Mouse Embryonic Stem Cells (ES cells) have the capacity to generate any tissue in the organism; this remarkable ability is called naïve pluripotency. Intriguingly, when ES cells start to differentiate they undergo a striking shape change.
Figure 1: Shape and fate change are concomitant during early differentiation in mouse embryonic stem cells. Schematic and scanning electron microscopy images depicting the change of shape, from round to spread, of differentiating ES cells.
During my interview with Ewa (Paluch), my PhD supervisor, we were discussing potential projects for me, when she showed me some preliminary scanning electron microscopy pictures of differentiating ES cells. I was immediately struck by the idea of fate changes coupled to cell shape changes. ES cells are round and smooth, whilst differentiating cells are spread with many membrane folds. As cell shape is dictated by cell mechanics, this shape change suggested a profound mechanical change during differentiation. We wondered if the shape change was a consequence of the fate transition, or was it a cause? This sounded like a great question to investigate for a PhD, at the interface between two cool labs (Ewa Paluch and Kevin Chalut) and so a few months later I joined, ready to dive into mechanics of cell fate transitions.
When observing ES cell fate transition in more detail, we noticed cells blebbing (blebs are pressure-driven spherical protrusions) intensely before spreading. This was pretty interesting because such blebbing is often a sign of changes in the organisation of the actin cytoskeleton, and notably membrane to cortex attachment. We measured this attachment during early differentiation (using microscopy & Western Blots) and found striking differences between naïve and differentiated cells. This was an important result for us as we knew from the literature that membrane to cortex attachment is the main regulator of effective membrane tension, which has been shown to play an important role in regulating cell shape and function.
Figure 2: Using a tether pulling assay, we measured membrane tension in ES fate transition. We found that ES cells undergo a drop in membrane tension as they spread during early fate transition. We could prevent this drop using mutants that maintain high membrane tension during this transition
The next step was to measure membrane tension during early differentiation; however, we didn’t have the setup ready for it just yet. In this frustrating interregnum, we decided to push ahead anyway and to try mutants and drugs that should generate defects in membrane tension. These preliminary experiments turned out to be a key milestone of this project: drugs and constructs increasing membrane tension led to major differentiation defects.
By the time we got these results, we had set up our system to measure membrane tension: a tether pulling assay using an optical trap. This technique is great because it enables precise membrane tension measurements without disturbing cell shape. As we hypothesized, we found that as cells differentiate, they undergo a dramatic drop in membrane tension. Furthermore, we were able to verify our membrane tension mutants, and establish that maintaining high membrane tension during fate transition (using drugs or mutants) leads to significant early differentiation defects. We also used our mutant cell line to show that maintaining high membrane tension resulted in developmental defects in gastruloids and cultured embryos.
Figure 3: Preventing the decrease in membrane tension results in early developmental defects. On the left is depicted an immunofluorescence assay in which we mix WT and mutant cells (which maintain high membrane tension during differentiation). On the right are pictures of our gastruloids formation assay, which shows that maintaining high membrane tension results in major morphological defects.
The next goal was to identify a mechanism by which changes in membrane tension could regulate differentiation. We tried to perturb various mechanosensors to no avail until one day Kevin had the idea of looking at endocytosis. Endocytosis is negatively regulated by membrane tension and is also a major regulator of signalling. We measured endocytosis in ES cells and found that it increases sharply as cells decrease their membrane tension and change shape. We next figured out a way to increase endocytosis levels in cells with high membrane tension (mutants) and found that this was sufficient to rescue their differentiation defects. At that stage we knew endocytosis was downstream of membrane tension but what was downstream of endocytosis? In other words, what was the link between increase in endocytosis and ES fate transition?
We decided to look at ERK activation, because ERK activation is required for early differentiation and recent work by the Scita lab beautifully showed that ERK is, in part, activated in the early endosome (Palamidessi et al., 2019). The Scita lab kindly provided us with a FRET sensor which allow to measure ERK activity in early endosome. We found that ERK activity sharply increased in cells as soon as they spread, which correspond to their drop in membrane tension and increase in endocytosis.
When we put the story together for preprint, we agreed that the main missing bit of the story was that we didn’t know what was triggering the observed change in membrane tension. After a few exploratory experiments we found out that this drop in membrane tension was triggered by Beta-catenin degradation. This degradation is coupled to a significant drop in RhoA activity, which is a known regulator of cell mechanics and notably membrane tension. This result was pretty incredible because it tied together two key pluripotency pathways, beta-catenin and ERK, via an intrinsic change in cell mechanics (membrane tension).
Figure 4: Membrane tension gates early differentiation. (Belly et al., 2021)
One aspect I really like about this paper is that it highlights the role of intrinsic cell mechanics in regulating cell mechanics. Before working on this, I always considered the role of mechanics from the environment point of view, for example how different substrate stiffnesses could regulate fate. But here we could show that cells can intrinsically modulate their mechanical properties, independently of their environment, and in turn regulate signalling. This change is clearly tied to changes in cell state, and speaks to another level on which cells tune their own receptiveness to signalling.
A mutation from a human patient with a rare metabolic disorder has been replicated in the Japanese rice fish. Researchers from the Centre for Organismal Studies Heidelberg, Germany, have developed a fish model to study disorders caused by a deficiency in the process of adding sugar molecules to proteins. These findings, published in the journal Development, provide a system to study the causes of complex metabolic disorders in humans.
In Alg mutant embryos, rod cells are initially born but not maintained and undergo programmed cell death indicated in magenta (TUNEL staining). Credit: Clara Becker.
Human cells are kept healthy by the activity of millions of proteins. These proteins are modified in different ways, such as by adding sugar molecules to them, which can be crucial for them to function properly. Given this importance, defects in the sugar-adding process are often lethal at the very early stages of development. In rare cases, however, patients can develop sugar-adding deficiencies that result in a range of metabolic diseases, known collectively as ‘congenital disorders of glycosylation’ (CDG). These disorders are caused by defects in the enzymes involved in the sugar-adding process. For example, ALG2-CDG (or CDG-Ii) is a disorder caused by mutations in the ALG2 enzyme, which combines sugar molecules together. ALG2-CDG patients appear unaffected at birth, but later develop problems in different organs, such as the eyes, brain and muscles.
The rarity, variety and complexity of these disorders has made them difficult to study, especially in the context of the whole body. Now, scientists have developed the Japanese rice fish (also known as the medaka) as a model system for studying such disorders. “Fish are particularly good models for these disorders because they develop outside the mother, making them very suitable for studying early embryonic defects,” said Professor Joachim Wittbrodt from the Centre for Organismal Studies, who led the study with Dr Thomas Thumberger. “The medaka is particularly well suited to this type of research, because we can edit the genome with high efficiency and we can utilise genetically identical lines.”
The team used CRISPR/Cas9 genome editing to introduce mutations in the medaka’s alg2 gene, in the same region where mutations had been found in a patient with ALG2-CDG. The scientists found that many of the symptoms of the patient, such as neuronal problems, were replicated in the fish. “We basically discovered a large fraction of the symptoms that had been described in the patient. Unlike studies of cells in a dish, the fish model provides the full spectrum of different cell types in an organism, which produced some unexpected results. For example, even though all the cells lack ALG2 enzymatic activity, only some cells respond, while their neighbours do not. In the fish eye, the cone (colour-sensing) cells are unaffected, whereas rod cells (which are required for vision in low light) form initially, but are then eventually lost. This defect, known as retinitis pigmentosa, is a symptom of many patients with congenital disorders of glycosylation,” explained Professor Wittbrodt. “We want to identify the proteins that require ALG2 in rod cells to understand their involvement in maintaining rod-cell function,” he added.
Importantly, these defects could be prevented by supplying fully-functional Alg2 to the fish eggs. Moving forward, the researchers plan to use this animal model to study the effects of human ALG2 mutations further. Professor Wittbrodt said, “the fact that this disorder can be efficiently rescued opens the door for understanding how different mutations in ALG2 affect its function. We especially want to study the cell type-specific responses in the context of a whole organism.”
The Marín-Juez laboratory, at the CHU Sainte-Justine Research Center, is recruiting a PhD student and a postdoctoral fellow (4-year fully funded positions). Our laboratory is interested in the cellular and molecular mechanisms regulating cardiac regeneration. The successful applicant will join the Marín-Juez laboratory at the CHU Sainte-Justine Research Center, where s/he will have access to state-of-the-art facilities and technology platforms including Advanced imaging platform (light-sheet, spinning-disc confocal, multiphoton, STED super-resolution, etc.), genomics (DropSeq, 10x, Illumina Novaseq) and bioinformatics platforms. CHU Sainte-Justine Research Center provides a thriving scientific environment where the successful applicant will have the opportunity to work with multidisciplinary scientific teams and to collaborate with talented clinicians and researchers.
Research project description
For this project, we are particularly interested in understanding how the cardiac endothelium regulates different aspects of cardiac regeneration and how alterations in the coronary network formation impact the ability of coronary vessels to support tissue replenishment. We have recently found early coronary regeneration as a key determinant of heart regeneration (Marín-Juez et al., PNAS 2016), and identified mechanisms regulating coronary network replenishment to form a vascular scaffold that supports cardiomyocyte regeneration (Marín-Juez et al., Dev Cell 2019). We now seek to define how the different components of the cardiac endothelium regulate tissue replenishment and identify the different mechanisms involved in their regulation of CM proliferation and migration.
Required training and profile
Ph.D. student position: Applicants should have training in vascular biology, molecular biology, cell biology, or related fields. Suitable candidates should be enthusiastic about regenerative and vascular biology. Previous research experience with zebrafish and/or heart regeneration is desired.
Postdoctoral position: We are looking for candidates with a Ph.D. in the biological sciences and laboratory experience in tissue repair/regeneration, cellular, molecular biology, or genetics. Previous experience working with zebrafish, imaging and histology are highly valued but not essential.
Both positions: Candidates with experience in confocal/light-sheet imaging and/or genome engineering are strongly encouraged to apply. Preference will be given to applicants with excellent collaborative and communication skills. The Marín-Juez lab and the CHU Sainte-Justine Research Center subscribe to the principle of equal access to opportunities and encourage women, members of visible and ethnic minorities, persons with disabilities and Indigenous people to apply.
Submit your application
Candidates must send the required documents before 07/31/2021 to Rubén Marín Juez at ruben.marin.juez.hsj@ssss.gouv.qc.ca
Please provide: Curriculum vitæ, Cover letter and References (2 or 3).
Royal Society Publishing has recently published a special issue of Interface Focus entitled Interdisciplinary approaches to dynamics in biology organized by Rubén Pérez-Carrasco and Berta Verd, and featuring lots of content relevant to developmental biology.
We are happy to announce the upcoming EMBO workshop “The Evolution of Animal Genomes”.
The event will take place virtually, from 13-17 September 2021 (Registration and abstract deadline: 12th July 2021)
The development of novel tools to analyze and reconstruct entire genomes open exciting possibilities to understand the appearance of phenotypical traits. This workshop will bring together internationally recognized scientists with distinct, but complementary, expertise in interpreting the effects of genomic variability. The combination of such aspects allows a comprehensive overview that goes from fundamental principles encoded in genomes to their ultimate biological significance on the formation of living, evolving organisms.
An exciting line-up of speakers (keynote lecture by Mike Levine) will cover the following topics:
– Principles of genomic adaptation – Evolutionary impact of regulatory variation – From linear to spatial – The 3D genome – Transposable elements as drivers of evolution – Evolution at single-cell resolution
There will be short talks selected from abstracts, as well as ample time for networking.
In the latest episode of Genetics Unzipped, we discover how researchers have used genetic engineering to turn genes into lifesaving drugs such as insulin for people with diabetes, and monoclonal antibodies that are used to treat autoimmune conditions, cancer and infectious diseases like COVID-19.
If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip
preLights, our sister community site here at The Company of Biologists, just published an interview with developmental biologist and zebrafish aficionado Christian Mosimann. It covers his research history, career trajectory, and, as in this quote, his views on preprints in science:
“I only preprint papers that we send out for peer review at a journal and not to stake a claim on something, and I’ve found this to be very helpful for job searches and for grants….I think the more we normalise preprinting our work we are confident in, the more we can show everyone that nobody is going to scoop you the very next morning if you put your research on a preprint server.”
Oligodendrocyte precursor cells prune axons in the mouse neocortex JoAnn Buchanan, Leila Elabbady, Forrest Collman, Nikolas L. Jorstad, Trygve E. Bakken, Carolyn Ott, Jenna Glatzer, Adam A. Bleckert, Agnes L. Bodor, Derrick Brittan, Daniel J. Bumbarger, Gayathri Mahalingam, Sharmishtaa Seshamani, Casey Schneider-Mizell, Marc M. Takeno, Russel Torres, Wenjing Yin, Rebecca D. Hodge, Manuel Castro, Sven Dorkenwald, Dodam Ih, Chris S. Jordan, Nico Kemnitz, Kisuk Lee, Ran Lu, Thomas Macrina, Shang Mu, Sergiy Popovych, William M. Silversmith, Ignacio Tartavull, Nicholas L. Turner, Alyssa M. Wilson, William Wong, Jingpeng Wu, Aleksandar Zlateski, Jonathan Zung, Jennifer Lippincott-Schwartz, Ed S. Lein, H. Sebastian Seung, Dwight E. Bergles, R. Clay Reid, Nuno Maçarico da Costa
Size-dependent protein segregation creates a spatial switch for Notch signaling and function Minsuk Kwak, Kaden M. Southard, Woon Ryoung Kim, Nam Hyeong Kim, Ramu Gopalappa, Minji An, Hyun Jung Lee, Min K. Kang, Seo Hyun Choi, Justin Farlow, Anastasios Georgakopoulos, Nikolaos K. Robakis, Matthew L. Kutys, Daeha Seo, Hyeong Bum Kim, Yong Ho Kim, Jinwoo Cheon, Zev J. Gartner, Young-wook Jun
Effects of gestational age at birth on perinatal structural brain development in healthy term-born babies Oliver Gale-Grant, Sunniva Fenn-Moltu, Lucas França, Ralica Dimitrova, Daan Christaens, Lucilio Cordero-Grande, Andrew Chew, Shona Falconer, Nicholas Harper, Anthony N Price, Jana Hutter, Emer Hughes, Jonathan O’Muircheartaigh, Mary Rutherford, Serena J Counsell, Daniel Rueckert, Chiara Nosarti, Joseph V Hajnal, Grainne McAlonan, Tomoki Arichi, A David Edwards, Dafnis Batalle
A single short reprogramming early in life improves fitness and increases lifespan in old age Quentin Alle, Enora Le Borgne, Paul Bensadoun, Camille Lemey, Nelly Béchir, Mélissa Gabanou, Fanny Estermann, Christelle Bertrand-Gaday, Laurence Pessemesse, Karine Toupet, Jérôme Vialaret, Christophe Hirtz, Danièle Noël, Christian Jorgensen, François Casas, Ollivier Milhavet, Jean-Marc Lemaitre
Molecular diversity and lineage commitment of human interneuron progenitors Dmitry Velmeshev, Manideep Chavali, Tomasz J. Nowakowski, Mohini Bhade, Simone Mayer, Nitasha Goyal, Beatriz Alvarado, Walter Mancia, Shaohui Wang, Matthew Speir, Maximilian Haeussler, David Rowitch, Arturo Alvarez-Buylla, Eric J. Huang, Mercedes Paredes, Arnold Kriegstein
Novel epigenetic clock for fetal brain development predicts prenatal age for cellular stem cell models and derived neurons Leonard C. Steg, Gemma L. Shireby, Jennifer Imm, Jonathan P. Davies, Alice Franklin, Robert Flynn, Seema C. Namboori, Akshay Bhinge, Aaron R. Jeffries, Joe Burrage, Grant W. A. Neilson, Emma M. Walker, Leo W. Perfect, Jack Price, Grainne McAlonan, Deepak P. Srivastava, Nicholas J. Bray, Emma L. Cope, Kimberly M. Jones, Nicholas D. Allen, Ehsan Pishva, Emma L. Dempster, Katie Lunnon, Jonathan Mill, Eilis Hannon
Longitudinal dynamics of clonal hematopoiesis identifies gene-specific fitness effects Neil A. Robertson, Eric Latorre-Crespo, Maria Terradas-Terradas, Alison C. Purcell, Benjamin J Livesey, Joseph A. Marsh, Lee Murphy, Angie Fawkes, Louise MacGillivray, Mhairi Copland, Riccardo E. Marioni, Sarah E. Harris, Simon R. Cox, Ian J. Deary, Linus J. Schumacher, Kristina Kirschner, Tamir Chandra
In vitro models of the human esophagus reveal ancestrally diverse response to injury Daysha Ferrer-Torres, Joshua H. Wu, Charles J. Zhang, Max A. Hammer, Michael Dame, Angeline Wu, Emily M. Holloway, Kateryna Karpoff, Caroline L. McCarthy, Margaret S Bohm, Sha Huang, Yu-Hwai Tsai, Simon P. Hogan, Danielle Kim Turgeon, Jules Lin, Peter D.R. Higgins, Jonathan Sexton, Jason R. Spence
Microtubule re-organization during female meiosis in C. elegans Ina Lantzsch, Che-Hang Yu, Yu-Zen Chen, Vitaly Zimyanin, Hossein Yazdkhasti, Norbert Lindow, Erik Szentgyörgyi, Ariel Pani, Steffen Prohaska, Martin Srayko, Sebastian Fürthauer, Stefanie Redemann
Scaling of cellular proteome with ploidy Galal Yahya, Paul Menges, Devi Anggraini Ngandiri, Daniel Schulz, Andreas Wallek, Nils Kulak, Matthias Mann, Patrick Cramer, Van Savage, Markus Raeschle, Zuzana Storchova
Minian: An open-source miniscope analysis pipeline Zhe Dong, William Mau, Yu (Susie) Feng, Zachary T. Pennington, Lingxuan Chen, Yosif Zaki, Kanaka Rajan, Tristan Shuman, Daniel Aharoni, Denise J. Cai
“How do we do this at a distance?!” A descriptive study of remote undergraduate research programs during COVID-19 Olivia A. Erickson, Rebecca B. Cole, Jared M. Isaacs, Silvia Alvarez-Clare, Jonathan Arnold, Allison Augustus-Wallace, Joseph C. Ayoob, Alan Berkowitz, Janet Branchaw, Kevin R. Burgio, Charles H. Cannon, Ruben Michael Ceballos, C. Sarah Cohen, Hilary Coller, Jane Disney, Van A. Doze, Margaret J. Eggers, Stacy Farina, Edwin L. Ferguson, Jeffrey J. Gray, Jean T. Greenberg, Alexander Hoffman, Danielle Jensen-Ryan, Robert M. Kao, Alex C. Keene, Johanna E. Kowalko, Steven A. Lopez, Camille Mathis, Mona Minkara, Courtney J. Murren, Mary Jo Ondrechen, Patricia Ordoñez, Anne Osano, Elizabeth Padilla-Crespo, Soubantika Palchoudhury, Hong Qin, Juan Ramírez-Lugo, Jennifer Reithel, Colin A. Shaw, Amber Smith, Rosemary Smith, Adam P. Summers, Fern Tsien, Erin L. Dolan
We are happy to announce that the UK Chick Developmental Biology Meeting 2021 will be held online on Friday 10th September.
This one-day virtual meeting brings together researchers from Universities and Research Institutes across the United Kingdom to promote and further our in-depth strength in using the chick embryo as a model organism to study a variety of topics in developmental biology. The meeting itself is a mix of talks from junior and senior developmental biologists and aims to stimulate productive interactions between research groups and individuals from different subject areas to exchange knowledge and expertise. This meeting will also provide a platform of support for early career researchers to engage with the community during a difficult time.
We are delighted to have Prof Marianne Bronner (Caltech, USA) as our international keynote speaker to join other invited speakers from the UK such as Prof Kate Storey (Dundee), Dr Matt Towers (Sheffield), Dr Siobhan Loughna (Nottingham), Dr Mike McGrew (Roslin) and Dr Fengzhu Xiong (Cambridge).
In addition we will have talks from early career researchers (i.e. those just starting their labs, post docs and graduate students) – so please do let us know if you or someone in your lab has a nice story that they could present please contract and email Dr Gi Fay Mok (g.mok@uea.ac.uk) or Dr Eirini Maniou (e.maniou@ucl.ac.uk) to discuss and submit a short abstract. The deadline for abstract enquiries and submissions is Friday 15th July.
In this episode of the Genetics Unzipped podcast we’re taking to the night skies with a closer look at the genetics of bats. Usually the stuff of horror films and Hallowe’en, these fascinating mammals have many important genetic secrets to share with us about evolution, longevity, immunity and more.
If you enjoy the show, please do rate and review on Apple podcasts and help to spread the word on social media. And you can always send feedback and suggestions for future episodes and guests to podcast@geneticsunzipped.com Follow us on Twitter – @geneticsunzip
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