A fundamental property of all organisms is the remarkable consistency within the formation of the body plan during development. A handful of conserved molecular pathways are responsible for morphological patterning in embryos across different taxa, despite an enormous diversity of embryonic size in nature. Remarkably, morphological patterns are scaled to tissue size at the time of development and same structures are maintained in relative proportions to the overall size.

One classic mechanism of pattern formation is the morphogen model, where a molecule – the morphogen – is secreted from cells at one end of a developmental field, and diffuses into the target tissue (Lander 2007). At any given time point, morphogen concentration is different around cells that are closer versus further away from the morphogen source. The target tissue responds by activating distinct transcriptional programs in progenitor domains that are exposed to different morphogen concentrations, leading to establishment of different cell fates along the length of the tissue. Therefore, size of the target tissue is an integral component of the patterning process.

How is the morphogen mechanism adjusted, then, to trigger the same transcriptional responses in proportional domains across tissues of different sizes in closely related species? In many instances, morphological patterns are set when tissues size is similar: human embryos are roughly the same size during embryogenesis, and differential growth can account for why taller and smaller human individuals nevertheless share similar body proportions. However, in other cases members of related taxa have quite different sizes during embryonic development, when the morphological pattern is being established. How conserved pathways that regulate patterning are modified to generate a size-invariant output is a biological problem that still remains to be solved (Figure 1).

In our recent paper from the Tabin lab, we tackled this problem through the study of aves, a clad of organisms that show a great variation in size – think of the range of egg sizes from an ostrich egg to a hummingbird egg (Uygur et al. 2016). Yet, these animals are related close enough that we would expect patterning mechanisms to be highly conserved. This gave us the flexibility to study how scaling of the body plan is achieved across two organisms with a drastic difference in embryonic size, the chick (Gallus gallus) and the tiny zebra finch (Taeniopygia guttata) (Figure 2). The neural tube provided to be an excellent model system to explore this question in aves. Pioneering studies in the past two decades have shown that the sonic hedgehog (SHH) morphogen secreted from the notochord and floorplate induces distinct transcriptional programs and cell fates at different concentrations along the ventral half of the neural tube (Liem et al. 2000; Dessaud et al. 2007; Stamataki et al. 2005). As is the case with the morphogen model systems, tissue size and distance from source is critical to patterning onset in this context.

When we examined the establishment of pattern in the ventral neural tube of chick versus zebra finch embryos, we were surprised to find out that it is not only the tissue size that varies between the two species, but also the duration of the patterning process. Across highly similar developmental stages and timepoints, the zebra finch ventral neural tube is patterned much faster compared to the chick neural tube. However, when we compare the levels of morphogen secretion, we found out that the zebra finch notochord, which is drastically smaller than the chick notochord, is also generating a morphogen gradient of much lower amplitude throughout development. Even though the smaller species is producing less morphogen, it is patterning the tissue at a much faster rate. What is going on?

In the past, adjustments to the morphogen mediated patterning systems have been explored at the level of adjusting the morphogen gradient itself(Cheung et al. 2014; Gregor et al. 2005; Ben-Zvi & Barkai 2010). Even though this may still be an interesting aspect of the scaling of the neural tube patterning, our findings lead us to explore differences in morphogen responsiveness. According to our initial observations, patterning process in the smaller zebra finch tissue is completed much faster, with less morphogen in the tissue. When exposed to the same concentration of the sonic hedgehog morphogen, would cells from the zebra finch versus chick turn on the same transcriptional program and cell fates? We turned back to classical embryology to answer this question and generated chimeric embryos at a very early time point in development (at 12 hours, to be exact). By transplanting cells between transgenic lines from the two species, we generated embryos with chimeric neural tubes that were made up of both zebra finch and chick cells, where cells from the two species were exposed to the same developmental signals and received the same level of SHH morphogen from a single source. Remarkably, we observed zebra finch cells to be much more sensitive to the morphogen compared to adjacent chick cells that are same distance away from the source. An analysis on the expression of different transcription factors confirmed that the transcriptional profile of the zebra finch cells were of a higher level morphogen threshold, as if they had seen a higher concentration of SHH for longer durations. This shows cell autonomous differences in morphogen sensitivity account for differential response to the morphogen and altered rates of patterning (Figure 3).

The strength of using the neural tube as a model is that the readout for morphogen activity is very well characterized in this system. We took advantage of this to quantify the differential response of neural progenitors to Sonic hedgehog in the two species. In an explant in-vitro culture system, we analyzed response to different concentrations and durations of SHH in neural tube naive explants isolated from either zebra finch or chick embryos. Indeed, all morphogen thresholds that have previously been identified for chick cells were lower for zebra finch cells, meaning that the zebra finch tissue can induce same cell types at lower concentrations and lower exposure durations.

Our studies on the mechanism of this difference showed that the differential response persists even when the SHH pathway is activated directly through its Smoothened receptor via an agonist. This finding revealed that the cell autonomous differential response is an intra-cellular difference downstream of the patched and smoothend receptors. We further showed that differential levels of the sonic hedgehog transcriptional effortors may be responsible for the differential response, as the two species display different levels of the GLI3 transcript. When we manipulated the zebra finch embryos to express higher levels of GLI3, we observed the speed of patterning is altered and the domain ratios are not scaled to the smaller tissue size.

Our study offers a potential explanation to how tissues of different scales achieve proportional domain sizes when using the same signaling pathways for patterning. It appears that in the case of chick and zebra finch, two avian embryos of different sizes, cell intrinsic differences in morphogen sensitivity control scaling of cell fate patterns in the ventral neural tube. We hope this work will provide new venues to study pattern scaling in order to enhance our understanding of how evolutionary mechanisms accommodate adjustments to body size.

Ben-Zvi, D. & Barkai, N., 2010. Scaling of morphogen gradients by an expansion-repression integral feedback control. Proceedings of the National Academy of Sciences of the United States of America, 107(15), pp.6924–9. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2872437&tool=pmcentrez&rendertype=abstract [Accessed July 11, 2014].

Cheung, D. et al., 2014. Adaptation of the length scale and amplitude of the Bicoid gradient profile to achieve robust patterning in abnormally large Drosophila melanogaster embryos. Development (Cambridge, England), 141(1), pp.124–35. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24284208 [Accessed April 12, 2014].

Dessaud, E. et al., 2007. Interpretation of the sonic hedgehog morphogen gradient by a temporal adaptation mechanism. Nature, 450(7170), pp.717–20. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18046410 [Accessed March 21, 2014].

Gregor, T. et al., 2005. Diffusion and scaling during early embryonic pattern formation. Proceedings of the National Academy of Sciences of the United States of America, 102(51), pp.18403–7. Available at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1311912&tool=pmcentrez&rendertype=abstract [Accessed March 26, 2014].

Lander, A.D., 2007. Morpheus unbound: reimagining the morphogen gradient. Cell, 128(2), pp.245–56. Available at: http://www.sciencedirect.com/science/article/pii/S0092867407000517 [Accessed December 10, 2014].

Liem, K.F., Jessell, T.M. & Briscoe, J., 2000. Regulation of the neural patterning activity of sonic hedgehog by secreted BMP inhibitors expressed by notochord and somites. Development (Cambridge, England), 127(22), pp.4855–66. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11044400 [Accessed August 12, 2014].

Stamataki, D. et al., 2005. A gradient of Gli activity mediates graded Sonic Hedgehog signaling in the neural tube. , pp.626–641.

Uygur, A. et al., 2016. Scaling Pattern to Variations in Size during Development of the Vertebrate Neural Tube. Developmental Cell, 37(2), pp.127–135.

(5 votes)

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In this new feature, we conduct a trawl for preprints to find the latest developmental biology and related research from the previous month.

Scientists are increasingly using preprints as a way of releasing research to the community without having to wait for the peer review process, and we want to promote this content here. Several sites host biology preprints, most notably bioRxiv, but also arXiv, f1000 Research, and PeerJ Preprints (for more info on preprints, ASAPbio has a useful FAQ page).

This month we found content from bioRxiv, arXiv, F1000 Research and PeerJ spanning developmental biology research, including investigations into cell fate in preimplantation mouse embryos and worm larvae,  a new technique for bi-allelic gene knockouts, and a method for making midbrain-specific organoids.

Developmental Biology

• Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes. Simi Muraleedharan, Helen Skaer, Maneesha S Inamdar
• Drosophila sensory cilia lacking MKS-proteins exhibit striking defects during development but only subtle defects in adults. Metta B. Pratt, Joshua S. Titlow, Ilan Davis, Amy R. Barker, Helen R. Dawe, Jordan W. Raff, Helio Roque
• A Facilitated Diffusion Mechanism Establishes the Drosophila Dorsal Gradient. Sophia N Carrell, MIchael D O’Connell, Amy E Allen, Stephanie MSmith, Gregory Reeves
• KIF1A/UNC-104 transports ATG-9 to regulate neurodevelopment and autophagy at synapses. Andrea K.H. Stavoe, Sarah E. Hill, Daniel A. Colón-Ramos
• Antagonistic roles of Polycomb repression and Notch signaling in the maintenance of somatic cell fate in C. elegans. Ringo Pueschel, Francesca Coraggio, Alisha Marti, Peter Meister
• Dynamic changes in Sox2 spatio-temporal expression direct the second cell fate decision through Fgf4/Fgfr2 signaling in preimplantation mouse embryos. Tapan Kumar Mistri, Wibowo Arindrarto, Wei Ping Ng, ChoayangWang, Hiong Lim Leng, Lili Sun, Ian Chambers, ThorstenWohland, Paul Robson
• Kinetochore Microtubules indirectly link Chromosomes and Centrosomes in C. elegans Mitosis. Stefanie Redemann, Johannes Baumgart, Norbert Lindow,Sebastian Fuerthauer, Ehssan Nazockdast, Andrea Kratz, SteffenProhaska, Jan Brugues, Michael Shelley, Thomas Mueller-Reichert
• Probing cytoplasmic viscosity in the confined geometry of tip-growing plant cells via FRAP. James L Kingsley, Jeffrey P Bibeau, Zhilu Chen, Xinming Huang,Luis Vidali, Erkan Tuzel
• RNase reverses segment sequence in the anterior of a beetle egg (Callosobruchus maculatus, Coleoptera). Jitse Michiel van der Meer
• Brachiopods possess a split Hox cluster with signs of spatial, but not temporal collinearity. Sabrina M. Schiemann, Jose M. M. Martin-Duran, Aina Borve,  Bruno C. Vellutini, Yale J. Passamaneck, Andreas Hejnol
• Nanog fluctuations in ES cells highlight the problem of measurement in cell biology. Rosanna C G Smith, Patrick S Stumpf, Sonya J Ridden, AaronSim, Sarah Filippi, Heather Harrington, Ben D MacArthur
• Trace elements during primordial plexiform network formation in human cerebral organoids. Rafaela C Sartore, Simone C Cardoso, Yuri V Lages, Julia M Paraguassu, Rodrigo F Madeiro da Costa, Marilia ZP Guimarães, Carlos A Pérez, Stevens K Rehen

Techniques

• Rapid, one-step generation of biallelic conditional gene knockouts. Bon-Kyoung Koo, Amanda Andersson-Rolf, Roxana C. Mustata,Alessandra Merenda, Sajith Perera, Tiago Grego, Jihoon Kim, KatieAndrews, Juergen Fink, William C. Skarnes
• A novel approach to derive human midbrain-specific organoids from neuroepithelial stem cells. Anna Sophia Monzel, Lisa Maria Smits, Kathrin Hemmer, SihamHachi, Edinson Lucumi Moreno, Thea van Wuellen, Ronan MTFleming, Silvia Bolognin, Jens Christian Schwamborn

Disease modelling

• Zika Virus in the Human Placenta and Developing Brain: Cell Tropism and Drug Inhibition. Hanna Retallack, Elizabeth Di Lullo, Carolina Arias, Kristeene A.Knopp, Carmen Sandoval-Espinosa, Matthew T. Laurie, Yan Zhou,Matthew Gormley, Walter R. Mancia Leon, Robert Krencik, Erik M. Ullian, Julien Spatazza, Alex A. Pollen, Katherine Ona, Tomasz J.Nowakowski, Joseph L. DeRisi, Susan J. Fisher, Arnold R. Kriegstein
• Hipk is required for JAK/STAT activity and promotes hemocyte-derived tumorigenesis. Jessica A. Blaquiere, Nathan B. Wray, Esther M. Verheyen

In silico modelling

• The Evolutionary Trade-off between Stem Cell Niche Size, Aging, and Tumorigenesis.  Vincent L. Cannataro, Scott A. McKinley, Colette M. St. Mary
• Conditions for cell size homeostasis: A stochastic hybrid systems approach. César Augusto Vargas-García, Mohammad Soltani, Abhyudai Singh
• Collective motion of cells crawling on a substrate: roles of cell shape and contact inhibition. Simon Kaspar Schnyder, Yuki Tanaka, John Jairo Molina, Ryoichi Yamamoto

Science Communication

• Not Communicating science? Aiming for national impact. Andreas Prokop and Sam Illingworth

Is there anything we missed for June? Anything in the list that catches your eye? Let us know in the comments section.

(6 votes)

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Here are the highlights from the current issue of Development:

A pioneer role for PBX1 in neurogenesis

The adult rodent subventricular zone (SVZ) is a neurogenic niche that provides new neurons and glia to the brain. A number of transcription factors, including MEIS2 and PAX6, are known to be required to promote the neuronal cell fate. PBX family proteins can interact with both MEIS and PAX family factors, and PBX1 is known to be expressed in the adult mouse forebrain. Dorothea Schulte and colleagues therefore set out to test the role of PBX1 in SVZ neurogenesis (p. 2281). They show that Pbx1expression is found in rapidly proliferating SVZ progenitors, as well as in subsets of their progeny in the olfactory bulb. Its ortholog, PBX2, is more widely expressed in the forebrain. Deletion of both genes in the adult SVZ leads to a reduction in neurogenesis and a concomitant increase in oligodendrogliogenesis – an alternative fate for SVZ progenitors. Although the authors can detect PBX1 binding to MEIS2 and PAX6, the phenotype is distinct from functional blockade of these factors, suggesting an at least partially independent function. Intriguingly, PBX1 appears to bind its targets before they are transcriptionally activated, indicating a putative pioneer factor function for PBX1. These data identify PBX1 as an important new regulator of SVZ neurogenesis.

A tyrosine kinase for FERtilisation

Mammalian sperm need to undergo capacitation to become competent to fertilise the oocyte. In vivo, this occurs in the female reproductive tract, whereas in vitro it can be triggered by culture in defined media. One of the key events during capacitation is cAMP-dependent tyrosine phosphorylation of various proteins. Although this phenomenon has been well described in many species, the kinase responsible has been elusive. Various candidates have been proposed, including SRC, FAK and PYK2; notably, inhibitors for the latter two enzymes block tyrosine phosphorylation in human and horse sperm. However, definitive evidence for the identity of the kinase has been lacking. Now (p. 2325), Pablo Visconti and co-workers show that, while FAK and PYK2 inhibitors also block tyrosine phosphorylation in mouse sperm (without blocking PKA activation), neither is the responsible factor. Instead, it is the FER kinase – also targeted by the same inhibitors – that carries out capacitation-associated tyrosine phosphorylation. However, and surprisingly, FER is not required for fertilisation in vivo, suggesting that the presumed involvement of tyrosine phosphorylation in acquisition of sperm competence may need to be revisited.

Coordinating morphogenesis and differentiation with WNT

The mammalian salivary gland is a valuable model for analysis of the morphogenetic and differentiation events that occur during branching morphogenesis of organs. Following a period of growth and branching, epithelial cells of the salivary gland differentiate down ductal or acinar routes. What are the signalling mechanisms that control morphogenesis, differentiation and lineage choice, and how are these processes coordinated to ensure appropriate size and composition of the final organ? The FGF pathway is known to play key roles in salivary gland development, including via the activation of KIT signalling – which promotes the expansion of distal (future acinar) progenitors. Moreover, WNT signalling has been implicated in salivary gland morphogenesis, although its function is unclear. On p.2311, Akira Kikuchi and colleagues use genetic and pharmacological approaches, both in vivo and in vitro, to demonstrate that mesenchymally derived WNT signals inhibit acinar differentiation and maintain end bud cells in an undifferentiated state, to promote secondary and tertiary duct formation. Mechanistically, WNT activity promotes MYB-dependent inhibition of Kit expression; KIT directs acinar differentiation through AKT. As WNT pathway activity is dynamically regulated, the authors propose this as a means by which salivary gland morphogenesis and differentiation can be spatiotemporally coordinated.

PLUS!

Cell behaviors and dynamics during angiogenesis

Vascular networks are formed and maintained through a multitude of angiogenic processes, such as sprouting, anastomosis and pruning. Only recently has it become possible to study the behavior of the endothelial cells that contribute to these networks at a single-cell level in vivo. Here, Markus Affolter and colleagues summarizes what is known about endothelial cell behavior during developmental angiogenesis, focusing on the morphogenetic changes that these cells undergo. See the Review on. p. 2249.

Generation of intestinal surface: an absorbing tale

The vertebrate small intestine requires an enormous surface area to effectively absorb nutrients from food. Morphological adaptations required to establish this extensive surface include generation of an extremely long tube and convolution of the absorptive surface of the tube into villi and microvilli. In their Review, Deborah Gumucio and colleagues discuss recent findings regarding the morphogenetic and molecular processes required for intestinal tube elongation and surface convolution in different species. See the Review on p. 2261.

Dental mesenchymal stem cells

Mammalian teeth harbour mesenchymal stem cells (MSCs), which contribute to tooth growth and repair. These dental MSCs possess many in vitro features of bone marrow-derived MSCs, including clonogenicity, expression of certain markers, and following stimulation, differentiation into cells that have the characteristics of osteoblasts, chondrocytes and adipocytes. Here, Paul Sharpe outlines some recent discoveries in dental MSC function and behaviour and discusses how these and other advances are paving the way for the development of new biologically based dental therapies. See the Review on p. 2273.

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Unraveling Development
Embryonic development is a complex and regulated spatiotemporal ensemble of signaling cues that control cell differentiation. Most of what we now know comes from experimenting directly on embryos. This provides biological realism, but involves a sea of uncontrolled/unobserved variables that sometimes obscures the basic underlying mechanisms. More recently, in vitro models, based on the differentiation of stem cells, have come into favor. These sacrifice some biological authenticity but have the distinct advantage of greater experimental ease and control over variables. Nevertheless, the culture dish or titer plate falls short of what could reasonably be considered an ideal imitation of an embryonic environment. As a result, in vitro models often give inconsistent or variable results.

Our goal has been to develop an experimental platform that captures some of the realism of in vivo models but with the ease and experimental control of in vitro methods. Our mantra is, “If you create the same physiochemical environment in vitro that occurs in vivo, cells will respond in the same way.” This not only includes the creation of molecular gradients and substrates, but the ability to alter these over time. To create this “ideal” in vitro environment, we’ve been using microfluidics.

A (very) brief history of microfluidics
Microfluidics is a growing field that deals with the fabrication of devices to control the flow and handling of small liquid volumes ranging from several hundred microliters to several picoliters. The actual physical size of any single microfluidic component can vary between several millimeters to several micrometers (about the size of a single cell) depending on the intended use, i.e. channels, valves, pumps, etc. .Examples of common microfluidic devices include: microbioreactors¹, microsensors², microanalysis³, lab-on-chip⁴, etc.

The field traces its origins to 1959 and the first demonstration of an integrated electronic circuit by Noyce and Kilby⁵. This discovery ushered in the microelectronics era and fueled the development of a plethora of high precision microfabrication technologies. Microfluidics “borrowed” these technologies to micromachine microstructures that comprise channels, valves and pumps. One of the first commercial applications of a microfluidic device was in 1979 when Hewlett-Packard invented the inkjet printer.

Because of its ancestral ties to microelectronics, most microfluidic systems were initially fabricated in silicon, but both technologies and materials have since expanded past their initial origins. More details of the history of silicon micromachining and microfabrication can be found here.

The role of microfluidics in developmental biology
Despite some setbacks, microfluidics are finally being applied to biology. Applications involving drug research and so-called “Organs-on-Chip” have enjoyed considerable attention. However, using devices to address fundamental biological questions has been slower to evolve. We find this rather surprising because developmental studies are particularly well suited to the strengths of microfluidics. Recent reports have revealed the incredible ability of embryonic stem cells to self-organize given appropriate instructive environment^6-9. These are at present limited to bath applications of soluble factors. Microfluidic technologies can provide a much needed additional layer of control for more biologically relevant experiments.

This background was what prompted us to develop a microfluidic device that could mimic morphogen gradients. The device is comprised of a cell culture chamber flanked by supply channels reminiscent of how tissues are perfused by capillary beds. Soluble chemicals can freely diffuse into the cell chamber establishing long-term, stable gradients. Using this simple microfluidic device and only three morphogen gradients, Shh, BMP and RA, we were able to recapitulate much of the neural tube development including motor neuron spatial organization¹⁰. However, it is clear that not all neural types are represented in our little artificial spinal cord. Something is missing. Whether this is another morphogen or signal, electrical gradients or just wrong timings for the signals we’re using, the search should be extremely interesting. For example, the anteroposterior axis develops with its own morphogen gradients concomitantly with the dorsoventral axis. It’s likely that cells in the early spinal cord simultaneously integrate signals from both anteroposterior and dorsoventral axes.

We should be able to test this. The microfluidic device we have engineered is capable of creating almost any “designer” chemical landscape¹¹ with any number of arbitrary morphogens. So there’s a whole set of experiments we are currently doing to explore some of these ideas. It’s also an example of how useful the technique could be to to developmental biology. All that is needed is an interesting question and a microfluidic design to test it. Although we haven’t yet faithfully replicated the physiochemical landscape in vitro, I think we’ve taken an important first step. I’m not sure where the journey will end but I’m hoping that we learn some interesting biology on the way. Fortunately, there is still plenty of research to be done.

Figure 1: Mouse embryonic stem cells cultured in a four-port microfluidic device for 9 days. GFP expression indicates the presence of post-mitotic motor neurons, which clearly favor a defined spatial region as dictated by instructive cues supplied to the cells. Cells are superimposed with a red soluble fluorescent dye to illustrate the shape of the gradient formed in the microdevice. Overall chamber dimensions approximately 1.5mm at the widest point.

This work was performed at the University of Maine, MicroInstruments and Systems Laboratory (MISL), under a grant from the National Science Foundation, [IOS-1145949].

Our full paper can be viewed at here.

Press releases can be found from University of Maine and the Francis Crick Institute. The article was also briefly mentioned in Science.

1. Grünberger, A., Wiechert, W. & Kohlheyer, D. Single-cell microfluidics: opportunity for bioprocess development. Curr Opin Biotechnol 29, 15–23 (2014).
2. Erickson, D. & Li, D. Integrated microfluidic devices. Anal Chim Acta 507, 11–26 (2004).
3. Reyes, D. R., Iossifidis, D., Auroux, P.-A. & Manz, A. Micro Total Analysis Systems. 1. Introduction, Theory, and Technology. Anal Chem 74, 2623–2636 (2002).
4. Haeberle, S. & Zengerle, R. Microfluidic platforms for lab-on-a-chip applications. Lab Chip 7, 1094–1110 (2007).
5. Kilby, J. S. Miniaturized electronic circuits. (1964).
6. Warmflash, A., Sorre, B., Etoc, F., Siggia, E. D. & Brivanlou, A. H. A method to recapitulate early embryonic spatial patterning in human embryonic stem cells. Nat Meth 11, 847–854 (2014).
7. van den Brink, S. C. et al. Symmetry breaking, germ layer specification and axial organisation in aggregates of mouse embryonic stem cells. Development 141, 4231–4242 (2014).
8. 3D Reconstitution of the Patterned Neural Tube from Embryonic Stem Cells. 1–34 (2014). doi:10.1016/j.stemcr.2014.09.020
9. Poh, Y.-C. et al. Generation of organized germ layers from a single mouse embryonic stem cell. Nat Commun 5, 4000 (2014).
10. Demers, C. J. et al. Development-on-chip: in vitro neural tube patterning with a microfluidic device. Development 143, 1884–1892 (2016).
11. Smith, R. L., Demers, C. J. & Collins, S. D. Microfluidic device for the combinatorial application and maintenance of dynamically imposed diffusional gradients. Microfluidics and … 9, 613–622 (2010).

(2 votes)

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I woke up this morning to a Facebook reminder of where I was 5 years ago. I was in Lille, France, on a 2 month sabbatical at Université Lille 1 from my PhD at the University of Cambridge, UK. It was supported by an EU collaborative grant to promote scientific interaction between member states.

By all measures, it was a roaring success – far better than we had dared to hope. I was working with one of the world’s least purifiable proteins, a transcription factor called Neurogenin2, and trying to study its phosphorylation status by NMR (nuclear magnetic resonance) imaging. We had gone in with the expectation that nothing would work but that it was worth a go, and I came out with an entire thesis chapter based on 2 months work, and what we would later publish as, “Phosphorylation in intrinsically disordered regions regulates the activity of Neurogenin2.”

Not only was it directly useful as a scientific endeavor, it was also a great experience. I learned about some of the intricacies of the French system, working with an independent researcher at CNRS who did not run the lab, but was nonetheless a researcher in her own right. My Parisian-French-with-a-Northern-Irish-accent proved useless in the face of a local dialect, Ch’ti, on the French-Belgian border. I went to Waterloo near its anniversary; I ate vast amounts of cheese; I marvelled at the holidays the French seemed to have every other week. I lived in dank student accommodation (about which my hosts were very apologetic) but I have nothing but fond memories for the 2 months I spent there. In the space of the last week, I have realised how much I took for granted that this was an opportunity available to me.

On June 23rd, the UK voted in a referendum to leave the EU.

I’m not going to comment on whether that is or isn’t going to happen, what it does or doesn’t mean for democracy in the UK – there’s enough on that already. What I’d like to do here is briefly address some areas crucial to science: funding, international collaboration and the mobility of scientific researchers. The implications for science from Brexit are both uncertain and unlikely to be clarified soon. And scientific research, as an enterprise, does not deal well with uncertainty.

Funding

Immediately after the results were certain, which I watched live, I gave my initial response and thoughts to a call from Nature for reactions from scientists:

What did I mean by “a science funding system already strained”? Last August, I made a brief visit to the UK, gave some talks on my research and scoped out the situation for academic jobs, as this was still the career track I was primarily pursuing. It became clear pretty quickly that funding was a key concern – and this was before Brexit was even really in anyone’s mind as an issue. Science funding by the UK has stagnated for years, and it was only through the major “Science is Vital” campaign that funding for science was not cut by the coalition government of 2010 – but neither was it increased. EU funding currently makes up around 15% of the UK’s research and development funding. The UK is only just behind Germany in the amount of money received from the EU for science funding and despite being a net contributor overall to the EU, has benefitted financially in terms of research funding (see Table 1 and surrounding discussion, “Examining Implications of Brexit for the UK Research Base”).

The UK has become overly dependent on EU funding – and a situation has arisen where many have allowed the EU to make up for the UK’s own lack of investment. In 2012, the UK only spent 1.63% of its GDP on research, lagging behind other countries with large research output.

The BBC reported claims from Jo Johnson, UK Science Minister, that, “world class research would “endure” in the UK following a Brexit. He added that researchers should be “optimistic about the future.” But in the same meeting he could not provide any guarantees about EU funds. The UK will also no longer be able to shape research policy from outside the EU.

The official line at the moment is that nothing has changed; the UK is still an EU member for at least two years, and there seems to be a lot of certainty that the UK will continue to pay into the Horizon 2020 and EU research funding based on its GDP, much like Switzerland. However, I have not yet seen how this is easily reconciled with a major feature of the Leave campaign – immigration – and the Swiss have restricted their access to EU funds by restricting freedom of movement, particularly funds allowing young researchers to move around, such as Marie Skłodowska-Curie Research Fellowships and the Erasmus programme (if you click this link, note the first lines on the web page).

And this is crucial because the scientific enterprise is about so much more than just money; it is about people. More concerning, in my mind, than the issue of what will happen to funding, is what the introduction of added bureaucracy and complications will do to international collaborations, and what the question over mobility of researchers – and the recent spike in xenophobia and racism – will change in the minds of junior scientists looking to come to, return to, or stay in the UK.

Collaborations

Jo Johnson, fresh from the assurances given above about UK science, soon had to address the issue that UK researchers risk exclusion from collaborative projects in the EU. This is what I cannot reconcile in my mind about the assurances that legally, nothing has changed, and everything will carry on as before. Science funding is highly competitive. To obtain that funding, researchers will seek to minimize any risk of rejection of grant applications. The UK has stated that it is not going to trigger Article 50, the mechanism to leave the EU, until the new Prime Minister is decided, at the earliest in September. EU officials have in turn retorted that no assurances or negotiations will be carried out in that time; and negotiations will then take up to 2 years.

In this vacuum devoid of certainty, with no realistic assurances, I would be very surprised if researchers in the EU, albeit with a heavy heart, do not begin to rule out collaborations with UK researchers. It is even the case that although the official line has not changed, misinformation and requests to withdraw UK researchers from or to not involve UK researchers in projects may be filtering into the system. This is already happening anecdotally:

there is no consensus. People just don't know about future bids. I, too have heard of people being asked to leave H2020 etc

— Dr. Vicky Forster (@vickyyyf) June 29, 2016

I've been at an EU event for about 5 mins & have already been told that EU research project bids 'won't be looking for UK partners any more'

— Dr Laura Molloy (@LM_HATII) June 29, 2016

Another area where quantitative data is sparse, but anecdotal data is also emerging, is in the mobility of young researchers.

The mobility of junior researchers

Stephen Hawking noted that the strong record the UK has in attracting EU funds, and its ability to attract young international researchers with EU grants, encouraged the best young EU scientists to think about moving to the UK. Paul Nurse has urged that free movement of people is essential for UK science, and Venkatraman Ramakrishnan, President of the Royal Society, has called for assurances for residency for EU researchers from the government.

Buzzfeed interviewed a range of scientists at various stages on their opinions and in particular these young European scientists currently in the UK who are now reconsidering their future there. There is now the question of belonging, or being welcomed, in the UK.

The existence of an "eu-staff-members-non-uk" mailing list at @UniofBath makes it clear that unity has faded

— Aron (@lonepair) July 2, 2016

I titled this blog post using a direct quote from UK Justice Secretary Michael Gove, “people in this country have had enough of experts” (Michael Gove is one of the candidates for the leadership of the Conservative party, and therefore the position of Prime Minister). Xenophobia and racism have spiked recently in the UK. Reporting of hate crimes has increased. Issues of funding and academic job security aside, the UK is not presenting an attractive image to those of us outside it. There does not appear to be a desire to welcome those who are foreign or those with expertise, even in the very highest echelons of government. It is also not encouraging expats I have spoken with to return.

Anecdotal evidence exists that fellowship applications from the UK are being hit:

https://twitter.com/davecl42/status/749157977643425792

This also brings in the question of the effect on students and higher education. Current EU students and those entering in 2016 will have loan arrangements honoured, but there is no guarantee as yet with respect to entry from autumn 2017. Institutions are issuing statements along the lines of this from Vice-Chancellor Sir Leszek Borysiewicz at the University of Cambridge, and the President at University College London reacted to claims that UK universities will not suffer with fears that EU students may be lost from the UK, put off by tuition fees (students from the EU pay the same fees as UK students, and not international fees). UCL and other institutions in the UK have expressed dismay at the effects leaving the EU may have on their operation.

Concluding thoughts

In the process of negotiation, UK science and universities may end up with the relatively unchanged situation that many hope for. But that situation is a long way away, and fraught with uncertainty. Those who argue that all is fine and will continue to remain so, and that there is therefore not a problem, are not appreciating the incentives and risks that people consider as they move around to do science. Science is a highly mobile affair – look at Emmanuelle Charpentier’s movements around the world as a case in point – and the entire incentive structure for moving to the UK, and the risks involved for young researchers, have completely changed, if only temporarily, in the absence of any certainty of what is to come. Science is so competitive right now that many researchers just can’t afford to enter into uncertain positions. The longer the uncertainty continues, the more likely (and my own anecdotal evidence, admittedly small, overwhelming shows this) young scientists in the UK are to leave; and expats and foreign researchers are to stay away.

But perhaps these may not be factors that concern the UK in the end. After all, as we were told in no uncertain terms, “people in this country have had enough of experts.”

Next steps

The situation in the UK is unpredictable, to say the very least. Continuously monitoring the situation is necessary – here’s a summary of the first week, from Nature and coverage of the situation in The Atlantic for further reading.

Scientists for EU (@Scientists4EU), a campaign that previously discussed the possibilities of the effect of Brexit on science prior to the vote, are already trying to gather evidence for the effect of Brexit on UK researchers – if you have anything to contribute, know someone who does or just want to aid in data collection on this issue, please share their link on Monitoring Brexit’s impact: http://scientistsforeu.uk/monitoring-brexit-impact/

Converting the rumour mill into data! Any experiences of Brexit vote impact add here pls ->https://t.co/CjRdGyGims pic.twitter.com/2eHUBrO2EN

— Scientists for EU (@Scientists4EU) July 1, 2016

I am working on a follow-up post to this for the American Society of Cell Biology’s COMPASS blog in which I would like to include some more data as it appears, so please do help with data collection efforts.

On Tuesday also the Commons Select Committee on Science and Technology will hold a session on “Implications and opportunities for science and research examined”. You can watch at the link below:

http://parliamentlive.tv/event/index/f225ac03-d091-4215-b547-7b0050d33cca

The Node’s Question of the Month is about your opinions after the referendum – make sure to contribute there too.

Do you have thoughts or comments? Please feel free to get in touch below. Fact-checking and requests for clarification are particularly encouraged.

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Gary McDowell is the Executive of Director of Future of Research (FoR), a U.S. nonprofit that assists junior scientists in grassroots advocacy to promote solutions to problems they perceive with science, and the scientific enterprise. He spent 9 years discovering the joy of developmental biology working with the frog Xenopus laevis. He is currently a resident at the Moore Foundation-funded Manylabs open science skunkworks in San Francisco, CA.

(7 votes)

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This month we welcomed Aidan to the Node, who took over from Cat as Community Manager, and introduced himself here. He’s been enjoying settling in to the Company of Biologists office, and getting to know the site and its users.

We had 17 jobs and studentships posted on our jobs page,  on everything from chromatin to membrane dynamics. We also had a host of other posts covering a typically wide range of content:

Research

Julia Turan wrote about the visualisation of stem cell signalling in the gut, and how organoids are helping us understand the Zika virus. Alexa Burger told us about her Development paper on efforts to maximise the efficiency of CRISPR in zebrafish. Danny Llewellyn told us about how trichomes can bind plant organs together to constrain their shape, and how the story came about.

Research Methods

We posted an excerpt from a Disease Models and Mechanisms article focusing on the problems of reproducibility in histopathology (many of the issues raised apply broadly to developmental biology research). We also reposted an article from Nathalie Percie du Sert on the Experimental Design Assistant: an online tool aimed at scientists who use animals in their research.

Meetings/Courses

We heard about the Young Embryologist Network meeting in London from two perspectives: Thomas Butts, and Vicki Metzis & Katherine Exelby. From Michigan, Eden, Martha, Samahitha and David introduced the Developing Future Biologists programme, and the course they ran over the summer for Puerto Rican undergraduates.

Lab life

Our ‘A day in the life…’ series continued, and we first learned about what life is like in a gar lab. Martin Minarik journeys from Prague to Mexico to harvest eggs from these non-teleost fishes. We then shifted from the humid tropics to the clean room, where one PhD student fabricates microelectrodes for neuroscience applications.

Campaigns/Questions

We reposted a piece from the Genetics Society of America campaigning for the maintenance of funding for model organism databases (such as Flybase and ZFIN); you can sign the Letter of Support by following the link in the piece.

Finally, we asked our Questions of the Month, which really couldn’t have been about anything else: what does the UK referendum result mean to you as a scientist, and what can we as a community do about it? Get involved here. We also created a page with a bunch of post-referendum links.

Thanks to all our posters and readers!

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Stockholm University, Sweden, invites applications for one postdoctoral position in the laboratory of Professor Mattias Mannervik at the Department of Molecular Biosciences, The Wenner-Gren Institute (http://www.su.se/mbw). The position is scheduled to start as soon as possible.

Transcriptional coregulators are proteins that facilitate communication between transcription factors and the basal transcription apparatus, in part by affecting chromatin through post-translational modification of histones. As such, they contribute to generation of cell-type specific gene regulatory networks and epigenetic control of animal development (see Mannervik et al. Science, 284, 606-609, Mannervik Exp Cell Res. 321(1):47-57). This laboratory is using molecular, genetic, and transgenic approaches in Drosophila melanogaster to elucidate the molecular mechanisms of transcriptional co-regulator and chromatin regulator function during development. To investigate if histone modifications can be instructive in regulating gene expression this project uses two approaches. A histone replacement system is used to examine the effects of amino acid substitutions in the histones on organismal development, and a modified CRISPR/Cas9 system is employed to target chromatin modifying enzymes to endogenous loci.

The position is available immediately and requires a recent Ph.D. as well as extensive experience in molecular biology techniques. The successful applicant should have a high-quality publication record, and motivation to study underlying mechanisms of gene regulation in development. The position will be funded with a fellowship, and includes health insurance.

Stockholm University is one of the largest and most prominent universities in Sweden, located in the nation’s capital city, beautifully surrounded by the first national city park in the world. For further information, see http://www.su.se/english/ and http://www.academicstockholm.se/

Application: 
Applications marked with reference number SU FV-2.1.9-2187-16 should be submitted electronically as a single PDF file to mattias.mannervik@su.se and to registrator@su.se. The application deadline is September 1, 2016

Applications should comprise the following:

1) a personal statement describing research interests (1-2 paragraphs), research experience (1–2 paragraphs) and career goals (1-2 paragraphs)

2) curriculum vitae

3) bibliography

4) names, e-mail adresses, and phone numbers of three references

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On the 23^rd of June the United Kingdom held a referendum on whether to remain a member of the European Union or to leave. Prior to the vote, Nature reported that 83% of nearly 2000 polled scientists favoured remaining, and letters from Royal Society members and Nobel Prize winners urged the public to vote to remain in the interests of British research. The result was called early on Friday morning: the UK had voted to leave the EU, by 51.9% to 48.1% (around 17 million to 16 million voters). We’re now faced with the question of what this might mean for UK science, and, given how interconnected science is, also how it might influence science in the EU and the rest of the world.

So we’ve got two Questions of the Month for June, one personal and one more global:

What does the Referendum result mean for you, scientifically and career-wise?

Are there practical steps we as a community can do to ensure a bright future for UK and EU science?

We’re hoping to hear from as broad a selection of people as possible: UK and EU nationals working here in the UK, elsewhere in the EU or in the wider world; students, postdocs, PIs, and people who have left the lab bench!

Considering this is a fast moving topic, we’ve also created a page with various links to ‘Science After the Referendum’ content. We will try to keep this page updated, and welcome suggestions for pieces we’ve missed.

We recognise what an emotive subject this is, and would like to try to keep this discussion to the referendum’s impact on science and careers. Let us know what you think in the Comment boxes below, or via social media (we’ll also be collating these answers via Storify).

(1 votes)

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To complement our Questions of the Month, we’ve brought together some post-referendum science links

News and Comment

Nature reported on science’s reaction to the news, on post-ref limbo, bemoaned the lack of a leaving plan in an Editorial, and assessed the mood seven days later.

Science  documented the immediate reaction, ran an interview with Anne Glover, former EU science adviser, and discussed the role of science in future negotiations in an Editorial.

Times Higher Education has collated all of its post-ref content in one place, and recently documented the emerging worries of UK acadmics

Buzzfeed documented the worries of young scientists, and reported on the government’s attempts to reassure us about the future

The Royal Society of Biology reported from their annual Parliamentary Links Day, where the President of the Royal society delivered a speech

The BBC interviewed Paul Nurse, Director of the Francis Crick Institute, and Jo Johnson, Science Minister. Pallab Gosh, BBC science correspondent, reported on the consequences for science.

Jeremy Farrar, Director of the Wellcome Trust, gave his thoughts in an interview

A view from Australia from The Conversation

Opinion

Mike Galsworthy (Scientists For EU) wrote in the New Scientist about scientists’ anger and future strategy

James Briscoe (Crick Institute) shared his thoughts

Helen Rippon (Worldwide Cancer Research) addressed what the result meant for collaboration in The Huffington Post

Phill Jones (Digital Science) explored what the result meant for the UK’s knowledge economy from The Scholarly Kitchen

Athene Donald (Cavendish Laboratory) encourages UK scientists to continue applying for ERC funding

Statements from organisations and institutions 

The UK Minister of State for Universities and Science  

The Royal Society

The Biochemical Society

EMBL-EBI (and from the EMBL blog)

Universities UK

The European Commission

Campaign for Science and Engineering

Surveys

Scientists for EU is monitoring the impact on science, and is seeking examples of direct effects on career plans, investments, and roles in consortia.

The Parliamentary Science and Technology Committee is seeking written submissions addressing science and the referendum for an inquiry in July (Nicola Blackwood, Chair of the Committee, outlined why here)

Please let us know if you find any additional useful material.

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Last month saw the return of the Young Embryologist Network annual meeting held this year at the UCL Institute of Child Health. To settle into the long weekend, a number of us from the Briscoe Lab at the Crick Mill Hill site headed on down to central London to spend the day being inspired by talks from PhD students, Postdocs and several invited guest speakers. We were met with an impressive array of speakers, highly interactive poster sessions and an atmosphere buzzing with young embryologists demonstrating their clear passion for cell and developmental biology. With such excellent organisation and enthusiasm, we are happy to write about our thoughts of the meeting, and greatly anticipate the next event.

To kick off the day, our newest lab member, Teresa Rayon, opened the “Cell Fate Determination” session. Presenting her PhD project, Teresa demonstrated the importance of enhancer usage and developmental pathways that direct tissue-specific expression of Cdx2 in the early mouse blastocyst. Following this tour de force, the opening session only continued to satisfy our appetite for developmental biology, concluding with the two prize-winning talks of the day. Sarah Bowling (Rodriguez Lab), presented an elegant story on the role of mTOR signalling, a main component involved in cell competition in early embryos. Her studies showed the link between developmental signalling, metabolism and cell survival, for which she received the Sammy Lee Memorial Medal. This award recognises an early stage career embryologist, who not only carries out inspiring work but also has the ability to communicate their science in a personable manner, a skill that Sammy Lee was well known for. Following this, the session concluded with Sarah Harrison (Zernicka-Goetz lab), who was awarded a second place prize for her work on organoids, and how asymmetry occurs in the early embryo. At this point we were left with a clear feeling of excitement that in vitro systems such as stem cells have afforded developmental biologists, as more and more becomes possible at the tissue and organ level….

The second session of the day concentrated on polarity and asymmetry with the best place to focus being the zebrafish brain. The talks up until this point all had one thing in common: stunning imagery, which gave a natural build up into the first external speaker of the day, Dale Moulding from UCL Institute of Child Health. His talk covered new and emerging technologies in microscopy, covering a range of approaches from micro-CT, to OPT, SPIM and light-sheet imaging – a veritable repertoire of optical imaging solutions, which are continually pushing the boundaries of image resolution and depth of field.

After the lunch break the second guest session was filled with inspiring presentations on early embryonic development from Kathy Niakan (Francis Crick Institute) and Shankar Srinivas (University of Oxford). Kathy discussed mouse and human blastocyst development, focusing on the key transcriptional regulators involved in early lineage commitment. Shankar then followed with an examination of the cellular mechanisms involved in driving early AP patterning in the mouse, and in establishing a beating heart. Echoing earlier sentiments of the day, he demonstrated how light-sheet microscopy can be exploited to reveal impressive cellular-level detail in the context of whole organisms as they develop in real time. The generation of this type of live imaging data now poses new challenges to the developmental biology field, in how best to reconstruct individual cells and extrapolate the intricate details of developing organs. Such a feat will undoubtedly rely on the continued collaboration between biologists and physicists alike to develop the best possible data analysis solutions.

The final session, “Morphogenesis, Repair and Regeneration” had talks on cell matrix adhesions during contact inhibition of locomotion as well as cell movement in distal neuropore closure. To complete the session, Tapan Papilia (Hughes lab) gave an insight into Pax7 stem cells in muscles and their roles in regeneration and repair.

Closing the talks for the day, we welcomed Professor Paul Martin (University of Bristol), invited to give The Sammy Lee Memorial Lecture. Capturing the essence of YEN, Paul’s talk reminded us that it’s not only important to work hard, but to enjoy the work that you’re doing – an important aspect that can drive discovery. His captivating presentation highlighted that no matter what area of development you may find yourself in, the basic underpinnings of biology can have far reaching implications. Some of these are being realised in the field of tumour biology, where studies that began in fly have transitioned to vertebrate models of wound healing that are informing cancer treatments. Never knowing exactly where one will end up is definitely an attraction for many developmental biologists, and a motivation that drives us to work long (and sometimes tedious) hours at the bench or desk.

From cells to embryos, the YEN meeting makes a compelling case that the UK harbours many talented young scientists. Importantly, these individuals come from across the globe and are fuelled by a potent combination of excellent research leaders, high quality facilities and resources – a tradition that we are proud to be a part of now and into the future. An important conclusion that we take away from this meeting is that a successful “young embryologist” is in our view not only a creative, open-minded, cross-disciplinary individual, but also an excellent communicator, both to the public and to scientists alike. Events such as these are important to promote these aspirations. For this, we would like to thank the organising committee and sponsors who enabled this excellent meeting. We look forward to continuing our participation in future events organised by the young embryologists network and strongly encourage you to do the same.

Katherine Exelby & Vicki Metzis

For another view on YEN 2016, read Thomas’ report

(7 votes)

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