Dorsal view of the left hand of a 10-week old human embryo. The dorsometacarpales are highlighted: these muscles (like others described in this study) are present in adults of many other limbed animals, while in humans they normally disappear or become fused with other muscles before birth. CREDIT: Rui Diogo, Natalia Siomava and Yorick Gitton
A team of evolutionary biologists, led by Dr. Rui Diogo at Howard University, USA, and writing in the journal Development, have demonstrated that numerous atavistic limb muscles – known to be present in many limbed animals but usually absent in adult humans – are actually formed during early human development and then lost prior to birth. Strikingly, some of these muscles, such as the dorsometacarpales shown in the picture, disappeared from our adult ancestors more than 250 million years ago, during the transition from synapsid reptiles to mammals.
Also remarkably, in both the hand and the foot, of the 30 muscles formed at about 7 weeks of gestation one third will become fused or completely absent by about 13 weeks of gestation. This dramatic decrease parallels what happened in evolution and deconstructs the myth that in both our evolution and prenatal development we tend to become more complex, with more anatomical structures such as muscles being continuously formed by the splitting of earlier muscles. These findings offer new insights into how our arms and legs evolved from our ancestors’, and also about human variations and pathologies, as atavistic muscles are often found either as rare variations in the common human population or as anomalies found in humans born with congenital malformations.
Since Darwin proposed his evolutionary theory, scientists have argued that the occurrence of atavistic structures (anatomical structures lost in the evolution of a certain group of organisms that can be present in their embryos or reappear in adults as variations or anomalies) strongly supports the idea that species change over time from a common ancestor through “descent with modification”. For example, ostriches and other flightless birds have vestigial wings, while whales, dolphins and porpoises lack hind limbs but their embryos initiate and then abort hind limb development. Similarly, temporary small tail-like structures are found in human embryos and the remnant of the lost ancestral tail is retained as our coccyx. Researchers have also suggested that atavistic muscles and bones can also be seen in human embryos, but it has been difficult to visualize these structures clearly, and the images that appear in modern textbooks are mainly based on decades old analyses.
This is changing with development of new technology that provides high-quality 3D images of human embryos and fetuses. In the new study published in the journal Development the authors have used these images to produce the first detailed analysis of the development of human arm and leg muscles. The unprecedented resolution offered by the 3D images reveals the transient presence of several of such atavistic muscles. Dr. Diogo said: “It used to be that we had more understanding of the early development of fishes, frogs, chicken and mice than in our own species, but these new techniques allow us to see human development in much greater detail. What is fascinating is that we observed various muscles that have never been described in human prenatal development, and that some of these atavistic muscles were seen even in 11.5-weeks old fetuses, which is strikingly late for developmental atavisms “.
He further added: “Interestingly, some of the atavistic muscles are found on rare occasions in adults, either as anatomical variations without any noticeable effect for the healthy individual, or as the result of congenital malformations. This reinforces the idea that both muscle variations and pathologies can be related to delayed or arrested embryonic development, in this case perhaps a delay or decrease of muscle apoptosis, and helps to explain why these muscles are occasionally found in adult people. It provides a fascinating, powerful example of evolution at play.”
Cells are the building blocks of life. However, in multi-cellular organisms, millions of cells are subject to death due to injury, infection and ordinary cell turnover (Galluzzi et al., 2018). For example, epithelial cells in the small intestine rapidly renew every 2 to 6 days in most mammals, which is crucial to maintain proper function of villus epithelia (Mayhew et al., 1999). Moreover, during embryonic development, cell death serves as a crucial mechanism to remove unnecessary cells, adjust tissue size and shape, as well as correct developmental errors (Arya and White, 2015). In order to maintain homeostasis and avoid unwanted inflammatory responses, cellular debris is usually cleared rapidly by professional phagocytes, such as macrophages and microglia. However, during early neurogenesis when the neural tube develops and large numbers of neurons and glia undergo apoptosis, myeloid-derived professional phagocytes have not yet infiltrated the trunk of developing embryos (Herbomel, Thisse and Thisse, 1999, 2001; McGrath et al., 2003; Bertrand et al., 2013; Stremmel et al., 2018). How dead cells are removed from this region during early development remained largely unknown until recently.
In our recent paper entitled “Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System”, we demonstrate an unexpected behavior of neural crest cells (NCCs), which have been studied for more than 150 years, in debris clearance before the colonization of professional phagocytes (Zhu et al., 2019) (Figure 1).
Figure 1. Schematic illustration showing neural crest cells (green) phagocytose dead cells (red) in zebrafish embryos. Right panel shows zoomed view of the trunk.
Interestingly, not only did we not expect this novel function of NCCs, but the discovery was also made by accident! Our original intent was to study how glial populations coordinated their behaviors during early spinal motor nerve development. But early in our studies, Yunlu started to notice NCCs behaving in a manner that didn’t match what the literature described. Some migrated away from their migratory streams, and others appeared to interact with cellular debris. Here, we would like to share the story behind this work.
Neural Crest Cells Clear Debris
What are NCCs and what do we know about them? During the earliest stages of vertebrate nervous system development, the ends of neural plate rise and fold into the neural tube, which further develops into the central nervous system (CNS), including the brain and the spinal cord. During or after the closure of the neural tube, NCCs located at the edge of the neural fold go through an epithelial-to-mesenchymal transition and delaminate from the dorsal neural tube. Multipotent NCCs then migrate through highly conserved pathways and give rise to a variety of cell types, including skeletal tissues, pigment cells, and neurons and glia of the peripheral nervous system (PNS) (Mayor and Theveneau, 2013). Given that NCCs are highly migratory and colonize the entire developing embryo before the infiltration of professional phagocytes, we hypothesized that they may have the ability to clear cellular debris at early developmental stages.
To examine whether NCCs are capable of clearing debris, we performed in vivo, time-lapse imaging in transgenic embryos expressing fluorescent proteins in NCCs. Excitingly, in these movies, we observed that some fluorescently-labeled NCCs migrated towards dead cells located far away from their innate migratory pathways. Then, they reached toward dead cells and engulfed them, resulting in the formation of large engulfment vesicles inside NCCs.
To confirm that the NCC engulfment process was similar to phagocytosis in professional phagocytes, we imaged transgenic lines labeling early endosomes (PI(3)P sensor) and lysosomes (Lamp1-GFP) (Rasmussen et al., 2015). In these movies, we found that, similar to the maturation of phagosomes, NCC engulfment vesicles fused with early endosomes and lysosomes, leading to progressive acidification inside the vacuoles. Interestingly, we observed NCCs phagocytosing a variety of dead cells, including dead NCCs, neuronal debris, and muscle cells.
In these movies, we also observed that PNS NCCs migrated through motor exit point transition zones, where spinal motor neurons send their axons into the PNS, into the ventral neural tube, and phagocytosed CNS debris. Most of these CNS-located NCCs stayed inside the neural tube for 2 to 12 hours and returned to the PNS through motor exit point transition zones. To test whether PNS NCCs were recruited into the spinal cord by cellular debris, we induced CNS cell death by ablating radial glia using nitroreductase-mediated cell death (Smith et al., 2014; Johnson et al., 2016) and observed a significant increase in the number NCCs recruited into the CNS compared to control embryos. Therefore, we conclude that NCCs can migrate toward and phagocytose dead cells in both CNS and PNS.
Are There Subpopulations of Neural Crest Cells?
Interestingly, we noticed dead cells were not always cleared by the nearest NCC. Instead, under most circumstances, they were phagocytosed by NCCs that came from a distance. And that led us to wonder if phagocytic NCCs were a specific subgroup of the neural crest population. To examine whether phagocytic NCCs belonged to a specific lineage, we performed lineage tracing on phagocytic NCCs expressing a photoconvertible protein. Contrary to our expectations, we observed phagocytic NCCs differentiate into a variety of derivatives, including pigment cells, motor axon-associated cells, and dorsal root ganglia cells, suggesting that phagocytic NCCs are not lineage restricted.
Additionally, under physiological conditions, we only observed 5-10% of NCCs that were phagocytic. To examine whether more NCCs could be “activated”, we induced cell death using laser ablation. Surprisingly, immediately after the ablation, the majority of neighboring NCCs started to engulf cellular debris, resulting in the formation of massive numbers of phagocytic vesicles. These results demonstrated that not only could NCC phagocytic abilities be induced by acute cell death, but also that the majority of NCCs have the potential to phagocytose debris. Therefore, these data are consistent with the hypothesis that phagocytic NCCs are not a specialized subpopulation of the neural crest population.
Previous studies show that NCCs migrate along conserved, segmentally restricted pathways. However, we found that phagocytic NCCs move toward cellular debris and sometimes even crossed somite boundaries. So what is the mechanism that directs phagocytic NCCs toward dead cells? To answer this question, we designed an ablation assay to quantify NCC recruitment and inhibited a variety of signaling pathways. We found that NCC recruitment was compromised when we treated embryos with a Caspase-1 inhibitor or a interleukin (IL)-1 receptor antagonist, indicating NCC recruitment was mediated by the IL-1β signaling pathway. Interestingly, we observed IL-1β expression in both cellular debris and phagocytic NCCs. Moreover, the IL-1β level we observed after cell ablation was significantly lower than that in previous studies using spinal cord transection and bacterial infection (Bernut et al., 2014; Nguyen-Chi et al., 2014). Given that our cell ablation is more precise compared to manipulations performed in previous studies, the low level of Il-1β release supports the hypothesis that Il-1β secretion is tightly regulated and dependent upon the strength of the inflammatory stimulus (Lopez-Castejon and Brough, 2011).
Conclusion (Yunlu Zhu)
I joined the Kucenas lab at the University of Virginia in early 2014 because I was astonished by the beauty of live imaging in zebrafish embryos (Figure 2).
Figure 2. Two transgenic zebrafish embryos at 22 hour-post-fertilization expressing fluorescent protein in neural crest cells mounted back to back.
My previous focus was the development of perineurial glia and the role of cell-cell interactions in the development of spinal motor nerves. However, while imaging transgenic embryos labeling NCCs, I accidentally observed that some NCCs migrated ectopically away from their innate pathways and had spherical vacuoles with diameters of 3 to 10 µm. I discovered that these weird behaviors of NCCs had never before been described and was deeply attracted by this phenomenon. Therefore, I shifted my focus toward this NCC project.
This novel role of NCCs in debris clearance was quite unexpected but perfectly reasonable. However, the nature of these phagocytic NCCs is not well understood and we have many remaining questions. Given that the majority of NCCs are capable of phagocytosing debris, why don’t they respond to cell death under physiological conditions? What is special about those active NCCs that migrate towards dead cells from a distance? And what happens when professional phagocytes like macrophages colonize the trunk region of the developing embryo? Are their immune-NCC interactions? And what happens if one of these two populations is unable to clear debris? There are so many new questions that have come from this work, and I’m excited to see which ones the next students decide to pursue.
I would like to end this post with a quote from Yogi Berra, which, I think, precisely describes the beauty of live imaging and my experience in this project: “You can observe a lot by just watching”.
References:
Arya, R. and White, K. (2015) ‘Cell death in development: Signaling pathways and core mechanisms’, Seminars in Cell & Developmental Biology. Academic Press, 39, pp. 12–19. doi: 10.1016/J.SEMCDB.2015.02.001.
Bernut, A. et al. (2014) ‘Mycobacterium abscessus cording prevents phagocytosis and promotes abscess formation.’, Proceedings of the National Academy of Sciences of the United States of America. National Academy of Sciences, 111(10), pp. E943-52. doi: 10.1073/pnas.1321390111.
Bertrand, J. Y. et al. (2013) ‘Three pathways to mature macrophages in the early mouse yolk sac Three pathways to mature macrophages in the early mouse yolk sac’, Blood, 106(9), pp. 3004–3011. doi: 10.1182/blood-2005-02-0461.
Galluzzi, L. et al. (2018) ‘Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018’, Cell Death & Differentiation. Nature Publishing Group, 25(3), pp. 486–541. doi: 10.1038/s41418-017-0012-4.
Herbomel, P., Thisse, B. and Thisse, C. (1999) ‘Ontogeny and behaviour of early macrophages in the zebrafish embryo.’, Development (Cambridge, England), 126(17), pp. 3735–45.
Herbomel, P., Thisse, B. and Thisse, C. (2001) ‘Zebrafish early macrophages colonize cephalic mesenchyme and developing brain, retina, and epidermis through a M-CSF receptor-dependent invasive process.’, Developmental biology, 238(2), pp. 274–88. doi: 10.1006/dbio.2001.0393.
Johnson, K. et al. (2016) ‘Gfap-positive radial glial cells are an essential progenitor population for later-born neurons and glia in the zebrafish spinal cord’, Glia, 64(7), pp. 1170–1189. doi: 10.1002/glia.22990.
Lopez-Castejon, G. and Brough, D. (2011) ‘Understanding the mechanism of IL-1β secretion’, Cytokine and Growth Factor Reviews. Elsevier Ltd, 22(4), pp. 189–195. doi: 10.1016/j.cytogfr.2011.10.001.
Mayhew, T. M. et al. (1999) ‘Epithelial integrity, cell death and cell loss in mammalian small intestine.’, Histology and histopathology, 14(1), pp. 257–67. doi: 10.14670/HH-14.257.
Mayor, R. and Theveneau, E. (2013) ‘The neural crest’, Development, 140(11), pp. 2247–2251. doi: 10.1242/dev.091751.
McGrath, K. E. et al. (2003) ‘Circulation is established in a stepwise pattern in the mammalian embryo.’, Blood. American Society of Hematology, 101(5), pp. 1669–76. doi: 10.1182/blood-2002-08-2531.
Nguyen-Chi, M. et al. (2014) ‘Transient infection of the zebrafish notochord with E. coli induces chronic inflammation’, Disease Models & Mechanisms, 7(7), pp. 871–882. doi: 10.1242/dmm.014498.
Rasmussen, J. P. et al. (2015) ‘Vertebrate epidermal cells are broad-specificity phagocytes that clear sensory axon debris.’, The Journal of neuroscience : the official journal of the Society for Neuroscience. Society for Neuroscience, 35(2), pp. 559–70. doi: 10.1523/JNEUROSCI.3613-14.2015.
Smith, C. J. et al. (2014) ‘Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone’, PLoS Biology. Edited by B. A. Barres, 12(9), p. e1001961. doi: 10.1371/journal.pbio.1001961.
Stremmel, C. et al. (2018) ‘Yolk sac macrophage progenitors traffic to the embryo during defined stages of development’, Nature Communications. Nature Publishing Group, 9(1), p. 75. doi: 10.1038/s41467-017-02492-2.
Zhu, Y. et al. (2019) ‘Migratory Neural Crest Cells Phagocytose Dead Cells in the Developing Nervous System’, Cell, 179(1), pp. 74-89.e10. doi: 10.1016/j.cell.2019.08.001.
One of the biggest open questions in biology is how organisms can form complex patterns (limbs, organs, entire body plans) from initially disordered or very simple states. Every animal does this at the beginning of its life, forming its full complexity from a single cell. Some are capable of similar feats even after their bodies are fully constructed: starfish and flatworms survive when cut in pieces, the zebrafish restores lost fins, the axolotl regrows limbs and organs alike. How does either case work? How are those patterns established so accurately, and how is it that things so rarely go wrong?
I’m trying to approach this question with the help of one of the most impressive regenerators of all: Hydra. As a 1 cm long freshwater cnidarian it’s a lot less impressive than the monster whose name it bears, but what it lacks in scales and fangs it makes up for in raw regenerative ability. The mythological beast grew two heads for every one it lost. The real thing can regenerate its head, can regenerate from a ball of tissue as small as 150 microns across, and can come back from being dissociated down to single cells if enough of those cells are placed together. On top of this, the small size and simplicity of the creature make it very easy to study and manipulate compared to other model organisms. In fact, several theoretical models purporting to describe the patterning process have already been developed.
Of particular interest is the fact that these models propose mechanical forces as drivers of pattern formation. Mechanical forces are well known to be important in embryonic development but their role in regeneration is less clear. As a Hydra regenerates it first forms a hollow sphere, which then undergoes osmotically driven cycles of swelling and rupture. According to the literature, there is a characteristic switch in these oscillations from large amplitude and low frequency to small amplitude and high frequency, and this shift is linked to when the animal sets its body axis. Thus the pattern shift was proposed to represent a link between mechanics and biochemistry. I wanted to determine what the mechanism linking mechanical forces to biochemical axis specification was.
On observing many regenerating animals, one of the first things we noticed was that nearly half of the animals we imaged regenerated without a clear shift in oscillation pattern. As this called into question essentially all previous assumptions about the nature and relevance of the pattern shift, we turned to trying to figure out its exact cause.
It was previously proposed that the change in oscillation behavior might be due to the beginnings of a regenerated mouth. This idea makes logical sense, as adult Hydras open their mouths to relieve internal water pressure as well as to feed, but nobody had experimentally tested it. By using injected fluorescent beads to track the location of successive rupture sites, we determined that the spot where rupture occurs is random during large oscillations but conserved during small ones. These data would be consistent with the theory that the large oscillations are due to the tissue tearing under internal pressure, while small oscillations occur when the mouth is established to act as a vent. As confirmation, a tissue piece containing the mouth of the original animal also produces conserved rupture sites and only shows small oscillations.
To this point we had only shown that oscillation behavior and the mouth are somehow correlated. These experiments do not explain how the mouth might affect oscillation behavior. Thankfully, Hydra’s ease of manipulation offers a direct way to establish causality. It’s possible to eliminate all nerve cells from a Hydra, producing a nerve-free animal that is structurally normal but cannot actively move. The critical experiment was to use mouth-containing tissue pieces cut from such nerve-free animals. These contain a mouth that is fully formed and complete, but cannot open on its own. If the deciding factor for small oscillations to occur is simply that the mouth acts as a weak point and tears more easily, nerve-free samples with a mouth should show small oscillations exactly like their normal counterparts. If on the other hand mouth function is the key, they should show large oscillations.
We find that nerve-free mouth pieces show only large oscillations, indicating a causal link between active control of mouth function and a decrease in oscillation amplitude. This provides a concrete explanation for the oscillation pattern shift in cases where it occurs: it is caused by the animal’s ability to open its mouth at will.
Our study does not answer why some pieces are delayed in developing mouth function or provide exact developmental checkpoints. What it does give us is further constraints and parameters that can be used to improve existing models, and put us one step closer to understanding how Hydra regenerates. As Hydra shares many key biochemical pathways with more familiar animals despite its alien appearance, figuring out patterning here could one day be the basis of a similar understanding in humans.
We are seeking a PhD candidate to join our EvoDevo lab in the University of Barcelona to study our favorite chordate model Oikopleura dioica, in which we are currently interested in heart development, 4D imaging of early embryo cleaving, and early developmental responses to environmental challenges. To meet our unique Oikopleura model -> Click here for a tour “A day in our lab” posted in The Node.
We have also engaged a new EcoEvoDevo line investigating if the developmental mechanisms of marine embryos are ready to respond to climate change, including the effects of biotoxins derived from algal blooms. Click here for a tour on this new EcoEvoDevo adventure.
Our approaches include Single-cell transcriptomics,RNAseq, RNAi Knockdowns, CRISPR and Fluorescent-Microscopy
PhD fellowship Call OPEN (FI Catalan program): October 1st-14th 2019 (contact for enquiries as soon as possible canestro@ub.edu)
REQUIREMENT: to have finished a Master degree
CONTACT: please send an email to Cristian Cañestro (canestro@ub.edu), including a brief letter of interest, and the final scores for the degree and Master (indicating the scale), all together in ONE single pdf file.
Dolly the Sheep via Flickr, Toni Barros (CC BY-SA 2.0)
In this episode from our centenary series exploring 100 ideas in genetics, we’re looking at mergers and acquisitions – but in a biological rather than a financial sense. We find out what happens when two cells decide to move in together, unpack the history of genetic engineering and bleat on about the story of Dolly the Sheep.
If you enjoy the show, please do rate and review and spread the word. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com
The Department of Biological Sciences at the University of South Carolina (UofSC) invites applications for two tenure-track Assistant Professor positions in neurobiology. The successful candidates will be expected to establish an independent, extramurally funded research programs in: 1) Molecular or Cellular Neurobiology relevant to neural development and/or disease focused on cell-cell interactions; or 2)Molecular or Cellular Neurobiology using animal models to understand the pathophysiology of Autism or neurodevelopmental disorders (see below). Minimum qualifications include a Ph.D. or M.D. and post-doctoral training in a relevant area. The successful candidates will be responsible for teaching courses relevant to their area of expertise, as well as mentoring research training for graduate and undergraduate students.
1) Molecular or Cellular Neurobiology focused on Cell-Cell Interactions (position # 67536): We are interested in applicants focusing on how interactions between different cells, including cellular interactions with axons, contribute to the biology of nervous system development, disease, or response to injury. This individual will closely interact with research groups in the SmartState Center for Childhood Neurotherapeutics, which includes neurobiologists focused on molecular mechanisms of axon growth in development and after neural injury, and the broader UofSC Neuroscience Community. Applications are made online at http://uscjobs.sc.edu/postings/67536. For questions or further information, please contact Dr. Fabienne Poulain (fpoulain@mailbox.sc.edu).
2) Neurobiology of Autism or Neurodevelopmental Disorders (position # 67556): This position is part of a university-wide initiative to enhance research on Autism and Neurodevelopmental Disorders and establish a Center of Excellence (USCAND) to accelerate interdisciplinary efforts in neuroscience. There is a parallel faculty search in the Department of Psychology for this initiative, and several additional USCAND faculty hires planned over the next few years in complimentary disciplines. This individual will closely interact with research groups in USCAND, as well as the SmartState Center for Childhood Neurotherapeutics, Institute for Mind and Brain,andResearch Consortium on Children and Families. Applications are made online at http://uscjobs.sc.edu/postings/67556. For questions or further information, please contact Dr. Jeff Twiss (twiss@mailbox.sc.edu).
Review of applications will begin by November 1, 2019. The review process will continue until the positions are filled. Qualified individuals should submit a curriculum vita, research statement (3 pages), teaching philosophy (1 page), and the names, email addresses and phone numbers of at least three references to http://USCjobs.sc.edu/postings (with position # and links as above).
The Department of Biological Sciences is a multidisciplinary unit of approximately 1,600 undergraduate students, 50 graduate students, and 35 tenure-line faculty representing a broad range of research areas (www.biol.sc.edu). UofSC has a highly interactive neuroscience research community that encourages and precipitates collaborations. UofSC in Columbia (www.sc.edu) is the state’s flagship university (founded in 1801 and currently one of the top 50 “Best Colleges” according to U.S. News and World Report).
Columbia, SC enjoys more than 300 days of sunshine annually and has ready access to pristine beaches, lakes, rivers and mountains.The city hosts historical and cultural attractions, festivals, performing arts and sporting events, parks and outdoor recreation including Congaree National Park and 50,000-acre Lake Murray.
The University of South Carolina is an affirmative action, equal opportunity employer. Minorities and women are encouraged to apply. The University of South Carolina does not discriminate in educational or employment opportunities on the basis of race, gender, age, color, religion, national origin, disability, sexual orientation, genetics, veteran status, pregnancy, childbirth or related medical conditions.
We are seeking to recruit an outstanding group leader who aims to explore bold new areas of biological inquiry and carry out interdisciplinary research to investigate multicellular development at all scales. How organisms respond and adapt to their environment, during development and throughout their lifetime, is of central interest.
The Developmental Biology Unit seeks to understand the general principles and mechanisms underlying the development of multicellular organisms. Researchers in the unit combine the power of genetic model organisms with quantitative imaging and -omics technologies, synthetic biology, reduced (in vitro) systems and theoretical modelling, to create a cross-cutting approach to modern developmental biology.
Research in the Developmental Biology Unit is firmly embedded within the overall EMBL environment, with extensive in-house collaborations, access to outstanding graduate students and postdoctoral fellows, and support from cutting-edge facilities, including genomics, transgenesis, metabolomics, mass-spectrometry, and microscopy.
Your role
You will lead a research group to pursue highly ambitious and original research at the frontier of developmental biology. In general, EMBL appoints group leaders early in their career and provides them with a very supportive, collaborative environment and generous work package for their first independent position. Significant core funding and limited teaching responsibilities allow you undertake a farsighted research program.
You have
The successful candidate will present a highly original and ambitious research plan that concisely describes the background and status of the questions that will be addressed, the experimental strategies and methods that will be employed, and the ultimate goals. A PhD degree in the Natural Sciences is expected. Candidates with a background in physics and modelling of developmental processes are also encouraged to apply.
Why join us
EMBL is an inclusive, equal opportunity employer offering attractive conditions and benefits appropriate to an international research organization with a very collegial and family friendly working environment. EMBL is committed to achieving gender balance and strongly encourages applications from women. Appointment will be based on merit alone. The remuneration package comprises a competitive salary, a comprehensive pension scheme, medical, educational and other social benefits, as well as financial support for relocation and installation, including your family, and the availability of an excellent child care facility on campus.
What else you need to know
We are Europe’s flagship research laboratory for the life sciences – an intergovernmental organisation performing scientific research in disciplines including molecular biology, physics, chemistry and computer science. We are an international, innovative and interdisciplinary laboratory with more than 1700 employees from many nations, operating across six sites, in Heidelberg (HQ), Barcelona, Hinxton near Cambridge, Hamburg, Grenoble and Rome.
Our mission is to offer vital services in training scientists, students and visitors at all levels; to develop new instruments and methods in the life sciences and actively engage in technology transfer activities, and to integrate European life science research. The working language of the institute is English.
In your online application, you will be asked to include a cover letter, your CV, the names and contact details of 3 referees and a concise description of research interests & future research plans, typically not exceeding five pages.
Further information about the position can be obtained from the Head of Unit, Anne Ephrussi (anne.ephrussi@embl.de).
Information on Group Leader appointments can be found here http://www.embl.org/gl_faq.
Interviews are planned for 18, 19 and 20 December 2019.
An initial contract of 5 years will be offered to the successful candidate. This is foreseen to be extended to a maximum of 9 years, subject to an external review.
Rapid turn over of sex determination mechanisms provides biologists with an elegant study system connecting sexual selection to molecular evolution. Striking examples of this turnover are found in African cichlids, where multiple sex determination signals exist not only within the same genera, but sometimes within the same species [1]; in the common house fly, where the primary sex determiner varies along a longitudinal gradient [2], and in the Japanese frog Rana rugosa, a species in which sex chromosomes segregate geographically but whose signals can be overridden by environmental (hormonal) intervention [3].
Downstream of the primary sex determination signal is a developmental pathway that also presents an intriguing puzzle for evolutionary biologists. While the sexual differentiation pathways of mice and humans are fundamentally similar, and while the downstream elements of this pathway are conserved between bees and flies, there are no shared genes in the sex determination pathways of mammals, arthropods, and nematodes with the striking exception of the Doublesex Mab-3 Related Transcription factor (DMRT) family. Furthermore, the fundamental biochemistry of sexual differentiation in these taxa is distinct. In mammals, signaling molecules establish feedback loops that promote one sexual identity and repress the opposite one [4]. In insects, sex-specific RNA splicing determines sexual identity [5]. And in nematodes, a cast of genes including a phosphatase and protease control the sexual fate of a cell [6]. Thus the puzzle: how does a pathway which appears stable on the order of hundreds of millions of years diverge so distinctly between different animal taxa? The Kopp lab was interested in how it is that the sexual differentiation pathway changes on a macro-evolutionary time scale.
In insect developmental biology Drosophila is the point of reference for all other species. Sex differentiation is no different, where two genes at the bottom of this pathway — transformer (tra) and doublesex (dsx) — were first described in the fruit fly. doublesex (a member of the eponymous Doublesex Mab-3 Related Transcription factor family) is a transcription factor with male- and female-specific isoforms. Both isoforms contain the same DNA binding domain, while alternative, sex-specific splicing at the 3’ end of dsx transcripts changes the effect the male and female dsx isoforms have on their targets [7]. In Drosophila, the male isoform of dsx is produced by default. Active intervention by Tra changes the pattern of dsx splicing from a male to a female isoform. tra itself is also sex specifically spliced, such that a premature stop codon appears in the male, but not the female, transcript [8]. The outgroup to insects with the best studied developmental model is the class Branchiopoda, containing the model crustacean Daphnia magna. In Daphnia, dsx is upregulated in males and lacks male and female isoforms. It is required for male, but not female differentiation [9]. tra is not sex-specifically spliced in this species [10].
To investigate how this insect-specific pathway of sexual differentiation based on sex-specific RNA splicing evolved from an ancestral state without male and female isoforms, we had to look beyond the Kopp lab’s traditional Drosophila domain. Way beyond it. Drosophila-like splicing and function have been documented in Coleopterans and in Hymenopterans, suggesting that the tra–dsx axis of sexual differentiation was present in the common ancestor of Holometabola [11,12,13,14]. The phylogenetic interval between crustaceans and Holometabola is filled in large part by the paraphyletic group of hemimetabolous insects — or those that go through a partial metamorphosis. And because insect pests are more likely to have sequenced genomes, we settled on the following delightful trio of hemimetabolous insects: the kissing bug Rhodnius prolixus (order: Hemiptera), the louse Pediculus humanus (order: Pthiraptera), and the German cockroach Blattella germanica (order: Blattodea). Because R. prolixus and P. humanus are both obligate blood feeders, they cannot be cultured in a lab without strict regulations. Thus, we were reliant on the kindness of Ian Orchard’s lab in Toronto for R. prolixus tissue shipped in RNALater. For P. humanus, our tissue collection was somewhat more exciting — we struck up a relationship with a local louse collector, who is hired to comb nits and lice from the heads of infested humans. In contrast, my experience getting to know German roaches was much more civilized. I was lucky to receive an NSF EDEN grant to study in Barcelona for two months with Xavier Belles, where I learned how to knock down genes in B. germanica. The Belles lab has done extensive work developing B. germanica into an evo-devo model.
Double stranded RNAi targeting transformer masculinizes females. From Wexler et al, eLife 2019;8:e47490.
For all three species, we investigated the isoforms of tra and dsx present in males and females. In the German cockroach, we knocked down both genes to probe their functions. The phylogenetic breadth of our samples allowed us to observe the evolutionary trajectory of dsx and tra across a large evolutionary interval. We discovered that the holometabolous pattern of tra splicing, with a male-specific premature stop codon, evolved after tra evolved a functional role as a regulator of female development. Our results from R. prolixus, where we isolated tra with a male specific stop codon, showed that the “canonical” pattern of sex-specific splicing of this gene did evolve before the common ancestor of Holometabola. In contrast, it appears that the familiar holometabolous pattern of male- and female-specific dsx isoforms differing at their 3’ end evolved before dsx became a key regulator of female differentiation. So for each gene, we observed a different story: with dsx, the “holometabolous” splicing pattern evolved before the “holometabolous” functional role; for tra, this pattern was reversed.
Summary of tra and dsx expression and function in the three hemimetabolous insect orders in comparison to the canonical holometabolous mechanism and to Daphnia. From Wexler et al, eLife 2019;8:e47490.
The knockdown experiments lead to some very fun collaborations as we attempted to probe the resulting phenotypes. After I returned from Spain and set up my own B. germanica colony at UC Davis, I was reliant on the generosity of Coby Schal, who shared a few roaches from his colony, allowing me to set up my own. The Schal lab specializes, among other things, in the study of insect behavior. When I attended the conference of the International Congress of Entomology with Coby a year later, I excitedly sat down to show him images of the masculinized females we observed upon knocking down tra. Coby then offered to quantify the degree to which these roaches’ behavior was masculinized. The experiments he did with his lab technician Ayako Wada-Katsumata revealed that like in Drosophila, sex-specific behavior of the German roach is under the control of tra. Separately, I began chatting with my teammate from the UC Davis cycling team about the work. This teammate and friend, Matt Amicucci, also happened to be a graduate student in the chemistry department, who then offered to help investigate the oligosaccharides produced by masculinized females in their tergal glands. Thanks to Matt’s expertise, we learned that tra also controls the chemical physiology of German roaches.
The breadth of the questions motivating this project and the number of questions that came out of it; the diversity of skills among the paper’s authors, and the generous support of everyone involved really made this work a wonderful and memorable PhD.
Above: Wild type males respond to the stimulus of a wild type female antenna by raising their wings and presenting their tergal gland.
Above: Females treated with dstra respond as wild type males do to the stimulus of a wild type female antenna. These treated females raise their wings in a stereotypical male courtship display.
The research group headed by Werend Boesmans and Veerle Melotte is looking for a highly motivated and talented PhD student to investigate the role of the intrinsic innervation of the gut in cancer. The enteric nervous system, also known as the second brain, is a mostly ignored member of the tumor microenvironment but is linked to the development and progression of colorectal cancer. In the current project, the involvement of specific neural cell types in colorectal tumorigenesis will be investigated in both in vivo and in vitro models, and combined with advanced optical microscopy, genetic lineage tracing, marker gene expression analysis and single cell transcriptomics.
This PhD project takes place within the School for Life Sciences (SLS) of the Transnational University of Limburg (tUL), a unique collaboration between Hasselt University (UHasselt, Belgium) and Maastricht University (UM, The Netherlands). The student will perform research at the Biomedical Research Institute (BIOMED, https://www.uhasselt.be/biomed) and the School for Oncology and Developmental Biology (GROW, https://www.maastrichtuniversity.nl/research/school-oncology-and-developmental-biology) embedded in the Department of Pathology of the Maastricht University Medical Center (MUMC).
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Story behind our recent paper in Current Biology “Cadherin-Mediated Cell Coupling Coordinates Chemokine Sensing across Collectively Migrating Cells” (Tugba Colak-Champollion, Ling Lan, Alisha R. Jadhav, Naoya Yamaguchi, Gayatri Venkiteswaran, Heta Patel, Michael Cammer, Martin Meier-Schellersheim, Holger Knaut)
Guided cell migration is a crucial event in many biological and mechanical processes. During development, orchestrated movements of cells pattern tissues and organs. Wounds in our bodies close by the migration of cell sheets. Cell migration is one of the weapons of the immune system, which sends leukocytes to the site of infection to fight against pathogens. But cell migration also contributes to several pathological conditions, such as the dissemination of cancer cells to the sites of metastasis in the body. Thus, detailed studies of mechanisms of cell migration are likely to improve our understanding of animal development, homeostasis, and disease progression.
Cell migration occurs in two major modes: single cell migration and collective cell migration. Single cell migration can be defined as a cell migrating on its own upon extracting directional information and polarizing and moving towards the target site. While collectively migrating cells also extract directional information and polarize toward the target site, they also have the additional task of coordinating with each other to move in the same direction and at the same speed.
How do cells move as a coherent collective following guidance cues? In the latest study from our lab [1], my coauthors and I investigated how directional sensing in collectively migrating cells is organized. We used the Zebrafish lateral line primordium (primordium) as a model for our study. The primordium is a collective of tightly adhering ~100 cells, which originates behind the ear of the fish at around 18 hours post fertilization (hpf) and migrates to the tip of the tail by 42 hpf.
The primordium migration is guided by chemokine signaling. For proper migration, the primordium requires the chemokine Cxcl12a and its receptors Cxcr4b and Cxcr7b. Importantly, the signaling receptor Cxcr4b is expressed in every primordium cell, while the Cxcr7b expression is restricted to the rear of the collective, where it acts as a sink receptor and generates a Cxcl12a gradient across the tissue [2-5].
Schematic of primordium migration and chemokine signaling system in primordium
Primordium migration in wild-type embryo
In collective cell migration, a prevailing model suggests that there is a division of labor between two groups of cells: leaders and followers. These two groups of cells have both topological and functional attributes. For example, leaders are located in front of the collective, whereas followers are located in the rear. In terms of functionality, the model suggests that leader cells explore their surroundings extracting directional information and relaying this information to the follower cells.
It was proposed that the primordium migration also follows a leader-follower model. An elegant study by Haas and Gilmour generated supporting evidence for this model using chimeric analysis [6]. They placed wild-type cells, which can see the chemokine signal into cxcr4b mutant primordia whose migration stalls prematurely. When at least a few wild-type cells ended up in the front region, the migration was restored – albeit at a slower speed than the speed of wild-type primordia.
However, when I started my Ph.D. project in the Knaut lab, two studies that recently were published made my advisor Holger Knaut and me rethink the leader-follower model [4, 5]. These two studies demonstrated that there is a linear chemokine signaling gradient across the tissue that is essential for the directionality of the primordium. This gradient is available to both leader and follower cells, not just to the leader cells.
Meanwhile, Gayatri Venkiteswaran, a post-doc in our lab, made an interesting observation when she was scoring the primordium migration phenotype in chemokine receptor mutants. Previously, it was known that loss of Cxcr4b leads to primordium stalling. However, loss of the other Cxcr4 receptor, Cxcr4a, that is also expressed in the primordium cells [7] does not result in a primordium migration defect. Gayatri found that taking away one copy of cxcr4a from cxcr4b-/-primordia makes the cxcr4b-/- phenotype worse and taking away both copies of both receptors is much worse than the cxcr4b-/- or cxcr4a+/-; cxcr4b-/-phenotypes. This observation suggested that Cxcr4a also contributes to migration and a primordium that is cxcr4b-/- can still see a little bit of the chemokine signal. This meant the earlier study which suggested that the existence of a few chemokine-sensitive cells could restore the migration phenotype of chemokine-blind (cxcr4b-/-) cells was conducted using primordia that could still see some of the directional signal because they retained functional Cxcr4a.
Quantification of primordium migration in 48 hpf embryos of indicated genotypes
Primordium migration in cxcr4b-/- embryo
Primordium migration in cxcr4a-/-;cxcr4b-/- embryo
In the light of these two pieces of new information—existence of a linear gradient across tissue and Cxcr4a’s contribution to directional migration—we decided to take a closer look at the leader-follower model and the contribution of follower cells to directionality and speed of the primordium.
Our first question was whether a few chemokine-sensitive (wild-type) cells could restore the migration phenotype of completely blind (cxcr4a-/-; cxcr4b-/-) primordia. To answer this question, we used a classical developmental biology technique named chimeric analysis. The idea was putting cells from wild-type donor embryos into cxcr4a-/-; cxcr4b-/- host embryos at the blastula stage. The donor primordium cells are labeled with a red transgenic marker and the host primordium cells are labeled with a green transgenic marker. After transplantation, the embryos are grown until 30 hpf and the transplanted host embryos are screened for the presence of red donor cells in the primordium. Although chimeric analysis provides very clean and reliable data, it is an inefficient technique. Unfortunately, not every single embryo transplanted gets the donor cells in the primordium. Additionally, it is a difficult technique and takes a while to perfect it. Even when you become very good at it, you can still have some days when the host mortality is high for unknown reasons.
Schematic of blastomere transplantation
Despite the difficulty and inefficiency of chimeric analysis, we still chose to do it because it was the best way to answer our questions. There was an additional layer of difficulty which turned out to be a good thing later: to generate cxcr4a-/-; cxcr4b-/-embryos, we had to in-cross cxcr4a+/-; cxcr4b-/- fish because cxcr4a-/-; cxcr4b-/- adults are not viable. This meant only a quarter of our host embryos would have the desired genotype. Half would be cxcr4a+/-; cxcr4b-/- and a quarter would be cxcr4b-/-. We had no way of knowing the genotype of the chimeric host embryos until going through a genotyping protocol after imaging these embryos. Nevertheless, we got what we wanted and more.
We found that a few chemokine sensitive (wild-type) cells do not restore the migration phenotype of completely blind (cxcr4a-/-; cxcr4b-/-) primordia, an observation that is inconsistent with the classical leader-follower model. However, we had one embryo in which about half of the primordium consisted of wild-type cells, and the other half consisted of cxcr4a-/-; cxcr4b-/-cells, and this chimeric primordium migrated about half of its path. This made us consider the possibility that cells pull on each other during migration. When there are many cells which can see the chemokine, they might generate sufficient pulling forces in the right direction to drag their chemokine blind neighbors along—in the case of this chimeric embryo, half of the way.
We decided to follow up on this observation using a quantitative approach. We predicted that as we increase the number of chemokine-sensing cells in an otherwise chemokine-blind primordium, the distance migrated by the chimeric primordium should increase. To quantify this behavior, we needed a lot of samples. Using chemokine-blind primordia (cxcr4a-/-; cxcr4b-/-) as hosts posed two challenges. First, it is difficult to get them (remember that only one-quarter of the embryos are of this genotype). Second, it might require a lot of chemokine-sensing cells to pull their completely chemokine-blind neighbors along. To overcome these challenges, we considered using cxcr4a+/-; cxcr4b-/- primordia as hosts. These almost chemokine-insensitive primordia have only one copy of cxcr4a left. Gayatri’s quantification of the migration behavior showed that the migration of these cxcr4a+/-; cxcr4b-/- primordia are worse than cxcr4b-/-primordia and a little bit better than cxcr4a-/-; cxcr4b-/- primordia. Having just a little bit of Cxcr4a activity would help us resolve the relationship between migrated distance and chemokine-sensing cell number, we hoped.
A) Schematic of experimental design and predictions. B-D’) Examples of chimeric primordia with low, medium, and high donor cell contribution. E) Graph of the total number of wild-type cells in the chimeric primordia (total primordial wild-type cells include interneuromast, neuromast (nm) and primordium (prim) cells) versus the migration distance of cxcr4-deficient primordia. 48 hpf.
Luckily, we already imaged a lot of chimeric primordia that consisted of chemokine-sensitive (wild-type) and chemokine-insensitive (cxcr4a+/-; cxcr4b-/-) cells. So we went back to this data set and counted the number of wild-type cells and measured how far these chimeric primordia migrated. As we predicted, the chimeric primordia migrated further as the number of chemokine-sensitive cells increased. This observation was consistent with the idea of cells “pulling” on each other. Next, we asked what could facilitate this “pulling”. We turned to the obvious candidates: cell-cell adhesion molecules. And there are a few of them expressed by the primordium cells including E-cadherin (Cadherin 1, Cdh1) and N-cadherin (Cadherin 2, Cdh2) [8-10].
We first decided to focus on Cdh1 and Cdh2. Our first question was how cdh1-/- and cdh2-/- cells behave during migration. The simplest method of answering this question would be observing the migration behavior of cdh1 or cdh2 mutant primordia under a microscope. However, this was not an option for either of these two genotypes. The problem with cdh1-/- embryos is that they die during gastrulation before the primordium is formed. As for cdh2-/- embryos, they have problems with somite development that affect the proper expression of Cxcl12a (the directional cue). Thus, we went back to our favorite technique, chimeric analysis by blastomere transplantation.
Live imaging of cdh1-/- or cdh2-/- cells in an otherwise wild-type primordium showed that lacking either of the cadherins does not affect migration. These mutant cells co-migrated normally with their wild-type neighbors. This raised the next question: Are these cadherins acting redundantly? To answer this question, we needed to generate cdh1-/-; cdh2-/- embryos to be used as donors. However, this is a real challenge, as the cadherin mutants are not adult viable. To obtain the desired genotype, we had to in-cross cdh1+/-; cdh2+/- fish. The chance of finding a double cadherin mutant embryo is one in sixteen! Despite the odds, we did this experiment—repeatedly. We never found a chimeric embryo whose donor embryo was cdh1-/-; cdh2-/-. But we obtained a good sample size that made us confident to think that either such double cadherin mutant cells do not become primordium cells or they dissociate from the primordium very early on. Despite not being able to find what we set out to find, we encountered some interesting genetic scenarios. For example, cdh1-/-; cdh2+/- cells dissociated from the migrating primordium when they were placed at the tip of the primordium, whereas cdh1+/-; cdh2-/- cells fell off the primordium when they were located at the rear of the primordium. These findings were puzzling until the next observation.
To observe Cdh1 and Cdh2 expression in the primordium, we made BAC transgenic lines that expressed Cdh1-GFP and Cdh2-mCherry. A closer look at the primordia in these transgenic fish revealed that Cdh1 was expressed more in the front region of the primordium and Cdh2 was expressed more in the rear. This could be a possible explanation for the observation mentioned above, that cdh1-/-; cdh2+/- cells fell off from the tip positions and cdh1+/-; cdh2-/- cells fell off from the rear positions. Perhaps Cdh1 was needed more in the front and Cdh2 more in the rear?
The observation of cadherin deficient cells falling off from the primordium also suggested that cell-cell adhesion through cadherins couples the migrating cells. However, is such cell coupling necessary for cells to pull on each other? To answer this question, we transplanted cdh1-/- or cdh2-/- cells into cxcr4b-/-primordia. While cxcr4b-/- primordia have a migration defect due to not being able to see the chemokine signal properly, cadherin mutant cells still have the chemokine receptors and should migrate directionally. To our surprise, cdh1-/- cells located in the front region split away from the cxcr4b-/- cells. But cdh2-/- cells located in the front region pulled the cxcr4b-/- cells along. This finding was consistent with the differential expression pattern of Cdh1 (more in front) and Cdh2 (more in rear) that we had observed earlier. Additionally, a little bit of literature digging revealed that in vitro studies suggested that Cdh1 could withhold more force than Cdh2 [for example 11]. It is plausible that there is increased tension between the donor population, which moves persistently in a specific direction, and the host population, which moves in random directions. A strong physical attachment might be necessary to keep these two groups together.
These findings suggested that the physical coupling of primordium cells is important for the group’s directional migration. But how does cell-cell adhesion affect individual cell directionality within a group? Up on discussions of our data with Martin Meier-Schellersheim, who is a physicist at the NIH and a co-author, we decided to take a quantitative approach using nuclear movement as a proxy for cell movement.
Next, we utilized our favorite technique, blastomere transplantation, once again and placed donor cells (wild type, cdh1-/-, cdh2-/-, or cxcr4b-/-) labeled with H2A-GFP into wild-type host primordia whose cell nuclei were labeled with H2A-mCherry.
Using commercial image software, we tracked the individual donor and host nuclei in different primordium locations at high spatial and temporal resolution. To analyze the tracking data, we used custom scripts to assess directional sensing using three measures: neighbor-neighbor distance, directionality index, and directionality angle. The nuclear tracking analysis showed that directional sensing of cadherin and cxcr4b deficient cells was impaired based on all three categories, lack of Cdh1 having the most severe effect. Together these data showed that in addition to directional cue sensing, efficient migration requires cadherin-mediated cell coupling.
Our observations suggested that cadherin-mediated cell-cell adhesion is important for coordinating cell movements in the migrating primordium. To test this further, we decided to use a gene trap line that expresses functional alpha E-catenin tagged with Citrine (Ctnna1-Citrine) from the endogenous promoter that we recently obtained from Scott Fraser’s lab [12]. Ctnna connects Cadherins on the plasma membrane to the actin cytoskeleton; therefore, lack of Ctnna should impair cadherin-mediated cell-cell adhesion. Chimeric analysis using blastomere transplantation would be the way to approach this experiment since the ctnna-/- embryos die during somitogenesis, before the primordium develops. But luckily, our lab recently developed a protein degradation system named zGrad that could be an efficient alternative to chimeric analysis [13]. As expected, the time-lapse analysis showed that depletion of Ctnna1 resulted in primordia that migrate less directionally. Interestingly, cells separated from each other during migration forming large irregular gaps consistent with the idea that cadherin-catenin complexes mediate the adhesion among the cells in the primordium.
In summary, these results suggest that all the cells in the primordium interpret the directional information and are physically coupled to each other to achieve robust migration. This behavior is not unlike some Turkish folk dances, which are characterized by groups of dancers who hold hands tightly as they dance to a tune in a synchronized fashion. Just as each dancer needs to listen to the music, each cell needs to sense the directional signal in order to coordinate their movements. Through their tight connections, dancers and cells alike synchronize their individual motions, thereby moving in unison.
References
Colak-Champollion, T., et al., Cadherin-Mediated Cell Coupling Coordinates Chemokine Sensing across Collectively Migrating Cells.Curr Biol, 2019. 29(15): p. 2570-2579 e7.
Dambly-Chaudiere, C., N. Cubedo, and A. Ghysen, Control of cell migration in the development of the posterior lateral line: antagonistic interactions between the chemokine receptors CXCR4 and CXCR7/RDC1.BMC Dev Biol, 2007. 7: p. 23.
Valentin, G., P. Haas, and D. Gilmour, The chemokine SDF1a coordinates tissue migration through the spatially restricted activation of Cxcr7 and Cxcr4b.Curr Biol, 2007. 17(12): p. 1026-31.
Dona, E., et al., Directional tissue migration through a self-generated chemokine gradient.Nature, 2013. 503(7475): p. 285-9.
Venkiteswaran, G., et al., Generation and dynamics of an endogenous, self-generated signaling gradient across a migrating tissue.Cell, 2013. 155(3): p. 674-87.
Haas, P. and D. Gilmour, Chemokine signaling mediates self-organizing tissue migration in the zebrafish lateral line.Dev Cell, 2006. 10(5): p. 673-80.
Siekmann, A.F., et al., Chemokine signaling guides regional patterning of the first embryonic artery.Genes Dev, 2009. 23(19): p. 2272-7.
Matsuda, M. and A.B. Chitnis, Atoh1a expression must be restricted by Notch signaling for effective morphogenesis of the posterior lateral line primordium in zebrafish.Development, 2010. 137(20): p. 3477-87.
Revenu, C., et al., Quantitative cell polarity imaging defines leader-to-follower transitions during collective migration and the key role of microtubule-dependent adherens junction formation.Development, 2014. 141(6): p. 1282-91.
Kozlovskaja-Gumbriene, A., et al., Proliferation-independent regulation of organ size by Fgf/Notch signaling.Elife, 2017. 6.
Panorchan, P., et al., Single-molecule analysis of cadherin-mediated cell-cell adhesion.J Cell Sci, 2006. 119(Pt 1): p. 66-74.
Trinh le, A., et al., A versatile gene trap to visualize and interrogate the function of the vertebrate proteome.Genes Dev, 2011. 25(21): p. 2306-20.
Yamaguchi, N., T. Colak-Champollion, and H. Knaut, zGrad is a nanobody-based degron system that inactivates proteins in zebrafish.Elife, 2019. 8.
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