The Department of Pediatrics at the University of Colorado School of Medicine invites applications for a faculty position in the tenure track. Appointment is expected at the Associate Professor level, but more senior individuals may apply. Applicants must have a Ph.D. and/or M.D. degree and demonstrated excellence in research.
We are particularly interested in individuals studying cardiac cell diversity, cardiovascular cell lineages and embryonic origins, gene regulatory networks and systems biology, cardiac cell regeneration and reprogramming, cardiovascular matrix biology and developmental models of cardiac biology. Applicants should have a record of creative and cutting-edge research, consistent and impactful publication and external funding. Applicants also should have a strong commitment to graduate and medical education and a successful record of trainee mentorship. Individuals who can enhance the diversity and accomplishment of our campus academic community are especially encouraged to apply.
Dr. Bruce Appel, Head of the Section of Developmental Biology, is Chair of the search committee. Questions can be sent to bruce.appel@ucdenver.edu.
The Neoproterozoic Earth System and the rise of biological complexity Thematic Project (FAPESP 2016/06114-6) directed by Prof. Ricardo Trindade at the Instituto de Astronomia, Geofisica e Ciências Atmosféricas (IAG-USP) and in collaboration with A. Morandini, M. Custodio, and F. Brown at the Instituto de Biociências (IB-USP), and D. Galante at the Laboratório Nacional de Luz Síncroton (LNLS) is recruiting a postdoctoral researcher with experience in physiology, cell biology, and/or developmental biology. We know little about the relative roles of environmental and biological factors involved in the late Neoproterozoic events leading to the ultimate oxygenation of the ocean-atmosphere system and dawn of biological complexity. We intend to fill these gaps by integrating information about the chemistry of oceans, evolution of complex life, paleogeography and tectonics between the Cryogenian and the early Cambrian. The selected postdoc will examine the physiology, ecology and developmental behavior of several groups of metazoans to test hypothesized connections between changes in ocean redox, nutrients and the evolution of life.
The main objectives of the postdoctoral project include:
(1) To study the tolerance of modern metazoans to Neoproterozoic ocean chemistry. The postdoc will examine tolerance to extreme oxygen level, as well as other Neoproterozoic environmental conditions, in several species (e.g. sponges, cnidarians, acoels, platyhelminthes, nematodes, and tunicates) to evaluate conserved adaptive physiological or phenotypic responses.
(2) To search for conserved metabolic pathways in species adapted to distinct oxygen levels. Using a comparative genomics approach, the postdoc will search for ancestral vs. derived gene pathways involved in oxygen metabolism. He/she will evaluate selection on oxygen metabolic pathway genes.
(3) To carry artificial selection experiments in C. elegans. To what extent can extreme oxygen variations alone drive the evolution of novel phenotypes that originated early Metazoans? We will evaluate the evolution of morphological and phenotypic complexity, and also evaluate alterations or changes in tolerance of phenotypically plastic physiological, developmental, or behavioral responses during the life cycle of C. elegans, an model animal with extensive understanding of the genetic, cellular, and developmental processes that generate phenotypes.
The candidate will be mainly based at the IB-USP in São Paulo to work with cultures of live animals, and at the LNLS in Campinas to use the space environment simulation chamber that will be used to recreate Neoproterozoic Earth conditions. The research team maintains an international working environment, speaking Portuguese is not required but it would be advantageous.
It is ESSENTIAL that:
-the candidate has a doctoral degree in Biological Sciences or related fields
-the candidate has experience in any of the following fields (the more the better): physiology, cell biology, developmental biology, evolutionary biology, astrobiology or bioinformatics.
-the candidate can coordinate a highly collaborative and integrative research project
-the candidate is willing to co-supervise students together with the PIs involved in the project
-the candidate has excellent communication skills in spoken/written English.
The applicant should contact directly to Dr. Federico Brown (fdbrown@usp.br), Dr. André Morandini (acmorand@usp.br) or Dr. Marcio R. Custódio (mcust@usp.br), and provide a letter of interest, a CV, and contact information of three potential referees until May 15th. Start date is any time after August 2018.
Often, the most enjoyable research projects are the ones that were never planned; and so it was with the current study.
In developmental biology, genetic lineage tracing methods have become a standard tool to interrogate the lineage potential and fate behaviour of cells [1]. By activating fluorescent reporter genes in individual cells, qualitative and quantitative methods have been defined to translate the size distributions of marked cells and their progeny – clones – into information on the statistical fate behaviours of constituent cells [2]. These approaches have led to important insights into the dynamics and functional behaviour of stem cells and progenitors during the development, maintenance and regeneration of tissues, as well as the dynamics of tumour growth during cancer initiation and progression.
Unlike continuous in vivo live-imaging approaches [3], the interpretation lineage tracing data, based on the analysis of fixed samples, can be confounded by uncertainties in the history of clonal evolution: First, there is a question of whether the tracing assay targets a single (functionally equivalent) cell population, or whether there is heterogeneity within the induced cell fraction. Second, there is an intrinsic ambiguity in the lineage reconstruction since multicellular clones can typically be generated from a multitude of potential lineage trajectories: Different combinations and permutations of cell divisions leading to symmetric and asymmetric fate outcomes can generate clones of the same size and cell composition. Fortunately, to a degree, both problems can be mitigated post hoc by the nature of the clonal dynamics itself!
Figure 1. Surface of a mouse heart at embryonic day (E)12.5 showing clusters of cells derived from cell precursors marked by a multicolour fluorescent (confetti) reporter system under the control of a Mesp1-promoter at E6.5. For details, see the primary paper. Looking at the image, can you tell which clusters belong to the same clone…?
Previously, research by our group and others have shown that, while the short-term dynamics of labelled progenitors may be highly complex, in the long-term, the size distribution of clones anchored in equipotent cell populations typically converge to rigid “scaling” behaviours in which the chance of finding a clone larger than some multiple of the ensemble average becomes unchanging over time [4]. Indeed, in self-renewing adult tissues, the constraint of homeostasis limits strongly the variety of scaling distributions, so that information on cell fate behaviour can be recovered “rigorously” from the functional form of the scaling distribution.
However, the utility of these methods is heavily reliant on the integrity of clonal assignments; and therein lies the rub! During development, and in the course of adult tissue turnover, cell rearrangements due to active cell migration, or serial rounds of cell loss and replacement, can lead to the fragmentation or merger of clones, corrupting the data and, potentially, misleading efforts to interpret the underlying clonal dynamics. Yet, in a lineage tracing approach based on the analysis of fixed samples, we can’t know what were the individual histories of clones and their fragments. Developments of multicolour lineage tracing strategies, based on the “brainbow” or confetti reporter constructs, can mitigate the potential for such effects. However, in the context of tissue morphogenesis or tumour development, large-scale cell rearrangements caused by tissue remodelling can lead to the long-distance segregation of clonal fragments rendering the task of clonal reconstruction potentially infeasible. Worse, confronted with such challenges, many researchers may feel inclined to abandon attempts to reconstruct putative clones dispersed across the three-dimensions of a solid tissue and instead to characterize the size of a clone by its intersection with a thin section of tissue – “surely a rough indication of the complete clone size…?”
Unfortunately, such hopes are dashed by the inconvenience of a mathematical riddle – the corpuscle problem – which states that, without making detailed assumptions, the distribution of three-dimensional objects cannot be recovered (even in principle) from the statistics of their random intersections [5]. Yet, for those brave enough to try to reconstruct the entirety of the clone, the situation is still treacherous since, at first sight, it is difficult to know whether the clonal assignment is faithful. With the increasing prevalence of genetic lineage tracing assays, this is the situation that prevails – with legions of researchers (and reviewers…!) questioning whether assays are clonal. Of course, statistical analyses can help. At low enough clonal induction frequency, fragmented clusters of cells can be associated with defined statistical confidence. Yet, in a developmental context, this may require organs with as few one or two clones per animal, a daunting, expensive and arguably unethical endeavour! So, can anything be done to alleviate this problem?
With the benefit of hindsight, we should have asked ourselves whether, in common with “true” clonal dynamics, the statistical distribution of “putative” clones could, in of itself, provide evidence that the source data was corrupted by clonal fragmentation and/or merger events. But we didn’t! Instead, we stumbled into this idea through an intriguing empirical observation. Our collaborators in the Blanpain lab at the Interdisciplinary Research Institute at the Université Libre de Bruxelles were interested in resolving the potency and proliferative potential of early Mesp1-expressing precursors in the developing mouse heart. To trace the fate of these cells, confetti labelling was induced in a fraction of Mesp1+ cells early in embryonic development, and the size and regionalization of clonal footprints were scored on the developing mouse heart (Fig. 1) [6]. Notwithstanding the corpuscle problem, we (recklessly) constructed the size distribution of clonal “fragments” on the embryonic heart and found a size dependence that was strikingly broad yet was almost void of structure (i.e. information on the underlying clonal dynamics): The average sizes of labelled fragments differed vastly between different heart compartments and time points in development – consistent with the complexity of developmental programmes during heart morphogenesis. However, to our surprise, once we divided fragment sizes by their ensemble average, the resulting rescaled probability distributions became indistinguishable between different heart compartments (Fig. 2). What could it mean…? Did this reflect a rigid growth characteristic of precursors in the maturing mouse heart – with some cells destined to proliferate and expand prodigiously while others are set to exit cycle early? Or was there a less “glamorous” explanation, at least from the perspective of our experimental collaborators…?
Of course, during development, clones are subject to numerous intrinsic and extrinsic influences that adjust cluster size: Labelled cell clusters can expand through the division of constituent cells; they can also expand through chance merger events that bring together clusters of the same hue. However, clusters can also diminish in size through cell loss – either through cell death, or by cells leaving the field of view in a sectional characterisation of cluster size; they can also diminish through fragmentation events caused by collective cell rearrangements created by larger-scale morphogenic changes or simply from stochastic forces exerted by the surrounding cells. Finally, new clusters can appear as labelled cells that are “out of frame” move into a sectional view. In short, in a model that mirrors both qualitatively and quantitatively the non-equilibrium dynamics of aerosol suspensions, the time-evolution of the distribution of labelled cell clusters conforms to a fiendishly complex set of possible processes that includes all the factors above, and potentially many more – a seemingly unpromising starting point for any analysis…
However, the rigour of statistical physics (and a little bit of hindsight) came to the rescue!! Indeed, during tissue development or turnover, constituent labelled cell clusters will stochastically expand and contract in response to the factors above. Under these conditions, the different possible influences impact on the resulting cluster size distribution to widely varying degree. Therefore, to quantify the relative contributions of these disparate processes, we employed a concept that has shaped the field of statistical physics: By progressively moving to coarser and coarser length scales, we obtained mathematical equations describing the varying contributions of different processes on these scales. We reasoned that, on the largest scales, i.e. as development proceeds, these equations then give an accurate, but reduced, description of the dynamics shaping the cluster size distributions. Notably, using an analytical approach inspired by the “renormalization group” methods of statistical physics, we could show that, over time, only the processes of clonal merger and fragmentation impact on the shape of the resulting cluster size distribution; in the parlance of statistical physics, these were the relevant operators. Crucially, if perhaps worryingly for the biologists(!), information encoded in developmental fate programmes becomes progressively erased from the cluster size record, while the emergent dynamics signals only the existence of fragmentation and/or merger events. The observed cluster size dependence was, indeed, not a reflection of some underlying fate programme, but an inevitable outcome of the collective dynamics of heart development. As a corollary, the erasure of biological information suggested that cluster size distributions obtained from lineage tracing experiments should take the same form across different biological tissues and even species. To drive home the message, we confirmed that the cluster size distributions of the developing zebrafish heart, as well as liver and pancreas, also conform to common – physicists would say “universal” – cluster size distributions with the same hallmark (in this case, log-normal) size dependence.
So far, so good, we thought; and we set off to inform the stem cell community of our findings. Not surprisingly, we found that not all experimentalists were equally enthusiastic to learn that the outcome of often challenging lineage tracing experiments might be, by design, void of biologically significant information… However, perhaps more surprisingly, we found that the very concept of universality – a rigorously defined mathematical concept at the heart of critical phenomena in statistical physics – caused additional irritation among our biological peers. More than once we were informed that “nothing in biology is universal…!” Indeed, this warning betrays something of the cultural differences that continue to divide the physical and biological science communities. While the former are trained to look for commonalities between different systems, the inherent complexity of biological systems makes it more natural to focus on differences and details.
Indeed, we believe that these observations have value both to the biology and physics community. For the former, these findings have a practical value: Although one cannot use statistical distributions to confirm positively the integrity of clonal assignments, one can at least use hallmark scaling dependences of cluster size to quantitatively and, therefore, rigorously signal when seemingly clonal data has been corrupted by fragmentation and/or merger events. At the same time, understanding the origins of universal scaling behaviour allows biologists to develop experimental strategies that can unveil the developmental programmes of labelled cells. For the physicists, the situation is more encouraging with clonal dynamics presenting yet another example of where biological systems can provide an arena in which powerful concepts from statistical physics and mathematics can find novel and practical application.
Figure 2. Cumulative distributions of fragment sizes from different tissues and species (dots) collapse onto the same universal scaling form (dashed line) when rescaled by the average size. For details, see primary paper.
In these lines I share with you some details of our recently published Nature paper. I will comment how this project was started and details which are not included in the manuscript. Finally, I will briefly comment on some questions we are working on today and others we believe are worth addressing in the future.
Historical context of the project.
One of the questions that has enthralled biologists for many years is how a single cell can give rise to highly organised multicellular organisms with extremely complex forms. Although finding an answer to this question represents a big challenge, the advances made by the cell and developmental biology community over the last 50 years have been truly astounding. The advent of molecular biology in the last century, allowed embryologists to generate a strong body of evidence whereby ‘old’ ideas based upon cellular observations have found their molecular explanation. One of the most astonishing examples of this type of molecular re-description of a cellular process is the molecular characterisation of the Spemann–Mangold organiser. This tissue was initially described in 1924 as an embryonic region exerting inductive effects on cells (1). More than 70 years later, Edward De Robertis’ group generated cDNA libraries from manually dissected organisers and found genes that were specifically expressed in this territory (i.e., goosecoid (gsc)) (2). This was the first demonstration that this structure was a molecular identity defined by specific gene expression patterns. Currently, most of the embryonic tissues and about 4 organiser regions have been molecularly defined across embryos of different species (3,4). Indeed, this field has progressed to such a level that well-defined networks of signalling pathways are now widely accepted as coordinators of morphogenesis, and that their most common modus operandi is the delivery of positional information allowing interaction among neighbour cells (3,4). However, despite such vast advancements in our understanding of molecular players during morphogenesis, the role of other types of cues in this process is comparatively less understood.
Morphogenesis is a highly regulated biological process which involves constant rearrangement of tissues and deep mechanical changes. However, the role of these mechanical changes is only just re-starting to be considered when looking for a more comprehensive explanation of the morphogenetic process. I use the term ‘re-starting’ because in the early 1900’s, and prior to the molecular ’boom’, scientists studied embryonic development by using theoretical, morphological, chemical, and mechanical approaches. More recently, and ‘swimming against the tide’, several groups have focussed their research on developmental mechanics as a means of understanding those aspects of morphogenesis that molecular patterning cannot explain by itself. Work from these contemporary groups has been nicely reviewed in Lecuit, 2008 and Gilmour et al., 2016 (5,6). Considering the strong molecular frame-work and new advances in tissue mechanics, the field is starting to look at embryos as finely regulated machines where tissue rearrangements lead to mechanical changes that, in turn, feed back into the cells to modify their gene expression and behaviour, ultimately coordinating morphogenesis.
In this context, and taking advantage of powerful mechanical and molecular tools, we revealed that tissue stiffening owing to cellular rearrangements occuring in the mesoderm during Xenopus gastrulation triggers the epithelial-mesenchymal transition (EMT) and collective migration of the neural crest (NC) at neurula stages. More generally, we showed that tissue mechanics act as long-range cues coordinating the timing of two developmental processes that, until now, were considered as unconnected events (7).
Early steps of the project
The initial question addressed in this project was: what triggers the migration of the neural crest? The neural crest forms by inductive signals at the border of the neural plate (induction) where it remains as a non-migratory population until, at some point and for some reason, they undergo EMT, delaminate, and collectively migrate toward their target tissues (7,8).
The conception of this project occurred towards the end of my PhD in 2014. I was working on the transcriptional regulation of Twist, an EMT regulator in the neural crest (9). A persistent question at the time was why Twist and most of the neural crest EMT-related transcription factors are expressed at induction stages, 12 hours before the EMT and migration take place. At the same time, I was due to start a postdoc, requiring me to design and write a project. By 2012–14 there was a wave of in vitro studies showing that stiffer substrates favour migration and EMT in several cell types (10) but whether this was the case in vivo, was lacking a demonstration. That is how, with Roberto, we began to speculate that mechanics of the tissues surrounding the neural crest may change during development and that, consequently, the neural crest could feel this change and migrate. I started to read about the topic and I was impressed by the vast morphological changes that Xenopus embryos undergo in these 12 hours prior to neural crest migration, during which embryos pass from a spherical to a rather more elongated shape due to convergent extension (11,12). I even found an article showing that paraxial mesoderm stiffens towards the onset of NC migration (13). Many questions emerged at the time i.e.: Does the environment play a role in NC migration? Does NC actually reply to mechanical cues from its substrate? What are the mechanical properties of the NC microenvironment? How are these mechanical properties regulated? So, we put these questions down in a project, got the funding, and start working on this story. Not as simple as it sounds; I had to apply twice for funding, but I was finally awarded an EMBO and a Marie Curie postdoc fellowship.
Main findings
We can divide our story in two parts. First, we studied the mechanical interaction of the NC with its surroundings and the role of the temporal environment in controlling NC migration. Second, we sought to understand the mechanisms underlying mesoderm stiffening.
Before discussing the results, I would like to clarify the nomenclature we use for the different stages in which we analysed the NC. Non-migratory NC are defined as, even if it sounds obvious, cells that are not migrating and that instead resemble a more epithelial phenotype. These are normally analysed between stages 12–14 in our work. Pre-migratory NC are motile but a migratory stream is not yet observable, and we analysed them at stage 17–20. Finally, migratory NC, which are defined when migratory streams are evident, are typically analysed between stages 22–25 in our article. Stages as in Nieuwkoop and Faber (14).
I was forgetting this! When I talk about stiffness throughout the text, I am referring to the apparent elasticity of the tissue or the apparent elastic moduli contained in it, if you prefer.
Mesoderm stiffening is necessary and sufficient to trigger NC collective migration in vivo
As most of the molecules required for NC migration are already expressed at non-migratory stages, we asked whether non-migratory and migratory NC had the same migratory potential. To address this, we explanted non-migratory and migratory neural crests and evaluated their chemotactic response towards Sdf-1, a well-known NC chemoattractant (15). To our surprise, ‘non-migratory’, pre-migratory, and migratory neural crests equally migrated to SDF-1 indicating that, regardless of their stage, NC cells have the same migratory potential. Based on this in vitro observation we hypothesised that, in vivo, the temporal environment may be contributing to govern the migratory potential of the NC. Therefore, we performed heterochronic grafts, where non-migratory cells where grafted into migratory hosts and vice versa. It was nice to observe that non-migratory or migratory NC cells grafted into migrating hosts almost immediately start migrating. However non-migratory or migratory NC cells grafted into non-migratory hosts took more than 12 hours to start migrating, the same time taken by host embryos to reach migratory stages. These results strongly suggested that there is ‘something’ in the environment that changes from non-migratory to migratory stages and that this ‘something’ is very likely to be involved in the initiation of NC migration.
Personal note 1: experiments like these are highly motivating (at least for me). They do have this beautiful mixture of simplicity, creativity, and a touch of classic embryology… but with a high reward in terms of information.
As in vitro studies show that substrate stiffness triggers collective migration (10) we next characterised the mechanical environment of the NC (and yes, we did rule out other environmental factors as potential triggers of NC migration). Because the NC migrates sandwiched between the mesoderm and epidermis, we measured stiffness of these tissues in the region just in front of the NC at non-migratory, pre-migratory, and migratory stages. Our weapon of choice to measure tissue stiffness in vivo was a very novel and versatile setup of in vivo atomic force microscopy (iAFM). This method was developed by one of our co-authors, Kristian Franze (University of Cambridge) (16). After adapting this setup to our experimental requirements, we discovered that the mesoderm, used by the neural crest as a substrate, stiffens by 3 times from non-migratory to migratory stages (Figure 1). Our controls revealed that the epidermis has either similar or lower stiffness than the mesoderm. Until here, our results showed that there is a strong correlation between mesoderm stiffening and neural crest migration.
Figure 1. Mesoderm stiffens towards the onset of neural crest migration.A Schematic showing the region where the measurements were taken. NP, neural plate; NC, neural crest; ep, epidermis; NT, notochord; msd, mesoderm. B Chart summarising the spread of data of our iAFM results in two of the analysed stages. Each dot is an AFM indentation, data was analysed with Mann-Whitney test.
Based on this, our next question was whether this correlation plays a functional role in NC migration. We addressed this question by developing an ex vivo system consisting of 2D hydrogels containing stiffness values equivalent to those obtained in embryos at migratory (stiff) and non-migratory (soft) stages. NC plated on stiff substrates chemotax towards Sdf-1, disperse, and undergo a switch of E- to N-cadherin (EMT). However, these parameters were drastically reduced in cells plated on soft substrates. This was a nice control that showed us that cranial neural crest replies to changes in substrate mechanics.
As we learned that mesoderm stiffens and that NC behave differentially on substrates of varying stiffnesses, we tested the relevance of mesoderm stiffening for NC migration in vivo. We used a combination of ablation experiments and targeted injections of myosin contractility inhibitors to release tension and reduce stiffness in the migratory substrate of the neural crest. We controlled that our treatments effectively reduced stiffness by using iAFM. In all our conditions, NC migration was analysed by in situ hybridisation. Our results were very clear and we observed that migration of the NC was inhibited when the stiffness of the substrate was reduced by tissue ablation or targeted injection experiments (Figure 2 A–C). These results indicated that mesoderm stiffening is necessary to promote migration of the neural crest in a non-autonomous manner.
Figure 2. Mesoderm stiffening is necessary and sufficient to promote neural crest migration in vivo.A Representative images showing lateral views of embryos hybridised with a probe against a NC marker. Treatments as indicated. B Chart summarising the spread of data of our iAFM results. Each dot is an AFM indentation. C normalised NC migration. D Representative images showing lateral views of embryos hybridised with a probe against a NC marker. Treatments as indicated. E Tissue elasticity plotted as a function of the AFM indentation. F chart showing normalised NC migration.
We next asked whether substrate stiffening was also sufficient to trigger migration of the NC in vivo. For this, we used a novel ‘strain stiffening’ assay to promote early stiffening of the tissue in front of the NC. This is an AFM-based method in which we can exert a controlled force for a determined amount of time in a precise location (16). As a result, we observed that applying extrinsic stress was sufficient to stiffen the mesoderm and promote premature migration of the NC (Figure 2 D–F). This was a very surprising result and it represented one of the first demonstrations of premature migration of the NC. Whilst former studies showed delamination and ectopic migration of delaminated single NC cells upon cadherin inhibition, the premature migration of the NC by following its stereotypical migratory paths had not been previously shown.
Personal note 2: designing and performing these experiments was a great experience for me. It was something not related at all to my biological background and it is one of the major sources of new knowledge from my postdoc. I have no doubt that around this time I changed the way I address biological questions.
Taking together this set of results we concluded that stiffening of the mesoderm is necessary and sufficient to promote NC migration in vivo, very likely by triggering its EMT. We believe this is not only relevant for the NC. Numerous groups of cells and single cells are challenged by the mechanics of their surroundings when migrating in vivo. There are several types of mechanical challenges that cells need to sort in order to reach their target tissues. Hence it would be of great value to start exploring the mechanical interaction of other cellular systems with their surrounding tissues in diverse biological contexts.
PCP-mediated convergent extension is the driving force of mesodermal stiffening in vivo
Our next goal was to unveil the driving force of mesodermal stiffening. The first idea we explored was an accumulation of extra cellular matrix (ECM). In many systems, accumulation of ECM is one of the main components of tissue stiffening (17). It was disappointing to discover that in our system this could not be the case. We found out that i) there is no difference in ECM deposition among non-migraotry and migratory stages; ii) removing ECM under the NC did not affect mesoderm stiffness; and iii) collagen is not expressed at these stages in Xenopus.
Thus, we next turned our attention to actomyosin contractility, as it has been previously shown that this activity could play a role in paraxial mesoderm stiffening (13). We analysed expression of phosphorylated myosin and f-actin in the head lateral mesoderm (mesoderm used by the NC as a substrate). We observed no differences in expression levels or sub-cellular localisation when comparing non-migratory vs migratory stages. We even inhibited contractility once the stiffness was already increased in the head mesoderm. As measured by iAFM, only a very small and non-significant difference was observed when compared to control embryos, indicating that although it may play a role, contractility was not the main component of head mesoderm stiffness.
Personal note 3: These were very frustrating results at the time, I was running out of options and the explanation about what was the driving force of mesodermal stiffening seemed more complicated than what we thought. But if you ask me today I think that this complication is from where one of the most interesting parts of our discovery was originated; mechanical coordination among temporally unrelated embryonic tissues.
We did not give up and kept looking for options. At the time, Kristian’s lab was just publishing their work about differential cell density as the source of a stiffness gradient that guided the growth of the optic tract (16). We went back and looked into the anatomy of the embryo and realised that from non-migratory to migratory stages, embryos elongate and cells accumulate dorsally. We did a very simple sectioning experiment on wild-type embryos with colour-labelled mesoderm and NC. We quantified cell density in the mesoderm under the NC at non-migratory, pre-migratory, and migratory stages and observed a strong accumulation of cells and increased cell density that nicely correlated with mesoderm stiffening and onset of NC migration (Figure 3 A–C).
Figure 3. PCP-dependent CE is the driving force of the cell accumulation required for mesoderm stiffening. A Representative images showing transverse sections with labelled nuclei of neural crest (cyan) and mesoderm (magenta) cells. Stages as indicated. Dorsal to the top. B Chart showing the total number of nuclei under the neural crest. C Chart showing the density of nuclei at the area immediately under the NC. D Representative images showing lateral views of embryos hybridised with a probe against a NC marker. Treatments as indicated. E Chart summarising the spread of data of our iAFM results. Each dot is an AFM indentation. F chart showing normalised NC migration. Treatments as indicated.
To functionally test this correlation, we quantified cell density upon application of distinct treatments into the mesoderm without inducing a direct effect on NC migration. We found that treatments resulting in blocked NC migration correlated with low mesodermal cell density and low stiffness, whereas treatments promoting NC migration perfectly corresponded with high mesodermal cell densities and high stiffness values. This strongly suggested that one of the main components of mesodermal stiffness is the accumulation of mesodermal cells that lead to increased cell density.
This matched seamlessly with what we observed before and at this point we re-interpreted our myosin knockdown results, where early inhibition of myosin contractility in the mesoderm led to decreased stiffness and inhibited NC migration. It is well-known from John Wallingford’s lab work that polarised activity of myosin is required for mesodermal cell migration (12). Consequently, it makes sense to think that what our treatments were doing was inhibiting mesoderm cell migration which in consequence decreased cell density and stiffness under the NC.
As planar cell polarity (PCP) is a master regulator of convergent extension (CE) (11,12) we inhibited PCP in the mesoderm. As expected, mesoderm stiffness was decreased and NC migration inhibited (Figure 3 D–F). As depleting PCP has many biological consequences, this treatment represented an excellent scenario to test whether introducing extrinsic stress to stiffen the mesoderm of soft embryos was enough to promote NC migration. So, as a final experiment we inhibited PCP, applied extrinsic stress with iAFM and observed that NC migration was mechanically rescued (Figure 4). These results led us to conclude that the driving force behind the increased cell density that promotes mesoderm stiffening and NC migration are PCP-driven cellular rearrangements during CE, with a minor contribution of myosin contractility.
Personal note 4: Something which I believe is really worth mentioning is the role of myosin and ECM in mesoderm stiffening. There is a large body of evidence from excellent groups that demonstrate the requirement of Fibronectin deposition and myosin activity to ensure correct convergent extension movements. Hence myosin and Fibronectin do contribute to build-up mesodermal stiffening by allowing mesoderm migration and accumulation of cells under the NC, but they represent a minor contribution to stiffness once cells have already accumulated.
These results were very exciting as they demonstrated for the very first time that two embryonic processes that have been always studied as separate events, CE during gastrulation and NC migration, are connected via tissue mechanics. Our data is an example of how applying more integrative approaches to study morphogenesis can lead us to actually understand how the process organises itself– such an approach has the potential to deeply impact our advances in the study of ‘self-organising’ biological systems.
Figure 4. Ectopic compression rescues NC migration in soft embryos. A Diagram describing the extrinsic stress experiments used to stiffen the mesoderm after PCP inhibition. B Representative images showing lateral views of embryos hybridised with a probe against a NC marker. Treatments as indicated.
Open questions
Although we demonstrate in our article that integrin, vinculin and talin, well-known mechanosensors (18), are required for NC migration we are now actively studying the mechanism by which NC cells sense and translate mechanical stimuli in a cellular and eventually molecular response. Another question that we are addressing now is related to a potential role for durotaxis in vivo. Durotaxis is the process by which cells migrate from compliant to stiffer substrates. In this front, we are studying how stiffness gradients may emerge as a result of mechanical heterogeneities of embryonic tissues and whether these gradients can direct cell migration in vivo. In the future, it may be interesting to address whether substrate mechanics are just permissive cues allowing cells to exert traction and migrate or instead are a real source of signalling that ‘permit’ migration but also modify the genetic program of the NC. Similarly, a question that may be linked to environmental mechanics and that is relevant for development, regeneration, and cancer is how cells know when to stop migrating and differentiate once they reach their target tissues.
Acknowledgements
I would like to thank the co-authors of this article Roberto Mayor, Guillaume Charras, and Kristian Franze for their input and the funding sources EMBO (LTF-971) and Marie Skłodowska Curie (IF-2014_ST VivoMechCollMigra). Dr Ailin Buzzi (KCL) and Emily Atalar (UCL) for their comments on this post.
References
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10. Roca-Cusachs, P., Sunyer, R. & Trepat, X. Mechanical guidance of cell migration: lessons from chemotaxis. Curr. Opin. Cell Biol. 25, 543–549 (2013).
11. Wallingford J.B. & Harland R.M. (2001). Xenopus Dishevelled signaling regulates both neural and mesodermal convergent extension: parallel forces elongating the body axis. Development 128, 2581–92.
12. Shindo A. & Wallingford J.B. PCP and septins compartmentalize cortical actomyosin to direct collective cell movement. Science 343, 649–652 (2014).
13. Zhou J., Kim H. Y. & Davidson L. A. (2009). Actomyosin stiffens the vertebrate embryo during crucial stages of elongation and neural tube closure. Development 136, 677–688.
14. Nieuwkoop, P. D. & Faber, J. Normal Table of Xenopus laevis (Daudin): a Systematical and Chronological Survey of the Development from the Fertilized Egg till the End of Metamorphosis 2nd edn (North-Holland, 1967).
15. Theveneau, E. et al (2010). Collective chemotaxis requires contact-dependent cell polarity. Dev. Cell 19, 39–53.
16. Koser, D. E. et al (2016). Mechanosensing is critical for axon growth in the developing brain. Nat. Neurosci. 19, 1592–1598.
17. Moeendarbary, E. et al. (2017). The soft mechanical signature of glial scars in the central nervous system. Nat. Commun. 8, 14787.
18. Charras, G. & Sahai, E. (2014). Physical influences of the extracellular environment on cell migration. Nat. Rev. Mol. Cell Biol. 15, 813–824.
Two Postdoctoral positions are immediately available at the University of California, San Francisco (UCSF), in the laboratory of Sarah Knox.
Position 1: The applicant will be part of an NIH-funded investigation into the development, regeneration and aging of exocrine organs including salivary glands, pancreas and ocular organs. The project specifically focuses on nerve-stem cell communication and how these interactions govern organ morphology, homeostasis and regeneration throughout the lifetime of the organism. In addition to defining key mechanisms regulating organogenesis, we pay particular attention to the impact of aging on organs and their potential for rejuvenation by employing parabiosis based methods in combination with 3D imaging, deep sequencing (scRNA and bulk RNA), and mouse genetics (including CRISPR generation of mouse models).
Position 2: The successful applicant will be part of an NIH-funded, pre-clinical investigation to develop a novel therapy that restores ocular and exocrine gland function through modulation of immune-nerve-epithelial cell interactions. The candidate will use genetic mouse models of autoimmune disease in conjunction with ex vivo and in vitro systems to study the effects of inflammation on epithelial cell-nerve interactions in the cornea and other ocular organs.
For both positions, applicants with a strong background in developmental biology, stem cell biology, neuroscience, mouse genetics and/or immunology are preferred. Technical expertise in the areas of mouse handling and dissection, immunoassays, immunohistochemistry, adoptive transfer, flow cytometry, qPCR, RNAseq, bioinformatics, and confocal microscopy are a plus. Applicants must possess an M.D., Ph.D., or equivalent degree. The candidate should have the proven ability to independently design and execute experiments, as well as interpret and publish results. They should also possess excellent communication skills and the ability to work as part of a team.
To apply, submit a C.V., a cover letter with a brief description of research interests and experience, and the names of at least 3 references via email: sarah.knox@ucsf.edu
The Landsman Lab invites highly motivated students to join its effort to decipher the role of the microenvironment in beta-cell development and function. We combine transgenic mouse systems with physiological, morphometric and molecular tools to define cell-cell interactions within the pancreas, with the aim of facilitating cell replacement therapy for diabetes.
The lab welcomes outstanding international students with a Masters degree in life sciences and a passion for research. The lab provides full fellowships for qualified students.
The Landsman lab is located at Tel-Aviv University, which is the leading interdisciplinary research and teaching university in Israel. Tel-Aviv is the cultural and commercial heart of Israel. Situated on a beautiful coast of the Mediterranean Sea, it is a vibrant, young city, with great food and weather.
Lectures and courses are in English, and Ph.D. studies usually last four years. Housing on campus is an option.
Please apply by sending CV (including transcripts) and a cover letter detailing your research experience and interest through our website.
Today we are delighted to announce the winner: Zubair Nizamudeen, a PhD student in Virginie Sottile’s lab at the Wolfson STEM Centre, School of Medicine, University of Nottingham, UK. Zubair’s prize was free registration for the meeting, which he will report from for us!
Here Zubair gives us the background to his research and tells us what he’s particularly looking forward to in the meeting.
Our lab is based in the Wolfson Centre for Stem Cells, Tissue Engineering and Modelling (STEM), and are part of the Division of Cancer & Stem Cells in the School of Medicine of the University of Nottingham, UK. We focus on the genetic aspects of stem cell biology, and on the molecular mechanism regulating the differentiation ability of adult stem cells compared to embryonic stem cells. We isolate and grow adult stem cells from different origins, in order to identify the key factors involved in the acquisition of a specific lineage identity, around two central themes:
1) the control of mesenchymal stem cell differentiation, and its use for translational tissue repair,
2) neural stem cell distribution in vivo, as well as differentiation using in vitro models.
We are interested in investigating the relationship between stem cells, differentiation control, tissue repair and neoplasia.
As a final year PhD student focused on understanding the potential of adult neural stem cells in the mammalian brain, I am very excited to attend the esteemed ‘Adult Neurogenesis 2018’ conference in Dresden, Germany. In particular, I am looking forward to attending the talks related to regulation and dynamics of adult neurogenesis by F. Gage (US) and S. Jessberger (Switzerland). I am eager and passionate to learn about new research ideas and explore networking and career opportunities that are to be presented in this conference.
The story behind our paper:Cell-Intrinsic Control of Interneuron Migration Drives Cortical Morphogenesis. Carla G. Silva, Elise Peyre, Mohit H. Adhikari, Sylvia Tielens, Sebastian Tanco, Petra Van Damme, Lorenza Magno, Nathalie Krusy, Gulistan Agirman, Maria M. Magiera, Nicoletta Kessaris, Brigitte Malgrange, Annie Andrieux, Carsten Janke, Laurent Nguyen
The research behind this article is a good example of how looking beyond expected results can lead to unexpected discoveries. This story is not what we originally thought it was going to be about. But simplistic hypotheses failed to explain standard measurements and this lead us to a deeper question.
In the beginning:
In the 1970’s a Purkinje cell degeneration mutant mouse had been studied1 and the deficient gene responsible for the phenotype was later shown to be Ccp12. This gene codes for an enzyme, called carboxypeptidase 1, capable of digesting chained glutamates on tubulin (the building block of microtubules) or on a wide range of additional proteins3.
When we first started working in the lab of Laurent Nguyen, we were given access to the CCP1 conditional knock out mouse3. At the time, the lab was focusing on understanding the mechanisms underlying interneuron migration during cortical development. This migration is a fascinating process. The cortical interneurons are born in regions located far away from the residence of their future, mature selves. As the embryonic brain is forming, a steady flow of interneurons moves away from their birth place to reach the cortical plate. The developing brain environment gives cues to steer them in the right direction, both repulsing them from the zones they should not invade and attracting them towards correct paths. Each individual interneuron senses its environmental cues and adapts its movements accordingly. This information is translated to directed movements thanks to the dynamic remodelling of the cytoskeleton, which gives scaffolding structure to the cell but also generates contraction and pulling forces4. Microtubules and acto-myosin fibres are the main components of the interneuron cytoskeleton and their contractions and dynamics are tightly regulated. Two general levels of cytoskeletal regulation can be found: 1) gene-encoded signalling cascades regulate contraction or polymerisation/depolymerisation; 2) posttranslational modifications of cytoskeleton components can fine-tune the movement. One of these posttranslational modifications is the addition/removal of glutamate.
We reasoned that by removing Ccp1 and hyperglutamylating cytoskeletal proteins, we would subtly modify interneuron migration without stalling cells.
The phenotype observed:
Our primary observation was that too many interneurons were invading the cortex upon loss of Ccp1 expression during development. It would then take us about 2 years to understand why.
We first characterised cortical invasion by cortical interneuron in more detail. We showed that between E12.5 and birth, more Ccp1 cKO interneurons were invading the cortex of mouse embryos. This was an unusual phenotype since so far, cytoskeletal modifications lead to delayed migration and reduced number of interneurons in the cortex.
In parallel we characterized the cellular pattern of migration of these interneurons, using time lapse video microscopy. We were able to follow their behaviour as they moved toward their destinations.
GFP-expressing cortical interneurons migrating away from MGE explants prepared from E13.5 CCP1 WT embryos. Duration of recording is 5h.
Not only could we measure the average speed of displacement but we were also able to study the way interneurons cortical interneurons were moving. We observed that indeed, the behaviour of interneurons lacking Ccp1 was slightly changed despite having a preserved average speed of migration. Instead of alternating phases of nuclear pauses and large amplitude jumps called nucleokinesis5, mutant interneurons were pausing for a shorter amount of time and the amplitude of their nucleokinesis was reduced. Smaller jumps compensating shorter pauses, amounting to similar average speed.
The next question was to understand what could explain our observation of larger number of interneurons in the cortical plate?
The simplistic hypotheses:
To answer this question we tested a large number of hypotheses that yielded negative results and frustrating statistically non-significant histograms.
We first tested if more cortical interneurons were generated in upon loss of Ccp1. Our genetic recombination tool (Dlx5.6 CRE mouse line) was expressed in a small portion of dividing cells. We counted the number of mitotic cells, the number of cells replicating their DNA and the general cell cycle phases distribution of our CCP1 mutant cells and nothing was changed in our mutant.
We then reasoned that if more cortical interneurons were reaching the cortex, it could be explained by an increased survival, leading to fewer cells dying on the way. However, we did not detect any reduction of apoptosis in Ccp1 cKO cortical interneurons as compare to their controls.
Another plausible explanation was that mutant cortical interneurons would take “short-cut” pathways to the cortex and thus would be more efficiently reaching their destination. We counted the number of cortical interneurons crossing the striatum, a no-go zone for cortical interneurons and again, we did not measure any differences.
Finally, we tested whether loss of Ccp1 could affect cell fate and favour generation of cortical interneurons at the expense of oligodendrocyte that are born in overlapping regions of the forebrain from a common pool of progenitors. We showed that the mutant brain did not lack oligodendrocytes. To be sure that the conditional knockout of Ccp1 knock-down in cortical interneurons was not resulting in higher interneurons generation through a non-cell autonomous activity, we counted the number of recombinant cells in the whole brain using FACS. At E13.5 we measured the same total number of cells in Ccp1 cortical interneurons brains as compared to WT controls. This observation suggested that more cortical interneurons were displaced in the cortical compartment in Ccp1 cKO embryos.
None of the above hypotheses proved to be true. So why were there more cells invading the cortex?
The unexpected:
By this point, the project was 3 years old and although several hypotheses had been eliminated we were not closer to an explanation for our phenotype. We sat down and looked at the data. It was the moment to decide to either put the project aside or take a fresh look at the whole thing in a different light. What if the slight change of individual behaviour could influence the way the entire population moved? To help us with this new hypothesis we called upon our colleague Mohit Adhikari, a talented physicist, to help modelling the behaviour of our cells in silico. Using the parameters of speed and pause duration of migrating cortical interneurons measured by time lapse experiments, we generated trials of surrogate cells displacements. In this in silico experiment, the interneurons are challenged along the simplest path, a straight line on a 2D plane to isolate and test the effect of kinetic behaviour on the population displacement.
Displacement of cell surrogates with WT or Ccp1 cortical interneuron migration parameters. Displacement of all cell surrogates in both groups (Group A gray and Group B red); lighter marker shade indicates higher total displacement of a surrogate. Horizontal bars mark the highest displacement thresholds crossed by 75% of surrogates in each group. Duration of simulation is 600min.
The simulation allowed us to conclude that a difference in the kinetic properties of migration, such as shorter pauses and smaller jumps, increases recruitment at short distances from the starting point of CCP1 mutant interneurons in the developing cortex. We finally had a possible explanation for our phenotype, and the reason why had been staring at us from the start.
The molecular regulation:
In parallel to running in silico simulations of cortical interneurons displacement, we set out to understand the molecular regulation of migration linked with the Ccp1 mutation. We first found that acto-myosin fibres, a component of the cytoskeleton, were not contracting normally in upon loss of Ccp1 in cortical interneurons. This observation was done in time lapse video microscopy experiments when cortical interneurons were electroporated with a acto-myosin contraction fluorescent probe.
Migration of pLifeAct-Ruby electroporated E13.5 WT or cKO Ccp1 cortical interneuron homochronic mixed cortical feeder
Mutant cortical interneurons were generating almost permanent actomyosin-derived forces instead of pausing. These forces were, however, either not strong or not focused enough at the rear of the nucleus to grant correct nuclear jump. We looked at the phosphorylation state of the myosin light chain (MLC), the effector of acto-myosin contraction, and we observed hyper phosphorylation, confirming MLC over-activation. We then turned our attention to the kinase that phosphorylates MLC, MLCK3,6, and showed that it was not only a substrate of our enzyme Ccp1 in cortical interneurons but also that it was hyperactive in the mutant cortical interneurons. This showed a new molecular regulation of acto-myosin contraction and helped us understanding what lead to the abnormal movement behaviour of our mutant interneurons.
The cherry on the cake:
Finally our last question was: why is cortical invasion by cortical interneurons regulated during cerebral cortical development? We raised this question because we had noticed that the mutant brains were able to correct their number of cortical interneurons after birth by killing off the supplementary cells by apoptosis. If the problem can be corrected later why does cortical interneurons cortical invasion need to be regulated? To answer this question we looked at the cortex, the compartment receiving migrating interneurons. During our birth dating experiments, we injected BrdU to birthdate and track cohorts of cortical interneurons. We analysed BrdU injected brains at E13.5 and noticed an abnormal proliferation of the projection neuron progenitors, the intermediate progenitors in the cortex of Ccp1 mutant brains. This lead to an overproduction of projection neurons persisting in postnatal brains. This result was unexpected as our genetic recombination tool is specific to subpallial regions and does not target the cortical progenitor cell populations. Interestingly this increased proliferation was detected in regions with higher numbers of interneurons. We postulated that during development, the number of interneurons entering the cortex regulates the production of the projection neurons with which they will form connections later on. To test this hypothesis we analysed another mouse model (Nkx2.1 mutant). In these mutants brains no cortical interneurons entered the cortex and this resulted in a decreased proliferation of intermediate progenitors, suggesting again a regulatory link between the number of interneurons in the cortex and the proliferation of intermediate progenitors.
These observations are original and they unravel a regulatory crosstalk between cortical progenitors and interneurons regulating corticogenesis progression. This could be relevant for human diseases as increased cortical thickness are described in patients suffering from autism spectrum disorder 7,8.
Conclusion
Overall this work took 5 years to complete and in the end, it is far from what we expected it to be when we first started. The challenge of getting to an explanation beyond the obvious lead us to unexpected and novel results.
Attempting to understand the biology of a complex organ, like the brain, comes with an array of technical challenges. Those of us who would like to understand development in the context of a living organism are faced with an enormously complex arrangement of cells, which may be superficially indistinguishable from one another, and furthermore, are inconveniently hidden away inside the body of an animal. Typically, if we want to examine the molecular characteristics of one of the constituent cell types of an organ, we need to physically isolate these cells from their biological context before applying the relevant assay. Cell separation can be very technically challenging and has been recently reported to cause artefacts in gene expression [1]. Therefore, methods in which cell types within a complex tissue can be examined without the need to remove them from their in vivo environment are of great interest to biologists working on in vivo models. Measuring the accessibility of chromatin to external factors is commonly used as a way of detecting functional regions of the genome. Techniques such as ATAC-seq or DNAse-seq are widely used for this purpose [2]. However, before now, we had no way of performing these chromatin accessibility assays without cell separation. In our recent paper we attempted to address this problem [3].
TaDa! – Chromatin interactions the easy way:
In the Southall lab we make extensive use of the Targeted DamID system (TaDa), which circumvents some of these issues when assaying protein-DNA interactions. This is a development of the well-established DamID system in which a chromatin-binding protein of interest is tethered to the E. coli adenine methylase, Dam [4]. Dam activity is relatively promiscuous, and so methylates any DNA that it comes into proximity with. Once this methylation is detected, we can infer the binding sites of a protein of interest. Targeted DamID expands on this approach by expressing the Dam-fusion protein as a bicistronic transcript, downstream of a primary open reading frame (ORF). This allows for very low level expression of the Dam-fusion so that the animal does not experience toxicity related to high Dam expression, and the methylation levels are not saturating – allowing for a semi-quantitative readout of protein binding [5]. Crucially, this construct can be combined with tissue specific binary expression systems (e.g. Gal4/UAS), allowing us to profile chromatin-protein interactions in a cell type of interest. (A full review of DamID and its applications can be found here: [6]).
Chromatin accessibility profiling with CATaDa
For TaDa experiments to work it is necessary to perform a control experiment in which untethered Dam protein is expressed in the cell-type of interest. The data produced from this control is then used to normalise the Dam-fusion binding data so that a reliable DNA-interaction profile can be generated. (This can be considered to have an analogous function to the input chromatin in a ChIP-seq experiment). Having generated much of this control data in the course of performing TaDa experiments, we wondered whether it might have applications beyond just normalisation. As Dam is highly active, we reasoned that the signal obtained after expression of untethered Dam would represent interactions between Dam and any regions of chromatin that it can access. Therefore, we inferred that Dam-only data may be a good proxy for chromatin accessibility.
Given that we had gigabytes of Dam-only sequencing reads stored on our hard drives going unused, we were excited by the possibility that we could leverage these data into yielding further biological insights. To determine whether these data were comparable to established chromatin accessibility techniques, we compared some of our Dam data to previously published ATAC-seq or FAIRE-seq results [7]. We were excited to see that the signal from Dam methylation displayed many of the hallmarks of chromatin accessibility data that are observed with alternative techniques. For example, enriched signal at the transcription start site of genes. Overall, our data compared favourably to both ATAC and FAIRE-seq data. We found highly significant agreement between these datasets, and encouragingly, we saw that our data was comparable to ATAC-seq and out-performed FAIRE-seq in identifying previously validated enhancer regions. Therefore, we concluded that these data are appropriate for inferring meaningful chromatin accessibility data. We coined the term CATaDa (Chromatin Accessibility profiling using Targeted DamID) to describe this use of Dam methylation data for cell-type-specific profiling of chromatin accessibility.
Chromatin accessibility during neurogenesis
Our group is interested in understanding how the development of the nervous system is coordinated by changes to chromatin states. Therefore, we decided to apply CATaDa to the cells of the developing nervous system, including neural stem cells, their intermediate progeny (ganglion mother cells), and mature neurons. These data showed us that chromatin accessibility is incredibly dynamic between cell-types, with many regions becoming open or closed as development processes. Furthermore, we were able to examine regions of increased accessibility to detect enriched sequenced motifs which point towards factors that are involved specifically in various stages of nervous system development. We were also able to use our data to identify enhancers promoting cell-type specific gene expression.
It is often said that chromatin is more open in stem cells, then becomes less accessible when differentiated. This has been shown to be the case when comparing embryonic stem cells to (e.g.) neurons [8]. This feature is often stated to be a general feature of stem cells, and is often applied to somatic stem cells (such as neural stem cells), despite the fact that we could find no evidence for the existence of any data attempting to examine this question in vivo. We realised that with our CATaDa data, we could answer this question by looking at the distribution of sequencing reads across the genome in each cell type. We were able to confirm that chromatin was globally more open in neural stem cells, which then becomes restricted in fully differentiated neurons. Interestingly, we saw that intermediate cell types showed intermediate chromatin accessibility which was closer to that of their stem cell progenitors. At individual loci this was also observed – regions of high accessibility associated with stem cell genes were also seen to be accessible (albeit at lower levels) in their progeny, despite the fact that expression of some of these genes is tightly restricted. To see if these trends were consistent in other tissues, we also assayed cells of the adult midgut. We chose this tissue because in contrast to neurogenesis, intestinal stem cells continue to divide throughout the lifetime of the animal for maintenance and repair. The previously described trends were also seen in these cells. Together these data suggest that chromatin accessibility decreases during the development of somatic lineages and intermediate cell types may have unexpected plasticity despite being fully committed to their cell fate.
Conclusion
We hope that we have shown that CATaDa is a useful approach for assaying chromatin accessibility that will appeal to researchers who are interested in in vivo approaches. Although we have only demonstrated CATaDA in flies, Targeted DamiD has recently been shown to be effective in mammalian cells, therefore our method can reasonably be expected to be effective in any model organism in which transgenic animals can be obtained.
Dam-ed if you do and Damned if you don’t – When to use CATaDa:
Yes:
You want to profile chromatin accessibility of a subset of cells within a complex tissue / whole animal. (We typically start with dissected brains, but it is possible to use whole animals if the drivers used are specific enough)
You have a limited amount of starting material (approx. 1000 cells minimum).
You would like to profile the in vivo chromatin accessibility of a tissue, avoiding fixation or tissue dissociation artefacts.
You have done a DamID/TaDa experiment with a conventional dam-only control and want to get the most out of your data!
No:
You want to find the exact positioning of nucleosomes / enhancer sequences (CATaDa resolution is limited by availability of dam target motif – GATC).
You need to detect changes in chromatin accessibility over very short timescales. Dam must be expressed over a relatively long time period for sufficient methylation to be detected. Consequently, the output of the experiment in slightly different to that of e.g. ATAC-seq (i.e. ATAC-seq represents a snapshot of a population of cells frozen in time, whereas CATaDa will include signal representing all accessible regions over the period assayed).
We are currently seeking applications from post-doctoral candidates (LabEx-funded positions).
Project: Christophe Heinrich “Cellular Reprogramming and Brain Repair” was recently awarded a LabEx Attraction Package at SBRI. Our group focuses on direct lineage reprogramming of somatic cells into induced neurons as a novel cell-based therapy for brain repair. We previously showed that glial cells can be converted into functional induced neurons (Heinrich et al., PLoS Biology 2010, Nature Protoc 2011, Stem Cell Reports 2014, Nature Cell Biology 2015; Karow et al., Cell Stem Cell, 2012). We aim at reprogramming glial cells in the injured brain into neurons that integrate into endogenous networks and modulate the activity of these networks with beneficial effects.
Environment: The University of Lyon (IdEx Initiative of Excellence) and LabEx CORTEX represent an outstanding scientific environment. Research at SBRI is at the interface of development biology with a strong emphasis on stem cell biology, neurogenesis and functional neurobiology of the brain. SBRI benefits from state-of-the-art research platforms for stem cell bioengineering and provides a high-quality scientific, technological and medical environment.
Candidates: We are looking for enthusiastic and highly motivated scientists holding a PhD and willing to join a challenging research area at the edge of cellular biology and neurosciences. Applicants are expected to have a strong background and proven track record in neurosciences and/or cell biology. Experience in working with mice, cellular and molecular biology (cloning), and/or electrophysiology would be helpful. English proficiency is mandatory and strong communication skills and team spirit are expected.
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