The “Meet Our Scientists” video entitled “Our relative the fly” presents the research performed by Marco Millán on the cellular and molecular mechanisms underlying the regulation of tissue growth during normal development, tissue homeostasis, and tumorigenesis.
Marco Milán, ICREA research professor, leads the Development and Growth Control Laboratory at IRB Barcelona. In the video, he talks about the study of human diseases, such as diabetes and neurodegenerative diseases, in Drosophila melanogaster—the fruit fly.
As he explains, his research aims to understand how the genes that are involved in the regulation of normal growth are also those responsible for generating a tumor. In particular, the Development and Growth Control Laboratory is very interested in understanding the link between diabetes, nutritional deficiency, and cancer.
“Meet Our Scientists” comprises a series of 3-minute videos that present several of IRB Barcelona’s leading scientists. The videos seek to show the insight, passion, character and talent of the researchers that work at the centre.
Watch the video “Our relative the fly” (Subtitles available in Spanish, Catalan and English).
This report written by Justine Alford and highlighting a recent Development paper originally appeared on the CRUK Science Blog.
Over the past 12 months, the acronym CRISPR has been popping up in science news left, right and centre. And for good reason.
Hailed as a revolution in genetic engineering, this molecular toolbox lets researchers make remarkably precise changes to DNA. By observing the consequences of these alterations on cells, such as how they look or behave, scientists can begin to work out what certain genes do.
“CRISPR has completely transformed the landscape for how we study gene function,” says Dr Steven Pollard, one of our brain tumour experts from Edinburgh’s MRC Centre for Regenerative Medicine. “It’s opening up the human genome for us to be able to do what we want genetically.”
And because cancer is a disease of faulty genes, CRISPR has huge potential for studying a raft of different types of cancer.
Now, for the first time, a team of scientists led by Pollard has succeeded in using CRISPR to change genes in specialised neural stem cells, which are thought to play a role in how the most common type of brain tumour, glioblastoma, grows.
This important work, published in the journal Development, lays the foundations for future, more detailed investigations. But firstly, what are these neural stem cells that scientists are tinkering with, and why are they so important?
Astrocytes grown from brain cancer stem cells. Steven Pollard/Wellcome Images. Flickr/CC BY-NC-ND 2/0
The stars of the brain
If you’re unfamiliar with stem cells, we all begin our lives as a small bundle of them in the womb. Like the shape-shifting Mystique from X-Men, these amazing cells can change their appearance, morphing into every specialist cell in the body.
In our brains, a type of stem cell – called a neural stem cell – divides to become the complex mixture of highly specialised cells forming our ‘grey matter’, including cells called astrocytes. These star-shaped cells play incredibly important roles in the brain, offering protection for other cells and repairing those that become damaged.
During the transformation from neural stem cell into an astrocyte, the dividing cells can make a genetic ‘spelling mistake’, leading to a tumour made up of rogue astrocytes.
But some of the tumour cells become ‘locked’ in a neural stem cell state and continue to grow uncontrollably, never becoming specialised. These haywire cells, called glioblastoma stem cells, are thought to be important in fuelling and maintaining brain tumour growth.
And the similarities between the glioblastoma stem cells and neural stem cells means that researchers can study genes in neural stem cells to understand more about how glioblastomas form.
By introducing deliberate genetic mistakes into the neural stem cells’ DNA, scientists can track the role they may play in cancer. And thanks to the work by Pollard and his team, they now how a range of powerful new approaches to do this, all built upon CRISPR.
Scissors and homing devices
CRISPR is a two-piece toolkit that uses a homing device to seek out a specific region of DNA, and a pair of ‘molecular scissors’ to make a precise snip across the strands.
The cut DNA is then flagged to the cell’s own DNA repair system, which galvanises into action to sew the broken strands back together. Scientists then sneakily trick this system into making a mistake by providing a repair template that has a fault in it.
After this error is unwittingly copied into the DNA, scientists can track the consequences of this genetic change on the cell. Check out the graphic below to see how it works.
“Most groups have been using CRISPR to make random changes to genes, which is its simplest use,” Pollard explains. But the team’s latest study, he says, was focused on more sophisticated and precise changes made to neural stem cells in the lab.
For example, by stitching a fluorescent marker onto the molecule produced by a gene called SOX2, the scientists were able to track the journey that molecule takes around the cell. Precisely following molecules in this way could help researchers understand the role they play in cancer and find new targets for drugs.
“The obvious next step is finding out if we can use the same technology in cells taken from patients’ brain tumours,” says Pollard. By making important molecules glow inside these cells in the lab, Pollard believes they could “see which drugs are important in destroying that fluorescence”.
Making headway
After proving the editing prowess of CRISPR in neural stem cells, the scientists then moved on to their next challenge: could they deliberately introduce faults in genes already known to drive brain tumours?
The team focused its attention on 2 different genes. The first,p53, protects cells from becoming cancerous. It’s faulty in more than half of human tumours, including many brain tumours.
The second is a gene called H3F3A, which is commonly faulty in childhood glioblastomas. H3F3A helps package up our DNA into chromosomes.
The team managed to make faulty versions of both of these genes in human neural stem cells. And when they studied the cells harbouring the faulty version of p53 in the lab, they found that they divided faster – a hallmark of cancer.
While it’s still early days, this study has important implications for brain tumour research and therefore ultimately for patients. By demonstrating that CRISPR can successfully be used to create and study faulty genes, the team has opened the door for more in-depth research into what genes play a role in these brain tumours.
“It’s proof of principle that it works very efficiently,” says Pollard of the technique. “It’s a toolbox to show that, in the future, we can target other genes to study them and manipulate them in diverse ways. This will help us to understand their function and reveal how to manipulate them with new drugs.”
And by using CRISPR to understand how brain tumours grow, this could lead to more targeted treatments.
Although significant progress has been made in cancer medicine, with survival across all cancers doubling over the past 40 years, poor survival is a shared feature among brain tumours, making them one of our top priorities.
It’s clear that we urgently need to do more. And kick starting a greater understanding of brain tumours is needed, which is exactly what this study does.
Successful division was an essential criterion for establishing the cell as the basic unit of life on earth. Later, cell-cell adhesion made possible the evolution of multicellular life forms. These two fundamental cellular processes co-function throughout the life of an organism, during development, wound healing and tissue regeneration. In epithelial tissues this results in a curious situation. Epithelial cells, to fulfill their barrier function, need to attach strongly with one another via E-cadherin mediated cell junctions and not leave any gaps between them. However, to divide, cells must round up and form an ingression furrow and therefore detach from their neighbors.
Therefore a ‘cell division conundrum’ exists in the multicellular scenario, wherein the dividing cell is trying to furrow inwards by assembling a contractile actomyosin ring, while the neighboring cells are attempting to keep the tension intact at the boundary by forming cell-cell junctions. In such a scenario an inhibitory regulation between cell-adhesion and cell division machineries would enable the cells to defuse this ‘tug of war’ situation between the two forces.
Convergence on the idea
After completing my Ph.D. with Prof. Mohan Balasubramanian studying cytokinesis in the single cell S.pombe[1], I was eager to address the interplay between cell-cell adhesion and cytokinesis in a multicellular organism. I joined the group of Dr. Ronen Zaidel-Bar, who had recently moved to the Mechanobiology Institute, Singapore and setup his lab to study cell and tissue morphogenesis in mammalian cells and in C. elegans. Co-incidentally, during his post-doc Ronen had observed a genetic interaction between cell-adhesion and cell division mutants in C. elegans embryogenesis and had wondered about possible connections between the two force-generating cellular machineries. However, he never followed up on those results. Therefore, he was extremely receptive to my idea despite the fact that I had never seen a C. elegans nematode before then.
The ride
We decided to focus on the early embryo, where the cells are large and divisions are rapid. Additionally, the absence of cell-matrix adhesion in early embryos meant we could focus solely on cytokinesis and cell-cell adhesion. The ortholog of E-cadherin in C. elegans is called HMR-1 [2, 3], and so my first experiment was to deplete HMR-1 by RNA interference (RNAi) and measure the time it took for cytokinetic furrows to ingress during the first, second and third cell divisions (Fig. 1A)[4]. We were really excited to observe faster furrow closure upon HMR-1 depletion during these divisions. This suggested that HMR-1-mediated cell adhesion inhibited cytokinetic furrow ingression and in the absence of HMR-1, this inhibition was released allowing embryonic cells to accomplish faster cytokinesis.
Around the same time, three papers, published back to back in Developmental Cell, reported the existence of a ‘tug-of-war’ mechanism in Drosophila epithelial cells between E-cadherin-mediated cell adhesion and contractile forces of the cytokinetic ring [5-7]. On the one hand, we were pleased our hypothesis received strong reinforcement from Drosophila, but on the other hand we couldn’t help feeling “scooped”.
The First Division Problem
While most of our results were consistent with our hypothesis and the newly published papers from Drosophila, there was one inconsistent result: furrow ingression was faster in the first cell division when HMR-1 was depleted. How could the “tug-of-war between forces” model explain the speeding up of furrow ingression in the 1-cell zygote lacking any cell contacts? The fact was it couldn’t.
To answer this question I analysed the localisation and dynamics of HMR-1 in these early embryonic stages, by imaging Green Fluorescent Protein (GFP) fused to the cytoplasmic domain of HMR-1 protein [8]. As expected, HMR-1 localised to cell-cell junctions in the 2-cell and 4-cell stage (Fig. 1B, blue arrows). Additionally, I also detected HMR-1::GFP as distinct spots or clusters along the non-junctional surfaces in these stages as well as the 1-cell stage (Fig. 1B, orange arrows) [4, 9]. Thus, we concluded, it must be these non-junctional E-cadherin/HMR-1 clusters, which, even though not involved in cell-cell adhesion, slow down cytokinesis. But how do they do it?
Non-Junctional E-cadherin/HMR-1 Dictate a Change in Course
Our unexpected findings in the zygote led to a new question: how do non-junctional E-cadherin/HMR-1 clusters regulate cytokinesis? The first clue to this puzzle emerged when I examined type-II myosin NMY-2, in hmr-1 depleted embryos and control embryos. I detected a significant increase in the levels of NMY-2 at the cortex (Fig. 2) upon HMR-1 depletion and also observed that HMR-1 and NMY-2 excluded each other. Depletion of HMR-1 also resulted in up regulation of cortical RHO-1, the global contractility regulator, which acts upstream of NMY-2. At this juncture we realised that although our analysis of the 1-cell stage might not be along the ‘tug of war’ model, there definitely is an inhibitory relationship between the adhesion and cytokinesis machineries of the cell.
As the zygote initiates first cleavage by assembling a contractile zone bisecting the separating chromosomes, the actomyosin cortex rotates. I found this cortical rotation to be faster in embryos lacking HMR-1 compared to the control. Given that NMY-2 is the major force generator in cells, it seemed natural to attribute the faster cortical rotation as well as the accelerated cytokinesis to the increase in cortical NMY-2. However, when I tested this hypothesis by reducing the amount of NMY-2 recruited to the cortex in both control and hmr-1 (RNAi) embryos, I observed that even when NMY-2 levels were reduced to similar levels, hmr-1 (RNAi) depleted embryos furrowed faster than control, indicating that HMR-1 could influence cytokinesis independently of NMY-2 regulation.
HMR-1 Inhibits Cortical NMY-2 Localisation
The Discovery of an Unexpected Mechanism
The second clue came to light when I examined the relationship between HMR-1 clusters and cortical F-actin. I detected transient interactions between F-actin at the cortex and HMR-1 clusters embedded in the membrane, and showed that this transient interaction slows the cortical movement akin to stapler pins holding paper sheets from sliding. Since furrow ingression during cytokinesis is clearly a form of cortical deformation, one possible mechanism by which HMR-1 slows down cytokinesis independent of NMY-2 is by physically resisting cortex deformations.
Our studies uncovered a hitherto unknown facet of E-cadherin/HMR-1, wherein it regulates the actomyosin cortex through interactions of its cytoplasmic intra-cellular domain and not the adhesive abilities of the extracellular domain. The intracellular domain – cortex association maintains the structural integrity of the cortex and slows its movement. Compromising this association leads to weakening of the cortex, which under the influence of cellular contractile forces results in faster cytokinetic furrow ingression and in extreme cases can result in splitting of the cortex (Movie1).
Movie 1 – F-actin cortex splitting in hmr-1(RNAi) embryos
Wider Implications
In parallel with my work in the C. elegans zygote, other members of our group working with mammalian epithelial cells have found that non-junctional clusters of E-cadherin can be found all over the cell surface[10, 11]. Whether non-junctional cadherin in mammals regulates the cortex in the same way they do in C. elegans remains to be tested. Nevertheless, given the high frequency of loss of E-cadherin in cancer the possibility that it could affect cellular processes other than cell adhesion, such as cell division and migration, is tantalising.
References
Padmanabhan, A., Bakka, K., Sevugan, M., Naqvi, N.I., D’Souza, V., Tang, X., Mishra, M., and Balasubramanian, M.K. (2011). IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast. Curr Biol 21, 467-472.
Costa, M., Raich, W., Agbunag, C., Leung, B., Hardin, J., and Priess, J.R. (1998). A putative catenin-cadherin system mediates morphogenesis of the Caenorhabditis elegans embryo. J Cell Biol 141, 297-308.
Armenti, S.T., and Nance, J. (2012). Adherens junctions in C. elegans embryonic morphogenesis. Subcell Biochem 60, 279-299.
Herszterg, S., Leibfried, A., Bosveld, F., Martin, C., and Bellaiche, Y. (2013). Interplay between the dividing cell and its neighbors regulates adherens junction formation during cytokinesis in epithelial tissue. Dev Cell 24, 256-270.
Guillot, C., and Lecuit, T. (2013). Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues. Dev Cell 24, 227-241.
Founounou, N., Loyer, N., and Le Borgne, R. (2013). Septins regulate the contractility of the actomyosin ring to enable adherens junction remodeling during cytokinesis of epithelial cells. Dev Cell 24, 242-255.
Chihara, D., and Nance, J. (2012). An E-cadherin-mediated hitchhiking mechanism for C. elegans germ cell internalization during gastrulation. Development 139, 2547-2556.
Munro, E., Nance, J., and Priess, J.R. (2004). Cortical flows powered by asymmetrical contraction transport PAR proteins to establish and maintain anterior-posterior polarity in the early C. elegans embryo. Dev Cell 7, 413-424.
Guo, Z., Neilson, L.J., Zhong, H., Murray, P.S., Zanivan, S., and Zaidel-Bar, R. (2014). E-cadherin interactome complexity and robustness resolved by quantitative proteomics. Sci Signal 7, rs7.
Wu, Y., Kanchanawong, P., and Zaidel-Bar, R. (2015). Actin-delimited adhesion-independent clustering of e-cadherin forms the nanoscale building blocks of adherens junctions. Dev Cell 32, 139-154.
Here are the highlights from the new issue of Development:
A new mechanism in ESC lineage priming
Histone demethylases have recognized roles in the control of gene expression during development and disease, and are typically associated with the remodelling of the chromatin environment. Jmjd2/Kdm4 H3K9-demethylases cooperate in promoting mouse embryonic stem cell (ESC) identity, but their specific roles during the exit from pluripotency are still unclear. In this issue (p. 567), Véronique Azuara and colleagues uncover a previously unrecognized functional link between Jmjd2c recruitment to lineage-specific enhancers and ESC priming for differentiation. The authors show that Jmjd2c is required for the proper assembly of mediator-cohesin complexes at lineage-specific enhancer regions, and that differentiation is stalled at an early post-implantation epiblast-like stage in both Jmjd2c-knockout and Jmjd2c-knockdown ESCs. Interestingly, Jmjd2c-deficient cells were still able to differentiate towards extra-embryonic endoderm-like cells. At the chromosomal level, the authors showed how Jmjd2c-bound enhancers are co-occupied by the H3K9-methyltransferase G9a/Ehmt2 independently of its canonical H3K9-modifying activity, and suggest that Jmjd2c-G9a co-occupancy might facilitate the loading of Med1 and Smc1a molecules. This study is significant and novel as it reveals a new mechanism for the regulation of lineage priming in ESCs via Jmjd2c-mediated stabilisation of essential protein complex assembly at enhancers.
Tick tock goes the segmentation clock
Somitogenesis is the process by which the somites, blocks of mesoderm that give rise to tissues such as the vertebrae, skeletal muscle, cartilage, tendons and skin, are formed. The process occurs under the control of a ‘segmentation clock’: the oscillatory expression of a number of genes and proteins that control cell commitment. The protein paraxial protocadherin (PAPC) is a protocadherin that has been implicated in paraxial mesoderm segmentation; however, the way in which PAPC controls somite formation remains unclear. Now, on p. 664, Olivier Pourquié and colleagues investigate the role of PAPC in chick and mouse somite boundary formation, and demonstrate an entirely novel mechanism for periodic somite formation through the regulated endocytosis of N-cadherin (CDH2). The authors first show that PAPC is cyclically expressed downstream of the segmentation clock, and that PAPC expression colocalizes with CDH2 in the rostral half of the forming somite. In ovo overexpression of the short PAPC isoform in the presomitic mesoderm disrupts apical accumulation of CDH2 and interferes with proper somite morphogenesis. Mechanistically, the authors show how PAPC regulates the endocytosis of CDH2 in the anterior compartment of the forming somite, that is, in regions that have not yet epithelialized. In this way, PAPC regulates the segmental de-adhesion of the somites, which is crucial for their subsequent formation.
p53+ cells drive in vivo cardiomyocyte expansion
The mammalian heart has a limited capacity to regenerate. Under certain conditions, however, cardiomyocyte proliferation has been observed, for example in resected neonatal hearts and in response to certain cytokine treatments. Nevertheless, the extent to which cardiomyocyte proliferation occurs both in steady state and after injury in the postnatal mouse is hotly debated, as studies are limited by a lack of reliable genetic tracing tools. Now, on p. 580, Zhongzhou Yang and colleagues use a p53-based genetic tracing system to investigate postnatal cardiomyocyte proliferation and heart regeneration through neonatal, adolescent and adult stages. The authors observed clonal expansion of p53+ cardiomyocytes in the neonatal heart, as well as in pre-adolescent and adult hearts. Interestingly, some of the labeled cardiomyocytes also formed larger clusters if given a longer tracing time, suggestive of a selectively long-lasting proliferative potential. The authors also investigated cardiomyocyte proliferation after cryo-injury and showed that p53+ cardiomyocytes exhibit cytomembrane localization of the sarcomeric protein cTnT during heart regeneration, consistent with previous studies. Finally, the authors demonstrated that the p53 genetic labeling system reliably traced proliferating cardiomyocytes following not only in cryo-injury, but also in two additional types of cardiac injury models in neonatal mice. This study reveals the specific lineage contribution to mammalian cardiac repair and provides evidence for the heterogeneity of cardiomyocytes in mammalian heart.
PLUS:
Auxin 2016: a burst of auxin in the warm south of China
Auxin – a key plant hormone – plays a prominent role in regulating plant developmental processes, and delineating its role is therefore the subject of intensive investigation. In their Meeting Review, Teva Vernoux and Stéphanie Robert discuss discuss new insights into auxin-mediated signalling that were presented at the Auxin 2016 meeting, which was held in Sanya, China, in October 2016.
Metabolic remodeling during the loss and acquisition of pluripotency
Cellular metabolism plays a vital role in development, beyond the simple production of energy, and may be involved in the regulation of cell fate. In their Review article, Julie Mathieu and Hannele Ruohola-Baker review the metabolic changes that occur during the transitions between different pluripotent states, both in vitro and in vivo, and discuss the extent to which distinct metabolites might regulate these transitions.
Neural tube closure: cellular, molecular and biomechanical mechanisms
The process of neural tube closure is complex and involves cellular events such as convergent extension, apical constriction and interkinetic nuclear migration, as well as precise molecular control via the non-canonical Wnt/planar cell polarity pathway, Shh/BMP signalling, and the transcription factors Grhl2/3, Pax3, Cdx2 and Zic2. More recently, biomechanical inputs into neural tube morphogenesis have also been identified. In their Review article, Andrew Copp and colleagues review these cellular, molecular and biomechanical mechanisms involved in neural tube closure.
We are seeking to recruit a postdoctoral fellow to develop and apply high resolution light sheet microscopy in order to image cytoskeletal networks and adhesion complexes in developing embryos (e.g. Drosophila). We have recently developed a light sheet microscope for fast 3D imaging and we aim at including a new illumination scheme to achieve higher resolution and single molecule detection. The recruited postdoc will also develop new image analysis tools to analyze the generated high resolution data.
The candidates should have experience in computational image analysis and/or optical engineering (including software engineering for machine control).
The postdoc will benefit from an interdisciplinary environment with expertise in imaging, optical engineering, physics and cell developmental biology (Labex Inform, IBDM)
The postdoctoral fellowship is offered for a period of two years.
Applicants should send a CV, names of two referees, and a short outline of their research interests to P.-F. Lenne and T. Lecuit.
Live imaging of mouse peri- and post-implantation morphogenesis
Fixed-term: The funds for this post are available for three years.
ImageInLife is a Marie Skłodowska-Curie Innovative Training Network (MSCA-ITN) funded by the European Commission Horizon 2020 programme and focused on the training of European experts in multilevel bio-imaging, analysis and modelling of vertebrate development and disease.
In the context of the ImageInLife network, the Department of Physiology, Development and Neuroscience (PDN), University of Cambridge, has a vacancy for one Early Stage Researcher (ESR, PhD student) on the project detailed below.
Project description: Live imaging of mouse peri- and post-implantation morphogenesis
The ESR will work in the group of Prof. Magdalena Zernicka-Goetz with Drs Neophytos Christodoulou and Matteo Molè as co-supervisors. The project aims to uncover the morphogenetic events shaping the mouse embryo during implantation development in the synthetic and natural environment. The ESR will use the well-established ex-vivo culture system developed in the host laboratory, in combination with transgenic fluorescent reporter mouse lines and advanced confocal and multiphoton microscopy. Additionally, 4D cell motion and lineage tracking analysis will be performed to characterise how single cell behavior contributes to tissue wide morphogenetic events.
Applicants should hold a degree in biology, biophysics, or biomedical sciences and must comply with the eligibility criteria and transnational mobility rules for MSCA-ITN:
Early-stage researcher(ESR) will be appointed for three years as Marie Skłodowska-Curie Fellow. The Fellowship is offered in conjunction with a PhD position in the PDN, University of Cambridge and will be subject to the Fellow satisfying the University’s admissions requirements. At the time of recruitment, the ESR shall be in the first four years (full-time equivalent research experience) of his/her research career and have not been awarded a doctoral degree.
Full-Time Equivalent Research Experience is measured from the date when the researcher obtained the degree entitling him/her to embark on a doctorate (either in the country in which the degree was obtained or in the country in which the researcher is recruited or seconded), even if a doctorate was never started or envisaged.
Trans-national mobility (i.e. move from one country to another) is an essential requirement of MSCA-ITN. The ESR can be of any nationality. At the time of recruitment by the host organisation, he/she must not have resided or carried out his/her main activity (work, studies, etc) in the country of the host organisation for more than 12 months in the three years immediately before the reference date. Compulsory national service and/or short stays such as holidays are not taken into account.
The ESR will be employed at the host institute by a contract with full social security coverage. He/She will receive a salary of £35,000 per annum augmented by a mobility allowance of £5,600 per annum in line with the EC rules for Marie Skłodowska-Curie grant holders. The ESR will be liable to pay his/her fees (see www.graduate.study.cam.ac.uk/finance). The appointment will be made on educational background, research experience, fluency in spoken and written English, and motivation to take part in and contribute to the research and training programme of the ImageInLife consortium. Applications, in English, should include a covering letter, CV, detailed academic transcripts and two reference letters, which are all to be submitted through the on-line application system at https://www.imageinlife-application.eu. Additionally, please submit the same application documents by email to Drs Christodoulou (nc480@cam.ac.uk) and Mole (mam238@cam.ac.uk).
ImageInLife strives to recruit between 40-60% female researchers. For more information contact Prof. Magdalena Zernicka-Goetz (mz205@cam.ac.uk).
Decoding the network logic for resetting pluripotency – Collaborative Stem Cell Research PhD Studentship with Microsoft Research – re-advertised, revised closing date 31st March 2017
Outline Project Description:
Interdisciplinary project at the interface of stem cell research and computational modelling
Delineation of network trajectories for cellular reprogramming at single cell resolution
Combination of wet lab research with logical modelling
Collaboration between the laboratory of Prof. Austin Smith and Microsoft Research Cambridge
The Smith Group at the Medical Research Council Wellcome Trust Stem Cell Institute in Cambridge in partnership with the Computational Biology Group at Microsoft Research offers an exciting interdisciplinary 4-year PhD studentship commencing October 2017.
The pluripotent ground state of embryonic stem cells (ESCs) is governed by a self-reinforcing interaction network of transcription factors (Dunn et al, Science 2014). Combinations of factors within this network can induce somatic cells to acquire pluripotency, a process called molecular reprogramming (Takahashi and Yamanaka, Cell, 2006). Experimental and computational efforts have led to circuitry mapping of the key players in maintenance of the ESC state. However, how this molecular circuitry is launched and fully connected during reprogramming remains unclear.
This project is a cross-disciplinary investigation to address systematically how cells transit to the pluripotent ESC state at the molecular network level. The multi-step, heterogeneous and asynchronous nature of the reprogramming process presents technical challenges. This project is designed to overcome these challenges by using a minimal reprogramming system and integrating quantitative single-cell gene expression profiling at defined reprogramming stages with computational network synthesis and modelling. This approach will transform a temporal series of single-cell snapshots of network status into reconfiguring network trajectories. Predictions formulated from the synthesised trajectories will be tested experimentally and the results used for iterative refinement of the model set.
As part of the BBSRC doctoral training programme, this 4-year PhD contains tailored training courses in the first six months of the studentship. In addition, a key element of this project is that the student will spend three months at Microsoft Research Cambridge, under the supervision of our collaborator, Dr Sara-Jane Dunn, to develop wider training and skills.
For further details about our group and the institute, please visit:http://www.stemcells.cam.ac.uk/
Funding Notes
UK and EEA students who have, or are expecting to attain, at least an upper second class honours degree (or equivalent) in relevant biological subjects are invited to apply. The interdisciplinary nature of the project means that we welcome applications from students with mathematical and computing experience who are interested in using their skills to address biological questions.
Application details are available at http://www.stemcells.cam.ac.uk/study/otheropportunities/#BBSRC. Please ask your referees to submit references directly to the SCI Graduate Administrator: sci-phd@stemcells.cam.ac.uk, using “BBSRCiCASE student reference” in the subject header. The deadline is 31st March 2017 and shortlisted candidates will be interviewed in April. Please note: this studentship is being re-advertised. Previous applicants need not apply.
References
Dunn, S. J., Martello, G., Yordanov, B., Emmott, S. & Smith, A. G. Defining an essential transcription factor program for naïve pluripotency. Science 344, 1156-1160, (2014).
Martello, G. & Smith, A. The nature of embryonic stem cells. Annu Rev Cell Dev Biol 30, 647-675, (2014).
Yordanov, B., Dunn, S.-J., Kugler, H., Smith, A., Martello, G. & Emmott, S. A method to identify and analyze biological programs through automated reasoning. Npj Systems Biology And Applications 2, 16010, (2016)
The use of chimaeras to study developmental mechanisms: from lineage tracing to disease models
Under the sponsorship of the Anne McLaren Memorial Trust Fund and The Company of Biologists, the BSDB Autumn meeting organised by Jenny Nichols and Tristan Rodriguez took place in the Pollock Halls at the University of Edinburgh. The topic this year was: ‘Chimaeras and their use in studying developmental processes and disease models’. Chimaeras are made of cells from two or more different organisms of the same or different species. Since their first conception, chimaeras have been an essential tool to dissect cellular potential and are used to address a large number of questions in developmental biology using a variety of different model organisms, from plants to vertebrates.
Read here the meeting report by Carla Mulas and Juan Miguel Sanchez Nieto.
The meeting kicked off with plenary talks by Professors Nicole le Douarin and Sir Richard Gardner, both responsible for key innovations using avian and mouse chimaeric embryos respectively. Their ideas and work led to remarkable discoveries that have been essential not only for our current knowledge in the area of developmental biology, but also to shed light on key biological concepts such cell fate and plasticity. Nicole le Douarin presented her earlier work on the use of chick-quail chimaeras and the realisation that their different nuclear organisation could be used as a marker to distinguish host versus graft cells. She took the audience through the application of chimaeras to study the neural crest, revealing the large contribution of these cells to the development of anterior structures in vertebrates. This work remains relevant to the present day when mouse-human chimaeras have been used to trace neural crest in mammalian embryos (Cohen et al. 2016). Richard Gardner’s development of tools to enable injection of single cells into mouse blastocysts, and the successful development of the embryos thereafter, was essential to decipher clonal behaviour of cells during early mouse development – with important observations spanning the fields of embryology, epigenetics and embryonic stem cell biology.
Through the following two days, five sessions saw a wide range of applications of chimaeras discussed in various model systems. Overall, the talks were a mixture of traditional uses of chimaeras, recent innovations within this historical context and a broad range of other ideas and approaches – incorporating both the weird and the wonderful!
The first and largest session, LINEAGE TRACING AND POTENCY, focused on the use of modern labelling and imaging techniques in order to trace the descendants of specific cells, which were either labelled in situ or challenged by transplantation. What was particularly interesting in this session was how universal the application of chimaeras is in different organisms and at different developmental stages. For example, Claire Baker presented work on cells responsible for sensing hypoxia in vertebrates, trying to resolve, by a combination of lineage tracing and deletion studies, whether homologous cells in different organisms share a common embryonic origin. Both Janet Rossant and Berenika Plusa used chimaeras as tools to assess the changes in cellular potential within the mouse pre-implantation embryo as it undergoes the first two cell fate decisions. Janet explained the correlation between the plasticity of cells from the inner cell mass (ICM) and Hippo signalling pathway combining classic chimaera experiments with single cell sequencing technologies to probe deeper into questions of cell fate and cell potency in the pre-implantation mouse embryo. Berenika discussed the different roles of Sox2 and Klf4 in regulating the fate choice that ICM cells make between epiblast and primitive endoderm. Staying with the early mouse embryo, Josh Brickman argued the importance of specific nutrients in the media to support the maintenance of the naïve pluripotent and endodermal states as well as increasing the efficiency of chimaera formation. After this talk came another highlight of the meeting, Virginia Papaioannou’s tour de force on her analysis of T-box genes and their importance as an example of harnessing knowledge from the study of development to understand the human condition.
From here we moved back to avian models, where Mike Clinton, used mixed-sex chimaeras in chickens to study how the host influences the grafted tissue. He investigated how sex identity is specified, showing that somatic cells possess an identity which is cell autonomous. To round off the session, two speakers illustrated the enormous power of chimaeras and lineage labelling to uncover the boundaries for cell fate determination and cell plasticity, John West in the adult mouse cornea and Filip Wymeersch for the neuromesodermal progenitors.
In the SIGNALLING MECHANISMS session that followed, the speakers covered the influence and relevance of signalling pathways regulating cell fate choice. Claudio Stern’s hunt for a new universal organiser’s signature opened probably the most diverse session of the conference. Alexander Bruce presented his work, in which he identified p38 as a regulator of primitive endoderm differentiation in the early mouse embryo (Thamodaran and Bruce, 2016). Chris Thompson, conversely, used Dictyostelium as a model system to interrogate how genetically uniform systems can break symmetry and undergo differentiation. In this session there was also exciting insight provided by plant chimaeras. Nicola Harrison discussed the implications of the technique of grafting in apple trees and how our understanding of this process may affect the quality of the product and the yield of the crops. Kim Dale, the last speaker of this session, presented her work on how Notch amplifies Shh signalling pathway in the neural tube regulating the cell fate of neuro-ectodermal progenitors (Stasiulewicz et al. 2015).
On day two, the REGENERATIVE MEDICINE AND HUMAN DEVELOPMENT session was started with a talk by Iwo Kucinski. Iwo was awarded the first Dennis Summerbell Lecture Prize, and he presented his work deciphering signalling pathways favouring the elimination of unfit cells in the process of cell competition. Interestingly Iwo showed that unfit cells, identified by a variety of means, show a number of common signalling changes that are not detrimental for viability when surrounded by other unfit cells, but that trigger their elimination when in a competitive environment with fitter cells. Nicholas Tan presented a novel strategy (DNA Adenine-Methyltransferase Identification sequencing) to identify genome-wide transcription factor binding targets within single embryos or with samples that have only 1000 cells and Man Zhang discussed the importance of ESSRb for Nanog function. Unfortunately, Hiro Nakauchi could not attend the meeting but Hideki Masaki flew in from Japan to present his own work in collaboration with the Nakauchi lab. Very interestingly, they observed that primed pluripotent stem cells with acquired resistance to apoptosis can contribute to chimaeras when injected into blastocysts, a process that does not occur when attempted with wild-type primed pluripotent cells. These experiments suggest that not only the pluripotency status of the cells is important for efficient chimaera formation but also their apoptotic threshold, thus providing an avenue for efficient chimaera generation with cells with restricted developmental potential.
Throughout the last two sessions, DISEASE MODELS andGENE FUNCTION several speakers discussed the advances in modern techniques and their applications to developmental biology and potential regenerative therapies. For instance, Stephen Pollard and Bill Skarnes outlined CRISPR-based approaches to generate genetically modified adult and embryonic stem cells while Ben Steventon and Kenzo Ivanovich demonstrated beautiful applications of live imaging to study the development of neuro-mesodermal progenitors during axis elongation and the early stages of mouse heart development, respectively. Vasso Episkopou presented her work analysing how Arkadia modulates the levels of TGFb signalling during early mouse development and Elena Lopez-Jimenez discussed how Oct4 is not only a pluripotency factor, but can provide positional information by regulating the Hox cluster. To complement these approaches, Megan Davey gave a fascinating insight into how chick chimaeras can tell us not only about the signalling inputs that pattern the vertebrate limb, but also shed light into the evolutionary origins of our five digit structure. The grandiose finale of the meeting was Liz Robertson, who discussed the importance of Blimp1 in the control of mammary gland development and homeostasis. Interestingly she showed how important Blimp1 is for the organization of this epithelial tissue, providing new insight into the regulation of mammary gland tissue integrity (Ahmed et al. 2016).
Throughout the meeting there were many fond tributes to Anne McLaren, who, amongst her many other accomplishments, previously organised a chimaera-themed meeting in the early 90s. A great scientific atmosphere was created during all the poster sessions that ran throughout the breaks and during the evenings, where everybody had the chance to present their work, learn, discuss and network. Reflecting the beauty of developing systems, chimaera and embryo-inspired artwork was on display and available for purchase, designed by Mia Buehr [LINK] and Aurora Lombardo [LINK].
Overall, it was a brilliant and diverse meeting that took the audience literally through time, from the earlier discoveries and innovations presented by the keynote speakers, towards the current state, where modern techniques are allowing a new generation of developmental biologists to explore deeper into development and disease by using chimaeras.
Acknowledgements
We would like to thank the meeting organizers and sponsors, especially the Anne McLaren Memorial Trust Fund and the Company of Biologists. We apologize to all the speakers and references that are not mentioned directly owing to space limitations.
References
Ahmed M.I., Elias S., Mould A.W., Bikoff E.K. and Robertson E.J. (2016) The transcriptional repressor Blimp1 is expressed in rare luminal progenitors and is essential for mammary gland development. Development 143: 1663-1673 [doi: 10.1242/dev.136358]
Cohen M.A., Wert K.J., Goldmann J., Markoulaki S., Buganim Y., Fu D. and Jaenisch R. (2016). Human neural crest cells contribute to coat pigmentation in interspecies chimeras after in utero injection into mouse embryos. PNAS 113. 1570-75 [doi: 10.1073/pnas.1525518113]
Stasiulewicz M., Gray S.D,, Mastromina I., Silva J.C., Björklund M., Seymour P.A., Booth D., Thompson C., Green R.J., Hall E.A., Serup P. and Dale J.K. (2015) A conserved role for Notch signaling in priming the cellular response to Shh through ciliary localisation of the key Shh transducer Smo. Development 142: 2291-2303 [doi: 10.1242/dev.125237]
Thamodaran V and Bruce A.W. (2016) p38 (Mapk14/11) occupies a regulatory node governing entry into primitive endoderm differentiation during preimplantation mouse embryo development. Open Biol. [doi: 10.1098/rsob.160190]
A recent publication in Developmental Biology by (Armit et al., 2017) describes how the TRACER dataset can be spatially compared with in situ hybridisation gene expression profiles.
An EMAGE entry with the accompanying spatial map of a TRACER regulatory element reporter in the E11.5 mouse embryo. The original data images are shown in the upper panel. The lower panel shows spatial annotation as a colour-map, with strong expression shown in red, and moderate expression shown in yellow. Cyan denotes regions of the embryo where expression is not detected. The spatially mapped pattern can be used to query the EMAGE database of 30K gene expression patterns.
The TRACER dataset of transposon-associated regulatory sensors (Chen et al., 2013) utilises Sleeping Beauty lacZ transposons that have been randomly integrated into the mouse genome
Hundreds of insertions have been mapped to specific genomic positions, and the corresponding regulatory potential is documented through lacZ imaging of E11.5 wholemount mouse embryos
Through spatial mapping of the lacZ expression patterns, the EMAGE gene expression database enables co-localisation and co-expression of regulatory elements to be explored computationally
Spatial mapping additionally enables rapid identification of cis-regulatory elements that are expressed in a region of interest in the mouse embryo
Click here to access the spatially mapped TRACER dataset in EMAGE.
References
Armit C, Richardson L, Venkataraman S, Graham L, Burton N, Hill B, Yang Y, Baldock RA. eMouseAtlas: An atlas-based resource for understanding mammalian embryogenesis, Developmental Biology, Available online 2 February 2017, ISSN 0012-1606, http://dx.doi.org/10.1016/j.ydbio.2017.01.023
Chen, C-K, Symmons O, Uslu VV, Tsujimura T, Ruf S, Smedley D, Spitz F. TRACER: a resource to study the regulatory architecture of the mouse genome. BMC Genomics 14 (2013), p. 215, https://dx.doi.org/10.1186/1471-2164-14-215
9th May 2017 at the Institute of Child Health, UCL, London.
This year, YEN is honoured to have Dr Darren Gilmour from
EMBL Heidelberg present the Sammy Lee Memorial Lecture. We are also pleased to host two invited speakers, Dr Karen Liu (King’s College London), and Professor Michael Stumpf (Imperial College London). As well as three abstract-selected talk sessions and a poster session, we are holding a Q&A panel on the topic of science communication with Jenny Jopson and Jonathan Wood from the Francis Crick institute.
We are looking for talks and posters from PhD students and Post-docs on Evo-Devo, Stem Cell, and Developmental Biology, from both experimental studies andtheoretical modelling.
Whether you want to submit an abstract, or just attend the meeting, you can register here.
The deadline for abstract submission is midnight on 9th of April 2017.
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