I had heard legendary stories about the MBL and the Embryology course in the months leading up to it. Stories about the collaborations of minds that wouldn’t normally meet in the same lab, discoveries that could only have occurred at the MBL, it’s what made me apply. But once I was accepted the nervousness kicked in. The other ubiquitous tales are of the incredibly long days and equally long nights in the lab. I’m not a night owl, I’m an eight-hours-a-night sleeper and I was convinced I would be the first and go to bed every night. I worried that I wouldn’t be able to keep up with my classmates-valid, reasonable concerns I initially thought.
Boy was I wrong.
The atmosphere at the Embryology course is electric. I felt like I was buzzing for six weeks straight and didn’t want to, almost couldn’t, leave the lab. To be surrounded by incredible classmates that get just as excited about the idea of an impossible experiment as me, egged on by brilliant, hilarious and endlessly supportive faculty every night? Why would you go to bed?
I remember a night when I absolutely HAD to image primitive streak ingression in the chick embryo with mosaic labelled cells. Andrea Streit and Claudio Stern sat with me and coached me through flipping a paper-thin stage-three chick embryo and poking it delicately with a dye loaded needle before imaging it for 16 hours. I can’t describe how excited I was waking up at 8am to turn off the microscope before lecture. Only to miss breakfast because I was watching my movie over and over again as I watched labelled cells stream into the embryo. It’s also when I realized how amazing my classmates were. That morning, I walked bleary eyed into lecture and was promptly handed a bagel and a steaming cup of coffee.
I learned to trust my hands during the Embryology course. I had never dissected anything before and I found that I love it. I dissected anything I could get my hands on throughout the summer, culminating in a few of us dissecting ovaries out of every arthropod species we had on hand. We imaged structures that had only been described in the early parts of the 20th century.
Vanessa cardui egg chamber stained with DAPI and Phalloidin
It’s also where I learned to be fearless. The lack of adverse consequences and the adventurous atmosphere meant that I got sea urchin embryos drunk to see how it affected their skeleton, I transplanted cells into zebrafish embryos and I tried to image tardigrade gastrulation. We used Walmart items and state-of-the-art microscopes in the same experiments! I can confidently say that 75% of everything I tried failed, but that was part of the fun. Our class motto ended up being along of the lines of “in DAPI we trust”.
The course tested my intellectual endurance like nothing has before. There is NO pressure to produce or perform, no judgement, no deadlines. You have to find your own drive to learn and try and fail and try and fail and sometimes succeed. Thankfully, its not hard to find that drive. At least six lectures a week from the best minds in the field, who then join you in lab and encourage you to answer impossible unanswered questions. Faculty that are just as willing to give you moral support at the microscope as they are to come have a drink with you at the local pub. Faculty that agree to an hour-long “sweatbox” Q&A session after a two hour talk so we can pepper them with every question under the sun. How can you resist?
Every time it got too hard or too overwhelming, there was always someone to get pie with at Pie in the Sky, always someone willing to take a quick dip into the ocean and always someone willing to “hunt for lightning bug embryos” on a walk by the ocean. The science makes the Embryology course amazing and enriching, the people make it incredible and special. Last summer I learned what I wanted to do and be as a scientist but I also made friends for life #embryology2018.
We enacted convergent extension during the Woods Hole 4th of July parade. (2 votes) Loading...
The Santa Cruz Developmental Biology Conference happens every couple of years in UCSC’s beautiful campus, and is seen as a kind of West Coast Gordon Conference for the field. In August a hundred or so developmental biologists gathered for the 2018 version, organised by Bin Chen, Natasza Kurpios and Ed Munro.
It was a first trip to California for one of us (Aidan, who came representing the Node and Development), and a short trip from the office for another (Jordan, whoseUCSC lab works on gene regulation and evolution in C. elegans and parasitic nematodes). Aidan got a chance to visit the town of Santa Cruz before the conference – Blue Oyster Cult were playing on the beach, the boardwalk was full of families, pelicans were diving, sea otters and seals cavorting, all as a bank of freezing fog rolled in – quite a scene! He also got a chance to explore the campus with its giant redwoods, roving turkeys and repeatedly breath-taking view over Monterey Bay.
The conference’s tagline, ‘Old and new: modern answers to enduring questions in development’, was introduced beautifully by the opening three keynotes. Judith Kimble gave a fantastic career retrospective on her quest to understand how embryos get in and out of totipotent states. She described the 27 year search for Notch targets that maintained a naive stem cell fate, and how it lead them to two redundant low complexity proteins with no domains and no clear homologs in other species. This lack of conservation or functional domains dissuades many of us from working on such factors, but the importance of such proteins in development was a theme of the meeting, popping up in several talks.
Eric Wieschaus explained how genetically determined cell fates are translated into local cell behaviors, discussing how myosin tension is a regulator of morphogenetic sequences. He also nicely touched upon his scientific philosophy of reducing his work to the simplest model, seeing what it lacked, and then refining his model. Good lesson for trainees!
Vilaiwan Fernandes, who has just startedher own lab in UCL with a Wellcome Trust Sir Henry Dale Fellowship, won the SCDB Young Investigator Award. Her award lecture covered her time as a postdoc in Claude Desplan’s lab, where she worked on the establishment of retinotopy in the fly brain. She found that glia provide a key link between photoreceptors in the retina and target cells of the optic lobe. The differentiation of both types of cells needs to be tightly coordinated, and Vilaiwan found that glia are crucial to this process by forming a signalling relay, in part using insulin. Aidan got to interview Vil over a beer at a poster session – you can hear all about her research career and plans for the future in the associated interview.
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The next two days provided repeated demonstrations of why developmental biology is such an exciting field at the moment, as new tools unlock fundamental questions that have kept the field busy for decades. There were so many amazing talks, and we will cover select ones to give readers a flavor of the meeting.
Dominique Bergmann introduced the problem of how cells and tissues establish polarity. Plants, which evolved multicellularity independently of animals, do things quite differently, not just in terms of the components involved but also in terms of how processes like symmetry breaking are achieved. She shared wonderful, long-term videos of asymmetric divisions in the plant stomatal lineage, and explored the reasons why can the establishment of polarity can take hours in plants compared minutes in something like a C. elegans embryo. This talk was another place in which small proteins of low complexity, no domains or conservation played a starring role.
Sally Horne-Badovinac asked how cells coordinate their migration with one another when moving collectively as a sheet. Follicle cells of the Drosophila ovary migrate around the growing egg chamber using basement membrane as a substrate, and this migration leads to the rotation of the egg chambers in a process thought to promote their elongation (it’s just such a bonkers way of sculpting a tissue, evolution really getting creative). Sally showed that the coordinated migration of follicle cells is regulated by the same molecules used in axon guidance, an example of how neurons and epithelia are closer to one another than we might usually think.
Shuonan He, a graduate student in Matt Gibson’s lab who also won the People’s Choice Award for his terrific poster, addressed the role of Hox genes in Cnidaria, which lack a true A-P axis and therefore provide a bit of a conundrum for those of us who think about Hox genes only in relation to A-P patterning. To tackle this problem, Shuonan utilised shRNAs knockdown and CRISPR/Cas9 knockouts to test Hox functional roles, and, with beautiful pictures of Nematostella cross sections, showed that Hox genes control tissue segmentation and tentacle patterning. It was a wonderful evo-devo story, hastened by the use of modern gene editing tools.
Taking a break from the multicellular, Laura Landweber took us somewhere truly strange – the astounding genome rearrangements that occur in the ciliate Oxytricha trifallax during its sexual reproduction. Oxytrichia has two genomes, one of which comprises over 16,000 chromosomes, most of which encodes a single gene; this ‘macronuclear’ genome has to be made anew from a micronuclear precursor during sexual reproduction, in a process of lncRNA-regulated genomic acrobatics that made many heads spin in the audience. It’s an incredibly powerful system to investigate genome fidelity across generations, and it was wonderful to hear about this in a development meeting.
Another major theme of the meeting was the power of single molecule approaches. Jeff Farrell and Sean Megason both presented their single cell RNA-seq (scRNA-seq) approaches to map zebrafish developmental trajectory. Dozens of distinct cell populations could be found by 12 hours of development. This work promises to answer major questions in developmental biology, such as how do cell states change over time and what paths can cells take through development?
Alejandro Sanchez-Alvarado identified the elusive planarian neoblast — the stem cell that allows for the amazing regenerative capacity of this animal — using scRNA-seq. Stunningly, a single neoblast could rescue viability and restore all cells in a lethally irradiated animals, which would otherwise dissolve into goop. Fun fact he shared: the strain of planarian they work on has lost sexual reproduction through a single translocation and now reproduce by anchoring their tail, crawling away until a fragment of their tail rips off, after which both halves generate entire new animals. Kristy Red-Horse used scRNA-seq to understand cell dynamics during coronary development, revealing that there is a gradual, overlapping transition of veins to arteries. Long Cai described sequential fluorescence in situ hybridization (seqFISH), which allowed single cell, single molecule detection of hundreds of transcription factor mRNAs or over 10,000 genomic loci by intron seqFISH.
There were also numerous talks using other powerful new techniques. Ari Pani explored Wnt signaling in C. elegans, providing evidence that a particular Wnt was using a signaling gradient, not a contact-based mechanism such as cytonemes. He used a clever MorphoTrap to disrupt the gradient without altering receptor or ligand levels and found Wnt diffusion was essential for neuroblast migration. Maria Barna used cutting edge mass spec to explore ribosome diversity, providing evidence for ribosomal proteins that associate only with a subset of ribosomes and allow translation of specific mRNAs
Like potato chips in sandwiches, the final keynote pairing of a talks on the morphogenetic Hox clock and evolution of forest mouse tail length was unexpectedly delicious. Denis Duboule gave an energetic, engaging broad overview of his work on Hox gene topologically associating domains. He also discussed the curious case of Hoxd13, which acts as a dominant negative to inhibit the other Hox proteins and seems to “mark the end of things, like digits”. Hopi Hoekstra’s keynote to close out the meeting started off exploring difference in forest mouse morphology, first observed by Osgood in 1909. Forest mice had longer tails due to both longer and more vertebrae, and these long tails were important for balance in lab tests. QTL mapping found three QTLs for length of longest vertebra, three for number of vertebra. And circling back beautifully to the first talk, these QTLs involved the Hox genes Duboule discussed in his talk. This session was a beautiful example of how the mechanisms we discover in the lab function in nature.
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On the final night we had dinner outside and a final chance to discuss old questions and new techniques under the redwoods, those giants that miraculously, like all of us, started life as a single, tiny cell. The fog rolled in from the Pacific to envelop campus and left some of us lost in search of the bar, but we got there in the end (thanks to the student volunteers for supplying the drinks and acting as deeply knowledgeable beer sommeliers!).
The next Santa Cruz meeting is scheduled for 2020 – we would urge anyone keen on a smallish, diverse and relaxed development meeting in a stunning location to apply.
Day 3 #SCDB2018 and I get to kick off the session under the perpetual vigilance of @ucsc Sequoia sempervirens, the sole living species of the genus Sequoia in the cypress family Cupressaceae. @ScienceStowerspic.twitter.com/SMyP16eI6M
At the meeting I was lucky enough to interview three amazing scientists, one of whom was Vilaiwan Fernandes, winner of the SCDB Young Investigator Award for her outstanding research achievements, who has just started her own lab in UCL. (Look out for interviews with Judith Kimble and Cassandra Extavour in future issues of Development.) I sat with Vilaiwan in a noisy and beery evening poster session and chatted about her career so far and plans for her new lab.
When did you first get in to science?
I grew up in Bombay (now Mumbai) in India, and I guess I was interested in science in school because it involved less memorisation than other subjects. I learned to scuba dive, which got me interested in conservation biology and I began volunteering with an organisation that held public education snorkelling camps. I tried to continue that when my family emigrated to Canada, and studied marine biology in my undergraduate. There’s a marine station on the west coast of Vancouver Island called Bamfield Marine Sciences Center, and I spent a lot of time there. Every summer in my undergraduate I would either do a course there or work as a research assistant on some project or other. There’s lots of amazing intertidal life there, particularly invertebrates, and we were lucky enough to explore tiny uninhabited islands and set up quadrats to see what we could find!
How do you go from that into something resembling developmental biology?
I was never interested in cell biology or development, because I liked being outdoors. At the end of my undergraduate I had a summer job but started panicking because I had no plan for once it was up. So I applied to a ton of random things, including an apprenticeship in Friday Harbor Labs in Washington, called ‘Computational Modelling of Biological Networks’. I had also studied math in my undergraduate, and assumed the apprenticeship was about computationally modelling ecological networks, which was wrong: I got there and found it was about gene networks. So that’s sort of how I got pulled in to the field – by accident.
I worked with an amazing postdoc, Kerry Kim, to try to understand the evolutionary benefit of being a haploid or a diploid that reproduces either asexually or sexually. We ran genetic algorithms to evolve populations under these different conditions for several generations. It was cool because we were able to represent haploidy and diploidy (and recombination) in a much more realistic way than other models had at the time. It was a purely computational project but a lot of fun and one I’m still very proud of.
And you then transitioned back again from dry to wet biology?
The apprenticeship was based in the NIGMS Center for Cell Dynamics, which was run by Garry Odell. His wife Victoria Foe and other amazing scientists like Ed Munro and George VonDassow also worked there. Garry was brilliant and encouraged me to try my hand at experiments. He had an engineering background and was really interested in size control and scaling. He convinced me to transplant nuclei between different sized Drosophila species just before cellularisation. The goal was to see whether the foreign nuclei would form cells staying true to the size of their own species or if they would switch to the size of the host species. Unfortunately, we didn’t finish this project as I left to start grad school, but it was the best introduction to experiments!
It sounds quite fiddly as an introduction to experimental work!
It was, but Victoria, who trained me, has amazing hands and she just taught me to be patient. It made me opt for lab work over computational biology in grad school, and I applied to McGill and joined Ehab Abouheif’s lab. Quebec didn’t suit me for a number of reasons and I ended up dropping out of that PhD programme and moving back to the West coast, where I re-enrolled in a grad student in a fly lab – Esther Verheyen’s in Vancouver.
My initial project with Esther was to work on Nemo, a rather promiscuous protein kinase. Thankfully, Esther convinced me to do a screen for morphogenetic furrow progression defects in the eye disc. That’s where we pulled out a kinase called Misshapen, which stood out because it sped up furrow progression in contrast to all our other hits. At that time, Sally Horne-Badovinac’s lab had just published Misshapen’s regulation of integrins in the follicle cells. I met her postdoc Lindsay Lewellyn at a conference, and following that we decided to test the role of integrins in the eye. Lindsay was super cool, and ended up on my paper! I guess the main point of that paper was to link the signalling pathways that affect transcription in the eye with the non-transcriptional, post-translational events, like cytoskeletal rearrangements and changes in adhesion, which are important in the morphogenetic furrow.
And then you go to a New York for your postdoc with Claude Desplan – I guess by that point you’d fallen for eye development?
Not quite – but every time I say I’m not going to do something, I end up doing that thing. I thought wanted to switch models and move away from neurobiology, but then I met Claude at a Gordon Conference and he suggested that I apply to his lab. The atmosphere in his lab was great, and I was lucky to get accepted. Claude is always, always excited about science and I could not have been in a better place.
My project was initially to try to understand how to make individual cell fates in the developing visual system, in particular how to generate cellular diversity in the lamina. When I started working on it we realised we didn’t actually know why neurons differentiate in the particular pattern that they do, and it seemed that this pattern was key to why they adopted different fates. Seeing how well glial cell processes entering the lamina correlated with the front of neuronal differentiation made us suspect glial involvement. You can read about where that story took us in my Science paper from last year.
So the next step was to look for jobs – you’d been a postdoc for a relatively short time, so what made you think about being a PI?
I had a lot of leads that I really wanted to follow up on, but felt that I wouldn’t be able to do all of it myself. I’m very excited to pursue all of them and see where they go. And Claude was very supportive.
When I applied for the Wellcome Trust Henry Dale Fellowships and got selected for an interview, UCL was very good at organising mock interviews – they ended up being more terrifying than the actual interviews! My very first mock interview was a complete disaster, and I cried for several hours afterwards, but I’m told that’s normal and I suppose it did the trick.
What are your plans for the initial years of your lab? Are you going to stick with glia?
Perhaps even more than glial involvement I want to focus more broadly on exogenous cues that affect neurons – the field has been very focused on neurons talking to neurons, and neuroblasts being intrinsically patterned, without really looking too much at exogenous cues. For now, I’m focusing on signals from glia.
In the long-term, one of the larger projects will use single cell sequencing of glia during development, with the goal of identifying secreted and membrane-associated factors that affect neuronal development. In theory, this approach should identify instances like the one we showed in our paper, where glia express insulins to induce lamina neuronal differentiation.
Finally, congratulations on your SCDB Young Investigator Award! How did you feel when you found out you’d won it?
It really was a huge honour to receive the award – I was very surprised and didn’t expect it!
At the very end of my talk I thanked the couple Garry [Odell] and Victoria [Foe] with whom I had started my research career in Washington. Garry died earlier this year, but I know he would have been really proud of me for this. I visited him in hospital before I went to my Henry Dale interview. He was adorable – he was there sick in bed but said ‘We have to practice your interview, get your slides out!’ He and Victoria had a can-do attitude in the lab – looking back at those early days I think I we were crazy to think we could transplant nuclei between species! But at the time, I didn’t think twice as to whether it was possible or hard. Gary was great for that, and was such a strong influence on my life and my science.
Radial growth in plants is driven by proliferating cells in the cambium that give rise to the vascular tissues of xylem and phloem, and increases plant girth. However, the identity and dynamics of the stem cells that drive this crucial process remain poorly understood. A paper in Development now characterises cambial stem cell activities in the hypocotyl of Arabidopsis. We caught up with first author Dongbo Shi and his supervisor Thomas Greb, Heisenberg Professor at the Centre for Organismal Studies in Heidelberg University, Germany, to find out more about the story.
Thomas (L) and Dongbo (R)
Thomas, can you give us your scientific biography and the questions your lab is trying to answer?
TG I started my career as a diploma and PhD student at the Max-Planck-Institute for Plant Breeding Research in Cologne, Germany. After spending 3 years as a postdoc in the lab of Caroline Dean at the John Innes Centre in Norwich, UK, I became a Junior Group Leader at the Gregor Mendel Institute in Vienna, Austria. From there, I moved to Heidelberg University, Germany, where I became a full professor in 2016.
Starting with my work as a diploma student, I was always fascinated by the question of how plant cells coordinate their activity in space and time, and how they respond to the environment. When we started working on radial plant growth, I realized that our lab had identified a process which allows us to focus exactly on those fundamental aspects of biology. Conveniently, the process is the most productive growth process on earth in terms of biomass accumulation, so it is clear that this work has quite some relevance in this regard.
And Dongbo, how did you come to join the Greb lab, and what drives your research?
DS I received my PhD in 2014 from Kyoto University, Japan, after working on a project in mouse oviduct development, which also ended up as an article in Development (Shi et al., 2014). The project was carried out in the National Institute for Basic Biology, Okazaki, Japan and co-supervised by Toshihiko Fujimori [National Institute for Basic Biology (NIBB), Japan] and Tadashi Uemura (Graduate School of Biostudies, Kyoto University, Japan). I was fascinated by the beauty of morphogenesis and wanted to understand the mechanisms behind it. After obtaining my degree, I was looking for a place to continue my career as a developmental biologist and to broaden my view on science by changing the topic and place. In both NIBB and Biostudies Kyoto University, you find a broad range of biological research focusing on animals and plants. Thus, I was regularly exposed to plant science and stimulated by the talks and seminars. The more I learned, the more I wanted to try it myself, and I felt that there were still many important questions to be answered. First of all, I felt that cambial stem cells should be identified more clearly. Consequently, I asked Thomas for an opportunity to apply for a fellowship to work in his research group: he not only agreed, but also rigorously helped me to strengthen the research proposal. As a result, the Alexander von Humboldt Foundation was brave enough to offer me an opportunity, and I took this chance to come to Heidelberg in 2016. In addition to the support from the Humboldt Foundation, I really appreciated that Thomas welcomed me. I think it was not an easy decision to accept a young scientist from a different field whom he had never met before. I think it is my personal interest that mainly drives my research. I know this is not always easy, but I want to follow my own interests for as long as I can.
The existence and activity of cambial stem cells were first postulated 150 years ago – why has it taken the field so long to experimentally analyse them?
TG Experimental analysis of the cambium has quite some history in woody species like Populus. However, identification of the underlying stem cells was always difficult due to the lack of definite cellular markers and efficient genetic tools. Only when it became clear that radial growth exists and is accessible in our favourite plant model Arabidopsis, did things moved forward more vigorously. Also, the fact that the cambium is embedded in all these differentiated and optically very dense tissues did not help to promote this kind of research.
DS After coming to Heidelberg, I had the chance to read two books. The first one was The Vascular Cambium by Philip Larson (Larson, 1994), and the other one was Esau’s Plant Anatomy by Ray Evert (Evert, 2006). I was so fascinated by these two books, and really admire the authors and other plant biologists for their detailed descriptions and deep thoughts about the cambium over the years. I imagine if they lived today and had access to modern biotechnical tools, they would perform the same analyses as I do, perhaps even in a more sophisticated way. On the other hand, I think these thorough descriptions and summaries prevented researchers from reviewing cambium dynamics or challenging it afterwards. Also, to me, it looked initially like a finished story. Excitingly, our findings prove that cambial stem cell activity is, to a large extent, as proposed, which emphasizes the brilliance of these former anatomists.
A cell clone originated from a single cambial stem cell contributed to both xylem and phloem. Maximum intensity projection of confocal images of hypocotyl cross sections, with cell wall visualized by Direct Red 23 staining (magenta) and cell clone visualised by Venus targeted to endoplasmic reticulum (green).
Can you give us the key results of the paper in a paragraph?
TG The basis of this work was the identification of promoters which are specifically active in different cambium domains. Although some of them were described before to be active in cambium stem cells, we discovered that they are active in stem cells and the wood-forming part of the cambium, but not in the bast (phloem)-forming part. By finding that the activity of the SMXL5 promoter labels the bast-forming half of the cambium, we had the tools in hand to perform a thorough cell lineage analysis targeting the bidirectional mode of tissue production by the cambium. We found that common stem cells producing both tissue types are located in a narrow and central cambium domain in which all those promoters are active. We also saw that those cells proliferate more than the immediate tissue progenitors. Thus, the cambium does not seem to hold transient amplifying cells as seen in other plant stem cell systems.
DS I think being able to analyse the history of distinct cells represented a substantial step forward in comparison to the snap shot analyses done so far.
Being able to analyse the history of distinct cells represented a substantial step forward
For any given division of a bifacial stem cell, what determines the fate of the daughter?
DS & TG This is certainly one of the most fundamental questions in the field. We know that gene expression profiles in the two tissue-forming regions are quite different, and that these profiles and individual regulators active in those regions are instructive in this regard. Currently, we think that, when a cell gets under the influence of those regulatory environments, they adopt the respective cell fates. However, we still do not have an integrated view on how these domains are established and maintained in the first place, let alone how they communicate with each other to balance tissue production and patterning.
How and why does cambial stem cell activity differ to other animal and plant systems?
DS & TG As mentioned above, we found that proliferation in the cambium domain mostly takes place in the common stem cell region. It looks as if, after they have divided, one daughter cell undergoes differentiation very soon and, if at all, divides only once or twice. This is different to stem cell systems sitting at the tips of shoots and roots, and also to most animal stem cell systems. There, tissue-specific progenitors divide relatively often, forming a transiently amplifying domain. Thus, we seem to have a special situation in this regard in the cambium. We think that this is because the ratio of stem cell number versus the number of differentiated descendants is relatively high in the cambium. So one given stem cell is only responsible for the production of a rather narrow radial sector in the stem or the root.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
DS The most exciting moment was when I got the data shown in Fig. 3. To be honest, I did not notice what it meant immediately while I was sitting in front of the confocal microscope. I was in a rush in collecting data because time slots available for working at the confocal microscope are usually short. After I went home, and it was probably a weekend, I still wondered what I observed. Then I noticed that I could only draw one conclusion: cambium-derived cells originate from cells sharing PXY and SMXL5 promoter activities. I questioned this conclusion again and again, checked the images many times, but I could not come up with any other reasonable interpretation. I could not wait to come to Thomas’ office and show him what I found. Before I could give my interpretation he said ‘Wait, Dongbo, let me think this through for a second’. He came to the same conclusion.
And what about the flipside: any moments of frustration or despair?
DS I would say this research went relatively smoothly, but since I started a new project in a new field and a new country, it took me a while until I got the first meaningful results. It was frustrating that, sometimes, things which took me several days in Japan took me several weeks in Heidelberg, due to misunderstandings, cultural or system differences, lack of experience with plants, and so on. And of course, I could not exclude the possibility that the experimental design simply would not work. I felt very relieved when I saw that the EdU incorporation and the inducible Cre-loxP system actually worked in Arabidopsishypocotyls. I really would like to thank Vadir López-Salmerón and other members in Thomas’ lab who supported me during that time.
So what next for you after this paper?
DS I think we are still at the very beginning of our research in radial plant growth. I want to describe the activity of cambium cells in a more quantitative manner and, of course, want to decipher the regulating mechanism of radial growth in terms of morphogenesis. As you asked, how the fate of daughter cells is determined after the cambial stem cell division is really an important and challenging question I want to address.
Where will this work take the Greb lab?
TG I think this work represents one milestone in our research because we were able to allocate various cambium functions to distinct spatial domains. Of course this opens many opportunities for characterizing these domains in more detail, and also for asking the question of how they are maintained.
Finally, let’s move outside the lab – what do you like to do in your spare time in Heidelberg?
DS Visiting towns and walking the streets. The culture and the society in Germany are of course very different from those in Japan. During the last two and a half years, I have visited many small towns around Heidelberg and joined many events like the city festival, the summer festival, the wine festival, and so on. Every town has its own atmosphere and the people there seem to be very proud of their towns. Fortunately, my family also likes it a lot, so we travel often together on weekends.
TG As Dongbo says, the Heidelberg region provides many opportunities for enjoying life. Because Heidelberg is small, nature is close and we do a lot of hiking with the family. Also, the many cultural events happening in the region, many adapted to young children, keep us busy.
Sperm development and differentiation are regulated by somatic cells and the extracellular signals they produce – often regulators of proteolysis. Premature or delayed differentiation can compromise fertility, and thus tight spatiotemporal control of the process is crucial. A paper in Development addresses how two secreted proteins control sperm maturation in the context of the C. elegans gonad. We caught up with the paper’s first author Daniela Chavez and her PhD advisor Gillian Stanfield, Associate Professor of Human Genetics at the University of Utah, to find out more.
Daniela and Gillian
Gillian, can you give us your scientific biography and the questions your lab is trying to answer?
GS Entering graduate school at MIT, I was interested in developmental biology: how can a single cell possibly transform into a complex organism? I was drawn to C. elegans for its genetics and simplicity, and the idea that one could analyse development at the level of individual, highly reproducible cell fate decisions and interactions, so I joined Bob Horvitz’s lab to work on the genetics of programmed cell death (apoptosis). Moving on to my postdoc, I became fascinated with germ cells, and now my lab uses the worm to study various aspects of sperm cell biology. We focus on two main areas – sperm differentiation and sperm competition – and how each of these processes is regulated to maximize reproductive success. In C. elegans, both sexes make sperm, but they use different genetic pathways to control their differentiation. Also, male sperm outcompete hermaphrodite sperm to fertilize oocytes, and we would like to understand the underlying cell biology that makes that possible.
And Daniela, how did you come to join the Stanfield lab, and what drives your research?
DC When I joined as a graduate student, I was already familiar with the research and with C. elegans because I had previously been an undergraduate student in the Stanfield lab. In fact, my graduate school lab rotations were all with other labs, but I returned for the opportunity to use genetics to study the cell biology of reproduction and for the rigorous training environment of the lab and department. My research is driven by my fascination with gamete cell biology and how their successes and failures can influence evolution and have big consequences for a species.
What was known about the roles of SWM-1 and TRY-5 in sperm activation before this paper?
DC & GS Previous work from the lab had shown that the serine protease inhibitor SWM-1 is required for male fertility. It ensures that sperm remain immotile within the male gonad. This is crucial because motile sperm are transferred inefficiently during mating. The TRY-5 protease had been shown to be a seminal fluid protein and sperm activation signal. While it was known that SWM-1 inhibits premature sperm activation via TRY-5, it was unclear which tissues expressed SWM-1 or where SWM-1 and TRY-5 might interact.
Can you give us the key results of the paper in a paragraph?
DC & GS We wanted to address the developmental question of where and when does SWM-1 function to inhibit premature sperm activation? We used integrated transgenes and a CRISPR-generated knock-in to determine SWM-1 protein localization and found that it is in seminal fluid: it surrounds sperm stored in the male and, unexpectedly, it is in body wall muscle. This led us to wonder which source of SWM-1 is important for inhibiting sperm activation, so we used tissue-specific expression in swm-1 mutants and found that the extragonadal source, body wall muscle, is the critical source of SWM-1. This was a surprising finding since body wall muscle is not a part of the gonad, and this result demonstrated that secreted factors outside the gonad can impact reproductive success. We also analysed SWM-1 and TRY-5 localization together and found that while TRY-5 is present at low levels near stored sperm, SWM-1 levels are higher. This suggested that, in the male, sperm are in an inhibitory environment, whereas in the uterus, after sperm are transferred, the balance is shifted toward activation. We were left wondering about the other source of SWM-1, in seminal fluid, and whether it has a role in the uterus. By overexpressing SWM-1 in seminal fluid, we found that high levels of SWM-1 in seminal fluid can reduce male fertility. While the role of SWM-1 in seminal fluid remains unclear, our experiment suggests that the level is regulated to achieve a balance that is optimized for sperm success.
What might be the benefits of producing such a crucial regulator of sperm activation in a distant tissue, rather than locally?
DC & GS It’s a great question. We think it may be beneficial to produce SWM-1 in a tissue that develops well before sperm are made to ensure that sufficient inhibitory signal is present as soon as meiosis is complete. After meiosis, sperm are poised to respond rapidly to activation cues. This is a critical feature because once they are transferred to the uterus, if they do not activate quickly they can be swept out of the uterus and lose the chance to fertilize an oocyte. We think that perhaps producing SWM-1 in a somatic tissue that develops prior to sexual maturity, rather than within the gonad, which develops coincident with sperm production, allows sperm to maintain their quick response characteristics without activating prematurely.
Do you have ideas of other roles for soma-to-germline (and germline-to-soma) signal exchange?
DC & GS Although there are other soma-germline signals described in C. elegans, we think there are likely many that are still unknown, given our and others’ findings that proteins can seemingly move freely from the body cavity to the germline. It will certainly be interesting to study the mechanism of exchange and to find other signals that can affect the germline this way.
Tail of an adult C. elegans male expressing SWM-1::mCherry, which is visible in muscle cells and in vesicles of seminal fluid within the vas deferens.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
DC I will always remember the first time I saw SWM-1::mCherry in a worm under the microscope. It’s a pretty amazing feeling to see something that no one else has ever seen before. Fluorescent SWM-1 is especially mesmerizing to stare at since it is in several cell types and has distinct localization patterns in each tissue.
It’s a pretty amazing feeling to see something that no one else has ever seen before
And what about the flipside: any moments of frustration or despair?
DC Troubleshooting the CRISPR editing was a maddening process. It took about a year and a half trying different strategies that did not result in a knock-in. Looking back, I’m not sure why I kept trying and continued to have hope after so many failed attempts, but I am certainly glad I didn’t give up.
So what next for you after this paper?
DC I hope to study gamete biology for a long time to come. I am currently a postdoctoral fellow in the Department of Reproductive Sciences at the Smithsonian Conservation Biology Institute studying the other gamete: oocytes. My work focuses on understanding oocyte developmental competence and developing fertility preservation methods geared toward banking gametes of endangered species. Mixing my interests in gamete biology with conservation is a wonderful combination of my passions so I hope to find ways to continue on this path.
Where will this work take the Stanfield lab?
GS There are many interesting new directions to consider. In the short term, we are thinking about what Daniela’s results mean for our current projects. For example, using genetic screens, we have collected a set of new sperm activation mutants, and we would like to identify the corresponding genes. We had considered it likely that those would be expressed in the germ line or gonad, but now we realize that this set of candidates may be too limited, even as a first pass.
Finally, let’s move outside the lab – what do you like to do in your spare time in Utah and Washington?
DC I love to snowboard, camp and get a massage to recover from pipetting and microscope time. I also like to cook food inspired by my family’s culture, which includes Costa Rica, Mexico and Utah. On occasion, I sew mediocre Halloween costumes or amoeboid sperm-shaped bean bags. In DC I am enjoying commuting to the lab on a bike and trying to eat all the varied food the city has to offer.
GS Utah is a fantastic place to spend time in the outdoors. Living in Salt Lake, we are surrounded by mountains – I can leave my house and be skiing in an hour. A little farther away, southern Utah has spectacularly beautiful desert scenery, and I camp and hike there whenever I can. But currently I spend most of my spare time chasing my 6-year-old son!
By Amie L. T. Romney and Jason E. Podrabsky from the Podrabsky Lab at The Center for Life in Extreme Environments at Portland State University
Environmentally-induced developmental plasticity provides an opportunity to explore one of the grand challenges of modern biology – identifying mechanisms that link genotype to phenotype. The annual killifish, Austrofundulus limnaeus is a particularly useful vertebrate system to address this question. Embryos of A. limnaeus are highly resistant to environmental extremes, and may in fact define the limits for vertebrate survival without oxygen. Yet, they remain highly responsive to environmental cues during development that can trigger a remarkable capacity for plasticity. Recently, we discovered that vitamin D3 synthesis and signaling directly links environmental cues into the developmental program of this species, and supports their ability to develop along two alternative developmental trajectories. This work provides a framework for exploring the role of nuclear receptors as master regulators of developmental plasticity and life history transitions across all animal taxa.
Biology of A. limnaeus A. limnaeus has adapted to life in a highly variable and often extreme environment. The ponds of its native habitat in Venezuela are ephemeral1, and form during the warm rainy season. High spatial and temporal variation in rain events produces ponds that are transient and can undergo multiple cycles of drying and flooding even within a single season2. When ponds dry, the entire adult and juvenile population dies, leaving only embryos to survive the dry periods3. Embryos can endure drought in a state of diapause for months while encased in the pond sediment under conditions that likely impose hypoxia/anoxia and severe desiccation stress4-6 (Figure 1). Diapause is a state of metabolic and developmental arrest that is characterized in this species by a severely depressed metabolism and heart rate, suppressed rates of proteins synthesis, the use of anaerobic pathways to support metabolism even in the presence of oxygen, and the induction of mechanisms that reduce evaporative water loss7.
There are three stages of diapause (I, II and III) possible during development in annual killifishes. Entrance into diapause II involves an alternative phenotypic trajectory that is unique morphologically, physiologically, and biochemically8, 9. Some embryos within the population bypass or “escape” diapause II and develop continuously toward hatching3, presumably completing their entire lifecycle within a single rainy season, while embryos that enter diapause II will have to wait for several months or perhaps years to hatch. Having mixed proportions of the diapause and escape phenotypes may provide an advantage for survival of the population in the face of unpredictable or fluctuating conditions. Because tolerance of extreme conditions is higher in diapausing embryos, there is a clear advantage for entering diapause II under times of extended drought.
Figure 1: Annual life cycle of Austrofundulus limnaeus. Adults spawn during the rainy season producing mixed proportions of diapause and escape embryos. Survival throughout the dry season is achieved by entrance into diapause II during embryonic development. When the ponds return with the rainy season, embryos continue developing toward hatching. Credit: Ignacio de la Higuera, December, 2018.
Vitamin D is not for Diapause
The developmental phenotype of killifish is regulated by two factors: an age-related maternal effect, and the incubation environment of the embryo. Typically, younger females produce a high proportion of escape embryos and older females produce diapausing embryos. However, offspring from a single spawning event, even when incubated under identical conditions, can differ in developmental outcomes. While phenotype can initially be determined by maternal patterns, the embryonic environment can override this programming9. The strongest effectors are exposure of embryos to light and incubation temperature, and there is a critical window (between 10 and 20 pairs of somites) prior to the entry into diapause II when these environmental factors cause commitment to a given developmental trajectory.
We recently identified temperature-dependent vitamin D3 signaling as a regulator of developmental phenotype in A. limnaeus10. When eggs from a single female are incubated at temperatures of 30˚C and 20˚C, embryos develop exclusively along escape and diapause trajectories, respectively, independent of maternal influences9. Under warmer conditions, embryos express a network of genes that code for enzymes responsible for synthesizing 1α, 25-dihydroxyvitamin D3, the active form of vitamin D3, and the signaling molecule that binds to the vitamin D receptor (VDR). Presumably, VDR binding to DNA response elements initiates a gene expression program that drives active growth and development along the escape trajectory (Figure 2).
Common knowledge of vitamin D3 synthesis and signaling is based largely on studies in humans and other terrestrial mammals, and spurred by the discovery of the importance of vitamin D3 in preventing rickets, a disease caused by perturbations in blood calcium homeostasis. Vitamin D3 is not actually a vitamin, but is rather a highly potent hormone that, under the right conditions, can be synthesized by humans and many other species from the precursor molecule 7-dehydrocholesterol (7-DHC). In humans, exposure of 7-DHC in the skin to UV-B light and increased temperatures results in the production of vitamin D3 (cholecalciferol)11. Vitamin D3 is then hydroxylated by two different enzymes to produce 1α, 25-dihydroxyvitamin D3. In many systems it appears that formation of vitamin D3 is a major limiting step in this synthetic pathway – thus linking seasonal changes in light and temperature to organismal levels of vitamin D3. It important to note that some species of fish can synthesize vitamin D3 using blue light and recent work suggests the potential for enzymatic conversion of 7-DHC to vitamin D3-like compounds in mammals12, 13.
Figure 2: Vitamin D synthesis and signaling with VDR. Sufficient vitamin D3 synthesis (magenta) is achieved in the escape pathway that, upon binding to the vitamin D receptor (blue) bound to DNA (orange and red), alters transcriptional pathways to promote growth and development. Otherwise, the receptor induces pathways for entrance into diapause. Credit: Vitamin D receptor bound to DNA, with vitamin D3 shown in magenta, David Goodsell, November 2012; doi:10.2210/rcsb_pdb/mom_2012_11.
Deep conservation for regulating life history decisions in animals
The VDR is a nuclear receptor (NR) – a transcription factor whose action is regulated by binding to specific molecules or ligands. Upon ligand binding, the VDR forms heterodimers with coactivating NRs such as the retinoid X receptor and the thyroid hormone receptor to drive cascades of gene transcription for calcium transport, hormone secretion, and cellular proliferation and differentiation14. The VDR is the vertebrate homolog of daf-12, a NR critical for regulating dauer dormancy – a state of developmental arrest similar in many ways to A. limnaeus diapause – in the nematode Caenorhabditis elegans15, 16. It is well understood that environmental induction of insulin/IGF-1, TGFβ, and cGMP signaling pathways converge on DAF-12 to inhibit dauer formation and promote active growth, development, and reproduction15. Thus, it appears that regulation of developmental arrest and dormancy is regulated by homologous pathways in nematodes and fishes, suggesting a deeply conserved mechanism for integration of environmental signals into animal developmental programs.
Maternal-embryo conflict: checkpoints for phenotypic plasticity
The ecological significance of light and temperature in vitamin D3 signaling and regulation of developmental trajectory is not fully understood in A. limnaeus or any other species of annual killifish. In their native habitat in the Maracaibo basin of Venezuela, embryos of A. limnaeus are exposed to a wide range of temperatures and may – especially during the dry season – be exposed to light. Thus, vitamin D3 synthesis can provide a direct link between variations in the developmental environment and regulation of phenotype. However, developmental phenotype in A. limnaeus is regulated at two separate life stages: through maternal influences during oogenesis and later by the environmental conditions experienced by free-living developing embryos (Figure 3). Having two checkpoints that offer a regulatory capacity for determining developmental phenotype may offer a distinct survival advantage in the highly variable and often unpredictable seasonal ponds.
Figure 3: Maternal versus embryonic regulation of phenotype in Austrofundulus limnaeus. The annual killifish utilizes a deeply conserved mechanism of secosteroid hormone synthesis and nuclear receptor binding in a manner that provides the species two life stages at which developmental phenotype can be regulated. At fertilization, (A) the embryo is provisioned with as of yet unknown maternal factors that can determine developmental phenotype, and (B) at approximately 10 pairs of somites, embryos can activate Vitamin D3 synthesis with can override maternal signals that would induce diapause (B). The nuclear hormone receptor, VDR, can initiate programs of gene transcription for either the diapause or escape pathway.
We hypothesize that maternal influences related to maternal age may help to integrate more reliable and predictable environmental conditions into the developmental program. Ecologically, this is logical because young females are usually found in newly formed ponds at the beginning of the season, and their early offspring have the greatest probability of completing the life cycle within a single rainy season. This may be especially important in ponds that experience multiple inundation and drying events during a single rainy season, allowing multiple generations or attempts at reproduction during a single season – a scenario that would be almost impossible if all embryos entered into diapause II. Maternal influences may also integrate other environmental factors that have yet to be explored in this system, such as food quality and quantity and social interactions.
Regulation of developmental phenotype during embryogenesis in a free-living embryo offers an opportunity to alter developmental outcomes in response to environmental cues that are inconsistent with maternal programming. Recent fieldwork suggests that embryos of annual killifishes remain in diapause I during the duration of the rainy season, and that development between diapause I and II occurs during the initial period of pond drying. Thus, the narrow window of development when temperature and light may affect development would allow for the embryo to “assess” environmental conditions when the ponds are drying and potentially alter developmental trajectory based on local environmental conditions. In this scenario, warm and moist conditions and/or exposure to light would result in active development while cool and dark conditions would favor entrance into diapause II. This mechanism could allow for embryos to respond to prevailing seasonal patterns in the environment, or to local microenvironmental cues. Perhaps embryos that are buried deep in the substrate remain cool and dark and enter into diapause II, while those in the upper layers experience warmer conditions with the chance for light exposure and thus develop along the escape trajectory. It is important to note that very little is known about the spawning behavior of annual killifishes, and it is possible that females may choose spawning sites, for instance in the shallow pond periphery, where the chances for exposure to higher temperatures and light are more likely. Thus, adding another layer of complexity to the potential for bet-hedging of developmental phenotype in this species.
Figure 4: Escape embryo of Austrofundulus limnaeus.Photograph credit: Daniel Zajic, December 2016.
Animals have evolved exquisite strategies to optimize developmental programs to prevailing environmental conditions to maximize growth, survival, and fitness. Here we have uncovered what appears to be a deeply conserved mechanism for integrating environmental cues into animal developmental programs with respect to entrance into developmental dormancy. This discovery suggests that many other developmental and life history transitions may be regulated in a similar manner. While it has been known for many years that nuclear receptors can be powerful regulators of phenotype, their role in developmental plasticity has received much less attention. We predict a major role for the VDR and other NRs in the regulation of other major vertebrate life history transitions such as smoltification in salmonids, metamorphosis in amphibians, and hibernation in small mammals.
Icons of evolution: Heliconius butterflies, Meyer A, PLoS Biology, Vol. 4/10/2006, e341 doi:10.1371/journal.pbio.0040341 CC-BY 4.0
In the latest episode of Genetics Unzipped, Kat Arney is reporting back from the Genetics Society’s Autumn meeting – Genotype to Phenotype to Fitness – asking whether street smart city-dwelling birds are genetically different from their country bumpkin relatives, how butterflies got their brightly patterned wings, and if today’s genetic research would have blown Darwin’s mind.
Plus, we have an interview with the new president of The Genetics Society, Professor Laurence Hurst, about his vision for the coming centenary year and beyond.
If you enjoy the show, please do rate and review and spread the word. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com
In contrast to nerves in the central nervous system, peripheral nerves are highly regenerative following injury. Regeneration is critically dependent on Schwann cells, the main glial cell type of the peripheral nervous system, but whether an additional stem cell or progenitor population also contributes has been a matter of contention. A paper in Development now addresses this issue with a characterisation of Schwann cell behaviour in the homeostatic and regenerating mouse peripheral nerve. We caught up with first author and PhD student Salome Stierli, and her supervisor Alison Lloyd, Professor of Cell Biology at the MRC Laboratory for Molecular Cell Biology at University College London, to find out more about the story.
Salome (L) and Alison (R) in the lab
Alison, can you give us your scientific biography and the questions your lab is trying to answer?
AL I trained as a biochemist at University College London (UCL), before studying for my PhD with Chris Marshall and Alan Hall at the Institute of Cancer Research, also in London. To work with Chris and Alan was the best possible start to a career in science and they remained my mentors until their untimely deaths in 2015. After my PhD, I did a couple of post-docs in Strasbourg and at the Imperial Cancer Research Fund in London, interspersed with a three-year break during which I had a baby and worked as an IP administrator with the Ludwig Institute.
I returned to UCL to start my own laboratory at the MRC Laboratory for Molecular Cell Biology in 1999, and I have remained here ever since. Throughout my career, I have always been a cancer biologist, and intrigued by how we can stay ‘normal’ for so long (homeostasis), and what causes these controls to break down (cancer). While initially I started by studying oncogenic signalling pathways in cells, it became increasingly clear that to answer these questions required an understanding of cell signalling within the context of tissue biology.
The peripheral nerve has become our tissue of choice for these questions, and the biology of this tissue enables us to address many fundamental biological questions. Peripheral nerves mainly consist of neurons and glia (Schwann cells), and the behaviour of each cell type is controlled by interactions between them. Crucially, peripheral nerve is a regenerative tissue, and we can use it to understand the multicellular response allowing tissues to switch between normal and regenerative states, which has important implications for multiple aspects of tumourigenesis. We continue to ask questions about how cells talk to each other to create a tissue and reach a homeostatic state, how the tissue is reprogrammed to regenerate following injury, and how this is relevant to the processes of tumourigenesis.
And Salome, how did you come to join the Lloyd lab, and what drives your research?
SS I joined the Lloyd lab as graduate student fascinated by the biology of the nervous system. Prior to joining the Lloyd lab, I had been working on glioblastoma signalling in the group of Brian Hemmings in Basel, and ever since that time I have been highly interested in understanding the mechanisms that drive tumour formation within the nervous system. The extensive expertise of the Lloyd lab in understanding the mechanisms of peripheral nervous system (PNS) regeneration and tumourigenesis, and the lab’s use of tissue regeneration as a model for multiple aspects of tumourigenesis, provided me with the best environment to pursue my research interests.
Why has there been controversy over the existence and activity of stem cells in peripheral nerve regeneration?
SS & AL Adult stem cells exist in most tissues and have important roles in maintaining these tissues and/or in their response to injury. In peripheral nerve, mature adult Schwann cells were known to be able to re-enter the cell cycle, but to many it seemed likely that a stem cell population either ‘helped’ in the regeneration of this tissue and/or was the cell of origin for Schwann cell-derived tumours. Such a progenitor population exists in the central nervous system (CNS); for example, oligodendrocyte precursor cells produce oligodendrocytes (the corresponding cell to the PNS Schwann cell) throughout life. Previous studies have suggested that skin stem cells could contribute to the regenerative process, whereas other studies have suggested that adult Schwann cells retain a certain level of multipotency. However, most of these studies have been performed in vitro or in relatively non-physiological conditions.
Can you give us the key results of the paper in a paragraph?
SS & AL We show that peripheral nerve regeneration is underpinned by the proliferation of mature cells rather than the activation of a stem cell population. In particular, we demonstrate that although Schwann cells are highly quiescent, stable cells in the adult nerve, they all retain the capacity to dedifferentiate to a proliferating, migratory, progenitor-like Schwann cell following nerve injury. Moreover, these progenitor-like cells remain restricted to the Schwann cell lineage with both a tumorigenic mutation (loss of Nf1) and a conducive microenvironment required to enhance their plasticity.
A transverse section of an adult sciatic nerve from a mouse in which myelinating Schwann cells have been labelled with Confetti fluorophores (P0-CreERT2:Confetti).
Why do you think there are such distinct mechanisms for maintaining the myelinating cells of the PNS and CNS, cells that seem to do similar jobs in similar environments?
SS & AL We can only speculate upon this, but it is likely to reflect a trade-off between plasticity and stability: the CNS requires increased plasticity to myelinate new axons during processes like learning, for example, while the PNS requires stability to transmit signals from the CNS to tissues and organs. Moreover, although a continually proliferating progenitor population provides a rapid source for new myelination, it is also a pool susceptible to tumour development. This has been shown for other tissues, where the presence of proliferating stem or progenitor cells was correlated to enhanced susceptibility to tumourigenesis. Consistent with this, malignant tumours are more frequent in the CNS compared with the PNS, which perhaps reflects the presence of a more susceptible, proliferating progenitor population.
Does your work offer any clues for how we might enhance peripheral nerve regeneration in a clinical context?
SS & AL Yes, it suggests that stem cells are unlikely to be beneficial for improving peripheral nerve repair; encouraging the nerve’s ‘natural’ regenerative processes is likely to be the best approach. In addition, we suggest that the function of a regenerated tissue could be improved by clearing the matrix that accumulates but isn’t removed naturally following an injury.
When doing the research, did you have any particular result or ‘eureka moment’ that has stuck with you?
SS One of the most exciting moments in the lab was when we performed correlation light electron microscopy (CLEM) in order to clarify the plasticity of myelinating Schwann cells (mSCs) following nerve injury. The protocol took a while to perfect, but the moment we first managed to visualise the mSC-derived cells associated with small calibre axons was so rewarding that all the long hours and the effort was forgotten.
The moment we first managed to visualise the mSC-derived cells associated with small calibre axons was so rewarding
And what about the flipside: any moments of frustration or despair?
SS Yes of course: in general behind each scientific paper there are years of hard work that includes many failed experiments and some particularly frustrating moments. During this project, very frustrating moments involved spending many days preparing precious samples only to encounter technical issues with confocal microscopes. I have also experienced the difficulties of working with live animals, as this involves the impact of factors that are completely out of your own control.
So what next for you after this paper?
SS I am about to complete my PhD and I am thinking about doing a postdoc in in the field of cancer biology. Prior to that, I am planning to write a review on the plasticity of myelinating Schwann cells. I have not decided yet which lab I will join for my postdoctoral training, but in general I am very interested in extending my skills, for example by learning cutting-edge techniques such as in vivo imaging in order to elucidate the processes driving the early stages of tumour formation.
Where will this work take the Lloyd lab?
AL This work has increased our understanding of the cellular environment of the peripheral nerve, and has provided us with a tool-box to gain a greater understanding of how the environment of the nerve can be regenerative but also how it can provide a tumourigenic environment. We are currently exploring how the microenvironment of the nerve can both promote and inhibit tumourigenesis, and this work is a fundamental starting point to that question. This work has also shown that the reprogramming of a mature Schwann cell to a progenitor cell is a remarkably efficient process. We don’t understand how this works and that is an important question for the lab. In this paper, we have also characterised a new cell type in peripheral nerve, which we have called tactocytes. I want to know what they are doing!
Finally, let’s move outside the lab – what do you like to do in your spare time in London?
SS I must confess that in the last year of my PhD, spare time outside the lab has been fairly rare. However, when I got some time outside of the lab, I particularly enjoyed exploring the vibrant art and music scene in London and simply having a good conversation with friends over a nice meal. I also enjoy exploring the countryside outside of London and love to travel to new places.
The ability to sense and respond to light is fundamental to plant development. As seedlings move from the soil to the air, a switch in developmental program occurs to promote light capture and autotrophic growth. A paper in Development now provides a molecular analysis of the proteins that regulate this transition in Arabidopsis. We caught up with first author and PhD student Vinh Ngoc Pham, and her supervisor Enamul Huq, Professor of Molecular Biosciences at The University of Texas at Austin, to find out more about the work.
Enamul (L) and Vinh (R)
Enamul, can you give us your scientific biography and the questions your lab is trying to answer?
EH I received BSc and MSc degrees in Biochemistry from the University of Dhaka, Bangladesh, in 1987 and 1988, respectively. As a graduate student in Thomas Hodges’ lab in Purdue University I worked on characterizing hypoxia-inducible gene expression in rice. I then did my post-doctoral research with Peter Quail at UC Berkeley, where I isolated and characterized phytochrome interacting factors (PIFs) in Arabidopsis.
I began my academic career as an Assistant Professor of Molecular Cell and Developmental Biology at The University of Texas at Austin in 2003, and am now a Professor of Molecular Biosciences. Research in my lab is focused on understanding how plants sense, interpret and respond to environmental light conditions that regulate almost every aspect of the life cycle, from seed germination to flowering time. Specifically, we focus on the red/far-red light photoreceptors (phytochromes) and their interacting PIFs.
And Vinh, how did you come to join the Huq lab and what drives your research?
VNP I have been fascinated by plants and how they respond to light signals since I was an undergraduate student. I did a student internship at POSTECH University in South Korea, working on PIFs. I decided to pursue my Master’s degree in POSTECH, and that was a great opportunity for me to learn the biology of light signalling. I first met Enamul during the International Plant Molecular Biology Conference in South Korea in 2012, and was interested in his research about PIF regulation. Being a Fellow of the Vietnam Education Foundation (VEF), an educational exchange program between Vietnam and the USA for PhD programs, I decided to apply to Enamul’s lab to continue my research in the light-signalling field.
What makes skoto-to-photomorophogenesis such a crucial developmental transition in a plant’s life?
VNP & EH Germinating seeds and young seedlings are very vulnerable, just like human babies at young ages. A proper transition from the dark-adapted developmental program called skotomorphogenesis to the light-adapted developmental program called photomorphogenesis is crucial for their survival. Skotomorphogenesis is defined by seedlings having long hypocotyls, appressed small cotyledons and an apical hook. This morphological pattern ensures the safe emergence of the seedlings, protecting their apical region in the subterranean darkness. Conversely, photomorphogenesis is defined by seedlings having short hypocotyls and open, expanded and green cotyledons that help plants capture maximum sunlight for photosynthetic energy production and autotrophic growth. Scientists have been using this transition to study light-signalling pathways for decades, and have isolated many mutants that mimic light-grown plants when grown in darkness. These are called constitutive photomorphogenic (cop) mutants, and have tremendously helped decipher mechanisms of light-signalling pathways.
Can you give us the key results of the paper in a paragraph?
VNP & EH In this paper, we provide multiple lines of evidence to explain why the cop1, spaQ and pifQ mutants display constitutive photomorphogenic phenotypes. Previously, it was shown that the so-called positively acting transcription factors were more abundant in cop1, spaQ and pifQ mutants compared with wild type, resulting in the cop phenotype. However, we now show that the cop phenotypes are not only due to a high abundance of the positively acting transcription factors, but also due to a reduced level of PIF protein levels. In addition, a high abundance of HFR1 in the cop1 and spamutants also reduces PIFs transcriptional activity. Strikingly, the gene expression signature of cop1 and spaQ overlaps with pifQ in the dark, with a preferential targeting of PIF direct target genes. All three activities are tightly linked to each other, contributing in concert to the cop phenotypes.
How do you think the COP1/SPA complex regulates PIF abundance and activity?
VNP & EH We think that the COP1/SPA complex regulates PIF abundance in both dark and light conditions. In the dark, HFR1, an atypical bHLH factor, is more abundant in the cop1 and spaQ mutants compared with wild type. HFR1 heterodimerizes with PIFs and induces degradation of this heterodimer through the COP1/SPA complex in darkness. In addition, HFR1 also sequesters PIFs from binding to DNA, thereby inhibiting PIF activity in darkness. In response to light, PIFs are phosphorylated in a phytochrome interaction-dependent manner. The phosphorylated forms of PIFs are then recruited by the COP1/SPA complex in a CUL4COP1-SPA E3 Ubiquitin ligase complex for ubiquitylation and rapid degradation through the 26S proteasome pathway.
Seedlings following PIF overexpression in a cop1-4 mutant background
When doing the research, did you have any particular result or eureka moment that has stuck with you?
VNP In our lab, we do a lot of biochemistry and molecular genetics. At the beginning, I spent a lot of time studying genomic data analysis and enjoyed applying genomic data to the big picture of plant light-signalling pathways. Therefore, applying transcriptomic and gene expression tools in cop1, spaQ and pifQ mutants in this paper gave us an integrated view of how gene expression is regulated in light-signalling pathways. The interesting part of this paper was when we analysed the RNA-Seq data and figured out that more than 40% of PIF-regulated genes are significantly regulated by COP1 and SPA. After that we became more confident about our hypothesis of the cop phenotypes due to the regulation of PIF level and PIF transcriptional activity.
And what about the flipside: any moments of frustration or despair?
VNP I became frustrated many times working on the PIF degradation experiments. PIFs are degraded very quickly in the light so we have to do all the PIF protein experiments in the dark. That was not easy at the beginning but when I got used to it, I really enjoyed doing experiments in the dark room. It also gave me a chance to take an occasional nap!
It took us a very long time to generate the PIF5 overexpression line in the spaQ background, as this is a quadruple mutant and a tiny plant. spaQ mutants do not make a lot of seeds for the next generation, so we spent a lot of time genotyping and waiting for enough seeds to do all of our assays.
So what next for you after this paper?
VNP I am very excited about applying computational biology methods to study regulatory gene networks in light-signalling pathways. I hope this will be a great resource for other researchers and will provide a holistic view of the transcriptional regulatory mechanisms in Arabidopsis light-signalling pathways. After this project, I would like to find a postdoctoral position where I can apply and develop both my molecular genetics skills and computational biology to understand biological networks.
Where will this work take the Huq lab?
EH We are still focusing on the intersection between the COP1/SPA complex and PIFs. A simple Pubmed search on ‘COP1’ resulted in over 550 papers – including many from us – published in the last few years, and we really think they hold the key to light-signalling pathways in plants. On a molecular level, SPA proteins have kinase-like domains at their N terminus, but a kinase activity has not been demonstrated yet. If SPAs do function as protein kinases, the COP1/SPA complex might function as a unique cognate kinase-E3 Ubiquitin ligase complex for rapid phosphorylation and degradation of their substrates. There is so much more to be learned about these complexes and their biochemical functions in regulating plant development.
There is so much more to be learned about these complexes and their biochemical functions in regulating plant development
Finally, let’s move outside the lab – what do you like to do when you’re not working?
EH Austin is a lovely city. We really enjoy hiking and exploring the trails and parks nearby. In addition, Austin is the music capital of the world as well as a kind of second Silicon Valley with many high-tech companies established here. With increasing international population on campus as well as around the city, Austin has a variety of great food and culture throughout the year.
VNP When I have free time, I try to cook great food, and I think I am a good pastry chef too. I think doing science is like cooking, following new recipes (protocols) to come up with interesting new outcomes!
The Company of Biologists (biologists.com) is looking to recruit an experienced Scientific Copy Editor to work across our portfolio of five life-science journals. This temporary position is expected to last for up to a year (starting March 2019).
The role entails copyediting articles to a high standard, compiling author corrections, overseeing the journal production process, and liaising with authors, academic editors, external production
suppliers and in-house staff to ensure that articles are published in a timely and professional manner.
Candidates should have a degree (ideally a PhD) in a relevant scientific area. Previous copyediting experience is essential. Additional requirements include excellent literacy skills, high attention to detail, a diplomatic communication style, good interpersonal and IT skills, a flexible approach and the ability to work to tight deadlines.
The position gives an experienced copy editor the opportunity to work on our highly successful life-science journals and offers an attractive salary and benefits. The position will be based in The Company of Biologists’ attractive modern offices on the outskirts of Cambridge, UK.
The Company of Biologists exists to support biologists and inspire advances in biology. At the heart of what we do are our five specialist journals – Development, Journal of Cell Science, Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open – two of them fully open access. All are edited by expert researchers in the field, and all articles are subjected to rigorous peer review. We take great pride in the experience of our editorial team and
the quality of the work we publish. We believe that the profits from publishing the hard work of biologists should support scientific discovery and help develop future scientists. Our grants help support societies, meetings and individuals. Our workshops and meetings give the opportunity to network and collaborate.
Applicants should send a CV to recruitment@biologists.com, along with a covering letter that summarises their relevant experience, why they are enthusiastic about the role, and their current
salary.
All applications must be received by Monday 11 February.
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