Waddington, whose writings on the epigenetic landscape continue to influence developmental biology to this day, called the developing embryo “the most intriguing object that nature has to offer”(Waddington, 1966). The mechanisms of pattern formation and morphogenesis have fascinated biologists for centuries. One question that is difficult to answer is what are the minimal requirements for generating an embryo? To ask a more specific question, at gastrulation stages, the cells of the mammalian embryo separate into three germ layers in a precise spatial arrangement. Is the information for this arrangement present in the cells themselves or do they need cues from the surrounding environment? With the ability to culture mammalian embryonic stem cells (ESCs), we can now begin to tackle these questions by asking how much of embryonic development we can recapitulate with ESCs in a culture dish.

Nearly every paper on ESCs touts their potential for regenerative medicine and there is a large literature that seeks to differentiate ESCs to pure populations of useful cells for just this purpose(e.g. Chambers et al., 2009; D’Amour et al., 2005; Kattman et al., 2011; Ozair et al., 2012). However, embryos, and by extension ESCs, are not in the business of making pure populations but have evolved to self-organized into patterns. Thus, given the right conditions, it is possible that embryonic patterns can be generated spontaneously in culture starting from ESCs. In a recent paper in Nature Methods(Warmflash et al., 2014), we asked whether the embryonic organization of the germ layers in the very early embryo can be achieved in a culture dish beginning with pluripotent human cells.

To begin to explore this idea, we began by simply adding BMP4, a part of the embryonic signaling cascade that induces gastrulation and patterning(Arnold and Robertson, 2009), to cultures of human ESCs grown under standard conditions. The cells readily differentiated, however, the results lacked reproducible organization at the level of fate. This negative result raised the question – why do ESCs fail to generate reproducible patterns in contrast to their embryonic analogs? One difference between the embryonic and culture environments is that the embryo provides a reproducible geometric structure while typical stem cell colonies take on arbitrary sizes and shapes (Figure 1A). We thus aimed to see how much of embryonic patterning could be restored simply by controlling the colony geometry (Figure 1B). To do so, we used micropatterning technology to grow cells on 1mm or smaller patches of cell adhesion molecules (Matrigel or Laminin) surrounded by a lawn of an inert substance to which cells and protein will not adhere. While such assays had been previous employed to evaluate the effects of varying colony size on cell fate(Bauwens et al., 2008), this work did not report any spatial structure in the differentiated colonies. To our astonishment, simple geometric confinement was sufficient to almost fully restore patterning. Two days after the cells were induced to differentiate with BMP4, four distinct cell populations emerged in concentric rings along the axis of the colony. From the outside in, these layers are extraembryonic, endoderm, mesoderm, and ectoderm (Figure 1C). This is precisely the arrangement found in the mammalian embryo at the conclusion of gastrulation.

A. Scan of a large region of a standard hESC culture showing variable colony organization. B. Scan of a large region of micropatterned hESC culture. C. A single micro patterned colony (corresponding to the largest colonies in B) stained for markers of trophectoderm (Cdx2), mesoderm(BRA), and ectoderm (Sox2).

Since the differentiating signal is added at high levels to the medium, it is presented homogenously to all cells. Thus, the presence of four cell-fate territories implies that induced signaling between cells overwhelms exogenously supplied signals. How do cells use these induced signals to define position inside a colony 1 mm in size and containing approximately 2000 cells? A clue emerged from examining smaller colonies patterned onto the same slide. The smaller disks lost the inner-most fate, but preserved the outer three at the same position as measured from the colony edge! Thus, cell fates are defined using the colony border as a reference.

What signals are responsible for this long ranged communication? The direct response to BMP4 is measured by the nuclear accumulation of the activated transcription factor Smad1. Persistent BMP signaling is only seen in a narrow ring at the border of the colony and specifies the extraembryonic tissue found there. Inside the colony, a broader ring of activated Smad2, an upstream component of the Nodal signaling pathway, is responsible for inducing mesendoderm. The inner ectoderm territory is defined by the absence of both signals. Several decades of work in developmental genetics have revealed the identity of the inhibitors that restrict BMP and Nodal signaling in the embryo. When the combinations of inhibitors that give a robust embryonic phenotype(Bachiller et al., 2000; Perea-Gomez et al., 2002) are knocked down, cell fate markers spread in the expected way. Thus the boundary is defined by the loss of BMP inhibitors, while Nodal inhibitors restrict the range of the mesendodermal territory. This conclusion is reinforced by differentiating the colonies in mini-wells with high walls: when the inhibitors are prevented from escaping, the entire colony adopts the central ectodermal fate.

Beyond simply providing a reliable geometric structure, colony confinement also has profound effects on the fates that form. Unpatterned colonies form mesoderm and extraembryonic tissue (consistent with previous studies on BMP4 differentiation) but largely fail to form endodermal and ectodermal tissues. We speculate that the increased cell density that results from confinement is necessary both to achieve high enough levels of Nodal signaling to form the endodermal region, and high enough levels of inhibitors to establish the ectoderm.

But what is the relevance of a circularly symmetric colony to an embryo? The ring of mesodermal cells defined by Brachyury expression becomes mounded and displays a collection of markers such as Snail and activated Erk that are characteristic of the primitive streak (PS). Thus the micropatterned colonies appear to form a radially symmetric PS region. This is reminiscent of mutant mouse embryos that are unable to initiate the formation of an anterior-posterior (AP) axis and show a proximal and radially symmetric primitive streak(Nowotschin et al., 2013).

These experiments raise many questions. In the mouse embryo, the signals that initiate AP axis induction reside at the distal end of the visceral endoderm, the extraembryonic epithelial layer, and move to the future anterior side during gastrulation. Will the same signals, presented locally to a micropatterned colony, restrict the PS-streak like region and induce an axis? How does endogenous signaling compete with exogenously supplied BMP4 in the culture medium? The effects of secondary signals can be directly examined by growing cells an imposed flow with microfluidics to remove such signals(Moledina et al., 2012; Przybyla and Voldman, 2012). It is typically very hard to study how signals spread in intact embryos at endogenous levels, so synthetic systems permit one to test hypothesis and then return to the embryo with more targeted experiments.

Much of what we know about the mechanics and biochemistry of signaling comes from cell culture experiments, which have at best only a temporal dimension. But the spatial patterns formed by morphogens and their secondary inhibitors are a biologically relevant phenotype, for which a simple in vitro assay has been lacking until now. Our system is very amenable to long term time lapse imaging, and it will be possible follow markers for cell fates as they emerge in time, and ask, for instance, whether the speckled patterns seen with immunofluorescence are merely cells following the same trajectories but at different rates. It is also an ideal system in which to quantify signaling in space and time and thus in a more predictive way engineer tissues and organs.

Arnold, S. J. and Robertson, E. J. (2009). Making a commitment: cell lineage allocation and axis patterning in the early mouse embryo. Nat Rev Mol Cell Biol 10, 91–103.

Bachiller, D., Klingensmith, J., Kemp, C., Belo, J. A., Anderson, R. M., May, S. R., McMahon, J. A., McMahon, A. P., Harland, R. M., Rossant, J., et al. (2000). The organizer factors Chordin and Noggin are required for mouse forebrain development. Nature 403, 658–661.

Bauwens, C. L., Peerani, R., Niebruegge, S., Woodhouse, K. A., Kumacheva, E., Husain, M. and Zandstra, P. W. (2008). Control of human embryonic stem cell colony and aggregate size heterogeneity influences differentiation trajectories. Stem Cells 26, 2300–2310.

Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M. and Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27, 275–280.

D’Amour, K. A., Agulnick, A. D., Eliazer, S., Kelly, O. G., Kroon, E. and Baetge, E. E. (2005). Efficient differentiation of human embryonic stem cells to definitive endoderm. Nat Biotechnol 23, 1534–1541.

Kattman, S. J., Witty, A. D., Gagliardi, M., Dubois, N. C., Niapour, M., Hotta, A., Ellis, J. and Keller, G. (2011). Stage-specific optimization of activin/nodal and BMP signaling promotes cardiac differentiation of mouse and human pluripotent stem cell lines. Cell Stem Cell 8, 228–240.

Moledina, F., Clarke, G., Oskooei, A., Onishi, K., Günther, A. and Zandstra, P. W. (2012). Predictive microfluidic control of regulatory ligand trajectories in individual pluripotent cells. Proc Natl Acad Sci USA 109, 3264–3269.

Nowotschin, S., Costello, I., Piliszek, A., Kwon, G. S., Mao, C.-A., Klein, W. H., Robertson, E. J. and Hadjantonakis, A.-K. (2013). The T-box transcription factor Eomesodermin is essential for AVE induction in the mouse embryo. Genes Dev 27, 997–1002.

Ozair, M. Z., Noggle, S., Warmflash, A., Krzyspiak, J. E. and Brivanlou, A. H. (2012). SMAD7 Directly Converts Human Embryonic Stem Cells to Telencephalic Fate by a Default Mechanism. Stem Cells 31, 35–47.

Perea-Gomez, A., Vella, F. D. J., Shawlot, W., Oulad-Abdelghani, M., Chazaud, C., Meno, C., Pfister, V., Chen, L., Robertson, E., Hamada, H., et al. (2002). Nodal antagonists in the anterior visceral endoderm prevent the formation of multiple primitive streaks. Dev Cell 3, 745–756.

Przybyla, L. M. and Voldman, J. (2012). Attenuation of extrinsic signaling reveals the importance of matrix remodeling on maintenance of embryonic stem cell self-renewal. Proc Natl Acad Sci USA 109, 835–840.

Waddington, C. H. (1966). Principles of Development and Differentiation. New York: Macmillan.

Warmflash, A., Sorre, B., Etoc, F., Siggia, E., & Brivanlou, A. (2014). A method to recapitulate early embryonic spatial patterning in human embryonic stem cells Nature Methods, 11 (8), 847-854 DOI: 10.1038/nmeth.3016

(7 votes)

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This is the first of four posts relating to the Future of Research symposium which was announced in a previous blog post. Each of these posts will discuss a topic that is the focus of a workshop at the Symposium. Even if you can’t attend, please tweet @FORsymp with suggestions, or follow us to respond to our questions about what YOU, trainee scientists, think is important. The hashtag for this post on the Training workshop is: #FORtraining

Who is a “trainee”?

By training, we are referring to the period between obtaining an undergraduate degree getting a first faculty-level position: graduate students and postdocs.

At this point in time, the common thread to training consists of the accumulation of research experience, culminating first in a dissertation for defending a PhD thesis, and then to produce science (most directly in the form of publications) during postdoctoral research, designed to be a more independent research environment under the scope of a Principal Investigator. The goal is then for a trainee to be able to find an academic position, beginning their own lab.

How many trainees are there, and how has this changed over time?

The NIH Biomedical Research Working Group Report (2012) makes for sobering reading. The proportion of PhDs moving into tenured or tenure-track positions decreased from 34 % in 1993 to 26 % in 2012, whilst the proportion of non-tenured faculty has remained constant but increasing in terms of actual numbers. The number of graduate students has doubled in the same time. Numbers of postdocs are harder to track, because nobody seems to, but estimates are that these numbers have also doubled (the NIH Biomedical Research Working Group Report implies that even this could be under-estimated by as much as a factor of 2). Research Training in the Biomedical, Behavioral, and Clinical Research Sciences, by the National Research Council (2011), suggests that as of 2011 there may have been just over 50,000 trainees in the biomedical research system.

How long is one a trainee?

In the US system, PhDs can take anywhere between 5 and 10 years. Postdoctoral training has been identified by the NIH as only being 5 years on average, but the number of people doing up to 8, or even 10 and beyond, has been steadily increasing in recent years. Some institutions put a cap on how long someone can be a postdoc, with a requirement to then make them a research associate.

What trainees expect, and where they end up

The assumption is that everyone who goes into academia wants to end up an academic. Is this the case? Expectations have been shown to change over time as a trainee, with a recent study showing that a faculty position tends to become less attractive over the course of a PhD, although by the end it is still the preferred career path for 50 % of trainees (Sauermann and Roach, 2012). This is in spite of strong encouragement from advisor, who actively discourage other career paths (Sauermann and Roach, 2012).

And the jobs people are actually getting (taken from Polka, 2014) show that, actually, academia is one of the “alternative” careers, and not the default. Less than 10 % of entering PhD students become tenure-track faculty: contrast this to the 50 % identified previously as having academia as their primary goal (Sauermann and Roach, 2012).

Taken from Polka, 2014

What are trainees supposed to accomplish?

This workshop is first going to discuss the question, “In what way are trainees unprepared?” and then depending on what attendees come up with, will then ask, “What are the solutions to these problems?” Then attends will try to arrange these according to feasibility/difficulty of implementation. If there is time, the group will discuss how these fit into the overarching themes of making science efficient, and trying to figure out if these help achieve the greater “goals of science”.

References

National Research Council. 2011. Research Training in the Biomedical, Behavioral, and Clinical Research Sciences. Washington, DC: The National Academies Press.

NIH Biomedical Research Working Group Report (2012)

Sauermann H, Roach M (2012) Science PhD Career Preferences: Levels, Changes, and Advisor Encouragement. PLoS ONE 7(5): e36307. doi:10.1371/journal.pone.0036307

Polka J, (2014) Where will a Biology PhD take you?

(3 votes)

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During mouse preimplantation development, the zygote divides and forms three distinct lineages: one embryonic called the Epiblast (Epi) and two extraembryonic called trophectoderm (TE) and Primitive Endoderm (PrE). The first cell fate decision occurs at the morula stage (from 16-cell to 32-cell) between TE and the Inner Cell Mass (ICM) and the second cell fate decision occurs within the ICM that gives rise to the Epi and the PrE in a salt-and-pepper organization during blastocyst formation(Artus and Chazaud, 2014). The complete specification of these three lineages is crucial for correct developmental progression. Moreover, the ICM represents the stem cell niche of Embryonic Stem Cells (ES cells) derived in 1981(Evans and Kaufman, 1981; Martin, 1981). Studying ES cells specification in vivo is important for their better use in regenerative medicine.

Previous research shows that Gata6 and Nanog are key transcription factors for the Epi/PrE cell fate specification. Whereas these two markers are co-expressed in all ICM cells at morula stage, their expression becomes mutually exclusive, asynchronously leading to intermingled Gata6-positive PrE cells and Nanog-positive Epi cells (Figure 1). Moreover, the Fgf/RTK signaling is sufficient and necessary to modulate the salt-and-pepper organisation by controlling Gata6 and Nanog expression (Artus and Chazaud, 2014).

Figure 1 : Expression profile of Gata6 and Nanog during preimplantation development.

We re-examined the role of Gata6 and showed that this transcription factor is required for PrE specification (Figure 2). The PrE defects cannot be rescued by Fgf4 administration, meaning that the cells need Gata6 function to be able to respond to the Fgf signal. These series of experiments also showed that, like in vitro, Nanog expression can be inhibited by the RTK pathway independently of Gata6 function, suggesting a direct repression. Interestingly, by examining Gata6 heterozygotes we also show that the amount of Gata6 is important not only to determine the Epi/PrE ratio but also to regulate the timing of Epi specification and determination. Some of these findings were also reported by Schrode and collaborators (Schrode et al., 2014).

Figure 2: E3.75 immunofluorescence against Cdx2 (blue), Nanog (green) and Gata6 (red).

All these observations suggest that Nanog, Gata6 and the Fgf4/Fgfr pathway act as a regulatory network to specify Epi and PrE cells. Despite progress in understanding the basic principles of stem cell niches, other techniques are required to define the dynamic spatio-temporal induction of salt-and-pepper. One alternative approach is mathematical modeling, based on the translation of biological interactions into mathematical equations. The main feature is to follow the dynamics of factors through time in a defined context.

We developed a fruitful collaboration with the Theoretical Chronobiology Laboratory (ULB, Brussels- Belgium). The aim was to develop a mathematical model based on the regulatory network, using published data combined with our results on the analysis of Gata6 mutants (also described in this publication). The model is defined by a set of differential equations. Three of them represent three variables corresponding to the level of intracellular proteins: Gata6, Nanog and Fgfr2. A fourth one represents the activity of Fgfr/Erk signaling, which depends on the extracellular concentration of Fgf4, the fifth variable of the system (Figure 3).

Figure 3: Representation of the regulatory network and its corresponding equations.

The model uncovers three stable steady states (tristability): an ICM-like state where Gata6 and Nanog are co-expressed, a PrE-like state where only Gata6 is expressed and an Epi-like state where only Nanog is expressed.

Simulations show that the model is sufficient to observe Epi and PrE cell specification and resume in vivo findings: 1/ Epi and PrE cells are organized in a salt-and-pepper manner, 2/ this organization is obtained by an asynchronous mechanism, 3/ Epi cells are specified earlier compared to PrE cells and 4/ Epi/PrE ratios obtained fit with in vivo results. The model also recapitulates cell behaviours in the different mutants (Nanog^-/-, Gata6^-/-, Gata6^-/+) (Frankenberg et al, 2011; Bessonnard et al, 2014).

After reaching the ICM state, a subset of cells favours Nanog expression and acquire the Epi-like state. In response to Nanog, cells secrete Fgf4 and modify their local concentration in the neighboring intercellular space. The local increase of Fgf4 is sufficient to push neighboring cells to favour Gata6 expression and reach the PrE-like state. Interestingly, simulations show that only one Nanog-expressing cell could be sufficient to induce the salt and pepper pattern.

The heterogeneity of Fgf4 distribution is crucial to resume the salt-and-pepper organization. It is inherent to the mechanism and causes the asynchrony in cell specification. Thus the model mathematically explains the in vivo observation in which Fgf4 mutants cultured in presence of recombinant Fgf4 cannot restore the salt and pepper pattern and act in a binary fashion (Kang et al., 2013; Krawchuk et al., 2013).

Even if we do not know how one or few cells favour Nanog expression, the following steps of the model are deterministic. Several hypotheses on how Nanog expression is promoted are proposed. The model suggests that it is the consequence of a pErk decrease, following the diminution and dilution of an early Fgf4 expression that was observed by Guo and collaborators (Guo et al., 2010). Alternatively, differences in pErk or Nanog expression could be influenced by cell position (Morris et al., 2010) or stochastically induced (Ohnishi et al., 2014; Yamanaka et al., 2010). More experiments will be required to answer this highly debated question.

In conclusion, this close collaboration enabled us to propose new insights for the salt-and-pepper induction in vivo that will be also useful for stem cell research. More generally, our study is a good example to show that experimental biology and mathematical modeling can synergize to provide insightful results and hypotheses.

Acknowledgments: I thank Dr. Claire Chazaud, Dr. Prudence Donovan and Pr. Albert Goldbeter for reading the article.

Artus, J., & Chazaud, C. (2014). A close look at the mammalian blastocyst: epiblast and primitive endoderm formation Cellular and Molecular Life Sciences, 71 (17), 3327-3338 DOI: 10.1007/s00018-014-1630-3

Bessonnard, S., De Mot, L., Gonze, D., Barriol, M., Dennis, C., Goldbeter, A., Dupont, G., & Chazaud, C. (2014). Gata6, Nanog and Erk signaling control cell fate in the inner cell mass through a tristable regulatory network Development, 141 (19), 3637-3648 DOI: 10.1242/dev.109678

Evans, M., & Kaufman, M. (1981). Establishment in culture of pluripotential cells from mouse embryos Nature, 292 (5819), 154-156 DOI: 10.1038/292154a0

Frankenberg, S., Gerbe, F., Bessonnard, S., Belville, C., Pouchin, P., Bardot, O., & Chazaud, C. (2011). Primitive Endoderm Differentiates via a Three-Step Mechanism Involving Nanog and RTK Signaling Developmental Cell, 21 (6), 1005-1013 DOI: 10.1016/j.devcel.2011.10.019

Guo, G., Huss, M., Tong, G., Wang, C., Li Sun, L., Clarke, N., & Robson, P. (2010). Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst Developmental Cell, 18 (4), 675-685 DOI: 10.1016/j.devcel.2010.02.012

Kang, M., Piliszek, A., Artus, J., & Hadjantonakis, A. (2012). FGF4 is required for lineage restriction and salt-and-pepper distribution of primitive endoderm factors but not their initial expression in the mouse Development, 140 (2), 267-279 DOI: 10.1242/dev.084996

Krawchuk, D., Honma-Yamanaka, N., Anani, S., & Yamanaka, Y. (2013). FGF4 is a limiting factor controlling the proportions of primitive endoderm and epiblast in the ICM of the mouse blastocyst Developmental Biology, 384 (1), 65-71 DOI: 10.1016/j.ydbio.2013.09.023

Martin, G. (1981). Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proceedings of the National Academy of Sciences, 78 (12), 7634-7638 DOI: 10.1073/pnas.78.12.7634

Morris, S., Teo, R., Li, H., Robson, P., Glover, D., & Zernicka-Goetz, M. (2010). Origin and formation of the first two distinct cell types of the inner cell mass in the mouse embryo Proceedings of the National Academy of Sciences, 107 (14), 6364-6369 DOI: 10.1073/pnas.0915063107

Ohnishi, Y., Huber, W., Tsumura, A., Kang, M., Xenopoulos, P., Kurimoto, K., Oleś, A., Araúzo-Bravo, M., Saitou, M., Hadjantonakis, A., & Hiiragi, T. (2013). Cell-to-cell expression variability followed by signal reinforcement progressively segregates early mouse lineages Nature Cell Biology, 16 (1), 27-37 DOI: 10.1038/ncb2881

Schrode N, Saiz N, Di Talia S, & Hadjantonakis AK (2014). GATA6 levels modulate primitive endoderm cell fate choice and timing in the mouse blastocyst. Developmental cell, 29 (4), 454-67 PMID: 24835466

Yamanaka, Y., Lanner, F., & Rossant, J. (2010). FGF signal-dependent segregation of primitive endoderm and epiblast in the mouse blastocyst Development, 137 (5), 715-724 DOI: 10.1242/dev.043471

(4 votes)

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Here are the highlights from the current issue of Development:

Modelling fate decisions in the early mouse embryo

In the early embryo, the first fate decision separates the trophectoderm from the inner cell mass (ICM). Subsequently, the ICM segregates into epiblast (Epi) and primitive endoderm (PrE), but how do cells decide which of these two fates to adopt? Claire Chazaud, Geneviève Dupont and co-workers address this question in the early mouse embryo (see p. 3637), using both experimental and modelling approaches. It is known that the FGF signalling pathway is involved in this fate decision and also that, while Epi cells express and depend on Nanog, PrE cells are defined by Gata6 expression. The authors complete the interaction network by demonstrating that Gata6 is not only expressed by the PrE but is also required for PrE specification. To understand the network in greater detail, the authors build a mathematical model incorporating the known interactions between these players. The model exhibits tristability, with steady states corresponding to cells displaying ICM, Epi or PrE fate, and is able to recapitulate experimental manipulations of the system, such as the removal of Nanog or Gata6 activity or the effects of altering FGF signalling. Moreover, this approach highlights a number of key features of the system – most notably the requirement for heterogeneity in FGF signalling levels and the self-organising nature of the process of fate determination in this context.

First bird born via ICSI

Intra-cytoplasmic sperm injection (ICSI) is a method of fertilisation that involves the injection of a single sperm into an oocyte. The technique has been applied successfully to generate viable offspring in human, mouse and other vertebrates, but never before has a live bird been produced using ICSI due to difficulties in mimicking avian polyspermic fertilisation. Now, on p.3799, Tomohiro Sasanami and colleagues report the birth of a live quail chick using ICSI and identify some critical parameters that hitherto prevented the successful application of this technique. The authors inject a single sperm together with sperm extract, of which they identify three different components essential for full egg activation and development: phospholipase Cζ, aconitate hydratase and citrate synthase. Importantly, the authors use this system to analyse Ca²⁺ dynamics during egg activation and show that, in contrast to mammals, avian egg activation requires two distinct patterns of Ca²⁺ flux: an initial transient rise and a long-lasting spiral-like oscillation. These findings represent an exciting step forward in the study of vertebrate egg activation and will provide new opportunities for the production of transgenic and cloned birds.

Brain keeps a stiff upper lip during development

Physical properties such as tissue stiffness have been shown to influence stem cell fate in vitro. It is possible that spatiotemporal changes in stiffness may influence tissue formation during development; however, it has been difficult to analyse this due to the technical limitations of measuring stiffness in embryonic tissues. In this issue (p. 3793), Yoichi Kosodo and colleagues overcome this problem and provide a novel approach to measuring changes in stiffness using atomic force microscopy combined with immunostaining in the embryonic mouse cerebral cortex during development. By combining these two techniques, the authors show that all layers in the developing cortex undergo significant changes in stiffness throughout the embryonic stages. In the ventricular and subventricular zones, stiffness gradually increases throughout development, while the intermediate zone and cortical plate show an initial increase in stiffness followed by a decrease before birth. Deconstruction of the role of cell and matrix in modulating stiffness in the developing brain reveals that tissue stiffness cannot be solely determined by the stiffness of the cells that constitute the tissue. This study not only sheds light on the spatiotemporal dynamics of stiffness in the developing mouse brain, but also provides a possible approach for understanding the global profile of physical properties in other developing organs.

Mesoderm gets a move on during Xenopus involution

Gastrulation occurs when a single-layered blastula transitions to a gastrula containing all three germ layers: ectoderm, endoderm and mesoderm. Many aspects of vertebrate gastrulation have been well-characterised using the model vertebrate Xenopus, but surprisingly little is known about how the dorsal mesoderm moves from the outer surface to the interior of the gastrula. Now, on p. 3649, Rudolf Winklbauer and colleagues analyse the cellular and molecular dynamics that are central to this involution process and provide several insights into the mechanism of Xenopus gastrulation. First, the authors show that the blastopore lip is capable of autonomous shape changes and that different domains, for example those marked by Goosecoid and Brachyury (Xbra), show differences in cellular migration patterns. Second, the authors investigate the molecular requirements during these events and show that the tyrosine kinase ephrin receptor EphA4 is transiently required to maintain appropriate levels of Xbra expression, but that this occurs indirectly via p21-activated kinase 1 (Pak1). These data highlight a role for Xbra during mesoderm involution in Xenopus and shed light on the general features of dynamic cell migration during gastrulation.

DMRT6 gives spermatogenesis the ‘go-ahead’

The mitotic-to-meiotic transition during spermatogenesis is essential for generating haploid spermatid cells from diploid spermatogonial cells. After differentiation and proliferation of spermatogonial cells, two rounds of meiosis are required to generate spermatid cells, but how the complex transition from mitosis to meiosis is coordinated remains unclear. In this issue (p. 3662), David Zarkower and colleagues investigate the role of theDoublesex-related gene Dmrt6 in male mouse spermatogenesis and show that without DMRT6 this process is severely compromised. By creating a null mutation in two different genetic backgrounds, the authors provide strong evidence that DMRT6 is required for spermatogonial differentiation (C57BL/6J strain) and meiosis (129Sv strain) and that inappropriate gene expression and chromatin events occur when DMRT6 activity is absent. Subsequent mRNA profiling and chromatin precipitation experiments suggest that DMRT6 functions both to suppress genes that promote spermatogonial differentiation and to activate important meiotic regulatory genes. These data provide novel insight into the molecular regulatory events that control and coordinate the mitotic-to-meiotic transition during mammalian spermatogenesis.

PLUS…

Adult neurogenesis: taking stock in Stockholm

In May this year, Stockholm hosted a Keystone Symposium on Adult Neurogenesis, attracting scientists from around the world despite the lack of customary snow. The symposium offered an extraordinary program, covering diverse topics that ranged from the neural stem cell lineage and regulation of neurogenesis to functional aspects of neurogenesis in homeostasis and disease, and even computational modeling. Here, Nambirajan Govindarajan and Gerd Kempermann describe some of the exciting presentations and emerging themes from the symposium, which reveal how much this young field has matured. See the Meeting Review on p. 3615

Poised chromatin in the mammalian germ line

< Poised (bivalent) chromatin is defined by the simultaneous presence of histone modifications associated with both gene activation and repression. This epigenetic feature was first observed at promoters of lineage-specific regulatory genes in embryonic stem cells in culture. More recent work has shown that, in vivo, mammalian germ cells maintain poised chromatin at promoters of many genes that regulate somatic development, and that they retain this state from fetal stages through meiosis and gametogenesis. We hypothesize that the poised chromatin state is essential for germ cell identity and function. Here, Bluma Lesch and David Page propose roles for poised chromatin in the mammalian germ line and discuss these roles in the context of recently proposed models for germline potency and epigenetic inheritance. See the Hypothesis article on p. 3619

Ultradian oscillations and pulses: coordinating cellular responses and cell fate decisions

Biological clocks play key roles in organismal development, homeostasis and function. In recent years, much work has focused on circadian clocks, but emerging studies have highlighted the existence of ultradian oscillators – those with a much shorter periodicity than 24 h. Accumulating evidence, together with recently developed optogenetic approaches, suggests that such ultradian oscillators play important roles during cell fate decisions, and analyzing the functional links between ultradian oscillation and cell fate determination will contribute to a deeper understanding of the design principle of developing embryos. Here, Akihiro Isomura and Ryoichiro Kageyama discuss the mechanisms of ultradian oscillatory dynamics and introduce examples of ultradian oscillators in various biological contexts. See the Review on p. 3627

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Every two years, the international Xenopus community gathers to meet. Two years ago I wrote about the meeting in Toulon, France and this time we crossed the pond to Asilomar, CA, just south of San Francisco. Cat Vicente recently wrote about Asilomar in the context of conference venues whilst attending the Genetics Society of America Xenopus conference for the Node.

The conference gives a chance for researchers in various fields, but with the common model system of Xenopus, to discuss their work. The meeting this year took a new direction in not having themed sessions, but rather mixing talks from very different fields into each session. This had the dual purpose of keeping everyone’s attention by switching between different subjects rapidly, but also ensuring people were more likely to stay around for talks outside their field. There was also a strong focus on getting junior faculty and postdocs to speak rather than just the usual senior researchers that are well-known. For these reasons, I personally found this the most engaging conference I have been to.

Keynote talks

There were special lectures given by John Gurdon (Gurdon Institute, University of Cambridge) and Marc Kirschner (Department of Systems Biology, Harvard Medical School), both senior figures in the Xenopus community. John’s talk in particular gave an historical perspective on the beginnings of Xenopus as a model organism, including the transport of frogs from South Africa to the UK by Lancelot Hogben. Apparently, in the early days he was forced to keep his frog colony in the crypt of a church and managed to balance the books by charging hospitals to use frogs as pregnancy tests: when injected with the urine of pregnant women, the hormones present would induce the release of eggs from the female frogs. And from such humble beginnings, the research community has grown, with of course achievements such as John’s Nobel Prize. John speculated on the possible future for research in the frog and the aspiration to be able to track the behavior of individual proteins, within a cell, in real time.

John Gurdon’s special lecture: a historical perspective on the field of Xenopus research.

The keynote talk was given by Rebecca Heald of University of California, Berkeley. Rebecca’s lab focuses on questions to do with size and scaling of cells. In particular, looking at frogs there are basic questions about different species and different stages of development. Xenopus laevis eggs are nearly twice as big as Xenopus tropicalis and yet they are both single cells and, upon fertilization, undergo cleavage in the same way. How does this relate to the DNA content of the cells and how is this remarkable feat of engineering, in an cell so large, achieved? Also, as frog embryos develop, cells continuously divide whilst the embryo as a whole does not grow. What are the implications of maintaining a similar mechanism of cell division, starting in a cell as large as 1 mm in diameter and ending up at a fraction of the size? Work in frog egg and embryo extract is a significant part of the Xenopus field but not often universally used by frog researchers and it was an overview of some of the amazing work that can be achieved with the simple yet versatile extract system.

Rebecca Heald’s tribute to the “Founding Froggers”. Photo courtesy of Cat Vicente.

The community

The frog community also has numerous resources that we simply couldn’t do without: you can check out all things frog at Xenbase and there are three resource centres around the world: the National Xenopus Resource (NXR) in Wood’s Hole, MA; the European Xenopus Resource Centre (EXRC) at the University of Portsmouth, UK; and the National BioResource Project (NBRP) in Hiroshima, Japan which have stocks of wild type and transgenic frogs as well as components such as DNA clones and antibodies. All of these groups presented updates on work they are doing to support the community, such as the genome-editing workshop planned for November 2014 at Wood’s Hole.

Two special workshops were also arranged. One for trainee researchers looking for career advice both in academic and non-academic areas; and one for junior faculty looking for advice on getting tenure. I attended the PhD/postdoc session (as did the Node’s Cat Vicente – as a speaker for non-academic careers) and although the outlook is, as ever, sobering, it was still good to hear some advice specific to people who had worked on frog and some key things to keep in mind. Especially that it’s never too early to be thinking about the next step.

These are both key examples of why I’ve enjoyed my scientific training in the Xenopus community. I started off after finishing my Master’s in Chemistry and moved into a frog lab, and have worked in 3 frog labs/projects since. The sense of community and support provided by the Xenopus field is really quite unique and many who have passed through frog meetings have commented on this. There are some key figures driving the support of more junior faculty and trainees, which is sadly not the norm across all fields of academia.

Looking forward

Work in the model organism Xenopus seems to be really taking off because of all the different techniques that are available to frog researchers. Genetics and genome editing, with TALENs and the CRISP/Cas system, have expanded recently, particularly using the diploid genome of Xenopus tropicalis, but even the pseudoallotetraploid genome of Xenopus laevis, for so long considered a disadvantage, is by its nature of being so unique and outbred generating a lot of questions that other inbred model systems simply can’t answer. The use of Xenopus extracts for in vitro biochemical work was showcased in particular by Rebecca Heald’s lab and the questions that can be answered by the cell-in-a-test-tube system that Xenopus provides still leaves many frog researchers surprised. The rapid development and easy manipulation of frog embryos also allows many simple but elegant in vivo experiments. So, in reality, frogs can be used to answer a whole lot of questions rapidly (and cheaply). I will try to expand upon some of these themes in the near future; needless to say I don’t have time here except to mention what an adaptable model system is across many fields, from bioengineering to biochemistry to biophysics.

Frog friends! Photo courtesy of Atsushi Suzuki, from the National BioResource Project, Japan.

This was my third Xenopus conference; each time I’m unsure if I’ll be at the next one, so I always make sure to enjoy the company of my froggy friends; we always make sure to have a good time!

If you want to keep up with frog-related updates, you can follow various twitter accounts for Xenbase @Xenbase, the National Xenopus Resource at the Marine Biological Laboratory at Wood’s Hole @XenopusNXR and I also blog at bewareofthefrog.tumblr.com and @BiophysicalFrog.

(7 votes)

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[Apologies for cross-postings!]

Call for Papers – IRCSHM 2014

October 15-16,

Dubai, UAE

Dear Colleague,

The unique idea behind International Research Conference on Science, Health and Medicine 2014 (IRCSHM 2014) is to provide an opportunity for leading academicians, scientists, researchers, doctors, medical practitioners, paramedical specialists and industry professionals from around the world to network and have scientific discussion on the latest advancements in the closely interlinked domains in science, medicine and health and it’s research benefits for each other’s domain progress.

IRCSHM 2014 will address multiple topics and issues of interest in the areas of science, medicine and health by practical exposure in the form of specialized sessions, poster presentations, plenary sessions and renowned speeches from the leading practitioners reinforcing the upcoming challenges to be faced and their potential solutions. .

We are emailing you this Call for Papers, hoping that you will consider submitting a paper for IRCSHM 2014 which will be held in Corp. Executive Al Khoory Hotel, Dubai, UAE during October 15-16, 2014. For further details, please visit www.ircshm.com.

The types of articles that will be considered for publication include case studies, reviews, as well as theoretical and empirical research. All submitted manuscripts are peer reviewed, and decisions about a manuscript are based only on the importance, originality, clarity, and contribution of the submission to knowledge in the conference scope.

According to this initiative selected invited extended version of best quality papers presented in IRCSHM 2014 will be recommended for publication consideration with Indexed Journals according to the participating Journal publishing policies and requirements.

1. International Journal of Emerging Technology and Advanced Engineering (IJETAE)(Special Issue) (ISSN 2250–2459(Online)), IJETAE is indexed in many professional databases, IJETAE Impact Factor of 1.932 as per International Society for Research Activity for the year April 2013.

2. International Journal of Recent Development in Engineering and Technology (IJRDET) (Special Issue) (ISSN 2347 – 6435 (Online)), IJRDET is indexed in many professional databases.

3. Medical Science (MS)(Special Issue) (ISSN 2321 – 7359; EISSN 2321 – 7367), MS is indexed in many professional databases.

4. Bioengineering and Bioscience (Special Issue) (ISSN: 2332-0028 (Online)), is is indexed in CABI, Index Copernicus, EBSCO A-Z, J-Gate, JournalTOC, Google Scholar, WorldCat many other professional databases.

The paper submission extended deadline is: 25th September 2014.  You can submit your research paper via email to ircshm@gmail.com.

The topics of interest for submission include, but are not limited to:

Please try to forward this message to as many potentially interested people as possible. For further updates, please visit www.ircshm.com or email to ircshm@gmail.com.

Best Regards,

Organising Secretary,

IRCSHM 2014.

(1 votes)

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The Node is on the road again, but this time not very far! We are going to attend the EMBO conference on interdisciplinary plant development, which starts this Sunday (21st September) at the Sainsbury Laboratory here in Cambridge (UK). We are planning on tweeting with the hashtag  #EMBOplantdev, and will try to include some photos of their beautiful building and the botanical gardens around it! If you are attending the meeting, our community manager will be there, and we look forward to meeting you! We are also looking for someone to blog about the meeting, so if you are interested drop us an email!

(1 votes)

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The Institut Pasteur (Paris, France) announces an international call for an outstanding mid-career/senior candidate wishing to establish a new research group in the field of “Stem Cells and Development”. The recruitment is part of the Revive Laboratory of Excellence(LabEx – http://www.pasteur.fr/revive) programme on “Stem Cells and Regenerative Biology and Medicine”. Candidates will be integrated into the cutting edge interdisciplinary environment provided by the Department of Developmental & Stem Cell Biology.

The successful candidate will develop a program specializing in the fields of stem cells, genetics, regeneration, translational research or ageing. He/She will be integrated in an internationally renowned Institute combining fundamental and translational research, in an attractive location in central Paris, in close proximity to other major research centres.

Applications will be evaluated on the basis of scientific excellence. A highly attractive package to match the experience of the candidate will be provided.

Candidates should send their formal applications by E-mail to the Director of Scientific Evaluation, Prof. Alain Israël, at the Institut Pasteur g5revive@pasteur.fr.

Application deadline: December 21, 2014

Short-listed candidates will be invited for interview.

Further information on the Institute and on-campus facilities can be found at http://www.pasteur.fr.

Applicants should provide the following (in order) in a single pdf file:

1. A brief introductory letter

2. A Curriculum Vitae, 10 most important publications and a full publication list

3. A description of past and present research activities (up to 2 pages with 1.5 spacing; Times 11 or Arial 10 font size).

4. The proposed research project (up to 4 pages with 1.5 spacing; Times 11 or Arial 10 font size).

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BSMB/BSDB Joint meeting – The Musculoskeletal System: from development to disease

1^st -3^rd September 2014, University of East Anglia

The first joint meeting of the BSMB and BSDB was, in our opinion, a great success. The meeting was held over three days and was packed with brilliant science from areas of musculoskeletal research from the talks, posters and general discussions. The conference was held in the newly built Julian Study Centre at the University of East Anglia, which boasts well-equipped lecture theatres and small meeting rooms, with inviting break out spaces.

The opening keynote speaker, Tom Rando, provided a brilliant start to the conference with a fascinating account of his group’s work into a primed state of quiescent stem cells, which turned out to be one of the highlights of the conference for me (DB), as it’s closely related to my research. This was followed by an exciting afternoon of presentations within the fields of signalling and development. Standout talks from this session included Gabrielle Kardon on the development of the diaphragm, Malcolm Logan on muscle and tendon formation in the limb and Christine Hartmann on bone development. One great feature of the conference was the opportunities offered to PhD students and post-docs to present their work should they be selected from submitted abstracts. I (GFM) had my abstract selected and was able to present my work during this session along with Anne-Gaelle Borycki, Dylan Sweetman, Susanne Dietrich, Clare Thompson and Sue Kimber. The first day ended with an evening poster session and reception.

The atmosphere at the poster session was exciting and stimulating, fuelled by a couple of glasses of free wine and nibbles, conversations around posters were bustling.  This was my (DB) first opportunity to present some of my PhD work in the form of a poster and the sessions allowed for brilliant discussions of my (DB) work with other students working in the field, but also with experienced researchers. It felt great to be able to discuss and get feedback on my work from my peers.

The second day consisted of two sessions, the first focusing on ‘Mechanobiology and Anatomy’. The session started with a fascinating talk from Eli Zelzer, with his group’s novel take on the mechanics of fracture repair. Other great talks included Mario Giorgi on the role of fetal movement in joint morphogenesis and Chrissy Hammond on jaw development in zebrafish. After a quick coffee break, I (GFM) was hugely impressed with talks by Ronen Schweitzer on tendon growth in the mouse limb and Andy Pitsillides on his group’s work on limb growth and joint formation.

The afternoon session was on ‘Human Genetics and Pathology’ with talks by Mike Briggs, Qing-Jun Meng, Madelaine Durbeej, Linda Troeberg and Veronique Lefebvre. The talks offered a great insight into the current research into various dystrophies and we think this was another ‘plus point’ for the meeting as it allowed the combination of developmental biologists and matrix biologists to share ideas with each other, thus initiating stimulating discussions and possible collaborations.

The conference dinner was held at St. Andrews Hall in the centre of Norwich, a grade 1 listed building dating back to the 14^th Century. This provided a great venue and atmosphere for more relaxed discussions of the days events. The three-course meal included salmon, pan-fried lamb and lemon tart (plus unlimited wine!).

The final day played host to the final session on ‘Transcriptional and Epigenetic Regulation’ with great talks by Cay Kielty on genotypes of fibrillinopathies and Simon Tew on gene regulation in chondrocytes. The BSMB also awarded the Young Investigator Award to Blandine Poulet and she gave a lovely talk on her work on modelling osteoarthritis. The final keynote speaker, David Glass, in my opinion (GFM) gave the most fascinating and entertaining talk on his group’s work in developing an antibody treatment for patients with muscular atrophies. It was a great talk to end a fantastic conference. The final session of the day concluded with prizes awarded for PhD students and post-docs for best talk and best posters judged by the invited keynote speakers – this was a great incentive for those who presented their posters and a great way to network.

We would like to give our thanks and congratulations to everyone involved in organising the conference (Ulrike Mayer, Andrea Munsterberg, Graham Riley, Ian Clarke and Tonia Vincent. It was a privilege to be involved in the first joint meeting of the BSMB and BSDB, which we thought was a great success between the two societies and their members, and hope that there will be future joint meetings again. We would also like to thank the BSDB/Company of Biologists for providing travel awards for us to attend.

Danielle Blackwell (PhD student) and Gi Fay Mok (Post Doc), Münsterberg lab, UEA

(3 votes)

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Last month I was lucky enough to attend the Xenopus meeting, which took place at the Asilomar conference grounds, close to Monterey, California. This meeting was fantastic, combining great science with a great venue. As there is already someone lined up to write about the scientific aspects of the meeting, I thought I would focus here instead on the venue.

Asilomar is one of the best conference venues I have visited so far. The grounds consist of cabin-style buildings (with fireplaces!) in a pleasant wood, which include accommodation, conference room and cafeteria. Just across the road is the beach and the beautiful californian coastline, there is a swimming pool, and we are surrounded by nature: deer and squirrels in the woods, and whales that can be seen from the coast! Though its natural beauty is definitely a winning feature, what I particularly liked about Asilomar conference grounds was its relative isolation. Conferences are as much about the talks as the interesting discussions that take place in between. When a conference takes place in a big city, attendees tend to stay in different hotels and go their own way after the afternoon sessions, in search of restaurants for dinner. This reduces quite significantly the possibility of chance encounters and hence the opportunity to meet new people and discuss science! This was not a problem in Asilomar, as all the attendees stayed at the grounds and ate all their meals there. And as it was not in a famous world city, there was less of a chance that anyone would skip a session to visit a nearby world attraction! The serene and relaxing location, where you could hear the sea in the nearby beach, kept you could focus on the science and on discussions with the other attendees. At the same time, Asilomar is close enough to Monterey, so that those who wanted to see the local sights (such as the famous Aquarium!) could do so in the free afternoon. And particularly important if you live in the UK, there was that beautiful (and reliable!) californian sun.

This is, of course, my personal perspective, and you may prefer a conference that takes place among the excitement of a big city! What do you think makes the perfect conference venue? Have you been to any conference locations that you thought were particularly good? Let me know what you think by leaving a comment below or on twitter using the hashtag #BestMeetingVenue

(4 votes)

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