Fluorescence images of some organoids I have grown in the lab. In the organoid on the left, we can see how different cell fates emerge (red and yellow), and on the right we can see the some of the cellular cytoarchitecture (green) in the organoid.
At university, I am also part of the Ambassadors for a Better Research Culture (ABRC), where we aim to improve the research environment on the medical school campus for postgraduate students and staff. Here, I am part of the ‘LGBTQIA+ Inclusion’ subgroup where we run monthly events to foster a community of LGBTQIA+ researchers at the medical school campus. We have also established a larger series called ‘Pride in STEM’, where we invite external speakers to discuss their experiences of being queer in different STEM career environments. I would like to carry this into my internship and compile some LGBTQIA+ voices for a post in the Honest Conversations blog series here on the Node. If anyone is interested in sharing their experiences of being queer in academia, please feel free to get in touch at ryan.harrison@biologists.com or thenode@biologists.com.
This year, 2024, marks the 10th anniversary of the first Development ‘From Stem Cells to Human Development’ meeting, and today is the beginning of the sixth meeting at Wotton House in the UK. The influence the meeting has had on the field is discussed in a recent article by science historian Nick Hopwood (Hopwood, 2024a), who suggests that human developmental biology has experienced peaks of attention and periods of neglect, fuelled by the productivity of technical innovations. In the current issue of Development, we have published a complementary Perspective article by Nick highlighting key aspects of the history of the field for an audience of stem cell and developmental biologists (Hopwood, 2024b). In addition, Development invited researchers from eight countries around the world to respond to these ideas and comment on how human development is perceived in their country of work, discussing how they believe their local legal, political, regulatory, societal and technological frameworks are influencing the field’s trajectory (Clark et al., 2024). The authors and some highlights from the Perspective are shown in the image below, and you can click the image to read the whole article.
Recognising that this article only manages to capture a small sample of the breadth of human development and stem cell research worldwide, we encourage you, readers of the Node, to share your opinion on human developmental biology in your country of work. Do you believe that interest in human developmental biology is cyclical, as suggested by Hopwood? If so, what lies ahead? Are we experiencing a boom or bust in support of human development research? How long might this trajectory lead before turning on its head? What societal undercurrents might contribute to maintaining or changing the field’s course? The floor is yours…
References
Clark, A.T., Goolam, M., Hanna, J.H., Long, K., Nicol, D., Petropoulos, S., Saitou, M., Tam, P.L., Wang, H. Human developmental biology – a global perspective. Development. 151(17). https://doi.org/10.1242/dev.203092
Hopwood, N. (2024a). Species Choice and Model Use: Reviving Research on Human Development. In Journal of the History of Biology. Springer Science and Business Media LLC. https://doi.org/10.1007/s10739-024-09775-7
Are you ready to be a group leader? Are you a multi-disciplinary biologist with interests in developmental cell biology?Join a robust, collaborative and supportive department with world-class research.
We seek to appoint a Lecturer (Assistant Prof Equivalent) in developmental, cell or stem cell biology, genetics or genomics with a focus on human disease modelling and/or craniofacial biology.
Who are you? You are a promising postdoc with an excellent publication profile, high-quality collaborative connections and ambitious plans for your independent research programme. You should be an early career scientists with an outstanding research track record and excellent potential to develop an internationally competitive research programme and to collaborate within the Centre and across King’s.
Who are we? The Centre for Craniofacial & Regenerative Biology at King’s College London is one of the leading centres for Craniofacial and Stem Cell Biology worldwide. Our Centre comprises 19 collaborative groups with interests in craniofacial and stem cell biology, innovative bioengineering strategies to regeneration and repair, and big data approaches to understand the complexity of development and disease. Our research spans basic, clinical and translational sciences. The Centre offers a vibrant, collaborative, and interactive research and teaching environment in the heart of London.
Successful candidates are expected to establish their independent research group in the Centre, to contribute to our educational programmes and to training the next generation of interdisciplinary scientists, and to support the strategic vision of the Centre and King’s. They will have access to a variety of PhD programmes, as well as mentorship and career development opportunities. They will work with outstanding scientists across King’s https://www.kcl.ac.uk/research and access our world-class Research Facilities: https://www.kcl.ac.uk/research/facilities
CLOSING DATE 6 October. Application link below. Informal inquiries are welcome to Head of Department Professor Andrea Streit or to relevant Faculty members.
Observing a cluster of migrating cells or a developing embryo through the lens of a microscope can be a visceral experience; one is struck by the ephemeral beauty, layered complexity, and alien intelligence displayed by such specimens. For those who seek a scientific understanding of these striking phenomena, it is also a humbling experience. There are so many moving parts here, so many subsystems within subsystems, so much noise, so much nonlinearity, so much contingency… how could we possibly hope to capture this in the simple yet powerful models that make scientific explanations so satisfying and useful?
I’ve been grappling with this question ever since my undergrad, and as anyone who does so, I have found plenty of reasons to be pessimistic about it.
Neural crest cells swarming through a zebrafish embryo are just one of countless phenomena in cell and developmental biology that are truly spectacular to behold. [Main panel: live-imaged AiryScan volume; by author. Bottom-right inset: fixed lightsheet volume for spatial reference; by Zimeng Wu, UCL.]
Though we have extensive knowledge of the molecular machines that form the building blocks of biological systems, putting this giant puzzle together from the bottom up seems an impossibly complicated task. Instead, the field’s still-dominant approach is to link particular perturbations to particular outcomes, usually by lifting out a handful of mechanisms or genes from the broader system, drawing arrows between them, and calling the result a pathway. But whilst the models this produces are of an appealing simplicity, they lack power; they often fail to explain or predict anything outside the narrow set of conditions and observations that were considered in the original study. At least we are indisputably making progress in developing new tools to collect more and better data, ever more quickly, ever more precisely… but alas, this progress is closely shadowed by the realization that it can only take us so far; more data does not yield more understanding if we don’t know how to ask the right questions.
With these problems permeating the field, it comes as no surprise that there is a measure of discontent in the community. Some argue that we have an attitude problem [1]; perhaps young researchers spend too much time on twitter and not enough time in the library? Others contend that we have an image problem [2]; perhaps we should be spending more time on twitter, reassuring each other and the wider public that our field remains essential – or even that it has recently entered, as some would have it [3], a “new golden age”? Like so many developmental biology papers, these viewpoints may not be entirely wrong, but they’re also not particularly compelling.
I’d like for us to entertain the possibility that we are in fact facing a science problem. That our progress is not bottlenecked by modern attitudes or public misperceptions, but by the profound intellectual challenge of finding new and better ways of thinking about the spectacles that play out under our microscopes. I’d like to take seriously the above reasons for pessimism and treat them as real scientific challenges for us to tackle and overcome. If the molecular details are intractable, we should search for new and better systems-level abstractions to subsume them. If the current standard of mechanistic explanation is inadequate, we should look to build new and better conceptual frameworks that set a higher standard. If it is hard to distill meaning from the deluge of high-throughput data, we should aim to develop new and better models that yield strong inductive priors for big-data analysis.
This is much easier said than done, of course, but in grappling with these issues I have also come across a few reasons for optimism!
Looking back in history, the challenge faced by Darwin and his contemporaries in seeking to unify the diversity of living organisms must have seemed no less daunting than our current predicament, yet they persevered and emerged with an entirely new understanding of life. Returning to modern times, a new theory of cell types established about a decade ago shows brilliantly that deep conceptual progress is possible even today [4]. And not only that; it also shows that such progress really does have the impact we would hope to see! For one, it has inspired new ways of analyzing and interpreting transcriptomics data (see e.g. [5]). For another, I have personally witnessed how much more productive the discourse on cell types can be within a group of researchers who know this theory (even if they don’t all fully endorse it) compared to a group who do not. These and other inspirational observations are always in the back of my mind as I explore my own ideas for tackling the field’s fundamental problems.
The C&P idea originated from discussions between first author Elisa Gallo and me on the prospects of discovering principles that generalizewell across different biological systems and phenomena. It is often implicitly assumed that the diversity of cellular and multi-cellular behaviors results from the contingent combination of various modular parts or subprocesses, much like sequence diversity on the molecular level. This would leave us with limited avenues to pursue explanations that generalize over many such contingent assemblies.
Mulling over this in search for alternative perspectives eventually led us to an almost metaphysical argument: if there do exist principles that can explain a diverse set of biological phenomena in a unified manner, then they must be generative principles, that is to say they must comprise a mechanism by which the explained diversity is generated. But are there any such mechanisms in cellular and developmental systems, or does contingency reign supreme?
As we started looking with fresh eyes, it turned out that many of the biological phenomena we are interested in (including cytoskeletal dynamics, reaction-diffusion patterning, different aspects of multicellular morphogenesis, and even embryo-scale processes like gastrulation) can indeed be decomposed into what we have come to call a versatile core and a function-specifying periphery. A versatile core is a mechanism that implements a generative principle and hence is capable of producing a wide range of different behaviors or outputs. The periphery, then, is what configures such a core to produce one particular function out of the many in its large behavior inventory.
A medley of examples of C&P architectures compiled from our recent paper. The same cores (orange disks) are reused with different peripheries (blue leaves) to generate different functional behaviors or outputs. [The exquisite illustrations in the paper were created by first author Elisa Gallo, UZH.]
Intriguingly, we expect systems with a C&P architecture to be highly evolvable because the core’s large behavioral space is readily accessible through modifications in the periphery. As a consequence, cores will tend to spread widely and become deeply conserved in evolution, even as their peripheries diversify to exploit the full range of the core’s versatility. If follows that a generative principle that describes how a core works will generalize across the many different systems and phenomena wherein that core is reused. In other words, the C&P decomposition helps us separate the generalizable (the core) from the contingent (the periphery).
A more systematic introduction and comprehensive discussion of what the C&P hypothesis proposes is of course found in the paper. For my ramblings here, what matters most is that working on this project has greatly increased my optimism, to the point where I now believe that it really is possible to discover human-interpretable yet powerful theories that capture the essence of complex living systems. It’s just that the structure of such theories may need to differ considerably from that of the classical mechanistic accounts we are accustomed to, which is what makes it so hard (and so exciting) to pursue them.
This pursuit requires conceptual work, which means reading widely, thinking deeply, and engaging in intense and interdisciplinary discussion. As it turns out, this is surprisingly difficult and time-consuming; it is real scientific work. Unfortunately, our current research ecosystem does very little to incentivize and support such efforts. Young researchers in particular feel the pressure to pipette and/or code as fast as we can, just to stay in place in an ever-accelerating academic rat race. Taking time to think outside established lines seems wasteful, let alone taking time to pursue an explicitly conceptual project. In my case, it was only through a combination of luck, privilege, and the generosity of a few individuals that I was able to take a sabbatical year and invest the time necessary to arrive at the C&P hypothesis as it now stands. If we want the pace of conceptual innovation to pick up, this will need to change. Fortunately, there are positive signals here, too, as some leading institutes are now building up new theory-focused research programs.
In conclusion, I see many serious obstacles that we must face on our quest to better understand the complexity, intelligence, and beauty of cells and embryos. But if we take these obstacles seriously, I dare hope that we can overcome them, and that the dawn of a new golden age is indeed on the horizon.
Many thanks to Elisa Gallo and Matyas Bubna-Litic for their feedback on a draft version of this post.
[1] C.D. Stern, Reflections on the past, present and future of developmental biology, Developmental Biology 488 (2022) 30–34. https://doi.org/10.1016/j.ydbio.2022.05.001. [2] J.B. Wallingford, We Are All Developmental Biologists, Developmental Cell 50 (2019) 132–137. https://doi.org/10.1016/j.devcel.2019.07.006. [3] P. Liberali, A.F. Schier, The evolution of developmental biology through conceptual and technological revolutions, Cell 187 (2024) 3461–3495. https://doi.org/10.1016/j.cell.2024.05.053. [4] D. Arendt, J.M. Musser, C.V.H. Baker, A. Bergman, C. Cepko, D.H. Erwin, M. Pavlicev, G. Schlosser, S. Widder, M.D. Laubichler, G.P. Wagner, The origin and evolution of cell types, Nat Rev Genet 17 (2016) 744–757. https://doi.org/10.1038/nrg.2016.127. [5] A.J. Tarashansky, J.M. Musser, M. Khariton, P. Li, D. Arendt, S.R. Quake, B. Wang, Mapping single-cell atlases throughout Metazoa unravels cell type evolution, eLife 10 (2021) e66747. https://doi.org/10.7554/eLife.66747. [6] E. Gallo, S. De Renzis, J. Sharpe, R. Mayor, J. Hartmann, Versatile system cores as a conceptual basis for generality in cell and developmental biology, Cell Systems (2024). https://doi.org/10.1016/j.cels.2024.08.001.
Fertilization is one of the most fascinating events during the development of an organism. In sexually reproducing multicellular organisms like animals and plants, fertilization involves the fusion of two gametes – a female egg cell and a male sperm. Gametes are highly specialized cells that, upon reaching maturity, await fertilization in a quiescent state. One way to achieve this is by inhibition of cell cycle progression, thus allowing gametes to arrest at a precise, stable stage. This aspect is crucial because uncontrolled gamete proliferation could have dramatic consequences, such as abortion of the progeny or a waste of resources.
Fertilization in plants is unique
When the egg cell and sperm fuse, their quiescent state is lifted and the cell cycle reactivated, so that the product of fertilization, the zygote, can initiate cell division. The molecular mechanisms that control the establishment of the quiescent state and its exit are still poorly understood.
In flowering plants, the fertilization event is rather unique as they produce two types of female gametes, called egg cell and central cell. During the process of double fertilization, the two female gametes are fertilized by one sperm cell each, giving rise to the embryo and endosperm, respectively, the latter being a placental-like, nourishing tissue that sustains embryonic growth.
Typically, the egg cell and central cell derive from consecutive mitotic events of the same haploid megaspore, making them genetically identical. However, despite their genetic similarity, the egg cell and central cell have distinct identities, unique transcriptomes, different DNA contents (the central cell is homodiploid at maturity), and behave very differently once fertilized.
The fertilized egg undergoes morphological changes soon after fertilization. It progressively elongates, and its nucleus strongly polarizes towards the apical domain of the cell. The first cell division occurs approximately 20-24 hours after fertilization, resulting in an apical cell (forming the embryo proper) and a basal cell (forming the suspensor). In contrast, the fertilized central cell takes a different rhythm, committing its first division to initiate endosperm production already about 6-8 hours after fertilization.
Figure 1. Ovule and developing seeds imaged with confocal microscopy. The central cell and the endosperm nuclei express a yellow fluorescent protein. The cell wall is labeled by propidium iodide. The first division of the central cell to produce the primary endosperm nuclei occurs just 4-6 hours after fertilization.
Cell cycle stage at which Arabidopsis gametes arrest
Over two decades ago, a hypothesis emerged suggesting the presence of a mechanism in the central cell that regulates the cell cycle, distinct from the one operating in the egg cell. The proposed idea was that a molecular brake prevents central cell division, and that fertilization acts as a trigger to release this brake, allowing division. This hypothesis stemmed from the observation of the rapid proliferation of the central cell after fertilization, as well as from the phenotypes exhibited by certain mutants where the central cell either divides in the absence of fertilization or is unable to divide once fertilized.
To understand fertilization’s impact on central cell quiescence, we initially determined the cell cycle stage at which the mature female gametes arrest. Quantifying DNA content in the female gametes is quite challenging as they cannot be collected in sufficient quantity for conventional ploidy analysis, such as flow cytometry. Our approaches involved propidium iodide staining to quantify DNA content, for which a reliable protocol was already established, and the imaging of histones tagged with fluorescent protein to infer the chromatin content in different nuclei of the ovule. These two approaches worked well and were reasonably straightforward. However, when it came to assessing DNA synthesis through nucleotide-analogue incorporation (EdU), well, we hit our head against a wall for about six months. It took a multitude of adjustments, trials, and a certain level of DIY attitude before we were able to establish a reliable, efficient protocol. But we made it!
It took a multitude of adjustments, trials, and a certain level of DIY attitude before we were able to establish a reliable, efficient protocol. But we made it!
The results of our ploidy analysis were both surprising and exciting. While we could confirm that the egg cell arrests in G2 as previously suggested, the central cell presented a completely different story. Its ploidy and behaviour suggested that its DNA synthesis (S phase) had initiated but not finished, and we could observe that fertilization was necessary for the S phase to be completed.
Figure 2. Ovules embedded in wax and sliced into 7μm thick sections. In this section, the central cell (cyan), the egg cell (pink) and the two synergids cells (orange) are clearly distinguishable. These sections are used for Laser Assisted Microdissection (LAM) microscopy, where single cell types can be cut with a laser and isolated. Here, we have used this technique for a transcriptome analysis of central cells at different time points around the moment of fertilization.
Finding the brake
Now that we knew the central cell is arrested in S phase, we wanted to identify the factor causing this arrest in DNA synthesis. Almost immediately, we considered RBR1, because it is a conserved cell cycle inhibitor known for regulating entry and progression through S-phase, and its absence causes central cells to proliferate in the absence of fertilization. The first confirmation that indeed RBR1 was our candidate came during a day at the microscope, observing the dynamics of an RBR1-YFP fusion protein during fertilization. For this type of experiments, we emasculated almost ready-to-bloom flowers by removing the stamens, so that self-pollination was avoided. The next day, we pollinated the pistils, marking the “0” time point. Then, after 4, 6, 8, 10, or 12 hours after pollination, we dissected the pistils and imaged the ovules using a multiphoton microscope. Normally, we pollinated between 8 and 9 in the morning, meaning that we had to spend quite some evenings at the microscope.
During these observations, we noticed that some central cells showed a RBR1-YFP signal, while others did not. After confirming the homozygosity of the RBR1-YFP line, it became evident that RBR1-YFP disappeared from the central cell only in fertilized ovules. This led us to the conclusion that something was degrading RBR1 at fertilization. Therefore, RBR1 acted as the brake, and fertilization somehow triggered RBR1 degradation, allowing the cell cycle to proceed.
Searching the signal that releases the brake
Just shortly after observing the turnover of RBR1 during fertilization, we received the sequencing results of transcriptomes from central cells at different time points before and shortly after fertilization that we had isolated by Laser-Assisted Microdissection (LAM). In practical terms, this technique allows us to isolate single cells from fixed, paraffin-embedded, and sliced tissues of interest. Completing this experiment took almost a year and a half for various reasons. The first significant obstacle was the global pandemic. We had just started to collect material when the institute went into a complete lockdown for about eight weeks, which meant that we lost at least two plant generations. The re-start was problematic too, because we had to do shifts to prevent overcrowding the labs, and experiments proceeded rather slowly. The second challenge was the time required make semi-thin sections of the material used for LAM. It takes approximately five days to gather enough material for a single replicate; our analysis covered four developmental time points, each performed in triplicate.
However, the results justified the long waiting time. Among the cell cycle-related genes potentially involved in RBR1 degradation, one D-type cyclin, CYCD7;1, caught our attention. Its expression peaked just around the moment when RBR1 is degraded in the central cell. Moreover, the literature indicated that CYCD7;1 is expressed only in stomata and pollen, and its ectopic expression in the female gametophyte was previously shown to induce proliferation of the unfertilized central cell. This led us to hypothesize that CYCD7;1 is paternally produced and stored in the sperm cells, and only upon fertilization, would CYCD7;1 be present in the same place and at the same time as RBR1, triggering its degradation. Observing CYCD7;1 messenger RNA location and delivery, as well as CYCD7;1 protein dynamics, confirmed our hypothesis. We also found that ectopic expression of CYCD7;1 in the central cell was sufficient to trigger RBR1 degradation and central cell division.
The only missing element was a visible phenotype. Mutant lines for CYCD7;1 (T-DNA and CRISPR-Cas9) were growing, and I (Sara) was confident in predicting the cycd7;1 mutant phenotype: paternal-effect seed abortion. This means that seeds would fail to develop when cycd7;1 mutant pollen was used as a male in a cross with a wild-type plant. Because RBR1 wouldn’t be degraded, the central cell wouldn’t divide, and no endosperm could be produced. However, upon inspecting the first cycd7;1 siliques under the microscope to evaluate the level of seed abortion, the result was hard to accept. All four cycd7;1 mutants I analysed exhibited a perfectly fine seed set – no seed abortion. We accepted the disappointing result that absence of paternal CYCD7;1 did not impact seed development. We went back to the LAM transcriptome, searching for alternative candidates, and stopped working on CYCD7;1. Sometime later, Ueli and I were having a meeting to discuss new hypotheses and strategies to further develop the project. As we revisited the CYCD7;1-related data, Ueli asked me which seed developmental stages I had been looking at for the phenotypical analysis, and he added “Do it again, look closer to the moment of fertilization”.
As we revisited the CYCD7;1-related data, Ueli asked me which seed developmental stages I had been looking at for the phenotypical analysis, and he added “Do it again, look closer to the moment of fertilization”.
That very afternoon, I sowed all the plant lines, and six weeks later, I made reciprocal crosses between wild-type and cycd7;1 plants again. This time, instead of looking at fully grown siliques, I sampled seeds at 12 hours after pollination, and the phenotype was evident: seeds generated by cycd7;1 pollen had fewer – or even no – endosperm nuclei compared to those derived from wild-type pollen. This meant that paternal delivery of CYCD7;1 is required for normal central cell division after fertilization. Central cells that receive a sperm cell lacking CYCD7;1 are blind to the fertilization event and do not divide immediately as they should. However, cycd7;1 mutant had no seed abortion, meaning that seed development can proceed normally even in absence of CYCD7;1 and, indeed, at 24 hours after pollination, cycd7;1-derived seeds showed endosperm proliferation. How can this happen? We hypothesized that other D-type cyclins, expressed from the maternal and/or paternal genome soon after fertilization, might compensate for CYCD7;1’s absence. This hypothesis turned out to be correct as we were able to delay endosperm proliferation even further when using pollen from plants mutated for four D-type cyclins.
Our results have not only addressed the fundamental question of how a cell determines the appropriate timing for division, but have also uncovered new and intriguing research directions. These include the understanding of how the central cell can arrest in S-phase, elucidating the mechanisms by which the CYCD7;1 messenger RNA is selectively stored in the sperm nucleus without degradation, and exploring the broader question of which other paternal or maternal signals regulate cell cycle arrest and progression in gametes. It also taught us the important lesson of formulating the right biological questions and designing the right strategies to address them. This is especially important when looking at developmental transitions, growth progression, and developmental processes in general: we cannot look at development if we do not take into consideration the time factor.
The 4 September 2024 Development presents… webinar was chaired by Development Senior Editor Alex Eve and featured two talks on the topic of cardiac development and regeneration. Catch up on the talks below.
On the topic of environment, evolution and development, chaired by Development’s Guest Editor, Karen Sears (UCLA).
Wednesday 2 October – 15:00 BST
Girish Kale (University of Hohenheim) ‘Elevated temperature fatally disrupts nuclear divisions in the early Drosophila embryo’
Natasha Shylo (Stowers Institute for Medical Research) ‘Gastrulation and Left-Right patterning in veiled chameleons’
Sergio Menchero (The Francis Crick Institute) ‘Understanding temporal diversity in mammalian developmental programmes using marsupial single-cell transcriptomics’
At the speakers’ discretion, the webinar will be recorded for viewing on demand. To see the other webinars scheduled in our series, and to catch up on previous talks, please visit: thenode.biologists.com/devpres
Timothy J. Duerr, Melissa Miller, Sage Kumar, Dareen Bakr, Jackson R. Griffiths, Aditya K. Gautham, Danielle Douglas, S. Randal Voss, James R. Monaghan
Bart Theeuwes, Luke TG Harland, Alexandra Bisia, Ita Costello, Mai-Linh Ton, Tim Lohoff, Stephen J Clark, Ricard Argelaguet, Nicola K Wilson, Wolf Reik, Elizabeth Bikoff, Elizabeth J Robertson, Berthold Gottgens
View ORCID ProfileSonja D. C. Weterings, Hiromune Eto, Jan-Daniël de Leede, Amir Giladi, Mirjam E. Hoekstra, Wouter F. Beijk, Esther J. M. Liefting, Karen B. van den Anker, Jacco van Rheenen, Alexander van Oudenaarden, Katharina F. Sonnen
Anna Fleming, Elena V. Knatko, Xiang Li, Ansgar Zoch, Zoe Heckhausen, Stephanie Stransky, Alejandro J. Brenes, Simone Sidoli, Petra Hajkova, Dónal O’Carroll, Kasper D. Rasmussen
Shreeta Chakraborty, Nina Wenzlitschke, Matthew J. Anderson, Ariel Eraso, Manon Baudic, Joyce J. Thompson, Alicia A. Evans, Lilly M. Shatford‑Adams, Raj Chari, Parirokh Awasthi, Ryan K. Dale, Mark Lewandoski, Timothy J. Petros, Pedro P. Rocha
Alessa R. Ringel, Andreas Magg, Natalia Benetti, Robert Schöpflin, Mira Kühnlein, Asita Carola Stiege, Ute Fischer, Lars Wittler, Stephan Lorenz, Stefan Mundlos, Lila Allou
Bohou Wu, Jae Hyun Lee, Kara M. Foshay, Li Zhang, Croydon J. Fernandes, Boyang Gao, Xiaoyang Dou, Chris Z. Zhang, Guoping Fan, Becky X. Xiao, Bruce T. Lahn
Sara Y. Guay, Prajal H. Patel, Jonathon M. Thomalla, Kerry L. McDermott, Jillian M. O’Toole, Sarah E. Arnold, Sarah J. Obrycki, Mariana F. Wolfner, Geoffrey D. Findlay
Yan Zhao, Andrea Fernández-Montoro, Greet Peeters, Tatjana Jatsenko, Tine De Coster, Daniel Angel-Velez, Thomas Lefevre, Thierry Voet, Olga Tšuiko, Ants Kurg, Katrien Smits, Ann Van Soom, Joris Robert Vermeesch
Atena Yasari, Monika Heinzl, Theresa Mair, Tina Karimian, Shehab Moukbel Ali Aldawla, Ingrid Hartl, Andrea J. Betancourt, Peter Lanzerstorfer, Irene Tiemann-Boege
Gal Finer, Mohammad D. Khan, Yalu Zhou, Gaurav Gadhvi, George S. Yacu, Joo-Seop Park, R. Ariel Gomez, Maria Luisa Sequeira-Lopez, Susan E. Quaggin, Deborah R. Winter
Meilin Fernandez Garcia, Kayla Retallick-Townsley, April Pruitt, Elizabeth Davidson, Yi Dai, Sarah E. Fitzpatrick, Annabel Sen, Sophie Cohen, Olivia Livoti, Suha Khan, Grace Dossou, Jen Cheung, P.J. Michael Deans, Zuoheng Wang, Laura Huckins, Ellen Hoffman, Kristen Brennand
Gal Finer, Mohammad D. Khan, Yalu Zhou, Gaurav Gadhvi, George S. Yacu, Joo-Seop Park, R. Ariel Gomez, Maria Luisa Sequeira-Lopez, Susan E. Quaggin, Deborah R. Winter
Yuki Ishii, Jessica C. Orr, Marie-Belle El Mdawar, Denise R. Bairros de Pilger, David R. Pearce, Kyren A. Lazarus, Rebecca E. Graham, Marko Z. Nikolic, Robin Ketteler, Neil O. Carragher, Sam M. Janes, Robert E. Hynds
Ian C Tobias, Sakthi D Moorthy, Virlana M Shchuka, Lida Langroudi, Mariia Cherednychenko, Zoe E Gillespie, Andrew G Duncan, Ruxiao Tian, Natalia A Gajewska, Raphaël B Di Roberto, Jennifer A Mitchell
Adam T Lynch, Naomi Phillips, Megan Douglas, Marta Dorgnach, I-Hsuan Lin, Antony D Adamson, Zoulfia Darieva, Jessica Whittle, Neil A Hanley, Nicoletta Bobola, Matthew J Birket
Luis G. Palma, Daniel Álvarez-Villanueva, María Maqueda, Mercedes Barrero, Arnau Iglesias, Joan Bertran, Damiana Álvarez-Errico, Carlos A. García-Prieto, Cecilia Ballaré, Virginia Rodriguez-Cortez, Clara Bueno, August Vidal, Alberto Villanueva, Pablo Menéndez, Gregoire Stik, Luciano Di Croce, Bernhard Payer, Manel Esteller, Lluís Espinosa, Anna Bigas
Vincent Truong, Jackson Brougher, Tim Strassmaier, Irene Lu, Dale George, Theodore J. Price, Alison Obergrussberger, Aaron Randolph, Rodolfo J. Haedo, Niels Fertig, Patrick Walsh
Nigel Kee, Mélanie Leboeuf, Silvia Gómez, Charles Petipré, Irene Mei, Salim Benlefki, Daniel W Hagey, José Dias, François Lallemend, Samir EL Andaloussi, Johan Ericson, Eva Hedlund
Abigail M. Guillemette, Guillian Hernández Casanova, John P. Hamilton, Eva Pokorná, Petre I. Dobrev, Václav Motyka, Aaron M. Rashotte, Courtney P. Leisner
Trevor Weiss, Maris Kamalu, Honglue Shi, Zheng Li, Jasmine Amerasekera, Zhenhui Zhong, Benjamin A Adler, Michelle Song, Kamakshi Vohra, Gabriel Wirnowski, Sidharth Chitkara, Charlie Ambrose, Noah Steinmetz, Ananya Sridharan, Diego Sahagun, Jill Banfield, Jennifer Doudna, Steven E. Jacobsen
Yijia Yan, Jaqueline Mellüh, Martin A. Mecchia, Hyung-Woo Jeon, Katharina Melkonian, Clemens Holzberger, Anne Harzen, Sara Christina Stolze, Rainer Franzen, Yuki Hirakawa, Ana I. Caño Delgado, Hirofumi Nakagami
Carlos Henrique Cardon, Victoria Lesy, Catherine Fust, Thales Henrique Cherubino Ribeiro, Owen Hebb, Raphael Ricon de Oliveira, Mark Minow, Antonio Chalfun Junior, Joseph Colasanti
Michael C Wilson, Alexander H Howell, Anika Sood, Youngwoo Lee, Pengcheng Yang, Heena Rani, Elena Yu, Eileen L. Mallery, Sivakumar Swaminathan, Corrinne E. Grover, Jonathan F. Wendel, Olga A. Zabotina, Jun Xie, Chelsea S. Davis, Daniel Szymanski
Martijn J. Jansen, Stuart Y. Jansma, Klaske M. Kuipers, Wim H. Vriezen, Frank F. Millenaar, Teresa Montoro, Carolien G.F. de Kovel, Fred A. van Eeuwijk, Eric J.W. Visser, Ivo Rieu
Shota Nakanoh, Despina Stamataki, Lorena Garcia-Perez, Chiara Azzi, Hayley L Carr, Alexandra Pokhilko, Lu Yu, Steven Howell, Mark Skehel, David Oxley, Simon Andrews, James Briscoe, Teresa Rayon
Gaspar Sánchez-Serna, Jordi Badia-Ramentol, Paula Bujosa, Alfonso Ferrández-Roldán, Nuria P. Torres-Águila, Marc Fabregà-Torrus, Johannes N. Wibisana, Michael J. Mansfield, Charles Plessy, Nicholas M. Luscombe, Ricard Albalat, Cristian Cañestro
Huanhuan Liu, Anupama Binoy, Siqi Ren, Thomas C. Martino, Anna E. Miller, Craig R. G. Willis, Shivakumar R. Veerabhadraiah, Abhijit Sukul, Joanna Bons, Jacob P. Rose, Birgit Schilling, Michael J. Jurynec, Shouan Zhu
Lisa K. Iwamoto-Stohl, Aleksandra A. Petelski, Maciej Meglicki, Audrey Fu, Saad Khan, Harrison Specht, Gray Huffman, Jason Derks, Victoria Jorgensen, Bailey A.T. Weatherbee, Antonia Weberling, Carlos W. Gantner, Rachel S. Mandelbaum, Richard J. Paulson, Lisa Lam, Ali Ahmady, Estefania Sanchez Vasquez, Nikolai Slavov, Magdalena Zernicka-Goetz
Caroline Hookway, Antoine Borensztejn, Leigh K. Harris, Sara Carlson, Gokhan Dalgin, Suraj Mishra, Nivedita Nivedita, Ellen M. Adams, Tiffany Barszczewski, Julie C. Dixon, Jacqueline H. Edmonds, Erik A. Ehlers, Alexandra J. Ferrante, Margaret A. Fuqua, Philip Garrison, Janani Gopalan, Benjamin W. Gregor, Maxwell J. Hedayati, Kyle N. Klein, Chantelle L. Leveille, Sean L. Meharry, Haley S. Morris, Gouthamrajan Nadarajan, Sandra A. Oluoch, Serge E. Parent, Amber Phan, Brock Roberts, Emmanuel E. Sanchez, M. Filip Sluzewski, Lev S. Snyder, Derek J. Thirstrup, John Paul Thottam, Julia R. Torvi, Gaea Turman, Matheus P. Viana, Lyndsay Wilhelm, Chamari S. Wijesooriya, Jie Yao, Julie A. Theriot, Susanne M. Rafelski, Ruwanthi N. Gunawardane
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Jialin Liu, Sebastian M. Castillo-Hair, Lucia Y. Du, Yiqun Wang, Adam N. Carte, Mariona Colomer-Rosell, Christopher Yin, Georg Seelig, Alexander F. Schier
Convergent evolution — the independent emergence of analogous structures among species whose last common ancestors lacked the trait— has long fascinated me. This phenomenon represents an exciting opportunity to study the genomic constraints that shape organisms during development to produce specific forms and functions. In the fall of 2018, as I was searching for a PhD program, I heard a talk by Dr. Ricardo Mallarino. He was interested in studying one such example of convergence, the evolution of the patagium, a skin membrane that, like a wingsuit, allows animals to glide through the air as a means of locomotion. Of particular interest was the patagium of a small marsupial possum: sugar gliders. The patagium has evolved many times across the tree of life including rodents, primates, marsupials, lizards, and frogs. Marsupials however, offered a uniquely tractable group to study how similar novel morphological traits evolved independently, as three closely related possums had acquired this adaptation. Here I will cover the findings of our recent publication.
Adult sugar glider extending its patagium (red arrow).
Biology strikes back: identifying candidate regulatory elements
Previous RNAseq-based exploration of the developing patagium indicated that a major difference between the early patagium and neighboring skin was the differential regulation of an early gene regulatory network1. Given this, I set out to identify cis-regulatory elements (CREs) which had similar patterns of evolution in gliding species, which may harbor the causative changes driving differential gene regulation, evidenced by previous work2,3. We produced 15 new marsupial genomes which included gliding (sugar glider, greater glider, and feathertail glider) and non-gliding species. To identify cis-regulatory elements in the developing patagium tissue, we used ChIP- and ATAC-seq, which when used in combination provided a candidate list of active and poised CREs. Then using both our biological data and our genomes, we measured the rate of nucleotide change across these identified cis-regulatory elements, giving us an indication of which CREs are experiencing selection as these species have evolved their patagium. Through this analysis, we identified thousands of candidate glider accelerated regions (GARs)— elements which showed a substantial increase in nucleotide substitution. By focusing only on the GARs that showed shared patterns across the three gliding species we analyzed, we hoped to find a smaller pool of CREs that could be involved in the evolution of the patagium. We were surprised to find that not a single CRE matched that description. Then in our darkest times, a moment of brilliance: what if the cause was not a single CRE but instead different CREs across species all regulating the same gene. We had previously conducted Micro-C on the developing patagium and thought to use this data and identify topologically associating domains (TADs) to inform an analysis of GAR distribution and abundance. Using these TADs, we assigned CREs to the genes that were in the same TADs and asked if any gene had an overabundance of GARs.
Return of a key gene
Previous work from our group uncovered that the sugar glider patagium develops through the deployment of a conserved network of genes, and that Wnt5a is heavily involved in the early development of the gliding membrane1. One of the other genes identified was the transcription factor Emx2. It just so happened, that our analysis for GAR enrichment identified Emx2 as our strongest candidate, having GARs from each of the three gliding species in its vicinity. We produced an shRNA lentivirus for Emx2 and began testing the effects of downregulating Emx2 in the developing patagium. Taking advantage of marsupial biology, that is they give birth to their young, or joeys, quite early in their development, we could then probe the early patagium. We conducted injections into the patagium primordium of sugar glider joeys and found that indeed downregulation of Emx2 caused a decrease in the area of the developing patagium. This was one of my favorite experiments and served to remind me how fascinating working in science can be. For some time, I was one of the only people in the world who knew that lack of Emx2 led to incorrect patagium growth. We then explored how Emx2 may be regulated. We had several candidates to test but initially focused on just two, one was positioned in what we presumed to be the promoter of Emx2 while the second seemed to be a distal enhancer located 1mb away from the Emx2 promoter but with a strong contact loop in the Micro-C dataset. The former was accelerated in the sugar glider and the latter accelerated in the feathertail glider. We wanted to test if the acceleration observed had an effect on the element’s ability to regulate expression and so we decided to use luciferase assays in an immortalized sugar glider cell line. This experiment works by placing your CRE of interest upstream of the luciferase gene (which originates from fireflies) to measure the amount of fluorescence produced by the cells to see if your CRE has regulatory function. In our case, our results indicated that the distal enhancer had accumulated changes that made it a stronger enhancer in the feathertail glider compared to its non-gliding sister and the sugar glider. We later found that the other gliding species, the greater glider, also had an enhancer that showed the same pattern.
Emx2 shRNA injection results in a decrease in patagium size
Duo of fate: Our two favorite genes are important for patagium development
Now we knew that Emx2 had a phenotypic effect on the patagium and had some clues as to how it could end up being highly expressed in gliding species, but I became interested in what was happening at the molecular level in the patagium when we disrupted Emx2 expression. We did another round of shRNA injections, and this time collected the tissue for RNA sequencing. We found that many of the genes that were normally upregulated in the patagium when compared to surrounding skin were now downregulated. Among the ~400 genes downregulated, Wnt5a was one of the more strongly affected genes. This prompted us to investigate if Emx2 regulates all these genes directly and specifically how it may be regulating Wnt5a. We did an Emx2-ChIP-seq experiment and found that many genes did indeed have Emx2 binding sites, and we were able to identify multiple binding sites in the Wnt5a promoter. An example of great peer reviewing led to another of my favorite experiments as we set out to test the Emx2 binding sites found in the Wnt5a promoter. We again used luciferase this time testing two versions of the same promoter, one was unaltered while in the second we mutated the Emx2 binding sites. We found that the loss of these sites led to a complete loss of regulatory ability. Then to see if Emx2 was responsible for activating this promoter we co-transfected an Emx2-producing plasmid with our luciferase reporter plasmids. This experiment showed that the wild-type promoter increased its production of luciferase nearly 4-fold when Emx2 was present.
Emx2 awakens a conserved pathway
Our final goal for the paper was to establish if the spatial expression and function of Emx2 was novel to sugar gliders or if it was conserved in non-gliding mammals. We decided to test this hypothesis in mice, a much more amenable system for testing overexpression of genes. We found that Emx2, as reported previously4, was expressed in mice in a similar spatial pattern as that of the patagium, however this expression was only present for ~2 days whereas in the developing sugar gliders it was present for at least 14 days. To test whether overexpression of emx2 was sufficient to produce early patagium phenotypes, like we had previously observed with Wnt5, we extended the duration of Emx2 expression in mice while maintaining its endogenous spatial pattern. This however resulted in mice whose forebrain grew uncontrollably and resulted in non-viability; previous work had implicated Emx2 in brain development5. Therefore, we restricted the overexpression of Emx2 to only the skin. We found that indeed this overexpression was capable of recapitulating phenotypes observed in the early patagium such as increased cell proliferation, density, and the thickening of the epidermis1. These experiments showed that Emx2 has a conserved role in driving proliferation, potentially via regulation of the Wnt pathway, further indicating that evolution has re-used existing cellular programs to evolve a new adaptation.
The last remarks
In my opinion, the key message of this paper is that the evolution of convergent traits can occur independently via similar pathways/mechanisms, but the path to get there can be different. Our work showed that the redeployment of a shared developmental pathway can be an effective mechanism by which adaptations evolve.
I am very happy to see this work published; it took many years to get to this point. There were many bumps along the way, and it was the culmination of the hard work of many people involved. I want to thank the reviewers for their candid and helpful words, truly they made the paper better than when we first submitted. As someone who finds great joy in working on emerging model systems and has received countless advice in the past to just work on “model” organisms, I am incredibly pleased with how this paper is received. I continue to work on non-model systems with fascinating biology now as a postdoc and advise anyone who is interested in working on new models to go for it! It is rewarding to work on questions that can only be asked in a new system and uncover and share new findings.
References
1 Feigin, C. Y. et al. Convergent deployment of ancestral functions during the evolution of mammalian flight membranes. Science Advances9, eade7511 (2023). https://doi.org/doi:10.1126/sciadv.ade7511
2 Booker, B. M. et al. Bat Accelerated Regions Identify a Bat Forelimb Specific Enhancer in the HoxD Locus. PLOS Genetics12, e1005738 (2016). https://doi.org/10.1371/journal.pgen.1005738
3 Capra, J. A., Erwin, G. D., McKinsey, G., Rubenstein, J. L. R. & Pollard, K. S. Many human accelerated regions are developmental enhancers. Philosophical Transactions of the Royal Society B: Biological Sciences368, 20130025 (2013). https://doi.org/doi:10.1098/rstb.2013.0025
4 Pellegrini, M., Pantano, S., Fumi, M. P., Lucchini, F. & Forabosco, A. Agenesis of the Scapula in Emx2 Homozygous Mutants. Developmental Biology232, 149-156 (2001). https://doi.org/https://doi.org/10.1006/dbio.2001.0159
5 Yoshida, M. et al. Emx1 and Emx2 functions in development of dorsal telencephalon. Development124, 101-111 (1997). https://doi.org/10.1242/dev.124.1.101
We need your help to optimise the SAFE Labs Handbook before it is disseminated throughout the academic community. Please complete our this survey to help optimize this tool for the academic community.
This handbook is an outcome of the 2024 SAFE Labs workshop, where new bioscience group leaders from across Europe discussed Starting Aware, Fair, and Equitable Labs. The primary goal of this workshop (funded through the UCL Global Engagement award) was to better-understand the common, and divergent, problems faced by new researchers trying to build successful, equitable, fair, and environmentally sustainable labs with a positive research culture. There were no scientific talks as part of the program (there are plenty of meetings for that!).
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