Applications are invited for a postdoctoral research assistant/associate to join the group of Dr Emma Rawlins at the Gurdon Institute, University of Cambridge to work on the regulation of human lung development with the long-term aim of developing strategies for regenerative medicine (http://www.gurdon.cam.ac.uk/research/rawlins). Our recent research has focused on cellular mechanisms of lung development, homeostasis and repair using the mouse as a model system (e.g. Balasooriya et al., Dev Cell 2016; Laresgoiti et al., Development 2016; Watson et al., Cell Reports 2015). We have now established novel in vitro systems for studying human lung development which will be the primary focus of our work over the next few years. We aim to recruit an outstanding individual who is interested in lung developmental mechanisms, their contribution to disease development and therapeutic potential. The research will combine the application of single cell techniques to human embryonic lungs with the in vitro genetic analysis of mechanisms using our organoid system.
Applicants should have a PhD in a relevant subject, or be close to completion of their degree. Expertise in general areas of developmental/stem cell biology including single cell biology, live cell imaging, image analysis and cell signalling mechanisms would be suitable for these positions. Experience of in vitro models and use of experimental animals would be an advantage.
Salary: £29,301-£38,183
Closing date: 15 June 2017
For further information and to submit an application for this vacancy visit
The recent bloom of genomic data from all of life’s kingdoms is revealing a novel perspective of gene loss as a pervasive source of genetic variation with a great potential to generate phenotypic diversity and to shape the evolution of gene networks. How do genes become dispensable and subsequently lost? Are patterns of gene loss stochastic or biased? What is the effect of gene loss on the evolution of gene networks? What is the influence of gene loss on the evolution of mechanisms of development (that is, ‘Evo-Devo,) and the diversification of species?
I am Alfonso Ferrández a PhD student working in the group of Prof. Cristian Cañestro and Prof. Ricard Albalat (@EvoDevoGenomeUB) in the Section of Biomedical, Developmental and Evolutionary Genetics in the Department of Genetics (@GeneticsUB), Microbiology and Statistics, and in the Institute of Biodiversity Research (@IRBioUB), of the University of Barcelona, Spain (Fig. 1). We are trying to address some of the previous questions related to gene loss in the field of Evo-Devo using a curious chordate called Oikopleura dioica, which despite having suffered an extreme process of genome compaction along with massive gene losses, still preserves a typical chordate body plan.
Fig. 1: The 2017 Barcelona Oikopleura team.
Why did we choose O. dioica?
Fig. 2: O. dioica inside the mucous house.
Ecological relevance: O. dioica is a peaceful filter-feeding and free-swimming planktonic organism, about 2-3 mm long. It lives inside a secreted mucous house that it uses as a food trapping device by filtering the water current propelled by its stylish and grooving tail movements (Fig. 2). O. dioica is the only known urochordate species that have separated female and male individuals, which are indistinguishable until maturity. O. dioica has a cosmopolitan world-wide distribution including seas of Europe, Asia and America and it is so abundant in the zooplankton community that plays a key role in marine trophic webs serving as food for fish larvae. Moreover, because of the small size of the pores of their mucous houses, they can also trap the smallest microalgae, thus creating a short circuit that accelerates the transference of organic matter both through the marine trophic web and towards vertical flux of carbon-rich organic material (i.e. marine snow) that sinks to the bottom of the oceans.
Phylogenetic position within our own phylum: O. dioica belongs to the larvacean class inside the urochordate subphylum, the closest sister group to vertebrates. Urochordates diverged prior to the two rounds of whole genome duplication (2R-WGD) that occurred early in the vertebrate lineage (Albalat and Cañestro, 2016; Cañestro, 2012), and consequently, the mutational robustness of most gene networks appears to be much lower in O. dioica than in vertebrates (Fig. 3). The availability of many deeply sequenced genomes of several other chordates –3 species of cephalochordate, 10 urochordate ascidians, and >100 vertebrates– provides a perfect phylogenetic framework for the identification of gene loss events in O. dioica by comparative genomics with other chordates.
Fig. 3. Chordate phylogeny. O. dioica belongs to the Larvacean class inside the Urochordate subphylum, sister group of vertebrates.
Genomic plasticity: O. dioica has the smallest chordate genome known so far with only 65Mb (even smaller than the 175 Mb of Drosophila or the 100 Mb of C. elegans), which results from an extensive process of compaction that has been accompanied by an extraordinary amount of gene losses. One striking example is the loss of all genes of the non-homologous end joining DNA repair system, plausibly one of the reasons that accounts for the elevated propensity for gene loss of this organism. Among key developmental genes, O. dioica has lost more than 30% of the homeobox gene groups, including all central Hox genes, and key genes involved in retinoic acid (RA) signaling (Albalat and Cañestro, 2016; Cañestro et al., 2006; Denoeud et al., 2010; Edvardsen et al., 2005; Martí-Solans et al., 2016; Seo et al., 2004).
A simple and transparent model for Developmental Biology: The embryonic development of O. dioica is very fast, and in less than 20 hours a transparent juvenile already shows a typical chordate body plan with organs that are unequivocally homologous to those in vertebrates, including a notochord anchoring muscle cells throughout a post-anal tail, a dorsal neural tube, brain, thyroid, pituitary, gill slits, pharynx, esophagus, gut and a heart (Fig. 4). In addition, O. dioica shares with other urochordate species, such as ascidians, a very similar embryonic developmental program, both at the morphological and molecular level, with the important difference that O. dioica does not suffer the drastic metamorphosis that ascidians do, and therefore, in contrast to ascidians, maintain all chordate features throughout its life.
Fig. 4. Organ homologies between an Oikopleura and a zebrafish larvae.
A day in the life of an Oikopleura lab
Our laboratory is one of the few laboratories in the world able to culture O. dioica all year round, which means almost unlimited availability of biological material (i.e. mature males and females, eggs, sperm, embryos and larvae) to carry out functional experiments. We maintain them at 19ºC, which results in a generation time of only 5 days (Fig. 5). Our lab has set up a low-cost maintenance regime by reducing as much as possible the amount of water, space and manpower to handle the animals (Martí-Solans et al., 2015). All these characteristics, together with its high fecundity and transparency makes it an attractive model for developmental studies.
Fig. 5. O. dioica facility in the University of Barcelona (Catalonia, Spain). (a) Animals were collected in the coast of Catalonia near Barcelona using a plankton net or directly with a bucket. (b) Seawater is filtrated at 50–20 µm (fSW) in the facility to remove excess of sand particles that could affect O. dioica buoyancy. (c) The production of the four microalgae for O. dioica feeding (Bouquet et al., 2009) was scale down in an adaptable fashion to the weekly needs of the facility (round-bottom glass flasks in upper shelves). Long-term stocks (100 mL Erlenmeyers in lower shelves) were renewed just once per month. (d-g) The use of agar plates provides an alternative method to maintain long-term microalgal stocks: (d) Isochrysis sp., (e) Chaetoceros calcitrans, (f) Rhinomonas reticulata and (g) Synechococcus sp. (h) O. dioica animals were maintained in suspension by the rotation of a paddle driven by a motor mounted on the lid of polycarbonate beakers. (i) Animal lines were maintained in a small room (5 m2) in four shelves (1.5 m2) at 19°C using a standard air conditioner device. (j) Protocol of husbandry. (Martí-Solans et al., 2015).
A typical day in the Oikopleura lab starts looking for ripe animals to mate and start a new generation (this happens early in the morning, since we have synchronized the animals to mature at that time of the day). At day 5 of their cycle, males and females are easily distinguishable. Males have a yellowish gonad full of sperm, whereas females have a refringent and translucent gonad full with 100 to 400 eggs (Fig. 6 K). Since O. dioica has external fertilization, to mate them we only have to put together about 20 females and 10 males, and wait for their spontaneous spawn that will give rise to the next generation. Both males and females naturally die after the spawning, which unfortunately does not allow us to keep the parental generations. Next day (day 1), we are normally happy to see hundreds of juveniles beating their tales inside their already inflated houses. Yes! They have a brisk development! The first division occurs just 20 minutes after fertilization; by 4 hours after fertilization (hpf) they break their chorion, and few minutes after the hatch we can already see them graciously twitching their tails in their first attempts to swim. By 5 hpf the tail movements are rhythmic and harmonious, which allow them to swim up and being suspended in the column of water. By 8 hpf, the heart is vigorously beating, and the ciliary rings are working at full speed creating the water to circulate through the pharynx. By 9 hpf, the animals do the tailshift, characterized by the shift of the tail to an acute angle relative to the trunk, flagging the end of embryonic development, and competence to secrete and inflate their first filter-feeding house (Fig. 6).
From day 1 to day 5, we need to feed them every single day (weekends and bank holidays included, aggh!), and to transfer them to fresh seawater to keep them happy, and not too crowded. Their diet consists of a mix of four different species of algae that we also grow in the lab, at different ratios depending on the size and needs of the animals each day of the culture (Fig. 5 c-g).
Functional approaches of gene knockdown or inhibition are amenable. The syncytial gonad of females is easy to inject with RNAi, morpholinos or even, the recent discovered DNAi (yes, dsDNA rather dsRNA of your target gene…cheap and effective), obtaining a massive generation of knockdown embryos (Omotezako et al., 2015). Moreover, permeability and small size of O. dioica embryos allow us to easily treat them with drugs or specific inhibitors of signaling pathways to modify their developmental programs.
Fig. 6. Embryo development in O. dioica is very fast. (A) Two cell estage embryo 30 minutes post fertilization. (B-D) From 2 to 4 hours post fertilization (hpf) we can identify the tailbud stage in which the embryo resides inside the corion. (E-H) At 4 hpf the embryo leaves the corion and became a swimming larvae during the hatchling stages. (I) The metamorphosis, that only consist in a 180º rotation of the tail, takes place 9 hpf giving rise to the Tailshift embryo. (J) Adult animal of 2 days of life. (K) Adult animals of 5 days of life. The female gonad contains hundreds of eggs, the male gonad is dark and contains the sperm.
Current research lines
To address the fundamental question of how gene loss affects the evolution of the mechanisms of development, as a case study, our research focuses on the analyses of the striking loss in O. dioica of the retinoic acid (RA) signaling pathway, which is conserved and essential for many developmental and physiological roles in all other known chordates. First, we have described a process of gene co-elimination of nearly the entire classic metabolic and signaling pathways. This analysis allowed us also to recognize surviving genes to the dismantling of those pathways, and to recognize processes of neofunctionalization and hidden pleiotropy as the probable causes that preserved the genes of vanishing. Currently, our focus of attention is on O. dioica heart development, since RA plays a fundamental role in all other chordates. Finally, two new lines of applied research are starting to fly in our lab, using O. dioica as an evolutionary knockout model to study the genetic bases of some human cardiomyopathies, as well as a model to better understand the limits of the genetic responses of chordate development towards environmental threats from anthropogenic origin such as heavy metal from industrial wastes or global warming (but these are two long new stories that would need another thread in the Node).
References
Albalat, R., Cañestro, C., 2016. Evolution by gene loss. Nat. Rev. Genet. doi:10.1038/nrg.2016.39
Research assistant position for subsequent appointment as PhD fellow in ‘Epithelial cell renewal’ to join the Sedzinski lab.
The Danish Stem Cell Center (DanStem) at Faculty of Health & Medical Sciences at the University of Copenhagen is looking for a Research assistant subsequent appointed as PhD fellow to join the Sedzinski group starting September 2017 or upon agreement with the chosen candidate.
The position as Research assistant is for 1 year. The position as PhD fellow is for 3 years.
DanStem comprises of two sections: The Novo Nordisk Foundation Section for Basic Stem Cell Biology, where we address basic research questions in stem cell and developmental biology (BasicStem). The second Section for Strategic Translational Stem Cell Research and Therapy (TransStem) is focused on the translation of promising basic research results into new strategies and targets for the development of new therapies for cancer and diabetes. Find more information about the Center at http://danstem.ku.dk
We are seeking a highly motivated and ambitious candidate to join the Sedzinski lab with the following project:
Job description
The Sedzinski lab (http://danstem.ku.dk/research1/sedzinski-laboratory/) is interested in understanding mechanics of epithelial tissue homeostasis and morphogenesis. Particularly, we want to determine both the mechanics and molecular regulation of epithelial cell renewal. For this, we study how forces generated by the actomyosin cytoskeleton and adhesion molecules shape and move epithelial cell progenitors within tissues. We use high-resolution microscopy to image dynamics of progenitor cells, biophysical and theoretical methods to describe the forces, and genetic to perturb the system.
We are seeking highly motivated and ambitious candidates to join our team.
Qualifications
Candidates must hold a master’s degree in biology, biophysics, biochemistry, bioengineering, bio-informatics, or similar, and possess a general understanding of cell and developmental biology.
Previous practical experience in quantitative biology, biophysics, computational biology, microscopy, and image processing is considered of great advantage.
Publications and practical experience are beneficial.
Good English communication skills, both oral and written, are prerequisite for the successful candidate
Terms of salary, work, and employment
The employment is for 4 years, as research assistant is for 1 year and as PhD fellow for the following 3 years, and is scheduled to start on September 2017 or upon agreement with the chosen candidate. The employment as a PhD student is conditioned upon a positive assessment of the candidate´s research performance and enrolment in the Graduate School at the Faculty of Health and Medical Sciences. The PhD study must be completed in accordance with the ministerial orders from the Ministry of Education on the PhD degree and the University´s rules on achieving the degree.
The place of work is at DanStem, University of Copenhagen, Blegdamsvej 3B, Copenhagen. Salary, pension and terms of employment are in accordance with the provisions of the collective agreement between the Danish Government and AC (the Danish Confederation of Professional Associations). In addition to the basic salary a monthly contribution to a pension fund is added (17.1% of the salary).
The application must include
1. Motivation letter
2. Curriculum vitae incl. education, experience, previous employments, language skills and other relevant skills
3. Copy of diplomas/degree certificate(s)
Questions
For further information about the position please contact group leader Jakub Sedzinski, jakub.sedzinski@sund.ku.dk
How to apply
The application, in English, must be submitted electronically by clicking APPLY below.
The University of Copenhagen wishes to reflect the diversity of society and welcomes applications from all qualified candidates regardless of personal background.
Only applications received in time and consisting of the above listed documents will be considered.
Applications and/or any material received after deadline will not be taken into consideration.
The application will be assessed according to the Ministerial Order no. 284 of 25 April 2008 on the Appointment of Academic Staff at Universities.
Assessment procedure
After the expiry of the deadline for applications, the authorized recruitment manager selects applicants for assessment on the advice of the Appointments Committee. All applicants are then immediately notified whether their application has been passed for assessment by an expert assessment committee. Selected applicants are notified of the composition of the committee and each applicant has the opportunity to comment on the part of the assessment that relates to the applicant him/herself. You can read about the recruitment process at http://employment.ku.dk
Application deadline: June 30 2017
Founded in 1479, the University of Copenhagen is the oldest university in Denmark. It is among the largest universities in Scandinavia and is one of the highest ranking in Europe. The University´s eight faculties include Health Sciences, Humanities, Law, Life Sciences, Pharmaceutical Sciences, Science, Social Sciences and Theology.www.ku.dk
Innovations in science are happening all over the nation! Visit the science videos at the 2017 STEM for All Video Showcase, funded by the National Science Foundation. Interact with the community by asking questions, getting answers, and voting for your favorite video! Support STEM education and let your voice be heard from May 15-22 at the STEM for All Video Showcase!
Your vote counts!
Did you see an amazing video at the showcase? Let us know! You can vote for your favorite video through Twitter or the showcase website. The video with the most votes will be given the “Public Choice” award on Tuesday, May 23, and the winning participants will be recognized by the National Science Foundation!
Do you support STEM? Let the world know!
You make the difference. Please share the STEM for All Video Showcase with your friends and colleagues, on social media, and with your local science centers. Help us spread the word about the amazing people working in STEM education and the awesome work they are doing for our communities!
More details on the event:
The Stem for All Video Showcase is funded by the National Science Foundation. This year’s theme is “Research & Design for Impact.” In short 3 minutes videos, leaders of STEM education initiatives describe how their projects are meeting some of today’s most pressing scientific and technological challenges. The STEM for All Showcase is a collaborative effort of the following NSF resource centers: MSPnet, CADRE, CAISE, CIRCL, STELAR, CS For All Teachers. It is funded by the National Science Foundation (#1642187).
The Showcase is powered by the Videohall.com platform developed by TERC, a STEM-focused education research nonprofit based in Cambridge, MA.
I had started to become a little worried when I didn’t see Eric on the opening day of the conference, but it turned out that his plane to Germany had been delayed by the snowstorms blanketing the Eastern seaboard of the US and he made it in the end. Between sessions later on, we found a slightly chilly empty computer lab to sit down in, and over the course of an hour talked about his history in research and views on science in general. My lasting impression was of an enthusiasm and passion for research that was by equal measure refreshing and infectious – it made me want to look down a microscope again!
Eric Wieschaus is a HHMI Investigator, and the Squibb Professor in Molecular Biology at Princeton University and the Lewis-Sigler Institute for Integrative Genomics. In 1995, he was awarded the Nobel Prize jointly with Edward Lewis and Christiane Nüsslein-Volhard for discoveries about the genetic control of Drosophila embryogenesis. We caught up with Eric at the joint meeting of the German and Japanese Societies of Developmental Biologists held in Kiel in March 2017, and discussed his career, his thoughts on the field and the impact the Nobel award had on his life.
Eric in the lab
I understand that after your first experience with Drosophila – as an undergraduate in Harvey Bender’s lab at Notre Dame – you said never wanted to see a fly again! What led you to change your mind?
I suppose the formative undergraduate experience wasn’t washing dirty fly bottles and making fly food, which is what I had done in Harvey Bender’s lab, but an embryology course where we looked at frog and chick embryos, which were the embryos people worked with at the time. I knew then that I wanted to be an embryologist, to study development. But I think I just didn’t know that flies had embryos! Anyway, I ended up at Yale in New Haven, and for various reasons I was assigned to the lab of Donald Poulson. Poulson had, in the 1930s and 1940s, first as a graduate student and through to his professorship, described all of Drosophila embryogenesis. So I knew that I wanted to study embryos, and I had by chance landed in the laboratory of the one person in the world who really knew how Drosophila embryos develop. Poulson was a very kind person: he took me in to his lab when he specifically didn’t want to have a graduate student. It took him a little while to find a young assistant professor who was willing to take me on, and that turned out to be Walter Gehring.
And as a graduate student with Gehring, you were trying to map cell lineage in Drosophila with somatic clones and disc transplantation. What was the drive behind this work?
Walter had developed a tool to culture embryos, based on the strategies people had used to culture imaginal discs. He was able to convince me, a naïve young graduate student entering the lab, that the best experiment in the world would be to isolate a single cell from the blastoderm, grow it in these culture conditions and measure its developmental capacity, to establish whether it had already undergone some determination or programming. And that was my project – I spent my first three years grinding up embryos and trying to get single cells to grow. I have to say that none of that ever worked, but at some point I decided that I needed to have a control – once I had these wonderful cultures going, and these cells displaying their inherent potential, I wanted to be able to compare them to what a blastoderm cell actually did if left in situ. I decided I would produce clones at the blastoderm stage and follow those cells, but I never really looked at them because I still believed my experiments would work. But at the end of four and a half years, when I realised that I wasn’t going to have cultured cells as part of my thesis, I decided to go back and look at the controls. Luckily, it turned out that what cells normally do in development is almost as interesting as what you can get them to do when you artificially manipulate them.
When Walter’s lab moved to Basel, you were brought into contact with Christiane (Janni) Nüsslein-Volhard. How did that work out?
Janni and I both finished our thesis work at around the same time – she was in Tübingen, and having done molecular biology was an attractive person for Walter to bring to his lab. She actually came to Basel specifically not to do molecular biology, but instead to learn how to look at embryos, and as I was the only person in the lab who was working with embryos, we bonded over that in the four months before I left. And later, we both were fortunate to get positions in Heidelberg – these were real group leader positions, but we didn’t know that we had to bargain for space! So we ended up together, sharing a lab, and we already knew kind of what we wanted to do; it was a perfect set of circumstances.
And in Heidelberg, you began work on the screen that would eventually win you the Nobel Prize. Screens obviously require a lot of time and effort: how did you stay focussed and cope with the practical demands at the time?
Well it was a lot of work, but the payoffs were real. We spent a certain amount of time figuring out how to do the screen, and that was maybe more frustrating than actually doing it. But once we started the big screen, scaled up and lasting about two and a half months, we discovered something new every day, and so focus was not at all hard. We would work from nine in the morning to around midnight, pretty much every day of the week, but the payoff was already there – not in understanding as such, but in the realisation that we were seeing stuff that no one had ever seen before, and thinking thoughts that no one had ever thought before. I don’t even remember being frustrated or wanting to slow down at all – my memory of that time is just of the fascination and excitement of doing it.
We were seeing stuff that no one had ever seen before, and thinking thoughts that no one had ever thought before
Was there a key lesson the screen taught you about how development works?
Before we did the screen, we really didn’t know what we would get out of it. The two dangers were that every gene that you mutated would mess up development, and that the phenotypes would be complicated and heterogeneous and impossible to interpret. But what actually happened was that the number of zygotically active genes we found was small – about 120 to 130 – and the mutants had unique phenotypes that identified specific processes, which meant you could immediately group things. This small number contrasted with the characterisation of RNA heterogeneity carried out at the time, which estimated there were thousands of different mRNAs present in the embryo, but most of these turned out to come from the mother. So the conceptual lesson was that the embryo uses transcription of a small number of genes to drive specific events forward. If gene products can be supplied to all cells, they’re provided maternally, but if you want to turn the gene on at a specific place or at a specific time, zygotic transcription does that. That was the power of the screen, to pick up these regulators: the genes controlling developmental decisions. It seems quite obvious now, but did I predict that at the time? Absolutely not.
Following the screen, you moved your lab to Princeton in 1981. What did you hope to achieve in the early days in New Jersey?
Initially, when we returned to Princeton, I helped Trudi (Schüpbach) a bit when she was setting up her maternal effect screen to find the determinants deposited in the egg by the mother. But what I really wanted to understand was morphology, and this very quickly became a cell biological problem, of how cells move and change shape and so on. This obviously required a lot of observation, and I remember at the time the great discovery was that there was something called phalloidin (which labels F-actin) – it was fluorescent, and you could buy it from a supplier! You could get a fluorescence microscope and just look – it really allowed you to see embryogenesis differently. So this period was also a time where, as well as advances in molecular biology, there were great advances in cell biological reagents, which you could begin to apply to embryos to think about these processes from a more cell biological perspective. It all came together – the observation of the real things that cells do, and their manipulation by genetics (a tool that cell biologists did not have at the time).
And how did you approach the cell biological side of development, particularly since many of the factors involved would have been missed by the screen?
The screen picked switches, decision-making mechanisms: most of these are transcription factors and the cell signalling pathways that govern them. Once you go downstream of decisions, it becomes more complicated and more synthetic, and less easily approachable by a genetic screen that looks for survival, for instance. I think that genetic manipulations are still really powerful here, but you can’t take shortcuts like only looking for genes essential for viability. A number of biological processes go into a change in cell shape, for example, and if you remove one of them, the cell shape transition may not occur in the same way, but the animal may be robust enough that a proportion of embryos survive and you’ll miss it. So understanding morphology turns out to be a process where you use genetics, but need other tools too, and much of this concerns imaging. I think that a major transformation in biology has been the development of microscopes of different kinds, whether confocal or light sheet, or whatever. And digital cameras! To those of us who can remember, images used to be things you had on film and you would spend artistic moments in the darkroom trying to bring out what you wanted; now they are just a matrix of pixels. And I think that aspect – doing microscopy on embryos, and doing it in a quantitative way – meant you learned stuff that you didn’t know before. It’s just transformed the field.
The Wieshcaus lab’s focus on the cell biology of development includes work on nucleolus assembly. In Falahati, et al. (Current Biology, 2016), the dynamics of the nucleolar protein Fibrillarin were tracked in a mutant embryo lacking rDNA repeats
To look at embryos with a quantitative eye, your lab has in recent years incorporated physics and mathematical modelling. From your background, to what extent do you personally need to understand the physics and the maths?
Well I’m totally dependent on having collaborators who have the patience to explain things to me three or four times, and also the tolerance to accept when I decide that I’ve got it, even though there might be a deeper level of understanding available that they feel I haven’t reached yet. But it’s also an interesting question to ask what your thresholds are for understanding: I think you set them, and that sets the style of science that you do. The fact that different scientists have different thresholds is one of the reasons why it is so valuable to collaborate, and also why the social nature of science is so important to its productivity. You profit from talking to people who are interested in working with you, but frankly not willing to invest the same amount of energy into your topic as you do. Of course there’s always danger in not understanding everything, but we live in a dangerous world. Scientifically, if you are going to be on the edge of discovery, you just take the best path forward even if you don’t understand everything in a deeply defendable way.
As someone who has worked on Drosophila for 40 years, what do you think this animal still has to teach us about development?
Science is hard, and in biology we are only at the beginning of a truly deep understanding. To understand development, you work on embryos, and you choose a model organism because to do things well, you need the technologies that everybody else has developed; you need to parasitise a field.
Working with Drosophila today, you have two avenues. If you want to strike out into some totally unexplored area, for instance in something behavioural or evolutionary, flies allow you to do this. And then there are the old areas, things that we think of as established and successful problems, like patterning in the early embryo. What we have there is actually a wonderful cartoon understanding of the process, and this is so rare and valuable in biology because it can provide the basis for building a deeper understanding. So if you want to understand transcription, for example, I think you’d be crazy not to work in flies! In flies you have this organism where you can study a real process with biological meaning that has been selected for in evolution, and which is primed by our existing knowledge for you to start. So the things that we know prime us to go deeper, and for the things that we don’t know, we’re in a position where we can profit from the community and the tools that are available.
In biology we are only at the beginning of a truly deep understanding
It’s now 22 years since you won the Nobel Prize along with Christiane Nüsslein-Volhard and Edward Lewis. What did the award mean to you at the time, and does it still have an impact on your academic life?
Of course it was a wonderful thing to happen for me and my family. It wasn’t really anything I’d foreseen, and then I got ripped out of my normal life for a week in Stockholm. But I also felt like it was wonderful for the community as a whole: at least from my perspective, my colleagues were happy because they saw it as recognition of a set of accomplishments that the field had made.
The other thing that it gave me was a certain power to control my work life. I’ve never assumed major administrative roles at the university – it gave me the power to insist that I would continue to work four hours a day at the bench myself, and meant that people were happy to have me around even if it forced some others to assume extra responsibilities. I could set certain standards for my life: I wanted to be a productive scientist, not to just run a lab and tell other people what to do. I wanted to be the person who did interesting things.
You’ve described yourself as a very visually oriented person, both in terms of science and as someone who loved to draw and paint in your youth. Does this dictate the way you do your science?
Since the public is giving their own money to support science, there are limited resources, an obligation to produce and a certain competition among scientists. So as a young scientist you have to ask yourself – what is my competitive advantage? What am I good at? And almost inevitably that means: what do I enjoy? And the answer will go deeper than science itself. So when I look at most things that I’ve done in the lab, they’ve always had a visual component to them. In the Heidelberg screen, we identified mutants based on our abilities to look at things and recognise what was worth following up on. And to look at gastrulation is a matter of reconstructing and visualising form, and form follows force, so an understanding of physics, of flow and force and mechanics, has, to my mind, at least a strong visual component to it. I am the scientist that I am because of the talents and particular proclivities I have, and clearly painting was another expression of that same quality.
And do you still paint?
I’ve got back in to painting, but it’s challenging because I think to be really good, you have to invest more time than I have. I’m still too hung up on the idea of getting a product at the end, getting the picture to work, rather than doing a hundred versions of it to figure out how to get it to work. Every step in becoming better costs time, which of course is not very different from science, where to be really great, you have to invest and risk, and do things a bunch of times. That’s why science is not a nine to five job: so much of what we do doesn’t quite work out!
What would Development readers be surprised to find out about you?
I am an aggressive card player, and I play to win (though I must say don’t win all the time!). Trudi and I have trained all of our children to be aggressive pinochle players, and also to play to win. Pinochle is different from poker: with poker there is an element of bluff, and while I am a theatrical enough person that I could pull that off, I feel so emotionally dishonest! So I tend towards card games like pinochle where you are confronted with a problem and have to figure out how to win with the cards you have. As long as I don’t have to be dishonest about it!
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A postdoctoral BBSRC funded position is available to study role of Par-3 in E-cadherin recycling and signalling in the group led by Dr Natalia Bulgakova http://www.sheffield.ac.uk/bms/research/bulgakova
The focus of the Bulgakova group is on discovering roles and regulation of cell-cell adhesion using Drosophila as a model organism. We have recently discovered that the protein Par-3 maintains the key cell-cell adhesion protein E-cadherin within the recycling route. The successful applicant will study the mechanism of this Par-3 function and its role in the development and physiology of the animal. The project will employ a combination of molecular genetics, live imaging, and protein biochemistry.
The successful applicant will have a PhD or equivalent experience relevant to studying cell-cell adhesion or intracellular trafficking, knowledge of working with genetic model systems and be highly motivated. Experience in using Drosophila, molecular biology, protein biochemistry, and confocal microscopy, and knowledge of cell-cell adhesion, cell polarity and intracellular trafficking will be an advantage. The successful candidate will be expected to have excellent interpersonal and communication skills, be highly independent, and committed to research in a fast-moving and competitive field.
Fixed-term with an immediate start date and an end date of 31 March 2020
For more information and to apply, please see http://www.jobs.ac.uk/job/BBL246/research-associate/
The publication of Marc Tessier-Lavigne’s seminal Cell papers (1, 2) in 1994 describing the identification of netrin1 (from the Sanskrit word, netr, meaning “one who guides”) was a defining moment in my graduate career. My friends and I talked about those papers for weeks, from the incredible technical feat, the biochemical purification of netrin1 from tens of thousands of chicken brains, to the commonality of the neural developmental mechanism, based on the homology of netrin1 with Unc6, a gene previously identified by Ed Hedgecock and Joe Culotti in a C.elegans screen for axon guidance defects (3).
A couple of years later, I interviewed for postdoctoral positions in San Francisco Bay area in neural development laboratories, and had arranged to stay with Marya Postner, a fellow Princeton graduate alumnus. Marya just happened to be married to Tito Serafini, who, with Tim Kennedy, was one of two lead postdocs on the seminal 1994 Cell papers. Tito was still working with Marc at the time. I asked him breathlessly about the status of the netrin1 project – did they have a netrin1 loss of function phenotype yet? Tito looked happy, triumphant even. “Yes!” he said. “We’ve knocked out the mouse homologue. Commissural axons stall before they reach the floor plate.”
This Cell paper came out a couple of months later (4) and cemented the idea of chemotaxis as the prevailing model of axon guidance (5): netrin1 is secreted from floor plate (FP) cells at the ventral midline of the spinal cord, and like a beacon in a harbor guiding ships in the night, orients commissural axons to grow towards it (Fig. 1A). This model fit with insight dating back as far as Cajal (6), including work performed in grasshoppers (7, 8) and then in vertebrates (9, 10). Together, these studies suggested that axons could be guided in a stepwise manner over long distances by chemotropic attractive or repellent cues diffusing from “guidepost” cells that they encountered along the way. The axon guidance field also recognized that axons also grew on local substrates provided by extracellular matrix components, such as laminin. But their contribution was generally considered prosaic, i.e. passive carpets that permitted or prevented axon growth. The more important and interesting contribution to neural circuit formation came from the chemotropic cues, the netrins, semaphorins, the slit/robo pathway and morphogens.
Problems with the chemotropic model for netrin1
I have taught the chemotropic model of axon guidance for years in my undergraduate and graduate lectures, with netrin1 as the centerpiece discovery. It was a beautiful example of scientific daring being rewarded with mechanistic understanding. I was able to trace a path from the first century-old scientific insight to the netrin1 mutant phenotype that strongly supported that hypothesis.
However after starting my own laboratory in 2004, I began to wonder about a reported, but underappreciated, feature of the netrin1 expression pattern. Textbooks often show the distribution of netrin1 in the chicken spinal cord, where netrin1 is expressed at high levels specifically in the FP. However, my first undergraduates – Joe Herrold and Anna Maglunog – found that mouse netrin1 is also expressed in the ventricular zone (VZ), which is the central compartment where the neural progenitor cells reside, oscillating back and forth on radial process as they proliferate. Spinal axons uniformly avoid growing in the VZ, staying rather at the “sides” of the spinal cord in a region that will ultimately segregate into the grey and white matter. Anna found that netrin1 expression extended into the dorsal VZ and appeared to strengthen, rather than diminish, over time (Fig. 1B).
This distribution had been accurately described in Serafini et al (4). But, it remained unresolved why spinal commissural axons first grew around the domain of VZ-derived netrin1 before growing towards FP-derived netrin1 (Fig. 1A). Were commissural axons unresponsive to ventricular netrin1? In that case, how did commissural axons then become responsive to FP-derived netrin1 to grow towards the ventral midline? Was there a molecular switch that controlled this process? Joe also noticed that in netrin1 mutant mice, while the vast majority of commissural axons of the Tag1+ subtype stalled as they entered the ventral spinal cord as published (4), there were always some Tag1+ axons that entered the VZ. Again, this latter phenotype was reported by Serafini et al, but it was not a major focus of the paper. We also wondered why spinal axons usually grew around the VZ. Was there a repellent in the VZ? Could netrin1 be that repellent?
I wrote some of these ideas into an R01 grant application that was eventually funded in 2008, which allowed me to bring a postdoctoral fellow on board to work on these questions. However, the project stalled for two years; the Tag1+ axon mispolarization phenotype was subtle and it remained stubbornly unclear whether these axons originated from within the spinal cord or from the dorsal root ganglia (DRGs) in the adjacent peripheral nervous system. Moreover, my plan to tackle the problem by recapitulating the putative VZ-repellent activity in a tissue co-culture assay proved challenging. No progress was made and alas, the postdoc left my lab. With time running out to fulfill this aim of my grant, I recruited a student, Supraja (Sup) Varadarajan onto the project with the idea that she could complete the characterization of the netrin1 phenotype. It would be a quick paper, I reassured her. One of the first ideas that we had was to characterize the netrin1 mutant using a wider range of axonal markers.
Figure 1. (A) In the canonical chemotaxis model, axons grow towards a diffusible source of floor plate (FP)-derived netrin1. (B, C) In our haptotaxis model, netrin1 is expressed by neural precursor cells in VZ (red domain) and then netrin1 protein is transported to the pial surface via their radial processes to form a growth substrate (green line). Axons extend adjacent to this substrate in a Dcc-dependent manner. (D) Axon growth is stalled, disoriented, and/or defasciculated in the absence of netrin1 (or Dcc). (E, F) Conditionally ablating netrin1 supports the haptotaxis model: VZ-derived netrin1, not FP-derived netrin1, is required to guide spinal commissural axons. Figure adapted from Varadarajan et al (11).
First moment of clarity: many types of spinal axons invade the VZ in netrin1 mutants
Not long afterwards, Sup called me over to her computer very excitedly: “LOOK at the pattern of neurofilament innervation!” she said. Neurofilament (NF) is an intermediate filament present ubiquitously in axons. Sup had made transverse slices of control and netrin1 mutant spinal cords and stained them with antibodies against NF. While control NF+ axons grew their usual orderly way avoiding the VZ, to our amazement we saw that NF+ axons were now profusely growing into the VZ in the netrin1 mutant (Fig. 1D, Fig. 2). Antibodies against another protein, Robo3, which labels all commissural axons, showed a similar phenotype; axons were no longer tightly bundled or fasciculated. Rather, the axons radiated in all directions, including into the VZ (Fig. 2). Thus, the observed stall of Tag1+ commissural axons appeared to be an anomaly: the loss of netrin1 resulted in other spinal axons extending wildly into the VZ. We suddenly had clear evidence that there might be a repellent in the VZ, with netrin1 as a top candidate for that repellent.
Sup then spent considerable time mapping domains of netrin1 expression in the mouse spinal cord at different stages of development. She found two general patterns of behavior: 1) spinal axons rarely grow on netrin1-expressing cells, and 2) spinal commissural axons grow precisely around the boundary of netrin1-expressing cells in the VZ. But were these activities mediated by a long-range activity from the FP or a more local, short-range activity from the VZ? Our genetic manipulations strongly supported the presence of a VZ-derived netrin1 repellent. First, we characterized Gli2 mutants, which have no FP (12) either singly or in combination with a netrin1 mutation. The result was clear-cut – it was not enough to remove the FP; NF+ axons only invaded the VZ in the absence of VZ-derived netrin1. Second, in collaboration with Jennifer Kong and Bennett (Ben) Novitch, we were able to ablate netrin1 expression from a stripe of neural progenitor cells, thereby creating two de novo netrin1(-):netrin1(+) boundaries. To our amazement, axons now detached from their normal trajectories and extended into the VZ to follow along one of the ectopic boundaries of netrin1 expression. Everyone in the Butler/Novitch joint lab meeting applauded when Sup showed these results for the first time.
Figure 2: Transverse section of a control (left of dotted line) and netrin1 mutant (right of dotted line) mouse embryonic spinal cord, showing NF+ (green), Robo3+ (red) and Tag1+ (blue) axonal staining. Figure adapted from Varadarajan et al (11).
Revised model: netrin1 provides a growth boundary for axon extension
Our first model was that netrin1, present in the VZ, was repulsive for axon growth. But our results were now suggesting a more complex activity. While spinal axons did generally avoid growing on netrin1-expressing cells, commissural axons appeared to grow preferentially along a netrin1(-): netrin1(+) boundary. Since there wasn’t an obvious term for this phenomenon, which was unwieldy to constantly explain, Sup came up with the concept of a “hederal” boundary. Ivy (genus, hedera) uses a wall (c.f. netrin1) as a necessary scaffold for growth, but it is unable to penetrate this wall as it grows. We wondered whether this hederal activity of netrin1 was more attractive or more repulsive, and tested this idea by examining mice lacking different classes of netrin1 receptors, sent to us by Artur Kania.
In the canonical model, netrin1 results in attractive or repulsive responses in axons by activating different receptor complexes. Thus, Dcc translates the attractive responses of netrin1 (13), whereas the Unc5 family mediates the repulsive responses (14). I was confident we would find that a member of the Unc5 family decoded the netrin1 growth boundary in axons, thus confirming that we were describing a repellent activity. But I was wrong: Sup found only minor axon guidance phenotypes in the Unc5 mutants, many of which had been reported before, and stemmed from the loss of Unc5 expression in the DRGs. However, the Dcc mutants looked just like the netrin1 mutants: Tag1 axons stalled, as already described (13) and NF and Robo3 axons dramatically extended into the VZ (Fig. 1D). Thus, Dcc appears to be the chief receptor that mediated the ability of spinal axons to avoid the VZ, and grow alongside a netrin1(-):netrin1(+) border. Moreover, these findings suggested that netrin1-Dcc might be working through an attractive, rather than repulsive, mechanism.
Second moment of clarity: netrin1 protein is deposited on the pial surface of the spinal cord where it acts as a haptotactic growth substrate
As we started to write the paper, Sup had a thesis committee meeting with Kelsey Martin, Alvaro Sagasti and Larry Zipursky where she was questioned skeptically about the feasibility of our hederal model. Larry, in particular, wanted to know more about the distribution of netrin1 protein. Larry, working with his postdoc Orkun Akin, had just shown that netrin had an adhesive, rather than chemotropic, role in the fly medulla (15). We grudgingly admitted that we had never looked, because we had no expectation that netrin1 protein would be anywhere other than the VZ. A couple of days later, Sup appeared at my door, looking worried. “The staining doesn’t look right,” she said. “There’s a lot of background staining on the pial surface of the spinal cord, and in axons.” I internally cursed the non-specificity of our antibody. “Try antigen retrieval.” I said, “That’s what Tim Kennedy had to do!” (16). Sup came back a day or two later, looking even more worried, and told me that the antigen retrieval protocol made the staining “worse.” Both the pial and axonal staining looked brighter than ever. “It looks real!” she said glumly. This distribution made no sense in our model, and I complained to Larry about his opening up a can of worms, when I saw him at a seminar that afternoon. “It’s a bad molecule,” Larry joked.
Sup was right; the antibody staining did look real. There were very low levels of netrin1 in the VZ as we had predicted, but there were much higher levels on most of the pial surface around the circumference of the spinal cord, and on axons (Fig. 1B). I showed James Briscoe the distribution pattern when he visited UCLA a couple of weeks later. “Of course it’s real,” he said, “The neural progenitor cells are making netrin1, and then depositing it on the pial surface using their radial endfeet.” We corralled Ben in from his office next door to mine to assess what he thought. He thought the staining was real too. The three of us peered excitedly at my computer screen, the realization of how the pattern of the netrin1 transcript related to the distribution of netrin1 protein slowly sinking in. We then discussed nothing else for the rest of the weekend.
Sup went on to show that indeed James’ supposition was correct. Through a trick of their cellular geometry, the neural precursors that make netrin1 then use their radial processes to deposit it as a growth substrate onto the pial surface (Fig. 1B, C). No netrin1 is made by the dorsal-most neural precursors, and indeed there is no netrin1 on the most dorsal pial surface. Sup pointed out that the resulting sharp on:off netrin1 boundary on the dorsal pial surface really suggested that netrin1, a member of a laminin family, could not be highly diffusible. In other exhilarating moments of understanding, we realized that spinal neurons specifically initiate NF+ axonal growth on this netrin1 pial-substrate. And that netrin1 only accumulates on commissural axons as they grow adjacent to the netrin1 pial-substrate, as if netrin1 can transfer from this substrate to axons. In a further remarkable result, Sup found that the putative axonal-transfer of netrin1 requires Dcc.
We finally understood how to model our results: VZ-derived netrin1 acts locally as a substrate to promote fasciculated axonal growth in an oriented manner towards the ventral midline. Netrin1 and Dcc appear to cooperate within axons as part of the mechanism that orients growth (Fig. 1C). The innervation of the VZ that we had observed in both netrin1 and Dcc mutants might in fact be randomized, or defasciculated, growth as a result of losing this adhesive interaction (Fig. 1D). VZ-derived netrin1 thus appears to act by haptotaxis, i.e. by the establishment of a local adhesive surface, the alternative model to chemotaxis. As a mechanistic side note: it remains unclear how this adhesive surface acts to orient and fasciculate commissural axon growth and whether there is an additional “no go” activity in the VZ. Is this mechanism functioning solely through haptotaxis? Or is there also a “hederal” component? Nonetheless, these activities – the pull of an adhesive substrate that promotes fasciculation, perhaps coupled with the push of a “no go” signal – permit axons to grow along the netrin1(-):netrin1(+) border, i.e. in a circumferential path precisely around the VZ.
Third moment of clarity: FP-derived netrin1 is dispensable for axon guidance
Our model – the ability of neural progenitor cells to deposit a haptotactic substrate of netrin1, which promotes ventrally-directed, fasciculated axon growth – was a notable departure from the textbook view of netrin1. But was this model the only mode of action? Or did netrin1 have long-range or short-range activities depending on the cell type? In his original papers, Marc Tessier-Lavigne argued the case for and against netrin1 acting by chemotaxis or haptotaxis (1), but ultimately settled on the idea, well supported by the data at the time, that netrin1 was presented as a long-range cue from the FP. I discussed this point vigorously and endlessly with Sup, Ben, Artur, Larry and Orkun. We had not doubted this model until now. Could our phenotypes still be explained by a long-range activity from the FP? Were the activities of short-range VZ-derived netrin1 in addition to the long-range activities? Or could FP-derived netrin1 really be dispensable for axon guidance?
In the end, the reviewers decided it. The beauty of the finding that neural progenitor cells lay down a substrate of netrin1 that orients and promotes the axonal trajectories of their own neural progeny was not considered sufficiently novel. It was clear what experiment needed to be done, thus we requested the conditional netrin1 lines from Holger Eltzschig and set about breeding them to Cre recombinase driver lines that would remove netrin1 specifically from either FP cells (ΔFP, Fig. 1E) or the dorsal VZ (ΔVZ, Fig.1F). Sup accomplished her breeding scheme astonishing quickly and the day came when she finally had the answer sitting on a microscope slide. I ran to the confocal room, both anxious and excited, to discover the result. “There’s no effect,” she said. “It doesn’t matter if you remove netrin1 from the FP!” In contrast, removing netrin1 from the VZ had profound effects. Axons grew in all directions, but only locally, specifically in the region where netrin1 had been removed from the VZ. Together with the Gli2-mediated FP ablation data, our studies had found no evidence for long-range chemotaxis in the spinal cord. Who knew? Perhaps Cajal’s model was wrong!
The paper was finally accepted at Neuron (11), and came out at the same time as a paper from Alain Chedotal in Nature (17), with complementary findings in the hindbrain. A few days later, I ran into a colleague on the street onside my lab. “I saw your paper in Neuron!” she laughingly scolded me, “But please don’t tell me I have to change my lecture on netrin1! I liked that lecture!” I liked my lecture on netrin1 too, but now I also have to change it. Netrin1, of course, remains the supreme architect of spinal circuitry, but now acts locally as a directional surface along which axons can extend, akin to the holds used by climbers to pioneer their way to the top of a mountain.
Samantha Butler
Department of Neurobiology,
Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research,
Intellectual and Developmental Disabilities Research Center
University of California, Los Angeles.
Here are the highlights from the current issue of Development:
A new model for lineage segregation
Lineage segregation during gastrulation has long been thought to be driven by differential cell adhesion and cortical tension among cells, which would together lead to a differential tissue surface tension (TST) and the spatial segregation of specific cell types. However, this long-standing hypothesis is mainly based on in vitro work, and it is as yet unclear whether it holds true in vivo. Now, on p. 1798 Carl-Philipp Heisenberg and colleagues assess the role of differential TST in lineage segregation and find that, contrary to in vitro work, differential TST is insufficient to explain progenitor cell segregation and germ layer formation within the in vivo gastrulating zebrafish embryo. In the study, the authors describe their unique version of video force microscopy called 3D CellFit, which allows them to analyse surface tensions in 3D within a living organism. Using this method, the authors show that ectoderm and mesoderm tissues do not, in fact, exhibit differential TST in the gastrula. They further present evidence that the apparent discrepancy between the in vitro and in vivo results is due to a difference in osmolarity between the culture medium and the interstitial fluid that surround the cells. Finally, by inhibiting the function of the small GTPase Rac, a key regulator of protrusion-driven cell migration, the authors show that directed cell migration, rather than differential TST, provides the major mechanism that determines the segregation of the germ layer progenitors.
Exciting input for inhibitory neurons
A crucial phase in neuronal development is the integration of newborn neurons into circuits. The right balance must be struck between excitatory and inhibitory neurons; however, the mechanisms that control inhibitory neuron integration and drive the maturation of inhibitory connectivity remain largely uncharacterized. In this issue (p. 1807) Michael Francis and colleagues identify a novel, non-cell-autonomous mechanism that regulates inhibitory neuron synapse formation at the neuromuscular junction (NMJ). The authors examine the electrophysiology and structural organization of GABAergic synapses at the NMJ in a number of different C. elegans mutants with developmental or functional defects in excitatory motor neurons. These analyses reveal that the activity of excitatory cholinergic motor neurons, during a period that coincides with the development of postembryonic GABAergic motor neurons, critically affects the size and distribution of GABAergic pre- and post-synaptic specializations. Furthermore, a severe reduction of cholinergic inputs to newly born GABAergic neurons reduces their synaptic density but increases the synapse size. This study makes an important contribution to our understanding of how neuronal activity impacts synapse development and highlights the functional relationship between excitatory and inhibitory neurons during circuit formation.
Heat shock protein regulates human hepatocyte differentiation
The directed differentiation of human induced pluripotent stem cells (iPSCs) into mature hepatocytes is a major goal of liver research. The approach relies on the recapitulation of developmental processes, and thus a better understanding of what regulates hepatocyte differentiation is essential in order to produce these cells more efficiently and to a greater maturity. In this issue (p. 1764) Stephen Duncan and colleagues identify heat shock protein 90 beta (HSP90β) as a novel regulator of endoderm-to-hepatocyte conversion in differentiating human iPSC cultures. The authors begin the study by conducting a screen for small molecules that modify the activity of master hepatocyte transcription factor HNF4A, identifying 132 candidate ‘hits’. They then focus on the role of molecular chaperone HSP90β and show how it acts at the post-translational level to stabilize HNF4A, thus controlling its half-life and availability. Targeted CRISPR-CAS9 mutations in the gene encoding HSP90 perturbs HSP90β levels, resulting in a dramatic reduction of HNF4A protein levels and reduced expression of HNF4A target genes. Moreover, these experiments reveal that HSP90β is specifically required for endoderm-to-hepatocyte conversion, and not for endoderm commitment generally. This study uncovers a new player in hepatocyte differentiation, and further highlights the utility of an iPSC differentiation platform coupled with chemical screens to uncover novel developmental mechanisms.
PLUS
The developmental biology of genetic Notch disorders
This Review discusses the developmental processes underlying Notch-related congenital disorders in humans, drawing on data from model organisms and genome-sequencing projects, on p. 1743.
An interview with Eric Wieschaus
In our latest interview, Eric Wieschaus tells us about his Nobel Prize-winning fly screens, his interest in the cell biology of development and his love of painting, on p. 1740.
Obituary: Tokindo S. Okada (1927-2017)
A retrospective on the life and work of the pioneering Japanese developmental biologist Tokindo Okada, whose research focussed on cell plasticity and transdifferentiation, on p. 1737.
Our lab is interested in the fundamental molecular mechanisms underlying lineage decision-making in stem cells and in vivo. We are fascinated by the question how defined and stable cell types are generated by the interplay of signaling inputs and gene regulatory networks. We study this question by precise quantification of the states of single cells in combination with bioinformatics analysis and machine learning. Based on this quantitative understanding we want to develop new ways to manipulate lineage decisions during in vitro differentiation in precisely controlled ways. Our group is highly interdisciplinary and works at the interface of biology, biophysics, bioinformatics and biomedical sciences.
References:
Semrau, S., van Oudenaarden, A., 2015. Studying Lineage Decision-Making In Vitro: Emerging Concepts and Novel Tools. Annu. Rev. Cell Dev. Biol. 31, 317–345. doi:10.1146/annurev-cellbio-100814-125300
Semrau, S., Goldmann, J., Soumillon, M., Mikkelsen, T.S., Jaenisch, R., van Oudenaarden, A., 2016. Dynamics of lineage commitment revealed by single-cell transcriptomics of differentiating embryonic stem cells. bioRxiv 068288. doi:10.1101/068288
Semrau, S., Crosetto, N., Bienko, M., Boni, M., Bernasconi, P., Chiarle, R., van Oudenaarden, A., 2014. FuseFISH: Robust Detection of Transcribed Gene Fusions in Single Cells. Cell Reports 6, 18–23. doi:10.1016/j.celrep.2013.12.002
Project and key responsibilities
The available postdoc project aims to create a single-cell atlas of the human embryonic kidney. Information about the transcriptional profiles and locations of all cell types in the embryonic kidney will improve our understanding of kidney development and will provide an important benchmark for kidney organoids. In this project you will be responsible for performing single-cell RNA-seq and single-molecule FISH measurements of human embryonic kidney samples. The necessary experimental techniques are established in our lab and samples will be provided by our collaborators. In particular, you will dissociate the tissue and prepare single-cell RNA-seq libraries with the drop-seq technique (Macosko et al., Cell, 2015). You will analyze the RNA-seq data (potentially together with a bioinformatics collaborator) and identify cell types using state-of-the-art machine learning tools. Based on these results you will define a set of marker genes that will allow you to locate cell types by single-molecule FISH in intact tissue sections. This comprehensive spatial molecular data set will then allow you, for example, to establish intercellular signaling networks.
Selection criteria
You hold a PhD degree in one of these disciplines: biology, biochemistry, bioengineering or related disciplines
You have a strong interest in experimental quantitative biology, in particular related to human development and stem cell differentiation
You have experience with molecular biology techniques, in particular NGS library preparation
Experience with programming in R or Matlab and relevant bioinformatics packages is a plus.
You are proficient in spoken and written English, and have good communication and writing skills
You are independent, creative and have team spirit
Research at our department
Our lab is part of the Leiden Institute of Physics (http://www.physics.leidenuniv.nl) and situated at the Leiden Cell Observatory (http://cellobservatory.leidenuniv.nl). The Cell Observatory is a highly collaborative community dedicated to the visualization and understanding of the fundamental molecular mechanisms of life, which is part of the core scientific profile of Leiden University. The Cell Observatory houses state-of-the-art bio-imaging facilities shared among the member labs, which actively develop new methods for the quantitative measurement of single-cell properties.
To apply for this vacancy, please send an email to Dr. Stefan Semrau (semrau@physics.leidenuniv.nl)until June 18. Please include your curriculum vitae, a letter of motivation and the names of 3 potential references.
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