From six-toed cats to cyclops lambs – and, of course, it’s fabulous name – the Sonic Hedgehog gene has a fascinating history, as well as a whole bunch of interesting developmental biology behind it.
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By Koichiro Uriu, Bo-Kai Liao, Andrew C. Oates and Luis G. Morelli
How local cell-cell communication can generate a global tissue pattern is one of the fundamental questions in developmental biology. Yet, studying this remains challenging, because developing tissues involve complexities such as cell rearrangement, heterogeneity along the body axis, and massive tissue shape changes. In our recent paper in eLife (Uriu, Liao et al. 2021), we addressed this question using the zebrafish segmentation clock as a model system. Our integration of experimental and theoretical approaches revealed that after desynchronization, the recovery of the iconic synchronized wave pattern in the segmentation clock is influenced by two distinct spatial and temporal scales. Firstly, there is the faster and local communication directly between the cells, and secondly, there is a slower and much longer-distance movement of the cells and the tissues in the embryo as the tissues change their overall shapes.
This work started about 10 years ago, when the four of us were all at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany. In fact, we all worked in the same research group, so local communication was relatively easy. This local discussion was triggered when one of us, Bo-Kai Liao, noticed an unusual pattern of disrupted body segments in zebrafish embryos as they attempted to re-synchronize their cellular clocks after perturbation. The thing that struck us was that the pattern really couldn’t be explained by the current models of re-synchronization.
We are sure you all recognize the situation: “Hang on a minute, that should not be happening…” and you just know you are going to find out something cool. Well, in this case, the journey from initial observation to an explanation would take us a decade, during which time all of us moved from the MPI-CBG. We each moved country at least once, and we changed jobs and titles and had families and even got some grey hair. The scientific story mirrored the personal: how local communication is affected by long-distance movements. To understand this story better, we need to go back to the beginning and start with the biology.
The precursors of the body segments of vertebrates, called somites, are formed rhythmically during embryonic development. Each segment buds off from the unsegmented tissue, presomitic mesoderm (PSM) one by one. The rhythm of segment formation is determined by an oscillatory spatial pattern of gene expression in the PSM and tailbud, termed the segmentation clock. In these tissues, the peaks of gene expression travel across the anterior-posterior axis. Cells in the tissue synchronize their gene expression rhythms with neighbors through Delta-Notch signaling, providing local integrity of gene expression patterns. Understanding this synchronization, and it’s role in forming body segments is what brought the four of us together in the first place.
Previous studies had shown that the treatment of an inhibitor of Delta-Notch signaling, called DAPT, led to formation of defective segments in zebrafish embryos. Some of these studies had also observed the recovery of normal segments after the washout of DAPT (Riedel-Kruse et al. 2007; Liao et al. 2016). These results had been interpreted in terms of desynchronization and resynchronization of oscillators: treatment of DAPT desynchronizes oscillators by inhibiting intercellular coupling through Delta-Notch signaling, and the hallmark spatial wave pattern of gene expression in the PSM is abolished due to the noise in the individual cellular oscillators. However, its washout restores coupling, letting cells gradually resynchronize their oscillators. When the synchrony level reaches a threshold, a normal segment reappears. This desynchronization hypothesis has quantitatively explained several experimental data, but there was a remaining gap in our understanding: how is the tissue-scale pattern reorganized through local coupling?
Figure 1. Intermingled segment boundary defects after DAPT washout. (A) Control embryo. (B) Embryo with late DAPT washout. (C) Embryo with early washout. Defective segments appear even after first recovered segment (FRS). ALD: anterior limit of defect. PLD: posterior limit of defect.
To address this, we analyzed segment recovery processes with different DAPT washout timing in zebrafish embryos. We unexpectedly found that washing out DAPT at earlier developmental stages caused intermingled segment boundary defects: a defective segment boundary was formed even after some normal segment boundaries were already formed (Fig. 1). This result suggests that the synchrony level fluctuates around the threshold, for some reason. We were very surprised by this result because we expected a monotonic recovery of normal segments based on previous theoretical works on coupled oscillators. Usually, once a population of oscillators gets synchronized, they remain synchronized and a large fluctuation of synchrony level hardly ever occurs. Thus, pattern recovery in the segmenting tissue seemed not so simple.
At this time, there were no transgenic markers of the zebrafish segmentation clock, nor were the microscopes, image processing, and data analysis developed to follow all these oscillating cells during the entire re-synch process (in fact, we are still working on this…).
Therefore, we decided to use a physical model for the segmenting tissue to analyze this phenomenon. We have previously proposed models for the entire presomitic mesoderm and tailbud tissues in lower spatial dimension (1D or 2D; Morelli et al. 2009) or in 3D space but only a part of the tissue (Uriu et al. 2017). This time, we chose to describe the entire tissue in 3D and tried to integrate some of the previous modeling efforts by us and others in this framework (Fig. 2).
Figure 2 Physical model of the PSM and tailbud. Cells are represented as particles and rendered as spheres. Color represents the phase of oscillation. R: right. L: left. The model includes four key aspects in the tissue shown in right schematics.
In numerical simulations of the model, we found a rotating phase pattern of oscillators, termed a phase vortex, in some situations of resynchronization (Fig. 3A, B). A phase vortex emerges in the tissue by local interactions of oscillators, moves through the tissue along the anterior-posterior axis by cell advection, and generates a defective boundary when it arrives at the anterior part of the PSM. A phase vortex can be formed posterior to the well-synchronized domain in simulation, so it can cause intermingled defective segments, as observed in the experiments.
Figure 3 Phase vortices and intermingled defects in simulations. (A) Snapshots of segments and phase patterns in the PSM. (B) Phase vortex in the PSM. Yellow arrow indicates the direction of rotation of the vortex. (C) Comparisons of time to first recovered segment (FRS) and time to posterior limit of defect (PLD) between embryonic experiment (exp.) and simulation (sim). twash-out: DAPT washout time. ss: somite stage.
It turns out that although the formation of a phase vortex is driven by the local interactions, its kinematics is determined by the larger-scale tissue properties. A phase vortex moves from posterior to anterior by cell advection caused by embryonic axis extension. We reasoned that the global tissue properties, such as cell advection and tissue length could affect the pattern recovery in the PSM. Moreover, these tissue properties change with developmental stages. Hence, our physical model predicted that the time to complete recovery, that is, when we do not see any further phase vortices, would depend on the developmental stage at which DAPT washed out. In fact, we observed good agreement between simulations and experiment for the time to complete recovery (Fig. 3C). So, are we done? No. The next key task is to test the theory by observing phase vortices in living tissues. This should be possible by the live imaging of reporters of both oscillatory protein and segment boundaries simultaneously, which requires the long-term imaging, tracking and analysis of the cells in the segmentation clock.
In summary, our study indicated that pattern recovery in the zebrafish segmentation clock occurs at two spatial and temporal scales: quick local synchronization and transport of local patterns through slow tissue shape changes. There’s a nice symmetry to the slow, long-range drift of the four authors, which certainly changed the dynamics of our local communication. Although we were already quite good at video conferencing when the pandemic struck, the available time interval when everyone on the team was awake and alert simultaneously in Japan, Taiwan, Switzerland and Argentina was rather short. Thus, a high signaling strength was an important asset. Indeed, working together on the project over such a long time and through so many life changes was a rewarding experience, and we hope you will enjoy the pattern that self-organized from the process as much as we do.
Uriu K., Liao BK., Oates A.C., Morelli L.G. (2021) From local resynchronization to global pattern recovery in the zebrafish segmentation clock. Elife 10:e61358. doi: 10.7554/eLife.61358.
References
Liao BK., Jörg D.J., Oates A.C. (2016) Faster embryonic segmentation through elevated Delta-Notch signalling. Nat Commun. 7:11861. doi: 10.1038/ncomms11861.
Riedel-Kruse I.H., Müller C., Oates A.C. (2007) Synchrony dynamics during initiation, failure, and rescue of the segmentation clock. Science 317(5846):1911-5. doi: 10.1126/science.1142538.
Morelli L.G., Ares S., Herrgen L., Schröter C., Jülicher F., Oates A.C. (2009) Delayed coupling theory of vertebrate segmentation. HFSP J. 3(1):55-66. doi: 10.2976/1.3027088.
Uriu K., Bhavna R., Oates A.C., Morelli L.G. (2017) A framework for quantification and physical modeling of cell mixing applied to oscillator synchronization in vertebrate somitogenesis. Biol Open. 6(8):1235-1244. doi: 10.1242/bio.025148.
The plant epidermis is a single layer of cells that forms a crucial barrier to the outside world, but the mechanisms that control epidermal differentiation – in particular the relative importance of position and lineage – remain incompletely understood. A new paper in Development tackles this question in Arabidopsis. To find out more about the story, we caught up with first author Kenji Nagata and his supervisor Mitsutomo Abe, Associate Professor at the University of Tokyo.
Mitsutomo (L) and Kenji (R).
Mitsutomo, can you give us your scientific biography and the questions your lab is trying to answer?
MA: As a PhD student I researched molecular genetics in Arabidopsis in the lab of Yoshibumi Komeda at Hokkaido University in Sapporo, Japan. I was excited to go to the lab every day, and so fascinated by the beautiful expression patterns of ATML1 and PDF2, twin genes that enabled me to get my PhD in 2001. After my PhD, I joined the lab of Takashi Araki at Kyoto University as an Assistant Professor and started the ‘florigen quest’ with great colleagues. I was fortunate to make a fundamental discovery regarding florigen in 2005, and I am very pleased that FT is now well known as an important component of florigen signalling. After this, I had a great experience working in the lab of Richard Amasino at the University of Wisconsin for two years, and very much enjoyed American life. I then moved to the University of Tokyo and started my own research group in 2009, and keep working on plant molecular genetics.
Our research group has a broad interest in plant development. Some members are involved in elucidating florigen function in Arabidopsis, others (like Kenji) are working on epidermal cell differentiation, and others are interested in the interaction between meristem identity and plant architecture. All have one thing in common: we are focusing on phenomena and molecules that are unique to plants.
Kenji – how did you come to work in Mitsutomo’s lab and what drives your research today?
KN: When I was an undergraduate student, I was very interested in two aspects of sessile plants: the phenotypic plasticity they show in morphology and physiology, and their developmental robustness in terms of patterning. So I looked for a lab that would allow me to explore these issues. I was fortunate to attend one of Mitsutomo’s lectures, where he described his lab’s work on flowering, which is mediated by signals from the external environment, and robust protodermal cell differentiation, which is not influenced by the external environment. I felt that working with him would provide me exciting research opportunities, and decided to join his lab. Curiosity about how the traits unique to sessile plants work and why they developed in an evolutionary context drives my research today.
How has your research been affected by the COVID-19 pandemic?
KN: Due to entry restrictions to the university, we were forced to suspend our experiments and stay at home. Fortunately, none of the members of our lab have caught the virus and all have stayed healthy so far. Although now there are still some restrictions, it is slowly getting back to (a new) normal.
MA: Like most universities and research institutes, only the minimum number of staff necessary to maintain plants and equipment was permitted to enter the lab from spring to summer. Since mid-July, research activities are allowed with the utmost care to prevent the spread of infection. But the number of infected patients in Tokyo is increasing recently, so I’m worried that the lab will be closed again.
Before your work, what was known about the relative roles of lineage and position in plant epidermis differentiation?
KN: It is widely accepted that cell position rather than cell lineage is important for plant cell fate decisions. For example, the inner cells, which derive from occasional periclinal divisions of epidermal cells, develop according to their position rather than their epidermal cell lineage. On the other hand, it is also known that, in shoots, the inner cells never adapt their epidermal identity even if they occupy the outermost position, suggesting that cell lineage is involved in the epidermal cell differentiation. However, it was recently shown that when cells in the inner cell lineage are displaced to the outermost position through laser ablation, they appear to acquire root epidermal cell fate. Thus, it is controversial whether cell lineage indeed affects epidermal cell fate decisions.
Can you give us the key results of the paper in a paragraph?
KN: In this paper, we found that ATML1, a master regulator of protoderm/epidermis differentiation, is only stabilized in the outermost cells derived from the outermost cell lineage. Furthermore, the stability of ATML1 in these cells is conferred by the interaction with its lipid ligand VLCFA-Cers. VLCFA-Cers appears to be polarly localized to peripheral domains in epidermal cells, and passed on to the outermost epidermal cells in a cell position- and lineage-dependent manner. Based on these results, we have proposed a novel model in which ATML1-VLCFA-Cers interaction is restricted to the outermost epidermal cells and consequently restricts protoderm/epidermis differentiation to the appropriate position.
MA: I think for me the key experiment in our paper is the transient induction of ATML1 by a heat shock treatment – I was very excited when Kenji first came to show me the GFP images.
Expression of gATML1-EGFP in two 16-cell embryos.
When outermost cells divide asymmetrically, the inner cells inherit ATML1 protein: what then stops them from differentiating as protoderm?
KN: This is because the ATML1 protein in inner cells is rapidly broken down, as shown in the careful observation in the 16-cell-stage embryo of gATML1-EGFP plants, or in our transient ectopic expression assay using HSP::NLS-mCherry; HSP::ATML1-EGFP plants in which the inner cells are not able to differentiate into protoderm. The rapid breakdown of ATML1 protein is due to the absence of VLCFA-Cers, in inner cells. Thus, we propose that VLCFA-Cers act as a landmark of the outermost cell position and lineage, and act as a post-translational signal that mediates positional information.
MA: Epidermis-specific expression of ATML1 and PDF2 is strictly regulated. Therefore, in addition to the mechanism we reported here, I believe that several other regulatory mechanisms are involved in epidermal cell differentiation. I’m really looking forward to the seeing this research progress in the future.
I was very excited when Kenji first came to show me the GFP images
Do lipid-transcription factor complexes mediate positional signals elsewhere in plant development?
KN: We would assume so. Kathrin Schrick and colleagues have shown that START domains from plant HD-Zip transcription factors bind lipid ligands to regulate transcription factor activity in a yeast system. Together with our findings, this suggests that lipids may mediate positional signals and modulate HD-Zip transcription factor activity elsewhere in plant development.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
KN: I think the most memorable moment was when I observed the outermost cell-specific ATML1-EGFP signal after heat-pulse treatment. This was an important moment when I was certain that our hypothesis was right.
And what about the flipside: any moments of frustration or despair?
KN: The purification of the START domain as a soluble form was challenging. I first tried to purify the full-length ATML1 protein as a soluble form, but I couldn’t obtain it, and ultimately it took over a year to obtain the soluble START domain.
What next for you after this paper?
KN: I am interested in how the ATML1 protein is broken down when VLCFA-Cers is absent – this will deepen our insights about lipid-mediated modulation of transcription factor activity in plants. On the other hand, from an evolutionary perspective, it is important to know whether lipid-transcription factor-based developmental mechanisms also work in the basal land plants or algae.
Where will this story take the Abe lab?
MA: As I mentioned in my biography, since my PhD I’ve been very interested in the molecular mechanism of epidermal cell differentiation. For the past 10 years or so, my lab has been focusing on florigen function and regulation, which involves FT, FD and FE. But I am very pleased that Kenji was interested in and restarted this project 5 years ago. In the future, Kenji and I hope to make exciting discoveries on epidermal cell differentiation.
Finally, let’s move outside the lab – what do you like to do in your spare time in Tokyo?
KN: I love to spend my spare time at the Onsen (a Japanese hot spring). After Onsen, I always drink a beer!
MA: In Spring 2019, our lab moved from Hongo to Komaba, which is a 20-min walk from Shibuya city. Shibuya city is a centre for modern culture and entertainment in Japan. I hope I will get to properly enjoy Shibuya life after the COVID-19 pandemic has settled down.
Development and Disease Models & Mechanisms welcome you to apply for our joint virtual meeting ‘Developmental Disorders: From Mechanism to Treatment‘, which aims to bring together developmental biologists, human geneticists and clinical researchers who are united in the goal of understanding and treating developmental disorders. The underlying causes of developmental disorders – genetic or environmental – are often not understood. Moreover, there is a disconnect between researchers working on animal models of developmental disorders, geneticists trying to identify the genomic lesion responsible, and clinicians hoping to treat affected patients. Given the resulting urgent need to improve communication between these groups, to promote basic research into congenital anomalies and to invest in translating this research to the clinic, this Meeting will focus specifically on building bridges from bench to clinic.
Find out more about the meeting from the organisers Phil Beales, James Briscoe, Monica J. Justice and Lee Niswander in the video below.
Speakers
Jeanne Amiel Institut Imagine, France Han Brunner Maastricht University Medical Center, The Netherlands Brian Ciruna The Hospital for Sick Children, Toronto, Canada Dagan Jenkins University College London, UK Nicholas Katsanis Rescindo Therapeutics Inc., USA Karen Liu King’s College London, UK Stefan Mundlos The Max Planck Institute for Molecular Genetics, Germany Emily Noël University of Sheffield, UK Eric Olson UT Southwestern Medical Center, USA Álvaro Rada-Iglesias IBBTEC, Spain David Rowitch University of Cambridge, UK Ian Smyth Monash Biomedicine Discovery Institute, Australia Lilianna Solnica-Krezel Washington University School of Medicine in St. Louis, USA Xin Sun University of California San Diego, USA Lori Sussel University of Colorado, USA
Organisers: Phil Beales, James Briscoe, Monica J. Justice and Lee Niswander Date: 14-17 September 2021 Abstract deadline: 16 July 2021 Location: Online
The Beddington Medal is the British Society for Developmental Biology’s major commendation to promising young biologists, awarded for the best PhD thesis in Developmental Biology that was defended in the year before the award.
The design of the medal, mice on a stylised DNA helix, is from artwork by Rosa Beddington herself.
This year, the Beddington Medal was awarded to Kristina Stapornwongkul, who did her PhD with Jean Paul Vincent at the Francis Crick Institute. Kristina presented her work at the BSDB/Genetics Society 2021 meeting and we caught up with her after the meeting to find out more about her life in science. Be sure to also check out the profile of Kristina – including a letter from JP Vincent and a list of Kristina’s selected publications – over on the BSDB site.
Where were you born and where did you grow up?
I was born in Giessen and grew up in Weil am Rhein. It’s a small town in Southwest Germany, directly at the border of France and Switzerland. It’s one of the sunniest places in Germany and I love to have a stroll through the vineyards whenever I go back there.
When did you first get interested in science?
I was always fascinated by technology and science, but I only developed a real passion for it in the last two years of high school. The realisation that each of our cells contains the information necessary to build an entire human, really blew my mind and made me want to learn more about how cells work.
How did you come to do a PhD in the lab of JP Vincent?
I think it all started with a zebrafish embryo and a stereo microscope. A few years later, at the end of my Masters studies, I knew that I wanted to do a PhD in the field of developmental biology. The UK has an outstanding developmental biology community and so I applied for the Wellcome Trust PhD programme in Developmental and Stem Cell Biology at UCL. As part of the programme, the students get the opportunity to rotate in three different labs. During my rotation, I realised that the Vincent lab was the perfect fit for me, both scientifically and personally.
Tell us about your PhD project: what were the main questions you were trying to answer?
The concept of morphogen gradient-mediated patterning has always fascinated me with its elegant simplicity: a single signalling molecule that can induce multiple cell fates depending on its concentration. How morphogen gradients form and what determines their shape are therefore important questions in order to understand how robust patterning is achieved in tissues. Several mechanisms by which morphogens might spread have been suggested over the years, with passive diffusion being the most parsimonious one. If such extracellular protein gradients form by simple diffusion, it shouldn’t be that difficult to engineer a morphogen gradient, no? At least that was the idea. So instead of further dissecting how natural morphogen gradients are generated, I wanted to test if an inert protein, such as GFP, could be transformed into a gradient-forming morphogen. Apart from probing whether diffusion is sufficiently reliable as a morphogen-transport mechanism, I was hoping that this synthetic approach would also help to uncover general principles and constraints that shape extracellular gradients. To do this work in vivo, I used the Drosophila wing pouch, one of the best studied model systems for morphogen gradient formation.
In your 2020 Science paper you describe your efforts to engineer a morphogen gradient, replacing Dpp with GFP. What did this technique reveal about how morphogens work?
The thing with engineering a synthetic morphogen gradient is that, even if it works, there is no guarantee that natural morphogens work exactly the same way. Nevertheless, our approach enabled us to show that protein gradients can, in principle, form by passive diffusion and that such gradients are reliable enough to pattern a tissue in vivo. It also made it feasible to specifically manipulate properties, such as binding affinities or expression levels, and test their effect on GFP gradient shape. Combining this with a modelling approach, we were therefore able to gain a good understanding of what each component was doing in our synthetic system.
Of course, we encountered several difficulties while building the GFP morphogen system and these were probably the most informative, because natural morphogen gradients that form by diffusion will encounter them as well. For instance, it became clear that secreted GFP can be lost from the tissue and end up in the larval blood, the hemolymph. This was really a big issue for the patterning performance of the GFP gradient. In fact, all secreted morphogens interact to a with components in the extracellar matrix and this is probably one important mechanism to regulate morphogenetic retention in epithelia.
If I had to summarise our findings in a sentence, I would probably say that a combination of high-affinity signalling receptors and low-affinity non-signalling receptors is sufficient to allow diffusing GFP to mimic the organising activity of a natural morphogen.
If you took one abiding memory with you from your PhD, what would it be?
As you can imagine this project involved many ‘trial and error’ experiments, and of course a good amount of luck. When you try to engineer something, it might not work for so many reasons. Even if your general design is good, expression levels might be too high or too low, or your synthetic receptor pair is not recycled efficiently (yes, that was an issue). So I think one of the most abiding memories of my PhD was when I saw for the first time that GFP in combination with GFP-responsive Dpp receptors was able to rescue growth and patterning of the fly wing pretty well. I expected a bit of a rescue, but I never thought that a two-component system could substitute that successfully an endogenous extracellular morphogen system, which not only consists of ligands and receptors but also of many extracellular regulators. My first thought was, ‘I must have messed up the genetics. The rescue is too good’. So, after checking everything three times, I went to JP and showed him the wing. His first response was, ‘Are you sure, you didn’t mess up the genetics?’.
You recently published a review making ‘the case for diffusion’. Why did you need to make this case?
Morphogen-mediated patterning has been studied extensively in a variety of model systems. However, the question of how morphogens spread in a tissue has remained quite controversial, especially in epithelial tissues. For instance, it has been suggested that diffusion is difficult to regulate and not reliable enough to generate robust extracellular gradients. As an alternative, active transport mechanisms, such as planar transcytosis or specialised filopodia (cytonemes), have been proposed. In our review, we try to give a comprehensive overview of the existing evidence from different model systems and conclude that there is strong evidence that morphogens disperse by diffusion-based mechanisms. In particular, we highlight how the tissue architecture and the ligand’s biochemical properties impose constraints on diffusion-based gradient formation and how components of the extracellular matrix help to overcome them.
So after your PhD you’ve recently moved to Barcelona: what are you doing there and how are you finding the city compared to London? I started as a postdoctoral fellow in Vikas Trivedi’s and Miki Ebisuya’s lab at EMBL Barcelona. We use aggregates of mouse embryonic stem cells as minimal model systems to study symmetry breaking and germ layer specification. London is amazing and definitely has a special place in my heart, but I have to admit that I am really in love with Barcelona. Being able to go for a swim after work and having tons of herbs that happily grow in the sun, is really amazing!
Longer term, do you know if you plan to stay in science?
Working in science is a huge privilege and I really appreciate the chance to interact with so many bright and inspiring people. Currently, I can’t imagine a more enjoyable job. However, being able to stay in science depends on many different factors and so I always try to stay open-minded.
Where do you think developmental biology will be in ten years?
I expect that we will have a much better understanding of the molecular mechanisms of human development. Already now, stem cell-based in vitro systems give us first insights into human organogenesis – a developmental stage in which functional studies were basically impossible before. As a consequence, we will probably also see a much stronger engagement of developmental biologists with the field of disease modelling.
Similarly, our research will depend less and less on the classical model systems. With CRISPR and stem cells, we will probably be able to widen our perspective on development by investigating anything from small insects to large mammals. I think it will be very exciting to see the differences and similarities we can find!
But I’m sure that’s not the only exciting direction developmental biology will take! I think developmental biology will be even more interdisciplinary (if that’s even possible) in ten years. Personally, I’m quite interested in the role of metabolism in development, but I’m sure there are also many more interesting intersections that we will further explore.
When you’re not in the lab, what do you do for fun?
I like to go climbing, swimming, hiking… pretty much all kinds of outdoor activities. Travelling is also a big passion of mine and I hope it will soon be possible again.
Some readers of the Node might already be familiar with preLights, another community site run by The Company of Biologists which aims to highlight new preprints from across the biological sciences. Most of the preLights community members are early-career researchers (PhD students and postdocs), but recently, preLights has found a new role as a teaching aid. Both the NYU Peer Review and Utrecht Protein Folding and Assembly courses have started using group preprint review projects as a tool to learn about critical reading and peer review. To learn more about how preLights has helped them teach these courses, preLights spoke to Gira Bhabha at NYU, and Tessa Sinnige at Utrecht University.
In many animal embryos, the tail bends ventrally as it grows, but the underlying mechanisms driving this multi-tissue deformation have been difficult to study. A new paper in Development uses the simple chordate Ciona as a model to study this widely conserved process. To find out more about the story, we met the paper’s two first authors, Qiongxuan Lu and Yuan Gao, and their supervisor Bo Dong, Professor at the Ocean University of China in Qingdao, China.
Qiongxuan (L), Yuan (C) and Bo (R)
Bo, can you give us your scientific biography and the questions your lab is trying to answer?
BD: I got my PhD from the Institute of Oceanology, Chinese Academy of Sciences (IOCAS), and then worked as a postdoc in the Sars International Centre for Marine Molecular Biology in the University of Bergen in Norway. After that, I went to RIKEN Centre for Developmental Biology (CDB) in Kobe, Japan, and worked on Drosophila tracheal tube geometry control. In 2014, I came back to the Ocean University of China (OUC) in Qingdao and established my own laboratory working on organ morphogenesis and evolution. My laboratory is principally interested in uncovering the cellular, mechanical and biochemical signalling networks that interact to drive the diverse morphogenetic processes during organ formation and tissue regression using marine ascidians and flies as models.
Qiongxuan and Yuan – how did you come to work in Bo’s lab and what drives your research today?
QL: I first met Bo in 2014 when he gave a lecture related to Ciona notochord tubulogenesis in 2014. From this lecture I was attracted to the field of morphogenesis, and the long-lasting question of how functional shape is generated. I then joined Bo’s lab two years later to investigate the mechanical role of the notochord in chordate embryogenesis. It was really memorable when I took a time-lapse movie on a Ciona embryo without a chorion from zygote to tailbud stage. Indeed, from this movie, we noticed and were curious about the phenomenon of the tail always bending ventrally after the initial tailbud stage, which led us to the story you see in the paper.
YG: Biomechanics has been recognized as the most promising direction of theoretical and applied mechanics. In the Institute of Biomechanics and Medical Engineering (IBME) in Tsinghua University, we focus not only on scientific mechanics problems in crucial biological problems at different length scales, but also emphasize the clinical issues of major diseases. As a PhD student majoring in biomechanics, I am particularly interested in how mechanical forces tune morphogenesis during development, and my PhD project is to develop physical/mechanical models to elucidate these underlying mechanisms.
The mechanisms behind embryonic tail bending in Ciona are so attractive. Thanks to the meeting of Prof. Bo Dong and my supervisor Prof. Xi-Qiao Feng, I was lucky enough to join in this project. Qiongxuan had performed a lot of experiments and obtained interesting and effective results. Based on this, I developed a physical model to further understand the mechanical role of each tissue during the tail bending process in Ciona embryos.
What is the current position of developmental biology and evo-devo research in China?
BD: Currently there is a pretty large developmental biology community in China. We have our own society and hold annual meetings. There are several hundred research groups working on developmental biology-related studies using either classical model animals such as zebrafish, Drosophila and Caenorhabditis elegans, or non-standard model organisms such as ascidian, amphioxus, ciliates and lamprey. Most aspects of developmental biology research – such as organogenesis, pattern formation, physiological metabolism, and regeneration – are categorised as basic research, so the main source of funding is the Natural Science Foundation of China. The evo-devo field is relatively smaller, but the increase in genomic data, new gene editing methods and the fast development of imaging techniques provides us with the opportunity to do evo-devo research in evolutionarily important animals.
Before your work, what was our understanding of how embryonic tail bending was controlled?
QL, YG, BD: Embryonic tail bending is an evolutionarily-conserved morphogenetic process in early embryogenesis for most invertebrates and vertebrates. This large-scale morphogenetic event has been long known about, but the underlying mechanisms have not been investigated. A possible reason is tissue-bending and tissue-folding at the embryo scale is difficult to study because of the anatomical complexity of many model animals. Before our publication, it was thought that embryonic tail bending is a passive process achieved by the physical barrier of the chorion that confines the tail, bending it during elongation.
Ciona embryo showing F-actin localization (green) and DAPI (red) in a longitudinal view. F-actin is asymmetrically enriched along the ventral side of the notochord.
Can you give us the key results of the paper in a paragraph?
QL, YG, BD: In this paper, we first show that in the urochordate Ciona, embryonic tail bending is not dependent on the chorion, but rather is a self-organized and genetically programmed active process. We then found that actomyosin is asymmetrically accumulated at the ventral side of the notochord, and cell proliferation of the dorsal tail epidermis is faster than the ventral counterpart during bending. Through a combination of genetic perturbation and chemical drug manipulation, we reveal that both asymmetrical notochord contractility and differential epidermis proliferation are required for the tail-bending process. We further developed a model with experimentally measured parameters to simulate the bending process. The simulation result shows that the asymmetrical notochord contractility is sufficient to drive the tail bending, whereas the differential cell proliferation is a passive response to mechanical forces. Thus, we reveal a mechanism of asymmetrical notochord contractility coordinated with differential epidermis proliferation that drives embryonic bending. The main implications of this work are not only revealing that embryonic bending within the chorion is driven by intrinsic forces, but also demonstrating how the different tissues of the tail interact and coordinate to sculpt the embryonic shape.
Do you have any idea about what causes the ventral enrichment of actomyosin in the notochord?
QL, YG, BD: This is a really interesting question that is worthy of further investigation. We actually have screened some candidate signalling molecules using in situ hybridization, but have so far failed to get positive results. We knew from the published literature that some proteins, such as those in the extracellular matrix, also show polarity during notochord morphogenesis. Interestingly, during notochord convergent extension, the notochord preferentially accumulates laminin, a basement membrane marker, dorsally, and atypical protein kinase C, an apical cell polarity molecule, ventrally, which might provide a polarizing cue for polarized actomyosin enrichment.
Bending appears to be conserved with many other vertebrate and tunicate embryos: do you think it serves a particular purpose for the embryo? And is the mechanism you’ve discovered in Ciona likely to also be conserved?
QL, YG, BD: Von Baer’s laws say that vertebrate embryos converge on a common physical structure and hence show a similar morphology during early embryogenesis, called the phylotypic stages. For example, at the beginning of neurulation, chordate embryos are commonly C-shaped. We think that bending of the embryonic tail could help embryos elongate continuously within the chorion without mechanical damage. It definitely saves space to contain the elongated embryos within the chorion.
In this paper, we found that the polarized contractility of the notochord plays a major role in shaping the bending tail at early tailbud stages, whereas biased epidermal proliferation ensures the robustness of tail bending at later tailbud stages. However, our data did not rule out the possibility that other tissues and their interactions also contribute to embryonic tail bending. Indeed other data suggest that the role of the notochord in driving embryonic tail bending depends on the synergistic effect of other tissues. In more structurally-complex vertebrate systems, we really do not know whether notochord contractility still plays the active role: further investigations are definitely needed.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
QL: I think the best moment for me was finding that actomyosin was temporarily enriched at the ventral side of the notochord. This interesting observation prompted us to investigate what role this polarised actomyosin might have in tail bending.
YG: A rational and effective model is only part of the way to success. The moments that most stick with me are when I find out a proper theory to depict the biological process. In this research, for example, we incorporated the active contraction of tissues into the model by using volumetric growth theory. Then it became easy to analyse the mechanical role of each tissue during tail bending.
And what about the flipside: any moments of frustration or despair?
QL: Sure, I definitely had a lot of them. For example, when we tried to confirm the role of the notochord in driving tail bending, our initial idea was to isolate the notochords from tailbud embryos and test whether they could bend spontaneously by the ventrally enriched actomyosin. This embryonic manipulation was rather challenging due to the tiny notochord located in the middle of the tail, surrounded by several tissues. We were stuck for a long time until we realized that we could try to think in an alternative way, and this transition led to the idea that physical modelling could also help us to address this question. Although I experienced so many failed attempts, I certainly learnt a lot from them, such as to always keep an open mind, and that interdisciplinary knowledge is essential for troubleshooting.
YG: As I am not a biology major, the technical terms sometimes become the obstacle to my understanding of the biological process. Likewise, my collaborators are not very good at mechanics and, at the first stage, it was a challenge to make the physical model understood. Fortunately, constant communication and discussions with my collaborators helped me overcome these difficulties.
Interdisciplinary knowledge is essential for troubleshooting.
What next for you after this paper?
QL: I am currently a postdoctoral fellow in the Umeå Centre for Molecular Medicine in Sweden, studying the neuronal basis of O2 sensing in C. elegans. I’m getting exposed to different fields, and I hope these interdisciplinary combinations will help me to explore more interesting questions.
YG: I will finish my dissertation in about six months. Meanwhile, I am looking for a postdoc position at present: the mechanical mechanisms underlying morphogenesis are intriguing and I hope to continue with this subject.
Where will this story take the Dong lab?
BD: Based on this and our previous work, we are recognizing the important roles of mechanical signalling in pattern formation during embryogenesis. For example, in this story, we believe that the faster cell proliferation in the dorsal midline epidermis can release the accumulated mechanical stress generated by asymmetrical notochord contractility. The follow up question is whether mechanochemical feedback exists between mechanical stretching and differential epidermis proliferation. If yes, how is the mechanical signalling sensed by the dorsal epidermis, and how does it respond?
Another question we are interested in pursuing is polarity signalling, which has important implications for tissue mechanics. Compared with the anatomically complex vertebrates, the Ciona embryonic tail is structurally simple, providing an excellent model to understand how polarity signalling impacts multi-tissue development.
Finally, let’s move outside the lab – what do you like to do in your spare time in Qingdao and Beijing?
QL: I spend most of my spare time either playing tennis, walking around our campus or climbing mountains nearby in Qingdao. These activities provide me a different kind of excitement outside of the lab, and more importantly, they enable me to balance my personal life and work.
YG: We are a big family in IBME. In our spare time, we often do sports together, like basketball and swimming. Besides, Beijing is an ancient and fascinating city, and I like to explore it with friends if we are free at weekends.
BD: I like to stay with my family during my spare time. We often climb Laoshan mountain and have weekend dinners together eating Qingdao’s delicious seafood. Sometimes, I also enjoy drinking Qingdao beer with my friends, which really can be relaxing, especially on summer nights.
Written by Shuangshuang Du, Rebecca Starble, and Lauren Gonzalez from the Yale Genetics Department.
We face a historical moment in which more and more women are pursuing scientific careers, but we have failed to support their success in leadership positions. This is in part because leadership styles that are authentic to their identities are not well represented by those who are currently in power. Opportunities for professional development could begin to offer young scientists techniques to overcome this gender barrier.
Despite this urgent need, training in interpersonal relationship skills is often absent in the graduate school curriculum. “Looking around the lab, I see talented graduate researchers undertaking challenging research projects who struggle not just with the science, but also because of the need to navigate the sparsity of female role models, confront cultural differences, and maintain self-belief,” said Shuangshuang Du, a Genetics student from Dr. Valentina Greco’s lab at Yale.
Du spearheaded the organization of a leadership workshop for graduate students in 2020, inspired by the first-ever female in science workshop for postdocs at Yale organized by Dr. Sara Gallini in 2019. She reached out to the Yale Biological and Biomedical Sciences (BBS) program to design a process by which this training could be accessed by the entire graduate student body, partnering with workshop liaison Dr. Jennifer Claydon to poll interests for such a workshop among BBS students and identify possible sources of funding. In December 2020, 16 Yale BBS women graduate researchers from five departments across the university participated in the inaugural iteration of this course.
16 biology PhD students and 2 coaches from hfp consulting met over Zoom in fall 2020 for an interactive course designed to help the students develop personalized leadership styles.
This course was taught by hfp consulting, a firm that specializes in leadership in science. Over the four half-day sessions, the facilitators and participants covered various skills critical to becoming empowered leaders in STEM, including effective communication, active listening, assertiveness, addressing imposter fears, and developing a peer support group. They approached these topics in a highly interactive way: participants were encouraged to engage with the content, the trainers, and each other through a combination of large-group and small-group activities to practice using these skills.
For example, in a module on conflict resolution, participants worked through their own real-life scenarios in small groups to get feedback from each other on how to deal with conflict using clear and respectful communication. This gave participants a safe, supportive environment to practice using these leadership skills, thus building participants’ confidence to apply these skills in their professional lives after the workshop ended.
This approach was transformative. “This course was incredibly valuable for my development as a female leader in science by enabling me to identify and take advantage of my strengths, learn what style of leadership is best for me, and expand my repertoire of interpersonal skills that are beneficial both professionally and personally” said Molly Bucklin, a PhD candidate in the department of Immunobiology. Renee Wasko, a PhD candidate in the department of Molecular, Cellular, and Developmental Biology, noted that although she is “someone who regularly attends other self-help/career development seminars, this was the first experience that felt eye-opening and realistically implementable.”
Participants also developed a strong community which didn’t end when the official course was over. “This program created a lasting support network for me,” said Wasko. “I still regularly connect with the other members of my cohort to discuss the topics and tools we learned and how they pertain to our lives currently.” This community has been especially valuable during the COVID-19 pandemic, when many students have felt disconnected from their support networks, and the pressures of graduate school have remained high.
Investing in young women early in their scientific careers is essential to preserve this diverse talent within academia because it provides them not only leadership skills, but also a space and a language to discuss their professional challenges and ambitions. This course serves as a blueprint to embed that learning within the Yale PhD curriculum, equipping young leaders with the tools to overcome systemic barriers and shift the culture of STEM towards one of inclusivity and empowerment.
In the latest episode of Genetics Unzipped, supported by the Institute of Genetics and Cancer (IGC) at the University of Edinburgh, we discover how researchers are using genetics to understand more about what’s going on in long-term debilitating conditions including myalgic encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and chronic pain, working hand in hand with patients to help to figure out who might be at risk and pointing towards new ideas for treatment.
We hear from Professor Chris Ponting from the MRC Human Genetics Unit (HGU) in the IGC who’s leading the DecodeME study together with patient representative Andy Devereux-Cooke, aiming to discover genetic variations that may explain susceptibility to ME/CFS and open doors to new therapies.
And we also speak with Professor Blair Smith from the University of Dundee and Professor Caroline Hayward from the MRC HGU who are sifting through the genomes of thousands of people enrolled in large cohort studies like Generation Scotland, in search of insights into chronic pain.
If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip
Today’s my last day working at the Node and Development. I started in June 2016, which really feels like a different world looking back now – at the first conference I went to, the SDB in Boston, the TV screens in the hotel lobby flipped between speeches from the two recently nominated Presidential candidates; at my second conference, also in Massachusetts but down in Southbridge, the first debate played in the bar to an eerie silence. I came here from the bench – I had done a PhD in Brighton and a postdoc in Cambridge but had always known that, as much as I love science, research (and/or being a PI) was not what I wanted to do with my life. The Node job allowed me to stay in touch with science and scientists. I’ve really come to appreciate the global community of developmental biologists.
Leaving has led me to wonder: what makes developmental biology such a rewarding field to be a part of? Of course, there’s the embryos, the time-lapses, the magic of it all. Those vertiginous shifts in scale – experiments that go from a misplaced nucleic acid to a funky protein structure to a misdirected cell to a novel tissue structure to a confused embryo – which you can then contextualise in the scope of evolution, ecology, physiology. It feels like we can have it all, not many disciplines can beat that (although try speaking to a misty-eyed cosmologist like my dad). Then there’s the field’s history, from bespoke experimental embryology in nineteenth century marine labs to the same embryos lit up by lasers and deconstructed by single cell sequencing; the golden ages keep coming, the old ways are repurposed. I’ve also always liked the mix between basic and applied research, a bit of a false dichotomy of course since one is not separate from the other; better to look at it as leveraging the rich body of developmental biology research to help understand and cure terrible diseases and make more food for the world – what’s not to love about that?
At the bottom of it though, I think it’s all about the people. On that maiden conference in Boston I got to do my first interviews for Development: Doug Melton, Dave McClay and the late Kathryn Anderson. Three totally different personalities, distinct career trajectories, but what tied them all together was a reverence for the embryo. Just as rewarding were the conversations later on in the poster hall, ten dollar beer in one hand and slice of pizza in another, with graduate students and postdocs, getting energised by their excitement. I wanted to showcase researchers young and old(er) in ‘The People Behind the Papers’, an interview series which started on the Node and has since moved to Development. Satisfyingly, my hundredth interview just came out, with tunicate researchers Izumi Oda-Ishii and Yutaka Satou (my last, number 101, will come out in the next few weeks). One of the hardest things about the pandemic for many of us has been the loss of personal contact without a screen in the middle, those chance encounters in conference bars…the people make the science, and it seems to me the people of developmental biology are a particularly good bunch.
It’s been gratifying to develop the Node, help a community journal promote the work of its authors, and work with a fantastic team in our not-for-profit publisher The Company of Biologists. Whatever you think about academic publishing, I’ll insist that we are one of the good guys. I’d encourage everyone to:
join the Node Network, our global directory of developmental biologists
Oh, and write for the Node – it’s free and easy, and could be anything from a job ad to an event notification to a behind the paper story to a polemical diatribe against funding inequity (honestly, we love it all). Why not look at our new topic pages for inspiration, up in the Archive dropdown
I’ll stop before I start excessively rambling. After this, I’m staying in science (communication), going to the Sanger Institute to be a science writer, combining my two favourite things. And you’ll still find me on Twitter, looking out for the next embryo time-lapse.
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