Are you interested in metabolism/nutrition/organ size control/developmental neurobiology? A postdoctoral researcher position is available in the Cheng Lab at the Peter MacCallum Cancer Institute from the beginning of 2019 in sunny Melbourne, to work on organ cross talk, metabolism, or developmental neurobiology related projects in Drosophila.  We are looking for postdoc candidates with expertise in metabolism/imaging or other areas of developmental/cell biology, experience with flies preferred but not essential. You need to be finishing or have recently finished a PhD in related subjects, with an excellent track record. The lab is located within the Parkville precinct, home to the University of Melbourne and other top research institutes. The position is for 3 year initially (with possibility of extension), internationally competitive salary (A$78k-A$94k) + benefits. This position is suitable for creative, ambitious, independent, highly organised, self-motivated and hard working individuals. Please contact: Louise.cheng[at]petermac.org for informal discussions and application.

(No Ratings Yet)

Loading...

www.irbbarcelona.org /@IRBBarcelona / www.facebook.com/irbbarcelona

(No Ratings Yet)

Loading...

Stockholm University offers a multicultural environment in one of the world’s most dynamic capital cities. With more than 60,000 students and 5,000 staff, the University facilitates individual and societal development by providing top quality education that is tightly linked to its internationally recognized research programs.

The Department of Molecular Biosciences, The Wenner-Gren Institute (MBW) unites 30 independent research groups pursuing fundamental questions in molecular cell biology, infection and immunobiology, and integrative biology. The department carries out experimental research primarily investigating the function of genes and cells in tissues and organisms.

Research project

We are looking for a highly motivated and focused young scientist to carry out postdoctoral research studies in Assistant Prof. Qi Dai’s research group. The research project aims at understanding how key transcription factors determine cell fates using Drosophila as a model system.

Qualification requirements

The successful candidate must have received a doctoral degree from an accredited college/university outside Sweden latest in 2016. The degree must be in the fields of developmental biology, genetics or equivalent. The ideal candidate should have documented experience in fly genetics, tissue manipulation and molecular biology skills. Hands-on experience in work with bioinformatics and in high-throughput assays will be an asset. Excellent English language skills, both written and spoken, are a requisite.

Terms of the fellowship

The terms of this fellowship are regulated by the rules for postdoctoral fellowships at the Stockholm University (http://www.su.se/mbw/mbw-internal/scholarship/guidelines-for-scholarship). The fellowship is for full-time postdoctoral studies during one year, with possibility for extension up to a maximum of two years. The starting date is negotiable and the fellowship is available to start immediately.

Additional information

Further information about the research project and about the conditions of this fellowship can be obtained from Assistant Prof. Qi Dai, qi.dai@su.se, tel. +46 8 16 4149.

Application

Applications should be sent electronically as a single PDF file to the following addresses:

qi.dai@su.se

The applications should comprise the following parts:

• Complete CV, including full contact information, date of birth, and copy of the PhD title
• List of publications
• Personal statement describing research interests (1-2 paragraphs), research
  experience (1–2 paragraphs) and career goals (1-2 paragraphs)
• Please provide also the names, e-mail addresses and telephone numbers of 2-3 references

(No Ratings Yet)

Loading...

Here, Mylène Lancino and myself will introduce our motivation to investigate and delve deeper into one essential and very peculiar process of Stem Cell Biology: the de novo genesis of hematopoietic stem cells, according to a process referred to as the Endothelial-to-Hematopoietic Transition (the EHT, named initially by Kissa & Herbomel 2010). The work led to a paper published in eLife at the end of last month (https://doi.org/10.7554/eLife.37355). We will explain the technical difficulties inherent to the live imaging and image analysis approaches that have been undertaken. We will also summarize some of our main findings and underline their impact on fundamental and conceptual challenges for the hematopoiesis topic.

In the year 2010, several important papers that unambiguously demonstrated the vascular origin of hematopoietic stem cells were concomitantly published, which shed light on a long standing debate initiated at the beginning of the 20^thcentury (Jordan 1917, Sabin 1917; and for a recent review on hematopoiesis reporting on the findings of 2010 by Bertrand et al; Boisset et al; Kissa & Herbomel; Lam et al, see Klaus & Robin 2017). The breakthrough was possible owing to the power of technical imaging approaches and in particular confocal time-lapse fluorescent microscopy on the living animal, more specifically the zebrafish embryo. This model organism offers unique advantages owing to its small size, its fast development and, last but not least, its transparency. This latter advantage offers unique opportunities for in-depth live imaging and was essential to achieve the goal that we had in the mind when we started our recently published work; i.e imaging the emergence of hematopoietic stem cells from the aortic wall at sufficient spatio-temporal resolution to capture potentially short-lived events and critical intermediate stages so as to be able to propose a comprehensive model of the EHT. In addition, we intended to perform an in–depth descriptive study of the sequential steps of the EHT without disconnecting it from its natural environment. Hence, we aimed at visualizing the morphodynamic changes of endothelial cells, meaning the whole aortic landscape surrounding hemogenic regions, concomitantly to the EHT. The rationale behind this is that some of the EHT unique features when the process is compared with any other cellular dynamic event leading to the extrusion of a cell from an organized epithelium (among which the very peculiar bending of the emerging cell, primarily visualized in Kissa & Herbomel 2010, see also figure 1), suggested that it is adapted to the biomechanical properties of the aortic wall, made of very flat endothelial cells subjected to high mechanical load and exposed to the multidirectional forces exerted by the blood flow. Beyond this and on more conceptual grounds stands also the fundamental issue of the influence of the blood flow on the fate of hemogenic and hematopoietic stem cells (see also later).

Figure 1: In vivo visualization of hematopoietic stem cell emergence via the EHT, from the aortic floor, in a live zebrafish embryo. The middle panels of the figure show the organization of the vascular system in a transgenic line that expresses eGFP under the control of a vascular promoter. The bottom panel shows a magnification of a region of the dorsal aorta in the trunk of the embryo and highlights the emergence of hematopoietic cells via the EHT (e1 and e2; e1 is a more advanced stage than e2).

Let’s come back to the work now and share with the reader our motivation to invest time and energy on the project as well as telling about its experimental challenges.

When Mylène started her PhD, she did not have a strong technical expertise on imaging approaches but was already well acquainted with the zebrafish model. She undertook however the challenge of imaging the EHT more in depth “I was fascinated by the way hematopoietic stem cells are born. I was also really determined to find out the most appropriate set up to visualize as clearly as possible the dynamics of intracellular components during the EHT within the live embryo. Here began a very long journey during which I learned from many imaging specialists and started to realize how tricky in vivo imaging is“, as she said. From my side, when I started with this topic, about a year after Mylène did, with a strong expertise in cell biology and biological membrane dynamics as well as a strong motivation to modelling the EHT, we joined forces and started to analyze the series of time-lapse sequences that she had accumulated in the meanwhile and that were aimed at focusing on two aspects of cell biological features of EHT undergoing cells: the dynamics of their luminal and basal membranes as well as intracellular actin organization (by using double transgenic fish line expressing the membrane marker ras-mCherry and the actin reported Lifeact-eGFP, respectively). But looking into the movies made us realize that few of them would actually allow visualizing the entire EHT process with enough resolution (in particular for the sealing of the aortic floor and the release steps) and be able to reproduce several times each significant observation. This is because we had to face a series of major issues: (i) the stochastic initiation of the EHT (EHT cells are born from a sub-population of endothelial cells belonging to the so-called hemogenic endothelium but you never know if and when a cell will initiate the EHT; to circumvent the problem, we often started imaging when the cell had already begun its typical bending (which we refer in our work as to the cup-shaped stage)), (ii) the stochastic length of the process (that varies very much, ranging from approximately 4 to more than 15 hours, hence raising the issue of phototoxicity after long periods of laser exposure, see our paper for more details), (iii) the growth of the embryos that triggers drifts as well as the movements associated with the beating of the heart. We also had to fight against mosaicism (not all hemogenic regions of the aortic floor were expressing the fluorescent marker of interest, thus decreasing the chance to image the relevant events successfully). Mosaicism hampered quite significantly the work when using transient transgenesis or another fish line that we had wished to use to follow intercellular contacts between EHT undergoing cells and their endothelial neighbours (and that finally brought us most valuable information; a transgenic line that expresses the junctional protein eGFP-ZO1 that Mylène had managed to obtain, after several rounds of selection (meaning 2 years of efforts to select the best expressing fishes)). So, on several occasions when the project was developing we have been thinking that the outcome of the fastidious work should better be worth the effort !!! At the end, when we made the final selection of time-lapses that would constitute the body of the publishable work, we used less then a 100 of them and had collected more than 500 over 4 years (which makes …. 5 TB to scrutinize !).

Apart from the visualization of the EHT sequential steps and key features, as mentioned above, our aim was also to picture the EHT in the developmental vascular landscape. This is not a simple issue if one wants to get a clear picture because during the EHT time-window (that lasts from approximately 30 hrs post-fertilization (hpf) to 60 hpf), the aorta shrinks in diameter, must adapt to the loss of cells constituting the hemogenic endothelium and aortic cells undergo cell-shape changes as they elongate to adapt to mechanical tension (Lagendijk et al 2017). To standardize as much as possible the analysis, we started imaging at 48 hpf (which is also the timing at which the EHT is culminating). When one looks through the Z-stacks of an acquisition, even with a 3D-rendering view, one realizes that our brain experiences difficulties embodying the information and morphometrics becomes quite tricky. Things become even more difficult if one wants to explore the dynamics and inter-relation of objects through time, which is what we wanted to do. To fulfil our aim, we initiated an essential collaboration with experts in image analysis and physics from the Pasteur campus, namely Jean-Yves Tinevez and Fabrice de Chaumont. This interdisciplinary collaboration was most fruitful. The team developed an algorithm capable of deploying the aortic wall to project the fluorescent signals onto a 2D-plane (see figure 2) and of accurately re-iterating the aortic projections through time (taking into account distortions of the aorta and its morphological changes). With this algorithm, we have been able to exploit several of our time-lapse sequences and in particular the ones performed with eGFP-ZO1 expressing fishes that allowed following the cell boundaries and inter-cellular contacts. With this, we managed to visualize unambiguously the symmetric division of EHT undergoing cells (an amazing event when you think about the tension that must be exerted on the emerging cells !) as well as the dynamic interplay between EHT cells and their endothelial neighbours. We observed that the number of endothelial cells contacting EHT ones decrease with time, most probably to minimize the risk of leakage upon sealing of the endothelium. Importantly, the clear dynamic views provided by the 2D-projections revealed that the cells from the hemogenic endothelium, around 48 hpf, are rather elongated (in comparison to the surrounding endothelial cells) and that progression of the emergence is characterized by the contraction of the interface with neighbours that proceeds along the antero-posterior axis (the interface delimitating the EHT cell apex facing the aortic lumen). This axis parallels the blood flow, raising in our mind the idea that the direction of the contraction, anisotropic since oriented preferentially along a specific axis, may be minimizing the exposure to hemodynamic forces. Finally, the 2D-mapping of our time-lapse sequences performed on embryos expressing the actin reporter Lifeact also revealed anisotropy in the organization of sub-cortical actin, with its densification at sub-plasmalemmal regions of hemogenic and EHT cells enriched in junctional proteins. This suggested that these regions (whose orientation is perpendicular to the blood flow) are the most exposed to mechanical tension. These interpretations are now awaiting being tackled by further modelling of the forces at play.

Figure 2: 2D-map representation of the dorsal aorta after its deployment using the TubeSkinner plugin of the ICY software. “e” highlights a cell undergoing the EHT. The transgenic fish expresses eGFP-ZO1, ZO1 being part of the complex building the tight junctions. For more details on the 2D-algorithm see our paper (https://doi.org/10.7554/eLife.37355).

Overall, our results strongly suggest that anisotropy, because it organizes according to the blood flow axis, may be dictated by the mechanical constraints imposed by the aortic environment and in particular the blood flow. Thus, what would happen if blood flow is inhibited from the very beginning ? To answer to this question, we prevented blood flow by blocking heart beating (amazingly, the zebrafish embryo can survive several days without heart beat and circulation, by passive diffusion of gas through the skin). In this situation, we observed the impairment of actin cytoskeleton anisotropic organization and destabilization of junctional complexes. Quite surprisingly, hemogenic cells were still capable of escaping from the aortic wall (with, surprisingly also, the apparent maintenance of their localization on the ventral side of the aorta). However, they managed to do so both toward the sub-aortic space – as they do under normal physiological conditions – and the aortic lumen. The intra-aortic emergence most probably took place because the force that normally applies to the aortic wall and that is perpendicular to the arterial axis (the so-called mechanical strain) does not act anymore to push the emergence toward the sub-aortic space. Hence, even in the absence of blood flow, a population of aortic cells retains the ability to escape from the endothelium. This was unexpected because the emergence of hematopoietic stem cells was shown to depend on the activation of a transcription factor essential for hematopoiesis and whose expression is induced by the blood flow (Runx1, see Adamo et al 2009; see also for a seminal paper on the influence of blood flow on hematopoiesis North et al 2009). However, we do not know if those cells, released in the absence of blood flow, do retain the bona fide properties of hematopoietic stem cells, meaning the capacity to differentiate and replenish the entire repertoire of immune cells of the adult body. It is probable that they don’t and that they will end up dying, which is what we observed for some of them, few hours after the release. What these results suggest in addition is that, even if essential pathways are impaired, cells programmed to become hematopoietic may retain some of their abilities, such as escaping from the endothelial environment. This is possibly the reason why in vitro settings aimed at producing hematopoietic stem cells for regenerative purposes and using pluripotent stem cells produce hematopoietic-like cells that ultimately fail to express full hematopoietic potential. Indeed, hematopoietic stem cells are among the rare ones that cannot be produced yet and our work reinforces the idea that the mechanical constraints of the aortic environment are required to produce bona fide hematopoietic stem cells. It would be very interesting in the future to address the question as of the influence, on the fate of hematopoietic stem cells, of mechanical forces taking place contemporarily to the emergence.

So, at the end, our investment was really worth the effort and we gleaned interesting results and ideas from the hundreds of time-lapse sequences that were accumulated by Mylène during her PhD. Currently, we hope that our work, beside its contribution to the understanding of the Cell Biology and Biomechanics of cell extrusion processes, will bring valuable knowledge for reproducing at will the genesis of hematopoietic stem cells.

Anne Schmidt & Mylène Lancino

Developmental and Stem Cell Biology Department

CNRS UMR3738

Macrophages and Development of Immunity

INSTITUT PASTEUR

25 rue du Dr. Roux

75724 Paris Cedex 15
France

anne.schmidt@pasteur.fr

mylène.lancino@pasteur.fr

_______________

References

Adamo, L., O. Naveiras, P. L. Wenzel, S. McKinney-Freeman, P. J. Mack, J. Gracia-Sancho, A. Suchy-Dicey, M. Yoshimoto, M. W. Lensch, M. C. Yoder, G. Garcia-Cardena, and G. Q. Daley.2009. Biomechanical forces promote embryonic haematopoiesis. Nature 459:1131-5.

Bertrand, J. Y., N. C. Chi, B. Santoso, S. Teng, D. Y. Stainier, and D. Traver.2010. Haematopoietic stem cells derive directly from aortic endothelium during development. Nature 464:108-11.

Boisset, J.C., van Cappellen, W., Andrieu-Soler, C., Galjart, N., Dzierzak, E., and Robin, C.2010. In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium. Nature464, 116-120.

Jordan, H. E.1917. Aortic Cell Clusters in Vertebrate Embryos. Proc Natl Acad Sci U S A 3:149-56.

Kissa, K., and P. Herbomel.2010. Blood stem cells emerge from aortic endothelium by a novel type of cell transition. Nature 464:112-5.

Klaus, A., and C. Robin.2017. Embryonic hematopoiesis under microscopic observation. Dev Biol 428:318-327.

Lagendijk, A. K., G. A. Gomez, S. Baek, D. Hesselson, W. E. Hughes, S. Paterson, D. E. Conway, H. G. Belting, M. Affolter, K. A. Smith, M. A. Schwartz, A. S. Yap, and B. M. Hogan.2017. Live imaging molecular changes in junctional tension upon VE-cadherin in zebrafish. Nat Commun 8:1402.

Lam, E. Y., C. J. Hall, P. S. Crosier, K. E. Crosier, and M. V. Flores.2010. Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells. Blood 116:909-14.

North, T. E., W. Goessling, M. Peeters, P. Li, C. Ceol, A. M. Lord, G. J. Weber, J. Harris, C. C. Cutting, P. Huang, E. Dzierzak, and L. I. Zon.2009. Hematopoietic stem cell development is dependent on blood flow. Cell 137:736-48.

Sabin FR.1917. Preliminary note on the differentiation of angioblasts and the method by which they produce blood-vessels, blood-plasma and red blood-cells as seen in the living chick. Anat. Rec. 13:199-204.

(3 votes)

Loading...

During early mouse development, a series of signalling interactions breaks the symmetry of the egg cylinder, spatially organising the embryo into territories that define the future axes of the body. Symmetry breaking can also be observed in embryonic stem cell (ESC) colonies cultured on micropatterned substrates, which thus provide a powerful system to test the role of signalling as well as other features of the cell population in early patterning. A recent paper in Development uses micropatterned colonies to test the role of geometry in symmetry breaking. We caught up with three of the paper’s authors – Guillaume Blin, Manuel Thery and Sally Lowell  – to hear all about the history.

Guillaume, can you give us your scientific biography and the questions that drive your research?

GB I did my undergrad in the south of France where I was trained as a biochemist and biophysicist. I spent one of my first lab experience with Prof. Catherine Picart (Material biophysicist) and Dr. Christian Roy (Cell Biochemist) in the DIMNP lab in Montpellier. I worked on a biomimetic system to model and quantify the interaction between the protein ezrin (known for its role in cell polarity) and PIP2 (a constituent of the plasma membrane). This work was truly interdisciplinary and as I built skills in good old biochemistry, I also had the chance to learn about techniques more alien to the biologist world I came from – zeta potential, DLS, FCS, QCM, this was fascinating.

Catherine, Christian, as well as Prof. Andrea Parmeggiani (theortical physicist and teacher who changed my understanding of bio-molecular interactions), succeeded in creating a synergistic environment spiced with intrinsic ‘cultural differences’ between actors which meant that the coffee table was often the scene of some of the most hilarious banter between physicists and biologists. I am very grateful to them as this experience gave me the will and confidence to keep on building my own interdisciplinary skill set later on.

A question that came out of this work is how macroscopic phenomenon emerge as a result of microscopic events and for this reason I next changed system scale and went on to study cell biology in the lab of Prof. R. Foisner as an Erasmus student in Vienna. Again I am grateful to the people who have trained me Dr. Andy Brachner and Dr Josef Gotzmann. I don’t think I would have adapted well to what would come next if I had not spent such a great time with them in the Max Perutz lab. My Viennese experience coincided with the publication of seminal papers from the D. Discher and C. Chen lab showing that the fate of stem cells could be largely influenced by the physical properties of their environment.

And so, I then started a PhD with Catherine who had elaborated a collaboration with stem cell scientist Dr. Michel Puceat, in order to test the influence of stiffness on the cell fate of embryonic stem cells. During my PhD, I was also involved in another project aiming at modeling cardiac development with ESC to isolate a cardiac progenitor population to use as a source for cell therapy. This jump from purely basic science to a more translational approach has also brought its load of teaching and I am grateful to Michel for introducing me to this important dimension of research.

While working with ESC I noticed that even a clonal population of cells would create a wide diversity of behaviors in the dish and at that time the literature was flourishing with examples of heterogeneous expression of genes associated with cell fate biases. As a result, the next question I became interested in is how can we reconcile the propensity of stem cells to diversify in a seemingly erratic fashion in the dish with the remarkable reproducibility and robustness of developmental patterning in vivo.

Inspiring work came out from the lab of P.W Zandstra who was using micropatterns to dissect endogenous signaling in ESC and the role of the micro-environment. This gave me the idea that we may be able to guide patterning with geometrical confinement. I was also very impressed with Manuel’s work and so I contacted Manuel to learn how to make micropatterns. Manuel was even more enthusiastic and supportive than what I could have hoped for. I learned a lot with him and I must say, his good mood and energy are very contagious, beware! The first preliminary results were obtained as early as 2010. However, this work also coincided with the end of my PhD and the work was held back by the writing of my thesis and also by some technical challenges I was facing (notably in imaging).

Motivated by the will to better understand the origin of the heterogeneous response of stem cells to differentiation signals, I then joined the lab of Dr. Sally Lowell in the MRC-Center for Regenerative Medicine in Edinburgh. I was awarded a 4-year Wellcome Trust post-doctoral fellowship in order to determine how changes in tissue geometry may feedback onto early cell fate decisions. As part of my post-doc project, I developed new image analysis tools to quantify patterning in complex 3D cell populations at single cell resolution. With this new computational tool in hand, we decided to resume the story initiated about 6 years earlier. Sally has been incredible at providing support to ‘resurrect’ this project. Like the dream PI for a post-doc, she provided the pitch-perfect environment to reveal the hidden potential of this work, a bit like geometry in our study!

So there I stand now, looking to establish my own group in order to continue identifying general principles about how the cells self-organise using synthetic and quantitative methods.

Sally, what are the main aims of the Lowell Lab, and what led you to investigate the role of geometry in patterning?

SL I’m fascinated by how cells talk to each other to generate patterns. We think that when these conversations are disrupted, for example through changes in adhesion and tissue morphology, this can help to reinforce differentiation decisions to make development more robust. Paradoxically these same mechanisms may well be confounding our ability to direct differentiation in cells in culture. My wonderful and creative lab members are working hard to understand how all this is happening.  When Guillaume joined the lab he set to work revolutionising the way we were able to approach these questions.  For example, thanks to a set of image analysis tools that Guillaume has developed we can now measure cell-cell interactions and changes in morphology at single cell resolution even within enormous (to us) post-gastrulation mouse embryos. Watch this space for a couple more of Guillaume’s papers which will report these tools to the community. The idea to investigate the role of geometry came entirely from Guillaume, who is the brains and the driving force behind this project. Indeed he very presciently began this project eight years ago during his PhD, taking inspiration from the work of Manuel Thery.  Manuel will be able to tell you more about how it all began…

And how did you become involved with the project Manuel?

MT I was working on cell architecture and developing micropatterning methods to play with cell shape and adhesion. Meanwhile I was reading the papers by Christopher Chen who has always been a great source of inspiration for me. He was constraining cell groups in crazy shapes where mesenchymal stem cells were differentiating toward adipocytes or osteoblast depending on their position in the group. Osteoblasts appeared at the periphery while adipocytes sat at the center of the colony. In a very thoughtful experiment he plated cells on sinusoidal avenues. All cells were close to the periphery, but osteoblast appeared along convex edges while adipocytes appeared in concave edges. The proof was shown that stem cells sense geometrical cues and not only the local cell density. I was super excited by this results and used it a lot as an example of the importance of geometry to cell physiology. But I could not work on it. In 2010 I was starting my group with two students and we were focusing on intra-cellular architecture. It was very frustrating. Then Guillaume run into my office and proposed to study the role of geometry in ES cell colony! I jumped on my chair like the wolf facing Little Red Ridding Hood in the nightclub in Tex Avery’s cartoons. We immediately started to draw shapes that could contradict the density dogma. The issue was that Guillaume was not equipped to perform microfabrication experiments in his lab. We had to set up a new protocol. Guillaume worked a lot and got very enthusiastic with what I would consider pretty unsatisfying results. So he insisted more and more, tried new chemicals and got it to work. From there I started reading a lot on ES cells but it was too complex to me. Guillaume was visiting me and teaching me mouse embryonic development. I learned so much! It was really great. I became fascinated by the gastrulation and started working on epithelial-to-mesenchymal transition.

What was known about the role of geometry in early mammalian patterning prior to your work?  

GBGeometry is known to have a strong influence during the 1^st cell fate decision, when the blastomeres become segregated either to the ICM or to the TE. Geometry dictates cell polarity which in turns define the activity of key transcription factors. This is a phenomenon that is now well documented.

Now for the developmental stage that we are looking at in our article (onset of gastrulation), two studies come straight into my mind: Mesnard et al. Current Biology, 2004 and Hiramatsu et al 2013, Dev Cell.

In the first article Mesnard and colleagues have shown that the axis defined by the anterior and posterior domains aligns with the longest axis of the embryo. They also show that this is a dynamic process as the AP axis initially aligns with the shorter axis early on.

In the second article, the authors grow pre-gastrulating mouse embryos ex-vivo within agarose microwells to mimic constraints from the endometrium. They show that the soft physical constraints provided by microwells facilitate the elongation of the embryo resulting in the most distal region of the embryo ‘moving’ far from the source of BMP signalling (the extra-embryonic ectoderm). According to the authors, the resulting decrease in BMP signalling in the distal region participates in the specification of the DVE.

Overall, I believe that  everyone has this intuition that geometry participate in defining the shape of signalling gradients, but there is still work to be done to understand how the cells sense the physics of their environment locally and how changes in geometry may feedback onto morphogenetic events.

Can you give us the key results of the paper in a paragraph?

GB, MT, SL We show that imposing physical boundaries to ESC colonies in culture drives the patterning of a T+ primitive streak-like population of cells which is initially present but spatially disorganised. Importantly, if we introduce asymmetry in the shape of colonies, the resulting positioning of the cells follows the asymmetry which raises the possibility that geometry plays an important role in guiding precise positioning of the cells at the streak.

We also show that  patterning is robust to changes in the relative number of T+ cells present initially. This is an important result because it indicates that the mechanisms which regulate the number of cells which will ingress into the streak may be distinct from the mechanism which regulates the precise positioning of the cells. To me, this is an indication that it is important to reflect on the notion of control during development and how processes are articulated with one another. Having a global signal triggering local events leading to patterning rather than tightly controlled signalling gradients micromanaging both patterning and specification is an elegant solution. The need for control is minimised and a certain level of robustness is achieved by the system. I can only recommend one of my favourite reviews on the topic from Arthur Lander ‘Pattern Growth and Control’

These ideas can also have great implications for our ability to engineer synthetic systems and actually one of our plans is to see if we can induce patterning in cells which do not normally sort.

Have you got any ideas for how your T+ cells end up at the periphery of the colonies?

GB/SL/MT There are quite a few hypothesis that we can formulate. A possibility is that the cells change fate if they end up in the ‘wrong place’. There may be some selective apoptosis going on as well. If the cells are surrounded by cells with a different level of T, maybe the cells are eliminated. If the cells reorganise, then again this can happen in various ways. As you suggest, cell sorting via differential adhesion may be involved or alternatively, T+ cells may become more motile and migrate faster and with more directional persistence than T- cells. This differential motility may be sufficient to drive sorting even in the absence of chemotaxic cues. It may be a combination of all of the above, we know that the embryo can use a diversity of cell behaviours in order to achieve patterning in the late blastocyst (Plusa et al, Development 2008). Our plan is to use mathematical modelling in combination with live imaging in order to further address this question.

Can you translate your findings to the early embryo – where might the guiding hand of geometry be acting?

GB, MT, SL This is a great and difficult question because it is still unclear how the shape of the streak is maintained as the embryo grows. In the chick, a community effect mediated by Nodal signalling and cell-cell interactions as well as Wnt-PCP,  have been identified as important elements regulating this process (Voiculescu et al., eLife 2014). The apical polarity regulator crumbs2 has also been involved (Ramkumar et al, NCB 2016). These are examples of molecular determinants which may be part of the machinery which enable the cells to sense the folding of the streak. This geometry sensing may be key to maintain the stable geometry of the streak as the embryo grows. However much work remains to be done to fully understand this process and to assess inter-species differences as well.

When doing the research, did you have any particular result or eureka moment that has stuck with you?

GB The first one is when I saw T+ cells localising at the tips of the ellipses. Despite variability between colonies, the patterning was very clear under the microscope! I remember thinking, I need to quantify this, maybe I am just imagining it, so when we found a way to create the density maps and saw the figure showing a clear result, I was both a little a bit euphoric and very intrigued.

And what about the flipside: any moments of frustration or despair?

GB I had moments when I felt the results were not coming as fast as I wanted, this was mainly due to the fact that imaging was a bottleneck in our initial setup. As a result I was spending very long hours at the confocal in the (cold and dark) room of the imaging facility, skipping activities with family or friends. This was particularly difficult in the autumn when the colours outside were so vibrant. Not being able to see the season pass by was frustrating. But in the end we managed to streamline the whole process and this solved the issue. This was not so bad but I am lucky to have a very understanding and supportive family.

What next for you after this paper?

GB I am currently working on publishing other outputs from my post-doc which include methodological papers describing computational tools for quantitative image analysis, I hope you will be able to hear soon about it and that my tools will be useful to the community. I am also trying to establish my own group in order to continue working on synthetic approaches to identify general principles about how the cells self-organise.

Any plans for future Franco-Scottish collaboration?

MT, SL Yes definitely! We will continue to work together, the combination of microfabrication and stem cells offers a fertile ground with almost limitless possibilities. We will look into remodellable materials and 3D solutions to further guide the development of the cells in vitro. Maybe this is a good place to advertise our recent review on the topic also (Laurent et al. NBE 2018).

Finally, let’s move outside the lab – what do you like to do in your spare time in Edinburgh and Paris?

GB I like to spend time with my family, whatever we do does not matter as long as my 2 daughters keep being cheeky with their sly faces hinting at their next mischiefs. When I find time I also enjoy cycling in the beautiful moors and hills around Edinburgh.

MT I like to get high, and I know many ways for it. Apart from the classical ones, I enjoyed art exhibitions very much. Artists observe the world, analyse it interpret it, and then try to fing original means to convey their thoughts into something that will question people imagination and help them to consider reality differently. Isn’t it exactly what we do every day in our labs? Except that we have much more tools to play with … ;-) Artists also want to understand how things work, how light makes things visible, how images print in our mind, how interactions change intrinsic properties, how context change identity. So I am pretty sure most of them would enjoy Guillaume’s paper very much. Too bad they don’t dare reading Development, while we all go to the Tate.

SL There is no such thing as spare time, but during my not-doing-science time my kids have been teaching me how to mix music on a computer. I’ve only had two lessons so far but probably I will soon become an internationally famous and unfeasibly wealthy DJ.  My other hobby is choosing theme tunes for papers that I like and then posting them on twitter, as my service to the developmental biology community*.

*The theme song for Guillaume’s Development paper, “Down on the Corner” by Creedence Clearwater Revival,  was suggested by new Development Editor in Chief and incurable hippy James Briscoe, for which we thank him.

Geometrical confinement controls the asymmetric patterning of brachyury in cultures of pluripotent cells
Guillaume Blin, Darren Wisniewski, Catherine Picart, Manuel Thery, Michel Puceat, Sally Lowell. Development 2018 145: dev166025 doi: 10.1242/dev.166025

This is #50 in our interview series. Browse the archive here.

(2 votes)

Loading...

Sha Wang, Deborah Gumucio

This article shares the story behind our recent Developmental Cell paper. It tells the history of this project and how three-dimensional (3D) observations at the individual cell level transformed our preconceived ideas and brought new insights into cell dynamics in the proliferative intestinal epithelium.

Epithelial tubes are present in many developing tissues and usually elongate to various degrees during organogenesis. Therefore, understanding how epithelial tubes elongate is an essential piece of the puzzle we need to solve in order to unveil the big picture—how the entire organ acquires a functional structure.

The fetal small intestine is a great model for this subject because it elongates rapidly to generate an incredibly long epithelial tube to meet postnatal nutrition demands. Interestingly, although its prominent elongation attracted the attention of many researchers, we actually knew far less than we thought we did about the underlying processes driving elongation. With the benefits of hindsight, the unique structure of the early intestinal epithelium indeed requires 3D information to reveal the critical details missed and misled by 2D imaging.

First impressions were firmly entrenched

Two decades ago, by examining fetal rat small intestinal structure on thin sections under the light and electron microscope, Mathan et al. concluded that the early developing epithelium has several cell layers, but only one cell layer is present at later stages (Mathan et al., 1976). It is easy to imagine that this reduction in cell layers could be a consequence of intercalations of cells from different layers. In a tubular structure, radial intercalations can drive convergent extension (CE) movements, leading to a longer tube with a smaller girth (Walck-Shannon and Hardin, 2014) (Figure 1). Indeed, in 2009, Cervantes et al. found that Wnt5a null mouse small intestines are severely shortened and proposed that the defect was caused by a failure of intercalation during CE-like movements (Cervantes et al., 2009). Shortly thereafter, Yamada et al. noted that Ror2, a Wnt5a receptor, is required for mouse small intestinal elongation, again, presumably by CE (Yamada et al., 2010). Additional studies indicated that the frog gut also elongates by radial CE (Reed et al., 2009). All these findings seemed to demystify the intestinal elongation process, but the details of cellular behaviors during CE were unclear.

To further probe how CE occurs at the cellular level, a previous graduate student in our lab, Ann Grosse, labeled a small subpopulation of cells in the small intestines of early fetal Cagg^CreERT2; R26^mTmG mice. After acquiring confocal z-stacks from thick sections (100 μm), she found that labeled epithelial cells are tall (up to 50 μm) and thin (1-4 μm); most cells touch both apical and basal surfaces, indicating that the early small intestinal epithelium (SIE) contains only one cell layer (Grosse et al., 2011). The earlier impression of multiple cell layers came from analysis of thin sections (5 μm) that seldom include an entire cell body. This newer appreciation of a single-layered structure clearly does not support the above-mentioned CE hypothesis. This discovery became a turning point, leading us to ask: what other cellular events could drive rapid intestinal elongation?

Brand new information provided by high-resolution 3D imaging tools

Does rapid elongation come from another type of unrecognized cell rearrangement that occurs within the single-layered epithelium? Up to this point, no one had examined how cell clones expand in early SIE and I thought that the spatial arrangement of clonal cells could provide some clues. Therefore I generated large clones in the early Shh^CreERT2; R26^mTmG SIE tube and acquired 3D images of these clones. In contrast to what I predicted, labeled clones were not patches of connected cells, but groups of separated cells with various shapes (Figure 2). These unexpected clonal patterns immediately opened the curiosity door. Why are offspring cells separated from each other? Why are they organized in such patterns? Why do cells adopt different shapes? Are any of these features related to the rapid elongation of the SIE tube? The key to all these questions was to learn how an individual cell behaves in the elongating SIE tube.

Based on previous studies in our lab, we knew that: 1) the early SIE is pseudostratified; 2) cell nuclei travel from the basal side to the apical side to divide (Grosse et al., 2011). However, we had no idea about how cells behave while their nuclei are cycling between the apical and basal surfaces. Therefore, I started to collect all different 3D cell shapes and bin them according to cell cycle markers. After observing hundreds or thousands of static 3D cell shapes, I noticed solid correspondences between the cell cycle phase and certain cell body shapes and was able to divide them into four major groups: a) interphase—the cell spans the apical-basal axis; b) mitosis—balloon-shaped cell body at the apical side; c) cytokinesis—a clear cleavage furrow between two nascent daughters; d) early G1—two apically connected cells (Figure 3). This assortment provided an initial framework of cell behavior over the cell cycle. By further comparing subtle differences within each group, I was able to fill in more details, especially how cells are dynamically connected to the basal surface, to rebuild the entire journey of individual cells.

Record it live

Even though we were quite confident with cell behaviors deduced from 3D shapes of fixed cells, the temporal information was still missing. Therefore, I started to focus on live imaging of small intestines. Due to the strong peristaltic movement of the small intestine, live imaging was challenging. After trying various conditions, with Dr. Cebrian’s help, I managed to apply a live imaging method for the embryonic kidney to our system (Costantini et al., 2011). The live recording data turned out to be extremely rewarding. It not only confirmed the temporal order suggested by static 3D images but also led us to discern two previously unrecognized modes of basal nuclear return in G1 phase. The two modes differ in the deployment of an asymmetrically inherited basal process, a thin filament that connects apical cell body to the basal surface.

The role of Wnt5a

In parallel, we were curious, if CE is not occurring, what makes the Wnt5a mutant gut shorter? By comparing epithelial cell behaviors in wild-types and Wnt5a mutants, we realized that the post-mitotic filopodial outgrowth is guided by Wnt5a, a cue from the underlying mesenchyme. Without Wnt5a, many newly divided cells cannot “see” the basal to establish a solid connection. Death of these rapidly proliferating cells (and loss of their future progeny) severely limits small intestinal elongation.

Looking forward

By following our own cues provided by 3D observations throughout this study, we uncovered a series of exquisite cell behaviors in the elongating SIE tube. These findings opened a new list of meaningful questions: Which features of this cellular choreography are broadly conserved in all pseudostratified epithelia and which represent tissue-specific adaptations? How do the filopodia follow Wnt5a cue to find the basal surface? What cytoskeletal machinery is engaged in this signaling process? Interestingly, mutations in FILAMIN A, a gene that acts downstream of Wnt5a, is associated with human congenital short bowel syndrome. That will be another interesting cue to follow for sure!

References:

Cervantes, S., Yamaguchi, T.P., Hebrok, M., 2009. Wnt5a is essential for intestinal elongation in mice. Dev Biol 326, 285-294.

Costantini, F., Watanabe, T., Lu, B., Chi, X., Srinivas, S., 2011. Dissection of embryonic mouse kidney, culture in vitro, and imaging of the developing organ. Cold Spring Harbor protocols 2011, pdb prot5613.

Grosse, A.S., Pressprich, M.F., Curley, L.B., Hamilton, K.L., Margolis, B., Hildebrand, J.D., Gumucio, D.L., 2011. Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis. Development 138, 4423-4432.

Reed, R.A., Womble, M.A., Dush, M.K., Tull, R.R., Bloom, S.K., Morckel, A.R., Devlin, E.W., Nascone-Yoder, N.M., 2009. Morphogenesis of the Primitive Gut Tube Is Generated by Rho/ROCK/Myosin II-Mediated Endoderm Rearrangements. Dev Dynam 238, 3111-3125.

Walck-Shannon, E., Hardin, J., 2014. Cell intercalation from top to bottom. Nature reviews. Molecular cell biology 15, 34-48.

Yamada, M., Udagawa, J., Matsumoto, A., Hashimoto, R., Hatta, T., Nishita, M., Minami, Y., Otani, H., 2010. Ror2 is Required for Midgut Elongation During Mouse Development. Dev Dynam 239, 941-953.

(No Ratings Yet)

Loading...

Hello there! This is Nora Braak and Nestor Saiz, we are based in Oxford and New York respectively and we study butterfly and mouse development. Last week we went to the BSDB Autumn meeting, which also happened to be the third workshop on Embryonic Extraembryonic Interactions. We enjoyed it so much that we wanted to share our thoughts with you [disclaimer: these thoughts don’t represent those of the BSDB, the organizers, nor, of course, our PIs’…]

– Hey Nora, do you know how many developmental biologists does it take to take over an Oxford University College…?

– Ha! Tell me… [eye rolls]

– Well, about a hundred apparently! Which is as many of us descended onto Corpus Christi College last week to chat about the most extra of all tissues: extraembryonic membranes…

– [Eye rolls, squared] ehhh… well, actually the Embryonic-Extraembryonic Interfaces ok…?? #bsdb2018EEI The third workshop on this piping hot topic already!

– Fine, fine… if you’re going to get all serious about it, do you want to tell us about some of the talks that you liked? It was a very exclusive meeting, I bet most readers did not get to go.

– It was a great meeting; it will be hard to pick highlights but it must be done. To start, it was the first extra-embryonic meeting where team Insect was properly represented and it was such a success we are thinking of getting t-shirts made for the next time #teamInsect. The meeting started off strong with a plenary talk from Liz Robertson. She gave us all a crash-course in early mouse development and all the essential genes in cell-lineage specification and TGFβ signaling. Their paper, still hot off the press shows how loss of both Smad2 and 3 alter the epigenetic landscape and activate extraembryonic gene expression in embryo-derived stem cells.

– After Liz’s keynote, I think Kristen Panfilio made it very clear to all of us mouse aficionados that #teamInsect was in the house. Turns out insects do have extraembryonic membranes, unlike what you might have heard from a certain famous fruit fly… She also showed some absolutely gorgeous movies of Tribolium‘s amnion and serosa breaking and retracting into the yolk to let the embryo develop further. You can see them and read more about their reporter and how the EE get themselves out of the beetle’s way in their paper.

– The rest of the Monday afternoon discussed how development meets bioinformatics; from Laura Banaszynski telling us about the function of the H3.3 histone variant to Sarah Teichmann, who wants to develop the ‘Google Maps Street View’ of the human body. The first day ended with a lovely drinks reception and a three-course sit down dinner in the beautiful hall of Corpus Christi, which made me wish I had dressed up a little. The dinner was followed for many by some more drinks in the Bear Inn, one of the oldest pubs in Oxford.

– Ah, the Bear Inn and its low ceilings… Shout out to Miguel Manzanares too (#teamMammal) talking about genome structure in the early mouse embryo and Federica Bertocchini, who is studying chameleon development, which is awesome because… chameleons?? Come on… Did you know chameleons take 200 days from laying to hatching? Did I say chameleon yet?

– Sorry, did you say it was about chameleons? I think I missed that…

– Tuesday was mouse day (#mousetastic). In the morning Ayaka Yanagida and I got the honor to present after two of my favorite embryologists, Jenny Nichols and Claire Chazaud! Jenny discussed their latest look at the Pou5f1 (aka Oct4) mutant, which is very close to her heart, then Claire doubled down on the mutants showing what happens when you knock out both Nanog and Gata6 in early mouse embryos. All of my favorite transcription factors!

– Yes it was a great morning, but after this early embryo overdose I was glad to switch to the lightning round of 3 minute presentations from all 24 poster presenters. They really piqued everyone’s interest, the poster sessions were so well attended – and not only because of the pastries and beer provided!

– I agree, it is the first time I see this pitching of the posters at a conference and I thought it was a very neat idea – though it probably only works in small settings like this workshop. Brief presentations are hard, kudos to the organizers for giving everyone a chance to practice!

– In the afternoon we had great talks from Takashi Hiragii and Veronique Azuara. I personally really enjoyed Matthew Stower’s talk, who used light sheet microscopy to study visceral endoderm migration, the pictures and the data analysis were amazing! Again the day ended in a 3 course sit down dinner in the beautiful hall of Corpus Christi, this time I was more prepared and knew which bread roll belonged to me and which fork to use for which course.

– I still don’t know how Matthew managed to take some of us on a pub crawl after lunch and then go and deliver his talk. Matthew you’re a total star.

– What did you think about Wednesday? It was an intense day.

– Yeah, Wednesday was packed. It started with all non-mouse mammal models. Berenika Plusa and Ania Piliszek presented their work on preimplantation rabbit development, whereas Stephen Frankenberg and James Turner engaged in their own marsupial cutey contest – for all of you dunnarts, possums and opossums out there: if you are interested in being the next top model organism, being cute will take you far! Jokes aside, theirs were some of my favorite talks. They had really nice data and ideas on the evolution of extraembryonic tissues and X-Chromosome inactivation in mammals that made me consider if I should switch model organisms.

@preg_lab contrary to recent rumours this is actually Ros John’s lab twitter account. Follow away science geeks! #bsdb2018EEI

— Harry Leitch (@HGLeitch) September 10, 2018

– I also really liked the talks about the placenta by Rosalind John and Myriam Hemberger. Their talks about the importance of the placenta in embryonic development and the influence it can have on the maternal behaviour were both thought provoking and well presented – here’s one of their papers.

The day ended with talks from Diana Laird, on the transgenerational defects of environmental damage, and from Elizabeth Duncan. She looks at bee and aphid reproductive control as a way to understand how animals respond to their environment. Did you know that aphids will change their mode of reproduction and development depending on the season?? Yet the embryos ultimately look the same!

– Elizabeth Duncan’s talk was so interesting! I think even Queen B would have agreed… (#RoyalJelly)

– Right… oh, in the evening we were welcomed in the Natural History Museum of Oxford, with bottomless gin and delicious bowl and finger food. The beautiful surroundings gave everyone a chance to mingle, share their enthusiasm about dinosaurs and admire a live bee colony which were even more of interest after Elizabeth Duncan’s great talk

BSDB night at the museum: Oxford natural history museum #bsdb2018EEI pic.twitter.com/y7kf6eWwjq

— Dr. Sophie Morgani (@Morgani_S) September 14, 2018

– Finally Thursday came around. It started with two great talks by #teamInsect, from both Maurijn van der Zee and yourself, Nora. You both seem to enjoy poking insects with infected needles… I guess it’s an effective way to trigger immune responses by the serosa – one of the many critical roles of the extraembryonic tissues in insects, as we had learned from Siegfried Roth’s talk on the evolution of Toll signaling! I personally loved Di Hu’s talk, from Shankar Srinivas’ lab, she had done some beautiful imaging of the early post-implantation mouse embryo and delivered like a pro. We also saw Zofia Madeja, Vasso Episkopou and Jaime Rivera, who is doing “dunkin’ transgenics!”. Delivering Cas9 to do CRISPR into mouse zygotes by bathing them in media with virus is definitely a slam dunk.

– Yes. The day was finished by two phenomenal speakers, Mariya Dobreva, who won the Dennis Summerball Award and presented her work on the role of Smad5 in the amniotic ectoderm, and Ali Brivanlou who wrapped up the meeting with some absolute eye candy on their work in in vitro models of human development.

– After that, awards were given to the three best poster presenters, Peter Baillie-Johnson, Matthias Teuscher and Berna Sozen. Well done them!

Extremely honoured and appreciative for receiving the poster 2nd prize from BSDB Embryonic-Extraembryonic conference. My lovely prize is my favourite book, Origin of Species✨
A huge thank you to all organisers for such brilliant 4 days! Looking forward to next one! #bsdb2018EEI pic.twitter.com/qPNkoSma4s

— Berna Sozen (@BernaSozen_) September 13, 2018

All in all, a wonderful little meeting – science, weather and setting all came together to make for a truly great week. Thanks so much to the organizers, Susana Chuva de Sousa Lopes, Kat Hadjantonakis, Kristen Panfilio, Tristan Rodriguez and Shankar Srinivas for putting it together and we’re looking forward to 2022!!

(5 votes)

Loading...

Are you an enthusiastic person who has the “start-up” mentality? Do you dream of working with like-minded people & build a leading global company? Are you looking to transition from academia into a leadership role and be part of an amazing experience?
If so, then why not apply to for this role and help us to build a global leading company together.

About Reagent Genie

Reagent Genie is a life science company with offices in Dublin & London. Founded by Colm Ryan PhD and Seán Mac Fhearraigh, it offers over 67,000 products across its 3 major brands, Assay Genie and ELISA Genie, Antibody Genie. Reagent Genie is a privately held company with global exports and operations in Ireland, UK, USA, Asia and Europe.

Job Overview

Reagent Genie is seeking a full-time ELISA Kit Product & Logistics Manager based in its Dublin office reporting to the CTO/CEO. Working closely with senior management, the primary responsibilities involve managing logistics operations and streamlining data handling & analysis to support sales and marketing functions. Full training in all aspects of the role will be given to the successful candidate.

Key Responsibilities

• Support and facilitate marketing & sales functions
• Work in partnership with senior management to implement standardized systems and processes for logistics and marketing
• Develop standardized data systems for the on-boarding & marketing of new products
• Organize Reagent Genie branded meetings in the UK & Ireland
• Ensure warehouse management standards support customer service demands
• Carry out warehouse planning to ensure efficient running of logistics functions
• Streamline the stock control system
• Responsibility for ensuring deliveries are organized and collected on time
• Assemble, package and label products while maintaining industry standards
• Measure logistics activity to ensure customer satisfaction and drive continuous improvement activities
• Assist in developing solutions and pricing for projects
• Engage will suppliers e.g. office supplies, transport etc. for best prices & solutions
• Provide technical guidance to customers
• Any other duties, within reason and capability, as determined by senior management

Competencies

• Possesses a high degree of initiative & willingness to learn
• Excellent communication & presentation skills with the ability to organise & deliver information effectively
• Ability to work in a multidisciplinary team environment & complete projects according to deadlines
• Is goal–oriented, committed and has a strong desire for success
• Possesses excellent negotiating & decision-making skills
• Positive attitude about company and marketplace

Qualifications

• An enthusiastic trainee with a Master’s degree or Ph.D. level degree is required
• Commercial experience desirable but not essential
• Understanding of work procedures and standards an advantage
• Proven planning and organizational skills
• Strong I.T. skills are essential

Application | Package | Closing Date

• Apply with cover letter & CV to info@reagentgenie.com
• Closing date for applications is 28^th September 2018
• Find out more at ELISA Genie Careers: https://www.elisagenie.com/product-and-logistics-manager/

(No Ratings Yet)

Loading...

Water is a fascinating substance. Its behavior sets a lot of interesting constraints on both how the surface of our world is shaped geologically and how life on said surface has adapted to optimize its use. Biology and geology, while vastly different in scale, share many commonalities that can we can learn from. Our work found one such connection: we found that the mammalian pancreas is like a river basin.

The mechanics behind the emergence and refinement of the ducts in the pancreas has been a mystery for some decades. First, the ducts form from unconnected microlumen in the pancreatic mass into a meshwork of interconnected tubes, connected seemingly at random (Fig 1). Later, as the pancreas nears maturity, it undergoes a transition from a mesh into a tree-like structure with no redundant ducts. The process does seem to have some stochasticity, as no two individuals has the exact same pattern of tubes when examined.  Yet the function of delivering pancreatic juices to the duodenum is the same across healthy specimens.

In nature, simple rules can often result in very complex behavior. A good example is Turing patterns (1), in which fur and skin coloration can elegantly be explained by reaction-diffusion equations. It is for that reason that physicists often try to use simple models and derive universal laws in order to get some conceptual understanding of the phenomena they are analyzing. And it was with that mindset that we set out to study pancreatic development. Three years later we had made a great discovery by applying network theory and fluid mechanics to describe the developing pancreas.

First, we needed quantifiable measures to describe how the pancreas tubes connect. We sought inspiration in network theory since the pancreas had all we needed – a network. Network (graph) theory is pretty simple at its core. Every network consists of nodes that serve as connection points and edges which are the connections between the points. The idea was to convert the pancreas tubes into edges and the tube intersections into nodes. The first snag we hit was that we found the images we had of the pancreas, while of excellent quality, were hard to automatically convert into a digitized network of nodes and connections between them. In the end we decided to go old school and digitize them by hand….

20000+ nodes later, we had an amazing dataset! We had digitized mouse pancreas networks at three different developmental stages: E12.5, when the network is very interconnected; E14.5, when the network is visually clearly beginning to change into a tree-like structure; and E18.5, when the network has almost fully transformed (Fig 2). What we found was stunning.  It turns out that while the networks are visually very different, most of the quantitative features of the network are strikingly similar. Furthermore some of these quantities change with each developmental stage.  This shows that the pancreas, while seemingly chaotic, must follow a set of rules when developing.  To a physicist like me there is no sweeter thing because then you get try and uncover these rules. In the end, development of the pancreatic ducts can be explained on a macroscopic level by two models. One model creates the mesh, the other matures it into the tree-like structure.

In order to model the creation of the interconnected network that appeared we tried to naively model what we thought we saw: that the microlumen that emerged simply connected to some of the nearest microlumens.  Our model therefore became the following:

1. Create a node close to the bulk of the already existing nodes
2. Form edges to some of the nearest nodes
3. Reiterate 1 and 2 until you have the desired pancreas size

With the addition of some noise, our model makes a very nice approximation to the real pancreas from a network viewpoint.

In order to model the maturation of the pancreatic network from mesh to tree-like structure, we first had to figure out which mechanism could be the driver of such a selection. It is here that we drew inspiration from the flow of rivers. At this time point, we had also noticed that at the end of development the width of the ducts was wider and wider close to the exit, as seen in river basins.

The concept of the path of least resistance is that every entity will select the easiest path from a to b when selecting from multiple potential pathways. Water exhibits such behavior. If you pour water on the top of a hill of sand, all the water will flow on some select trajectories. There is another phenomenon hidden in this analogy: once water has passed through the sand, the water following it will experience less resistance. Therefore water seems to stick with its initial choice. This is why water flowing down hill is confined to specific paths. During a flood the water from a river can branch off in new directions, and if these newly created river branches are more favorable than the original, the river will change shape as the old river branch dries out and the new branch is widened.  The end result is a river that is fairly optimal in delivering water from one or more source points to an end point.

Our idea is that the pancreas may follow similar rules. By doing so, we postulate that the delivery of pancreatic juice from multiple source points (the pancreatic acini) into a single end point (the exit into the duodenum) finds the shortest path. Forming redundant ducts may enable this optimization. When fluid then runs through, the pancreas changes the duct diameter to correspond to the flow (Fig 3a). When the fluid flow is higher than a given threshold, the duct widens. When the flow is lower than the threshold, the duct shrinks in diameter. The ingenious detail in this simple scheme is that if redundant paths exist between the exit and the acini, the suboptimal paths can be shrunk until they no longer exist and the cells lining them may be reused by the surrounding tissue (Fig 3b). If a given path is the only path, it will stabilize into a duct with a diameter perfect for the flow it receives. The end-result is a structure with one optimized path from every acini to the exit that is homogeneous in fluid pressure as every duct has the ideal diameter for the given flow.

The concept was easy enough to test as we had the digitized networks. The model we constructed did the following:

1. Let fluid run through the network either with the acini as the source or every point in the network as the source and wait until approximate steady state.
2. Remove the redundant duct with least flow.
3. Reiterate 1 and 2 until no redundant ducts exist.

The model can artificially mature an E14.5 into an E18.5 network, and optimize the average duct distance from every acini to the exit.

So there you have it: what the pancreas needs to optimize delivery of pancreatic juice is flow, a flow sensor, a duct diameter adjustment mechanism and a duct removal mechanism.  Here it should be noted that while the pancreas is the basis of our discovery its task of producing fluid and move it somewhere else is not unique in the body.  Other glands include the salivary (2) and lachrymal gland (3) and the mammary gland (4). It could be immensely interesting for future research to digitize these glands in the same way and see if any follows the same principle.

As a physicist I am happy to end here and say that the pancreas optimizes its own fluid delivering capabilities by locally following the path of least resistance, just like a river basin. My friends and collaborators who are biologists say that while we have seen that the exocrine cells actually secrete fluid as soon as they form a lumen, we have not yet measured flow, that we need to perturb it, that we only have hypotheses regarding the sensor and that we do not know the mechanisms leading to the disappearance of supernumerary loops … it will keep them busy for a while.

References

1 Turing, A. M. (1952). The chemical basis of morphogenesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 237, 37-72.

2 Patel VN, Rebustini IT, Hoffman MP. Salivary gland branching morphogenesis. Differentiation. 2006; 74(7):349–64. https://doi.org/10.1111/j.1432-0436.2006.00088.x PMID: 16916374

3 Gjorevski N, Nelson CM. Integrated morphodynamic signalling of the mammary gland. Nature reviews. Molecular cell biology. 2011; 12(9):581–93. https://doi.org/10.1038/nrm3168 PMID: 21829222

4 Dean C, Ito M, Makarenkova HP, Faber SC, Lang RA. Bmp7 regulates branching morphogenesis of the lacrimal gland by promoting mesenchymal proliferation and condensation. Development. 2004; 131(17):4155–65. https://doi.org/10.1242/dev.01285 PMID: 15280212

Link to our work

Deconstructing the principles of ductal network formation in the pancreas 
Dahl-Jensen SB, Yennek S, Flasse L, Larsen HL, Sever D, et al. (2018) Deconstructing the principles of ductal network formation in the pancreas. PLOS Biology 16(7): e2002842. https://doi.org/10.1371/journal.pbio.2002842

(No Ratings Yet)

Loading...

Vienna, Austria

July 2018

The use of salamanders in regeneration and developmental research has a long history filled with luminaries of the life sciences. Thomas Hunt Morgan, Hans Spemann and Hilde Mangold, Ross Granville Harrison, Inez Whipple Wilder, and August Weismann all employed salamanders in their work ^1,2. Current experimental research on salamanders largely focuses on studies of regeneration. Their abilities to regenerate limbs, brain, kidney, heart, and tail³ is enviable from the perspective of our limited human regenerative abilities. There is a large and growing toolkit of experimental manipulations, mutant and transgenic lines, and genomic resources that have advanced the use of salamanders to probe critical questions on the evolution of regeneration and its loss. These offer opportunities to discover mechanisms that could lead to new therapies for tissue repair.

The first “Salamander Models in Cross-Disciplinary Biological Research” meeting, organized by Elly Tanaka, Jessica Whited, Karen Echeverri, and Randal Voss, was held this past July (2018) at the Research Institute of Molecular Pathology (IMP) in Vienna, Austria. The meeting was hosted by the intrepid Dr. Tanaka, who is a Senior Scientist at the IMP and a leader in the regenerative biology field. This was a gathering of principal investigators working on various aspects of salamander regeneration, development, genetics, and genomics. The major goals included reviewing recent advances in research tools, along with nuanced tips for employing them, while also establishing a master “to-do” list for the field. Another objective was to lay the groundwork for organizing future salamander meetings intended for the broader community, including postdoctoral and pre-doctoral trainees and potentially representatives from funding agencies.

The Salamanders

Unlike “the” worm or “the” fly, there are multiple salamander models used in molecular studies of development and regeneration. These prominently include the Mexican axolotl (Ambystoma mexicanum), the Iberian ribbed newt (Pleurodeles waltl), the Japanese newt (Cynops pyrrhogaster) and to a lesser extent the North American eastern newt (Notophthalmus viridescens) and several species of lungless salamanders (Plethodontidae). Currently the most commonly used model is the axolotl, which has the longest captive history of any laboratory animal¹. This history includes the importation of a founder population to Paris in 1864, some of which contained the mutant “white” phenotype (an edn3 mutant), and deliberate introgression of an A. tigrinum locus found in 1962, which confers albinism through a tyrosinase mutation^4,5. These salamander species are representatives of three families (Ambystomatidae, Plethodontidae, and Salamandridae – the newts) out of the ten extant salamander families, which likely had extensive limb regenerative abilities at the base of the their clade⁶.

Genomics

Our sessions started with a review of the impressive new work in salamander genomics. Recent published genomes for both the Iberian ribbed newt (Pleurodeles waltl⁷) and Mexican axolotl (Ambystoma mexicanum⁸) provide tremendous new resources for the field. The axolotl genome is roughly ten times the size of the human genome, making it the largest genome to be sequenced and assembled to date (sorry loblolly pine). The publication of both of these genomes promises to help resolve the loci of multiple established mutant lines⁵, and offers the opportunity to establish future forward and reverse genetic screens that will improve our mechanistic understanding of regenerative processes. The community identified additional work needed to make consistent annotations, resolving 5’ ends of genes, and approaching a chromosome scale resolution of contiguous sequence. The latter was recently made available through a bioRxiv preprint from the Smith and Voss labs at University of Kentucky. The lack of whole-genome sequences in salamanders has been a major impediment to the field, and though the assemblies will still require extensive refinements, having these resources should prove to be enormously beneficial to labs currently working with these species as well as those interested in starting salamander research.

Tansgenics and Genome Editing

Genome editing approaches are now available for the axolotl^9,10, the Iberian newt^11,7 and the Japanese fire-bellied newt Cynops pyrrhogaster¹² using TALEN and CRISPR-based approaches. The most recent protocols developed by Ji-Feng Fei¹³ (now at the South China Normal University) for knock-out and knock-in approaches were reviewed. Current best practice techniques focused on improving knock-in strategies without relying on homology-directed repair. These include targeting introns for knock-in’s, screening injected embryo knockouts for efficient guide RNA’s prior to knock-ins, and non-homologous end joining approaches with “ORF Baits.”

The advent of CRISPR and TALEN approaches to genome editing in Pleurodeles was reviewed by Ken-Ichi Suzuki from Hiroshima University. The Suzuki lab is currently working to identify a ROSA-like locus for constitutive expression of knock-in constructs that would also provide a “safe harbor” for exogenous DNA. The intent of this approach is to develop a site that would both be minimally disruptive to normal cellular physiology while experiencing minimal interference from histone modifications in different cell lineages.

A wide range of transgenic and CRISPR edited lines are becoming available, especially in the axolotl. These include constitutive RFP and GFP reporter lines (available from the University of Kentucky’s Ambystoma Stock Center – AGSC), a pax7-mcherry muscle satellite cell marker, a neuronal marker, and a brainbow axolotl¹⁴. Discussions focused on prioritizing existing stocks and facilitating their dissemination through the AGSC (primarily in North America), Medipol University, Max Planck Dresden, and MPI (in Europe). The need to pursue financial resources to enable more extensive repository functions for salamanders, including cryopreservation to biobank lines, was also addressed. A long-term goal of the community is to secure resource funding for these types of valuable operations, which will be necessary to advance discoveries in regenerative biology through this growing research community.

Temporal control of transgene expression

Many genes implicated in regenerative processes have pleiotrophic roles in early development. These make knock out experiments in studying adult regeneration difficult as they can be embryonic lethal. Therefore both temporal and spatial control of gene expression is critical for furthering regeneration research.

There are several creative approaches available to address this potential stumbling block. These include inducible cre-lox systems¹⁵, constitutive cas-9 expression in genomic “safe harbors” with drug inducible guide RNA’s, and viral delivery of foreign transgenes into regenerative blastemas¹⁶¹⁷¹⁸. The latter approach has been championed by both Jessica Whited’s group at Harvard University and the Tanaka lab at MPI. These pseudotyped retroviruses have tremendous potential for further labeling and functional studies of the limb blastema, without raising transgenic embryos or modifying the expression of pleiotrophic genes outside the limb.

Open discussions evaluating approaches and avenues for future research allowed individual labs to share their own research objectives with a collective eye toward developing critical methodologies to advance the community.

Resources needed for the field

The real value of this PI-focused meeting was to allow researchers to define limiting resources they found most pressing for the field. Several additional needs were identified in addition to continued improvements to genome annotations and the temporal control of gene expression. These included detailed histological atlases of regeneration, master lists of validated antibodies, and more stable cell lines for in vitro experiments. One of the most obvious resources needed was the continued communication between labs with subsequent salamander research conferences that should continue to strengthen this growing research community.

Coordination and prioritization of a model system

Several research communities have benefitted from the intentional development and promotion of particular model organisms¹⁹. One-stop repositories of information such as Flybase, Wormbase, ZFinBase, Xenbase, and Sal-Site have all expanded the research capabilities of participating labs. While many of these labs focus on Developmental Biology, the reach of these model systems includes studies of neuroscience, evolutionary biology, physiology, and ecology. Emulating this deliberate approach will provide vital cohesion and an undoubted boon to investigators studying salamander regeneration and development. While salamanders are a remarkable “model” for regenerative research they are also remarkable organisms for their unique evolutionary histories, ecological roles, life history variation, and conservation biology. Development of tools and resources for the molecular biologists and biochemists working on salamanders will undoubtedly have unintentional spillover benefits into a wider range of research fields.

Outlook

Salamander models continue to be fertile ground for amazing discoveries on regenerative biology, cell differentiation, and development. This conference generated a tremendous amount of motivation for its participants. We have planned a broader 2019 meeting in Massachusetts, which will be a showcase for these recent developments and an iterative checkup on this rapidly growing experimental field.

References

1. Reiß, C., Olsson, L. & Hoßfeld, U. The history of the oldest self-sustaining laboratory animal: 150 years of axolotl research. Journal of Experimental Zoology Part B: Molecular and Developmental Evolution 324, 393–404 (2015).
2. Wilder, I. W. The Morphology of Amphibian Metamorphosis. (Smith College, 1925).
3. In Salamanders in Regeneration Research Methods and Protocols (eds. Kumar, A & Andras, S) (Humana Press, 2015).
4. Humphrey, R. R. Albino axolotls from an albino tiger salamander through hybridization. J. Hered. 58, 95–101 (1967).
5. Woodcock, M. R. et al. Identification of mutant genes and introgressed tiger salamander DNA in the laboratory axolotl, Ambystoma mexicanum. Scientific Reports 7, (2017).
6. Fröbisch, N. B., Bickelmann, C. & Witzmann, F. Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian. Proc. Biol. Sci. 281, 20141550 (2014).
7. Elewa, A. et al. Reading and editing the Pleurodeles waltl genome reveals novel features of tetrapod regeneration. Nat Commun 8, 2286 (2017).
8. Nowoshilow, S. et al. The axolotl genome and the evolution of key tissue formation regulators. Nature 554, 50–55 (2018).
9. Fei, J.-F. et al. CRISPR-mediated genomic deletion of Sox2 in the axolotl shows a requirement in spinal cord neural stem cell amplification during tail regeneration. Stem cell reports 3, 444–459 (2014).
10. Flowers, G. P., Timberlake, A. T., McLean, K. C., Monaghan, J. R. & Crews, C. M. Highly efficient targeted mutagenesis in axolotl using Cas9 RNA-guided nuclease. Development (Cambridge, England) 141, 2165–2171 (2014).
11. Hayashi, T. & Takeuchi, T. Gene manipulation for regenerative studies using the Iberian ribbed newt, Pleurodeles waltl. Methods Mol. Biol. 1290, 297–305 (2015).
12. Nakajima, K., Nakajima, T. & Yaoita, Y. Generation of albino Cynops pyrrhogaster by genomic editing of the tyrosinase Gene. Zool. Sci. 33, 290–294 (2016).
13. Fei, J.-F. et al. Efficient gene knockin in axolotl and its use to test the role of satellite cells in limb regeneration. Proc. Natl. Acad. Sci. U.S.A. 114, 12501–12506 (2017).
14. Currie, J. D. et al. Live imaging of axolotl digit regeneration reveals spatiotemporal choreography of diverse connective tissue progenitor pools. Dev. Cell 39, 411–423 (2016).
15. Khattak, S. et al. Optimized axolotl (Ambystoma mexicanum) husbandry, breeding, metamorphosis, transgenesis and tamoxifen-mediated recombination. Nat Protoc 9, 529–540 (2014).
16. Kuo, T.-H. & Whited, J. L. Pseudotyped retroviruses for infecting axolotl. Methods Mol. Biol. 1290, 127–140 (2015).
17. Khattak, S. et al. Foamy virus for efficient gene transfer in regeneration studies. BMC Dev. Biol. 13, 17 (2013).
18. Oliveira, C. R. et al. Pseudotyped baculovirus is an effective gene expression tool for studying molecular function during axolotl limb regeneration. Dev. Biol. 433, 262–275 (2018).
19. Abzhanov, A. et al. Are we there yet? Tracking the development of new model systems. Trends Genet. 24, 353–360 (2008).

(3 votes)

Loading...