he stresses that some of these ideas are still only conjecture and a lot of work will now need to be done to understand the relationship between maternal insulin, implantation and early development before researchers can make concrete recommendations.
Transmitter Substances Play a Main Role in the Discovery The researchers in the study have examined in detail two types of stem cell which resemble each other. One kind – the embryonic stem cells, also called pluripotent stem cells – have the ability to support the development of the entire foetus. These cells can develop into any types of cell in the body. The other kind is the extraembryonic cells, which produce a type of endoderm that makes tissues that support foetal development known as the yolk sac and also helps make the internal organs, for example the intestinal system.
Mouse embryo that has been injected with embryonic stem cells grown in Insulin showing these cells can make all cell types. The embryos are at the stage when they are making all the different basic cell types of the future body, gastrulation. The stem cells were labelled genetically so they fluoresce in red under the microscope.
“The mechanisms we have uncovered are very interesting. This suggests that different amounts of insulin cause cells to respond differently to the same signals. So far we have only done tests on mice, but the next step is to examine whether the same mechanisms are found in humans.
Post.doc. Kathryn Anderson
The researchers have studied two transmitter or signal substances (Nodal and Wnt), which play a main role in stem cell development. Here they discovered that the transmitter substances were able to promote cell division in the endoderm and, at the same time, supported cell division among the pluripotent cells, but that they could choose which cell they supported based on what insulin was telling them to do. If the researchers removed insulin, the pluripotent stem cells stopped dividing and endoderm cells replaced them. When Insulin was there, the pluripotent cells grew and retained the ability to become any cell in the body.
‘The mechanisms we have uncovered are very interesting. This suggests that different amounts of insulin cause cells to respond differently to the same signals. So far we have only done tests on mice, but the next step is to examine whether the same mechanisms are found in humans’, says post.doc. Kathryn Anderson.
Applications are invited for a Postdoctoral Research Scientist to join Dr Duncan Sparrow’s laboratory in the Department of Physiology, Anatomy & Genetics to work on a project focused on investigating the environmental causes of congenital heart. This post will be funded for up to 5 years by the British Heart Foundation. You must hold, or be near completion of, a PhD/DPhil in Molecular Biology, Biomedical Sciences, Genetics or a related science. You must have experience working with animal models of development and/or cardiovascular diseases, preferably experience with mice. You will have a high level of technical competence in developmental, molecular and/or cell biology methods and techniques, for example mouse embryology and histology, fluorescence microscopy, immunohistochemistry, in situ hybridisation, ability to communicate your findings in a scholarly manner in writing as well as orally in English. Must have, or be capable of obtaining, a Home Office PILB (or better) to conduct required procedures. The closing date for applications is midday on 23 October, with interviews likely to be held the week beginning 30 October
For further information, please contact Dr Duncan Sparrow (duncan.sparrow@dpag.ox.ac.uk)
To apply, go to: https://www.recruit.ox.ac.uk/pls/hrisliverecruit/erq_jobspec_version_4.jobspec?p_id=131117
George Daley is Dean of the Faculty of Medicine, Professor of Biological Chemistry and Molecular Pharmacology, and Caroline Shields Walker Professor of Medicine at Harvard Medical School. A former Howard Hughes Medical Institute Investigator and President of the International Society for Stem Cell Research (ISSCR) from 2007-2008, his lab works on the biology and clinical application of stem cells, with a particular focus on hematopoiesis. He was awarded the Public Service Award at the ISSCR 2017 meeting in Boston, where we caught up with him to discuss his move from the lab to the clinic and back again, his quest to derive human hematopoietic stem cells in vitro, and his advocacy for science in uncertain political times.
You’re here to receive the ISSCR Public Service Award, which recognises your role in the formation of the ISSCR guidelines for stem cell research. How did you come to be involved with these guidelines, and what does the award mean to you?
My involvement with the guidelines began around 2005, when we had been living through years of debate about embryonic stem cells (ESCs), and the US National Academy of Sciences (NAS) had established a set of professional guidelines for this work. Although these had some influence internationally, many in the ISSCR leadership felt that there were subtle but meaningful restraints in the US that were unique because of the pressured political environment at the time, and that if the issues were vetted more broadly, global practices might be a bit more liberal. So I appealed to the ISSCR leadership to convene a taskforce that would examine the NAS guidelines and cast them in a broader perspective for international audiences and constituents, and I ended up chairing this ISSCR guidelines taskforce. Ultimately, we endorsed the broad principles of the NAS guidelines but proposed subtle differences as well, for example around payment for egg donation: our language was less restrictive, and this let particular communities evolve their own responses. Subsequently, the ISSCR ethics and policy committee has written a white paper outlining the ethical frameworks on which payments for these kinds of donations can be ethically justified.
Then again, around 2007 it was clear that the issue of premature clinical translation of science and unproven interventions was becoming a real problem. I had been elected ISSCR president, and decided to convene and serve on the clinical translation guidelines taskforce – these deliberations produced a framework for thinking about the legitimate way to translate basic science, and about how to preserve prospects for innovation and flexibility on behalf of practitioners. In the years after the 2006 and 2008 guidelines, the field produced induced pluripotent stem cells (iPSCs) and, by 2014, people started wondering if the existing guidelines were out of date. So I acted as the board liaison to yet another taskforce chaired by Jonathan Kimmelman, which led to the 2016 guidelines. In the end, I’m accepting the Public Service Award on behalf of the many dozens of folks who worked on these guidelines with me for the last 11 years.
The ISSCR’s 2016 Guidelines for Stem Cell Research and Clinical Translation, available here
In such a fast-moving field, how do the ISSCR guidelines keep pace?
It was specified in the guidelines that they should be re-evaluated periodically – we didn’t specify how frequently, but in response to changing technology. What is challenging about working on those kinds of guidelines is that your goal is to articulate timeless principles – independence, peer review, autonomy of the individual, those kinds of things – so that you don’t necessarily have to tie the guidelines to particular technologies, which we know will evolve. When we wrote the 2006 guidelines, we anticipated various kinds of pluripotent stem cells, without exactly knowing that iPSCs would emerge. In the latest version, we knew CRISPR gene editing was possible, but since we can’t anticipate even more transformative future technologies, we framed a set of principles around the broader concerns of intervening in the germline, whether mitochondrial or nuclear. Like a constitution that is well written, the guidelines can provide principles that can be reinterpreted in different eras but, like the constitution of the US, they are supposed to be subject to amendment.
Let’s go back to the beginning: was there anything that got you into science in the first place?
I always wanted to be a scholar. I was a good student, I liked to read, to think, to solve problems. So I always thought I would end up in academia, but along the way I took a number of crazy turns. When I came to Harvard as an undergraduate, I was as interested in politics as science. I quickly became disillusioned by the anonymity of the large, introductory science courses, so I searched the course catalogue for the smallest class at Harvard and found a freshman seminar on spiders given by a professor and curator of arachnology at the Museum of Comparative Zoology that was limited to two people. I thought that I’d get a lot of attention there, but when I went to interview the professor said he’d already filled the class! It turned out that the two who had already signed up were identical twins! I argued to the professor that he’d never be able to tell them apart, so he should take me as a third; he thought this was quite funny, so let me in. In studying a particular class of animals I was introduced to taxonomy and the underlying principles of evolutionary biology. Meanwhile, I also became very interested in the philosophy of science and evolution, and initially declared my major in philosophy.
I got interested in laboratory science also quite by happenstance. As a freshman on financial aid, I had a work study job washing dishes in one of the dining halls. When I came back for the sophomore year, I figured I would find a more interesting job washing dishes, so this time I began washing glassware in a lab. Very quickly thereafter I ingratiated myself into the lab: once I’d done the beakers and test tubes I would be looking over the shoulders of the scientists to see what they were doing. That was a lot of fun, and I really loved the research angle – you were asking a question that no one else had ever answered. Research wasn’t just being told this is they way it is – you had to discover what the principles were.
How did you first get interested in stem cells?
I had a wonderful early supervisor and mentor, a postdoc named Beth Luna, who told me that if I was really interested in doing graduate work in biology, then I would need more intensive coursework, so I started taking more biology classes, and then ultimately switched my major from philosophy to biology. Then, the summer after my junior year, I decided to spend some time with a clinician just to see what being a doctor was like (initially more to prove to myself I didn’t really want to do it!). I spent time with a neurologist at Massachusetts General and the experience struck me as very compelling – the problems of human biology were deeply entrenched in medicine. This convinced me to apply to the MD PhD programme.
In grad school I ended up working on a problem that had been introduced to me in my hematology class: the molecular basis of chronic myeloid leukaemia (CML). I went to work for David Baltimore, the world authority on ABL, the gene that was disrupted in CML. That was really deep cancer and leukaemia biology, and I ended up making a mouse model of the disease that validated the BCR-ABL fusion gene as the driving molecular lesion in CML and as a target for treatment.
But even then I realised that there were a lot of leukaemias that didn’t have a defined molecular lesion, and that the broad platform for treating leukaemias – if one didn’t have targeted chemotherapy – was bone marrow transplantation. I was heavily influenced by a visiting professor from the UK, John Goldman, who was making his reputation expanding the use of unrelated donors for marrow transplants. We spent a lot of time thinking about hematopoietic stem cells (HSCs), the challenges of finding matched donors, and whether one could either generate a universal ʻoff-the-shelf’ donor cell, or an autologous cell that you could make from some non-leukaemic tissue. I was thinking about stem cell transplants when, once again by happenstance, Martin Evans, who had isolated mouse ESCs, was on sabbatical in the next lab. He introduced me to a paper from Rolf Kemler’s group describing the methodology of in vitrodifferentiation of ESCs to make embryoid bodies – one of the prominent differentiated tissues was blood. So even as a graduate student – and unbeknownst to David – I started culturing ESCs and seeing whether I could coax them to make blood. And I could, and this led to the notion that I could use ESCs as a platform to understand blood development.
But then I had to decide whether I was going stay in science or go back and finish my training in clinical medicine; because I had done work in a human leukaemia I decided to finish medical school and a medical residency and hematology/oncology fellowship. I was asked to spend an extra year as Chief Resident, which delayed my return to the lab but was an amazingly deep and wonderful experience. When I finally came back to the lab, my goal was to see if we could use ESCs as a platform for investigating the mechanisms of blood development. My scientific focus over the last 20-plus years has really been using mouse models in parallel with in vitro models comprising human and mouse ESCs and iPSCs to get at the core pathways driving blood development.
At the end of a recent paper you describe your ultimate goal in this line of research as ‘the derivation of bona fide transgene-free HSCs for applications in research and therapy’. How far are you from achieving that goal?
When I first laid out this goal in my proposal for becoming a Whitehead Fellow back in 1995, my hypothesis was that because pluripotent stem cells give rise to blood, they must transit through an HSC intermediate to get there; the dogma was that all blood cells arose from an HSC. That turned out to be wrong – the HSC isn’t there in the dish, unless you go through a different set of morphogen patterning to make a different type of mesoderm. That’s where developmental biology comes in – to inform directed differentiation in vitro.
The cells that we’ve made recently come from a two-step strategy: morphogen-directed differentiation gets us to what is probably the equivalent of lateral plate mesoderm, from which hemogenic endothelium and HSCs arise; then we direct cell fate specification with transcription factors in the manner of Yamanaka. The cells behave according to principles by which you define HSCs: they self-renew, engage multi-lineage engraftment over a long term (although not as long as a native cell), and they can do this on a single-cell basis. However, functionally we know our iPSC-derived HSCs are way below the standards and performance of freshly isolated human HSCs, and molecularly there are significant differences. I think we can continue to refine our strategy and get ever closer to a fully functional cell; then, the question becomes how native and how normal does it have to be? Does it need to be identical to a native HSC? This sort of goes back to philosophy again – it’s not really science!
Not only does developmental biology inform stem cells, but it also works the other way round
How important has developmental biology been for your research, and how do you see the relationship between developmental and stem cell biology?
I think all of stem cell biology is built upon the foundations of developmental biology. In the absence of the principles by which embryonic polarity is laid down, morphogenesis is induced and gastrulation is regulated, attempts at directed differentiation of pluripotent stem cells in a dish would be pure empiricism, pure tinkering, and not particularly intellectually satisfying. A large amount of directed differentiation is just that anyway, which is part of its well-justified criticism. But what’s so distinctive about stem cell biology is that you are observing every single step of the differentiation pathway – there are very few developmental systems that allow you that degree of direct observation and insight, especially when you get to more complex organisms. We work very closely with Len Zon, in part because the transparent zebrafish embryo allows direct visualisation and experimental manipulation of the earliest stages of commitment to both primitive and definitive hematopoiesis. That’s been enormously powerful, but we’ve had to then make the leap back to mouse and, crucially, to human; many of the principles are conserved, but the detailed mechanisms and specific gene sets might not be.
I’d like to make the point that not only does developmental biology inform stem cells, but it also works the other way round. In our recent work we started with a screen in vitro, taking iPSC-derived progenitors that make primitive forms of hematopoietic cells and asking whether we could identify epigenetic barriers to multipotency. Indeed, we identified a histone-modifying enzyme that broadly inhibits multipotency in embryonic blood precursors. Then, in order to really understand how this negative regulation works, we went back into a developmental system, the mouse. I think the real interest of this work is what it reveals about the regulation of the emergence of hematopoiesis in the embryo.
Back at the start of my lab, I saw an unmet medical need: there were, and still are, lots of patients who could benefit from bone marrow transplants but are never offered the chance, either because they didn’t have a suitable donor or due to the enormous toxicity of the transplant because of immunological dissimilarities. I have for many years thought that we could make a platform for combining gene therapy and cell replacement in a way that could open up a curative marrow transplant approach to dozens of diseases. But I always knew that clinical success would be a very high bar, and so what has really driven me, and the reason I’ve been able to sustain my lab for 20 years on the same model, is that it’s been a fabulous platform for learning about the pathways and mechanisms that contribute to blood formation in the embryo, and blood lineage development.
You were president of the ISSCR a decade ago. What have been the most significant changes in the field in that period, and where do you see the next ten years going?
When I was president, the revolution in reprogramming was just happening. It’s really interesting to see how that evolved: in the later part of the 1990s and early 2000s, many of us were thinking about reprogramming via nuclear transfer as a mechanism for generating customised or individualised patient-derived cells. There were even many who appreciated that genes such as Oct4 and Nanog were almost certainly involved in reprogramming. But Yamanaka’s great insight was that you could identify a manageable set of transcription factors that would change a cell’s fate, contrary to the expectation that reprogramming would be so complex that it could never just involve a small set of factors. There’s been an enormous amount of progress since then, and the number of disease models that have benefitted from reprogramming is staggering.
I think that over the last decade we’ve come to a much deeper understanding of the regulation of the genome, the mechanisms of gene regulation, but also chromatin organisation. Side by side with this, the technological advance that I think is so exciting and likely to dominate the next ten years is organoid biology. It’s just so exciting to see tissues forming because the information is preordained in the cells themselves, but perhaps not surprising given what we know about anatomy and historical experiments on tissue assembly after dissociation in animals such as sponges.
You have been a stem cell researcher and vocal science advocate since the years of George W. Bush. How did stem cell research cope when federal funding for human ESCs was barred?
It was discouraging and profoundly upsetting to be part of a scientific community that was seeing the limitless potential of a new field opening up and then to have it embroiled in a deeply divisive, fundamentally ideological debate around the nature of the human embryo and subject to a form of scientific censorship – the denial of federal funding. Science advocacy was not something I’d planned to get involved in, but the knowledge base that I had and the areas in which I was working made certain individuals in the political sphere seek me out for support and advocacy. I was a junior person at the time and several senior mentors were worried about the effect on my scientific career of being seen as too public; a lot of people told me to keep my head down, focus on the science. It was a struggle and I often wondered about this balance, and did turn down numerous opportunities to speak out more aggressively. But I think it really is a core responsibility of scientists to not simply be withdrawn behind their lab doors but to be willing to speak out on behalf of their science. It’s become obvious once again under the new administration that we have to stand up and justify the value of what we do in the context of lots of other competing priorities.
It really is a core responsibility of scientists … to be willing to speak out on behalf of their science
How do you see the prospects for state-funded research six months into the new administration?
The current President’s suggested budget included a roughly 20% cut in the NIH budget, and cuts for many other areas of discretionary scientific funding. Congress is unlikely to allow these cuts to stand, given that they have a broader base and interest in the importance of scientific research as a foundation not just for the future of human health but as an economic driver, a real creator of jobs, a way of priming the pump of biotechnology and the pharmaceutical industry. So I’m hopeful that the US’s traditional support for scientific research will continue. The other crucial element is that biotechnology is an area of international competitiveness, and the US is arguably the unchallenged leader. But without continued support, that leadership is subject to disintegration, and with the rise of China as a force in science, and the amount of investment they have committed to, international competition can be seen as an existential threat to the US. Trump is nothing if not hugely competitive, and this is an area where we certainly don’t want to lose out.
This year you started as Dean of Harvard Medical School (HMS). What do you hope to achieve during your tenure?
It’s an exhilarating and challenging new role. Before I took it up, I was Principal Investigator of a Howard Hughes-funded lab, a director of a small clinical division of bone marrow transplantation, a teacher of an undergraduate and a medical school course, a consultant to biotechnology and founder of a number of biotech companies. I had a very diverse professional life as it was, but I stayed within a fairly narrow realm of my own expertise. Becoming Dean has forced me to get up to speed with a tremendous array of different areas, both scientific and biomedical, and to some degree political. The Dean of HMS is seen as one of the prominent spokespersons for US medicine broadly defined, so I have to think about things like the economic aspects of healthcare delivery. HMS plays an enormous role internationally in global public health; we’ve got constituencies that are setting the pace in fundamental discovery, we have a community that is organised around translating biology into new medicines and new treatments, and we’re partners with biotech and pharma, which is increasingly concentrated in Boston. Clinical development and delivery of healthcare is an aspect that I now have to think about and influence. It has been exciting, and I hope that I can marshal my deep respect for science and my excitement and enthusiasm for clinical translation, together with a heightened sense of service as a clinician, to help with HMS’s fundamental mission to relieve the considerable suffering that disease inflicts on society.
In 2011 you were awarded the HMS A. Clifford Barger Excellence in Mentoring Award. Is there a ‘Daley’ style of mentorship, and who were your own important mentors in research?
I have to say that was one of my proudest awards, and in fact I was delighted as Dean to preside over this year’s awards just the past week.
I’ve had a phenomenal group of wonderful mentors who have taken an interest in me and helped me to develop throughout my career. I mentioned how important Beth Luna was very early on, and later there were people such as John Goldmann, Sam Lux and David Nathan, physicians who worked with me, and John Potts, who is in his eighties but who still meets and shares wisdom with me.
David Baltimore’s style of mentorship very much influenced the kind of mentor I want to be – the way he organised his lab and ran group meetings, demanding high standards and yet welcoming open candid critical debate about science. He emphasised the importance of the question and not just the technique, which fostered a sense of innovation: in chasing questions you marshalled whatever technology you needed, absorbing established techniques and developing new ones. Like David’s, the best scientific labs have a horizontal structure, so that everyone speaks equally and individually to assert a quest for the truth; that was very influential on me. So I have tried over the years to establish a climate in the lab where people are inspired to ask big questions, are empowered by adequate resources to chase those questions in a relatively unfettered way, and are open to collaboration. I also want to encourage a climate of having fun: we do a lot of social events and sing a lot of karaoke; if I can get up in front of a group, belt out a song and make an ass of myself, I feel it gives others a certain willingness to step forward and to take risks.
Finally: is there anything Development readers would be surprised to find out about you?
Outside of the lab, I’m passionate about cooking and collecting wine: I’m a foodie. It grew directly from travelling for science – going around Europe and being hosted by people who were eager to feed me their local fare and ply me with their local libation, whether wine or beer. My wife and I came together around cooking and we do a lot of that. One has to eat, so it’s a fairly efficient way of exploring new cuisines and experiences while still keeping your time commitments.
In the future diabetics might benefit from getting insulin-regulating beta cells transplanted into their body because their own beta cells are destroyed or less functional. However, according to new stem cell research at the University of Copenhagen, the current way of producing beta cells from stem cells has significant shortfalls. The beta cells produced have some features resembling alpha cells.
Beta cells release insulin in your blood, but when you suffer from Type 1 diabetes, you hardly have any of them left in your body. This is because the immune system attacks the beta cells.
The role of insulin is to reduce and regulate the blood sugar level when it is too high. People with diabetes do not have this function, and therefore need insulin injections in order to regulate their blood sugar levels.
Researchers are trying to produce beta cells artificially with the purpose of transplanting them to diabetic patients to regulate their blood sugar. A new research result from the University of Copenhagen and Novo Nordisk recently published in the scientific journal Stem Cell Reports provides a better understanding of how to improve the production of beta cells from human embryonic stem cells.
“At the moment, we can make stem cells develop into something that resembles proper beta cells. Our research shows that the current method produces cells that resemble alpha cells a little too much. However, the research has given us a better understanding of the steps stem cells go through when they develop into beta cells. In fact, we also show that the cells can develop along different paths, and still end up making the same type of beta cells,” says Anne Grapin-Botton, professor at the Novo Nordisk Foundation Center for Stem Cell Biology, DanStem.
Cells individually examined
The researchers have based their work on human pluripotent stem cells, which are able to evolve into any cell type in the body. Using known methods, the scientists analysed about 600 different cells on their path to beta cell differentiation and individually examined the cells to find out how much they molecularly resemble the beta cells.
In doing so, the researchers acquired important new knowledge about the way in which the cells develop and which genes play a role in this development. Notably, it was important that the genes NXK6.1 and MNX1 were activated for the cells to become beta cells in the end.
“This study takes an in-depth look at the molecular mechanisms on the cell level. We are not looking at what the average cells do, as other scientists have previously done – we are looking at all the individual cells. We are doing so in the hope that we can prevent cells from developing in the ‘wrong direction’. This work sheds light on the paths which the cells take in their development and how we human beings develop in the womb,” says Anne Grapin-Botton.
Start but do not complete the process
Alpha cells have the opposite function of beta cells. They must ensure that the body secretes the peptide hormone glucagon into the blood when the blood sugar level is too low. While the alpha cells cause the blood sugar level to rise, the beta cells ensure that it falls. And when the produced cells resemble the alpha cells too much, they are not optimal for treating diabetics.
“The cells definitely start the process of becoming either alpha or beta cells, but they don’t complete it. Here, we need to carry on researching to learn even more about how we can optimise the last step in the development of beta cells,” explains Anne Grapin-Botton.
The study was conducted in cooperation with project leader Christian Honoré from Novo Nordisk, and is supported by Innovation Fund Denmark, the Danish National Research Foundation and the Novo Nordisk Foundation.
Applications are invited for a Research Assistant position in the group of Prof Daniel St Johnston at the Gurdon Institute, University of Cambridge (http://www.gurdon.cam.ac.uk/research/stjohnston). The BBSRC-funded project aims to determine how epithelial cells organize apical-basal arrays of microtubules and how this is controlled by cortical polarity factors. Responsibilities will include establishing a method for the biochemical isolation of microtubule organizing protein complexes and their analysis by mass spectroscopy in collaboration with the Cambridge Centre for Proteomics and the generation of transgenic and mutant flies using CRISPR/Cas9.
Applicants must have a Bachelors or Masters level degree in a relevant area of Biology or equivalent experience. Expertise in protein purification, molecular biology and/or Drosophila transgenesis would be an advantage, although training can be provided where necessary. The post does not require a PhD qualification.
Our paper, like so many scientific findings, was brought about by a beer – or more specifically a discussion over a beer.
“I had a beer with David (Drechsel)” Jochen (Rink) said to me after one of our weekly scientific social events at the MPI-CBG. Over their beers they had discussed the challenges we were having imaging planaria and David had suggested we use Iodixanol as a supplement to increase the refractive index of mounting media – a suggestion which proved to be key to our success. While this particular problem and solution were quite straightforward, getting to the point where we could even recognize the problem and thus seek a solution involved a few more people (and a few more beers!) This is the story of how our paper, which describes a simple and straight forward method to correct for spherical aberrations of live tissues thus enabling significantly improved image resolution and quality in deeper tissue layers, came to be.
Tuning the refractive index of zebrafish cell culture media to RI 1.362 leads to improved signal to noise ratios along the z-axis. (Boothe et al, eLife 2017, https://elifesciences.org/articles/27240)
During my Ph.D. training in cell biology at the University of British Columbia in the laboratory of Jim (Johnson) I became used to thinking of microscopy as a tool rather than a challenge. This changed when I started my postdoctoral work, which focused on imaging cell dynamics during regeneration in planarian flatworms, in Jochen’s lab. Jochen warned me that this would “not be an easy” endeavor and, given how few tools were available for this relatively rare model system, I believed him. Once I started the imaging experiments and realized it was not possible to see any nuclear structure beyond the outermost cell layer however, I started to understand just how difficult the task I had taken on was. I took advantage of the lab’s species collection to obtain and test an unpigmented planarian species and exhausted the resources of our well-equipped light microscopy facility but the epithelium still seemed to act like a black-out curtain. At this point in time I did not have a well-developed understanding of optics in complex tissues however with the help of MPI-CBGs Moritz (Kreysing) and his student Alfonso (Garcia) I began to understand why a lack of pigment does not automatically mean that a tissue becomes optically clear. One of Moritz’ projects had seen a similar “black-out” effect when imaging dense retinal tissue and he had been able to overcome this challenge and image the deeper tissue layers by tuning the refractive index of their mounting media. While this was a bit different from what we were looking to do, as their work focused on fixed tissues, their results did suggest that the problem we were having could be caused by a significant difference in the refractive indexes of the planarian tissue and the aqueous mounting medium we were using at this time, and that if this were the case it might be possible to improve our images by tuning the refractive index of our mounting media. What we needed to do seemed clear however we still faced a challenge – while refractive index adjustment is a core component of state of the art clearing techniques in fixed tissues we had to find a component to tune the refractive index of our mounting medium to that of the sample without harming the live specimen.
I started by trying obvious candidates such as glycerol and sucrose but their high osmolality created a lethal environment for planaria. Halocarbonoils, which we tried next, worked well in fixed planaria but their hydrophobic nature made it impractical for an aquatic model system. After many trials and even more errors, I found myself at an institute social event sharing a beer and discussing these failures with Lennart (Hilbert). Lennart was studying DNA structure by live super-resolution microscopy in Nadine’s (Vastenhouw) lab at the time, and used BSA for refractive index tuning. While BSA was a promising candidate for planaria, the rather low refractive index tuning range and the viscosity and stickiness of saturated BSA solutions limited its usefulness. Lennart also found these limitations frustrating and so, while his approach at that time could not solve my problem, I did find myself a companion in the search for a live compatible refractive index tuning media supplement.
The postdocs had rounds of beers and discussions week after week but it was not until (of course) the PI got involved that we had our next breakthrough. At the time of Jochen’s forementioned beer with David we were focusing on epithelial cell dynamics because this was really the only cell type we could image in planaria due to the “black-out” effect. David, who was leading the MPI-CBG’s protein expression facility at the time, suggested over that famous beer we should give Iodixanol a try. Like many others he knew Iodixanol as a density gradient medium. While it was widely used for cell or cell organelle isolation, David was also aware though that the stock solution has a rather high refractive index which meant it might be able to meet our particular needs.
Refractive index tuning of planarian culture media by Iodixanol supplementation leads to a significant improvement of nuclei detection in deeper tissue layers. (Boothe et al, eLife 2017, https://elifesciences.org/articles/27240)
After performing some initial tests it became clear that Iodixanol was indeed the reagent we had been looking for. We were able to image past the first cell layer in planaria and significantly improved the live imaging quality in these specimens. Since we were not aware of any compound with similar properties we were eager to test it in other model systems. The diversity of MPI-CBG provided us with access to a number of different model systems and so we tried to improve live imaging in zebrafish embryos. Although this organism is thought to be easy to image, I knew of Lennart’s frustrations thanks to our earlier beers and was happy to share that there might be something which would help. Since imaging zebrafish was so far already of high quality Iodoxanol supplementation did not lead to the clear “day and night” effect we could observe with planaria. As I am a very self-critical (some would even call it pessimistic) experimentalist I started to doubt the broad applicability of Iodixanol but luckily Lennart has a more optimistic approach to things and just said: “Of course it works, it’s Physics.” – and sure enough it did.
In the end it was the diversity of the institute and the inter-lab interactions which led us to this story. We are happy that together we could establish with Iodixanol a compound which can now compensate for spherical aberrations in vivo and we are eager to hear how it works for the community – ideally over another case of beer!
Applied research team in Cape Town, South Africa, is seeking quick assistance from an adventurous soul who is a specialist in the expansion and differentiation of hESCs into neural progenitors and, ideally, also islet progenitors and hematopoeitic cells. The project is part of a clinical trial and will start in early October. We will provide a stem cell lab (we don’t have a bioreactor) and will cover travel and lodging cost and provide a stipend.
We are looking for a highly skilled and motivated candidate to join our group for a PostDoc position. In the Payer lab (http://www.crg.eu/bernhard_payer), we study epigenetic reprogramming in the mammalian germ line and the effects of ageing on fertility. In this project, which will be performed in collaboration with a fertility clinic, the prospective candidate will study molecular links between ageing and fertility decline in women.
We are seeking a candidate with a strong background in Mammalian Cell Culture, Stem Cell Reprogramming and Differentiation, Epigenetics, Reproduction and Molecular Biology. Excellent candidates from other related fields will also be considered.
Work Environment
Our lab is part of the Gene Regulation, Stem Cells & Cancer Programme at the Centre for Genomic Regulation (CRG) in Barcelona, Spain (www.crg.eu). The CRG is a vibrant International Research Institute with Research Groups working in diverse fields such as Genomics, Cell and Developmental Biology, Systems Biology, Stem Cells, Cancer and Epigenetics. English is the working language.
Eligibility
Candidates can be of any nationality, but must undertake trans-national mobility and must not have resided or carried out their main activity in Spain for more than 12 months in the 3 years prior to the call deadline. Furthermore, applicants working at CRG for more than 3 months before the deadline will not be considered.
Candidates must have a PhD degree from a recognized university, plan to obtain a PhD degree by the time of employment, or have at least four years of full-time equivalent research experience. Candidates who already hold a PhD degree at the time of application are eligible to apply only if they passed their PhD exam (or equivalent) in the four years prior to the call deadline. Exceptions up to 3 years for maternity/paternity leaves and other documented career breaks will be considered.
Candidates must have at least one publication as first author (either in press or published) at the time of the deadline
Candidates must provide two letters of reference
Fellowship
36 months by the INTREPiD Fellowship programme.
Applications are accepted exclusively online through:
We now seek to appoint a Research Technician in Molecular and Cell Biology to complement our existing expertise and fill a vacant position for the final 4 years of the “Cellular thyroid hormone availability: regulation of development and tissue repair, and pathogenesis of degenerative disease” project.
The Molecular Endocrinology Laboratory employs state-of-the-art high-throughput imaging and functional phenotyping, together with next generation sequencing and bioinformatics, in a whole organism and systems biology approach. You will receive comprehensive training in order to provide up-to-date technical expertise in cell culture, investigation of molecular mechanisms and signalling pathways, and skeletal phenotyping. You will also contribute to general administration and management of the laboratory.
Closing Date: Tuesday 3 October 2017 (Midnight BST)
Development often involves the asymmetric partitioning of cellular components to daughters, and this process is crucial for successful gametogenesis. Today’s paper, published in the current issue of Development, explores the cytoskeletal mechanisms of spermatogenesis in different nematode species. We met the multi-lab team behind the work, starting with Diane Shakes (The College of William and Mary in Williamsburg, VA), and then her collaborators André Pires-daSilva(University of Warwick, UK), Gunar Fabig and Thomas Müller-Reichert (Technische Universität Dresden, Germany), and Jessica Feldman (Stanford University, CA).
Diane, can you give us your scientific biography and the main questions your lab is interested in?
Diane Shakes
DS I got my start in research as a high school senior through a special program at NASA-Ames Research Center.There I was paired with a fantastic mentor, Patricia Buckendahl, who has talent for productively incorporating novice young scientists into her research quest which at the time was to understand the fundamentals of bone metabolism and why astronauts were losing bone mass in zero gravity. As an undergraduate at Pomona College, NASA-Ames continued to be my summer research home as I explored the breadth of biology in my coursework. Ultimately, I was captured by the wonders of cell biology and the big questions of developmental biology. It was also during this time that I developed an appreciation of the special insights that can be obtained by studying unusual organisms and cell types.
As a Ph.D. student at Johns Hopkins, I joined the research group of Sam Ward. This choice not only linked me not only to the early community of C. elegans researchers but also immersed me in the exciting research that was going on at the Carnegie Institution, Department of Embryology. In Sam’s lab, I worked alongside postdoctoral fellow Steve L’Hernault to isolate and phenotypically characterize a large collection of spermatogenesis-defective mutants in C. elegans. Through these studies, I developed an appreciation for genetics, a love for microscopy, and a life-long interest in the mechanisms of cell polarity. As I neared the end of my graduate studies, Ken Kemphues had just published his foundational study on the C. elegans PAR mutants, so I was excited to join his lab for my post-doctoral studies. My project was to analyze par-5, which like the other par proteins is required to establish proper asymmetries in the 1-cell C. elegans embryo, and ultimately was found to encode 14-3-3.
When I subsequently established my own lab, first at the University of Houston and subsequently at the College of William and Mary, I decided to use my combined knowledge of C. elegans sperm and oocytes to investigate a pair of C. elegans mutants that had been reported to exhibit both maternal and paternal effect defects.
Penny Sadler
Within my own lab, Penny Sadler discovered that although affected oocytes and sperm were both arresting in metaphase of meiosis I, the sperm continued to develop post-meiotically into anucleate sperm that could nevertheless crawl and fertilize oocytes.And in a fruitful collaboration with Andy Golden and a generous supply of mutants from the Bowerman and Seydoux labs, we showed that these and other mutants with the same phenotype were temperature-sensitive alleles of the anaphase-promoting complex. These studies that came out this work stimulated my interest in the interplay between the various cellular and developmental sub-programs of gamete development and drew me back into the analysis of C. elegans spermatogenesis, particularly in the stages leading up to meiotic divisions. The next set of studies – an analysis of the spermatogenesis-specific events during meiotic prophase – were carried out in collaboration with Diana Chu whose expertise in chromatin complemented my own in the cell cycle and cytoskeleton.
In addition to on-going studies in C. elegans, my group has also started using what we know about gametogenesis in C. elegans as a basis for comparative studies in other nematodes. A phone-call from André Pires da Silva got us specifically interested in the trioecious (male/female/hermaphrodite) species Rhabditis sp. SB347 (now called Auanema rhodensis), a lab cultivable nematode with strikingly non-Mendelian sex ratios. In many ways, these cross-species comparisons are analogous to studying a very informative mutant; but in this case, they help us distinguish highly conserved, fundamental processes from those that have been subject to variation over evolutionary time.
What was known about the cytoskeletal drivers of sperm development and asymmetrical positioning in worms before your study?
DS A conserved feature of sperm development in all organisms is that, following the meiotic divisions, sperm become streamlined by discarding unnecessary cellular components. In the early 1980s, Sam Ward’s group had shown, that in C. elegans, these unnecessary components included both actin and microtubules. This is only possible because nematode sperm motility is driven by a completely different cytoskeletal protein, the major sperm protein (MSP). Subsequent experiments with actin and microtubule inhibitors suggested that actin was more important than microtubules in this process of asymmetric partitioning. Subsequently, the L’Hernault and Titus labs showed that proper partitioning required the non-conventional myosin (myosin VI). Yet, no one had ever gone back to study the stepwise progression of events that underlies this wholesale swap of the cytoskeletal system during sperm development in C. elegans. Were there aspects of the process that could be better understood in light of new studies of asymmetric partitioning? Was the unusual partitioning event in R. sp. SB347 completely novel, or an informative variant of events that happen in all nematode sperm?
André – how did your collaboration with Diane come about, and why are different nematode species such useful models for the evolution of sex and reproduction?
André Pires-daSilva
APS Sex determination is a developmental switch prone to rapid evolution, but the causes and consequences for this pattern of evolution are poorly known. The existence of species with three sexes caught my attention because they are supposed to be extremely rare. In 2004 Marie-Anne Félix published a paper mentioning the free-living nematode strain SB347 (now named Auanema rhodensis), which produces males, females and hermaphrodites. Until that time, other free-living nematode species producing three sexes were not available in culture, or were parasitic nematodes difficult to work in the laboratory. Back in 2009, I contacted Diane Shakes to help me in characterizing the cytology of SB347 spermatogenesis, because we were trying to understand why males of this species generate so few males. This was especially puzzling, since heterogametic XO males should produce XX and XO progeny in equal proportions. However, we observed mostly XX progeny only. I contacted Diane because of her expertise in cell biology of C. elegans spermatogenesis and her recent interest in comparative work.
Gunar – I understand that an interest in C elegans sperm mutants brought you, Anna and Thomas into the collaboration?
Gunar Fabig
GF As a PhD student, I am working in the lab of Thomas’ on chromosome segregation in C. elegans male meiosis. Our lab has a strong expertise in live-cell imaging and electron microscopy. So, some years ago I started to image living C. elegans males by fluorescence microscopy to analyze the dynamics of meiotic chromosome segregation. We also started to characterize wild-type spindles of various stages at the ultrastructural level using electron tomography. During the initial phase of my project, I started to think about a comparison of wild-type and mutant data to study situations of impaired chromosome segregation. At the time, I was aware that Diane had a very interesting paper together with André, in which they characterized male meiotic spindles in Rhabditis sp. SB347 (now A. rhodensis). In this paper, they reported about a skewed sex ratio that was most likely caused by a changed meiotic “program” during male chromosome segregation. So we contacted Diane and André to ask whether they would be interested in collaborating with us on the ultrastructure of male meiotic spindles in Rhabditis sp. SB347.
Thomas Müller-Reichert & Anna Schwarz
TMR At the same time Anna started in my lab to work on her Master’s thesis and I proposed to her that an EM analysis of males of this species would be a very exciting project. She did a terrific job in preparing and analyzing SB347 males. Anna discovered the interesting patterns of organelles partitioning to the respective daughter cells.
Jessica – how did you get recruited, and how did your previous work on non-centrosomal microtubules fit into the story?
Jessica Feldman
JF One of the interests of my lab is to understand the mechanisms underlying non-centrosomal microtubule organization. C. elegans is a particularly good model in which to study this question as microtubule organizing center (MTOC) activity is completely reassigned from the centrosome in dividing cells to another site following mitotic exit. I had been exploring this switch in MTOC activity in a number of different cell types in C. elegans and started to focus on the germline, where MTOC activity is at the plasma membrane of non-dividing germ cells, and at the centrosome of mitotically dividing germ cells or meiotically dividing spermatocytes. I started to film this transition and found that microtubules and microtubule minus end proteins remarkably appeared to move from the centrosome of dividing spermatocytes to the residual body. This behavior appeared to mimic a similar movement of microtubules and microtubule regulators that I had previously seen in embryonic intestinal epithelial cells during their polarization, but due to the geometry of the movement in spermatocytes was much easier to visualize there. I presented this work at a conference that Diane was also attending. I think my live imaging data helped shed light on some of the observations Diane had been making about microtubules in fixed samples. In an incredibly gracious act, Diane contacted me to see if I would like to incorporate my data into this manuscript.
Can you give us the key results of the paper in a paragraph?
DS We knew from our earlier paper that the meiotic divisions of R. sp. SB347 male spermatocytes yielded a 50:50 mix of functional X-bearing sperm and residual bodies containing the other chromosomal complement. However we didn’t understand much about the details of the process or whether this pattern was an oddity of a single species. In fact, no one had described the stepwise process by which C. elegans sperm partition into residual bodies, not only unneeded cellular organelles but also their entire pool of actin and microtubules. In this study, we address all of these questions using the combined approaches of immunocytology, transmission electron microscopy, and live-imaging of GFP constructs. We show the first time that in C. elegans, the microtubules redistribute with their gamma-tubulin ring complexes intact from the centrosome to the sperm-residual body boundary in a process that resembles microtubule shifts in differentiating cells. At the same time, in a variation of normal cell division, actin reorganizes through a combination of cortical ring expansion and clearance from the poles. Relative to these cytoskeletal changes, organelles appear to partition in at least two phases; most partition just after the completion of anaphase chromosome segregation while others partition as the sperm detach from the residual body. In the much smaller spermatocytes of both R. sp. SB347 and its near relatives, the cytoskeletal remodeling events are restricted to the pole of the X-bearing chromosome set. Consequently, a partitioning process that is normally bipolar with two haploid sperm generating a central residual body becomes unipolar and generates one functional sperm and one DNA-containing residual body. Intriguing, this unipolar partitioning process also occurs in the XX spermatocytes of SB347 hermaphrodites. In contrast, partitioning is bipolar in the large spermatocytes of R. sp. SB347’s closest known male/female relative. Taken together, this study reveals two major insights. First, the process by which nematode sperm discard actin and tubulin into their residual bodies may be variations of common cellular and developmental processes. Second, constraints related to spermatocyte downsizing may have contributed to the evolution of a sperm cell equivalents of female polar bodies.
Fixed male gonad from C. elegans, from Figure 2 in the paper
What did the TEM bring to the story?
GF & TM Spermatocytes had to be visualized in whole worms, so we had to perform serial sectioning for this project. Anna mastered this without any difficulties. The beauty about electron microscopy is that one can get very detailed images of cellular organization (e.g. about centrioles, microtubules or the Golgi apparatus). For electron tomography, we prepared whole males of Rhabditis sp. SB347 by applying high-pressure freezing. Males were ultrarapidly frozen to liquid nitrogen temperature, freeze-substituted and embedded them in Epoxy resin. We took overview images of serial sections of several worms and counted the number of different organelles in numerous cells as worms contain many spermatocytes. This information enabled us to quantify the distribution of organelles during various stages of meiotic cell divisions in males.
Thin section EM and 3D models from serial electron tomographic reconstructions, from Figure 3 in the paper
What are the key open questions about the role of microtubules in the asymmetric cell division that makes nematode spermatocytes?
JF To me the most interesting questions about the microtubules in nematode spermatocytes are, 1) how is the centrosome inactivated as an MTOC?, 2) how are microtubules transported to the RB?, 3) what organizes microtubules inside the RB?, and 4) in Rhabditis sp. SB347, what controls the selective inactivation of MTOC activity at only one of the centrosomes? The answers to these questions will not only teach us about spermatogenesis, but will also answer major questions in the field of microtubule organization.
γ-tubulin localization during the separation phase of spermatogenesis in C. elegans. Movie 1 from the paper
What do you think the main evolutionary implications of the work are?
APS Three-sexed species are interesting, because they are probably evolutionary transitions between male/female and male/hermaphrodite mating system as known in C. elegans. They may help us understand in how mating systems evolve, a long-standing question in Evolutionary Biology. In this paper, we showed the mechanisms of ‘how’ males generate non-functional nullo-X sperm. This is one of the few examples in the literature describing cellular mechanisms of how heterogametic animals generate progeny of a single karyotype. There are examples for other nematodes and animals in other phyla in which crosses between XX and XO individuals generate mostly only XX progeny. Perhaps they use similar mechanisms as SB347, and this paper provides the foundation for testing this hypothesis. At the moment, however, we do not understand ‘why’ SB347 males get rid of their ‘male-making sperm’. According to evolutionary theory, this would happen if sibling matings are common. Unfortunately, we do not know much about the ecology of SB347 to test this.
DS From my perspective as a cell and developmental biologist, I think that this work highlights how a conserved set of cellular and developmental sub-programs can be co-opted for different outcomes. This study suggests that highly unusual process of partitioning microtubules into a residual body is not a novelty of nematode spermatogenesis but rather a variation of a common process during which differentiating cells lose their centrosomal microtubules as they establish a distinct population of non-centrosomal microtubules. Similarly, we show that the step-wise progression by which nematode sperm partition actin into the residual body exhibits many similarities to actin remodeling during the transition from anaphase to cytokinesis. Thus I predict that much of the underlying molecular machinery will be evolutionarily conserved with the notable exception of a novel regulator or altered feedback loop. Yet because these processes within nematode sperm are indeed a bit extreme, their analysis will yield important insights into related cellular and developmental processes.
Actin changes in R. axei spermatocytes, from Figure 5 in the paper
What next for the Shakes lab?
DS Our on-going analysis of R. sp. SB347 continues to surprise and delight us. There is still much to be learned about the molecular mechanisms of this partitioning event as well as other oddities of SB347 meiosis. For example, we also have a paper coming out this month in Developmental Biology, in which we describe how this clade evolved a distinct solution for acquiring hermaphrodite self-fertility, namely the simultaneous rather than sequential production of oocytes and sperm. So in the R. sp. SB347 realm, we have many interesting avenues to explore. At the same time, the C. elegans focused cohort in my lab are hard at work gleaning new insights from the existing collection of spermatogenesis mutants, some of which have been languishing in our liquid nitrogen tanks since my graduate days.
Any plans for future collaborations?
Yes, Andre and Diane’s lab are collaborating on a project that describes differences in meiosis in SB347 males, females and hermaphrodites, and the labs of Andre, Diane and Thomas are collaborating on a project to identify the signal that determines the directionality of the asymmetric division of the SB347 male spermatocytes.
Anything else you would like to add?
Ethan Winter
DS One voice that is missing in this interview is the lead author Ethan Winter, a former undergraduate honors student in my lab who helped spearhead this project through his cytological studies of R. sp. SB347 and its near relatives. Ethan continued on to Harvard to pursue a Ph.D. in Chemical Biology. Not only was he brilliant, hard-working, and tenacious, but he was also kind, patient, and possessed a wonderful quirky sense of humor. We all predicted that he was on track to develop into a beloved professor. Sadly, Ethan passed away last October (2016). I hope that this paper serves at least in a small way as a tribute to his scientific accomplishments and his too-short life.
Mouse embryo that has been injected with embryonic stem cells grown in Insulin showing these cells can make all cell types. The embryos are at the stage when they are making all the different basic cell types of the future body, gastrulation. The stem cells were labelled genetically so they fluoresce in red under the microscope.
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