In time of revision: of Wingless and morphogens

Alfonso Martinez Arias

The recent publication of the important work of C. Alexandre, LA. Baena and JP. Vincent on the molecular requirements for Wingless signalling in Drosophila (http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12879.html) offers an opportunity to consider the relationship between ideas and facts in modern developmental biology.

The work reports the surprising finding that fruit flies whose only source of Wingless is a membrane tethered form of the protein are viable and, except for a few minor defects in growth, morphologically normal. This is a very surprising result and, although earlier work had shown some hints in this direction during wing development, there is much that invites thought and reflection in this incisive and beautiful piece of work. However, to understand this, we need a small amount of background.

Wingless is the Drosophila homologue of Wnt-1, a member of a major family of signalling molecules that play significant roles in development and disease. Wnt signalling became to prominence because of work in Drosophila which, in the 90s, was deep in the hunt for Morphogens, molecules postulated to be able to instruct the patterning of fields of cells. There are two definitions of morphogens –and as critical results begin to emerge, the differences between the two becomes more and more important. The original one by Alan Turing refers to molecules that can trigger the generation form. A second definition, more popular and the one most widely known, emerged in the 70s and is associated with Lewis Wolpert notion of Positional Information (PI). According to this a Morphogen is seen as a diffusible substance that specifies pattern in a field of cells in a concentration dependent manner i.e. cells can ‘interpret’ different concentrations in different manners and activate different genes. It is this second definition that has served as a guide to interpret much of the molecular genetics of developmental processes in Drosophila. Wingless signals through an elaborate molecular device of which only one element needs to be considered in this discussion: like all Wnt proteins, Wingless effects its signalling activity through the nuclear activity of ß-catenin (Armadillo in Drosophila) i.e. Wingless can signal to other cells, but in the cells that see Wingless, what matters is the state of Armadillo/ß-catenin.

In the 90s, the finding that many of the molecules uncovered by the genetic analysis of pattern formation were diffusible led to identify many of them as “morphogens”, in the wolpertian sense. Naturally one of these was Wingless and much of the effort to make it a Morphogen focused on its function during the development of the adult, in particular within a structure called the wing imaginal disc, that will give rise to the wing and thorax of the adult Drosophila. For most of the development of this structure (over 40 of the 96 hours that lasts its development and patterning), Wingless is expressed in a thin stripe bisecting the growing disc. As ill defined (timewise) removal of Wingless generated defects in the growth and patterning of the wing, the notion emerged that it was the diffusion of Wingless from the stripe that was responsible for the pattern. A number of experiments were designed to test this hypothesis, including testing the effects of diffusible Wingless in contrast to membrane tethered Wingless and to those of its effector Armadillo/ß-catenin. Key in these experiments, as it is to any test of a wolpertian Morphogen, was the identification of direct response targets to the signalling activity of Wingless. Following a tradition, three were identified that during the patterning of the wing disc could be interpreted in this light; from high to low response thresholds: senseless, Distalless and vestigial. The experiments were interpreted to suggest (I am being careful in how I phrase this: ‘interpreted to suggest’ i.e. there was a fair amount of wishful thinking here) that there was a functional gradient of Wingless in the wing disc and that indeed high levels of Wingless triggered senseless expression, intermediate did Distalless and low elicited vestigial. Furthermore, membrane tethered Wingless could only signal to adjacent cells and Armadillo/ß-catenin could only elicit a response in the cells where it was expressed. And everybody, or almost everybody bought into it. In characteristic style Nature, Cell and Science broadcasted the news: Wingless was –and by the way still is- a Morphogen. However, looking at the data some of us had problems with these readings, the design of the experiments and the interpretation of the results. NCS was not interested and some of this questioning can be found in other journals where results have, on average, a longer shelf life and more information (see Development or Developmental Biology) as opposed to ‘cool experiments’ for morphogene enthusiasts, perpetuated in reviews. Part of the reason for these doubts was the existence of another view of the function of Wingless in the wing that, even though ignored, had a firmer base on the experimental results.

In the alternative view, Wingless did not (and DOES NOT) act as a Morphogen –in the wolpertian sense. Furthermore, a large number of experiments suggested that there was not much of a relationship between the long range diffusion of Wingless and the patterning of the wing. This work has been summarized elsewhere and in more general terms (for some details see http://amapress.gen.cam.ac.uk/?p=1191 and references therein). The gist of it is that

1. There are different phases of Wingless expression in the wing and that there is a need to correlate specific functions with this different phases. Particularly during a crucial phase in the growth of the wing disc, all cells appears to express low levels of wingless.
2. That removal of the stripe of Wingless expression had little or no effect on the growth of the wing.
3. That Wingless acted at different times by creating some sort of a memory (implemented by Vestigial and Distalless) that would be pass on to the next stage.

The new work of Alexandre et al is a very rigorous confirmation of these observations in the wing but goes beyond them, showing in a most convincing manner, that there is no requirement for Wingless long range diffusion at all during the development of Drosophila and providing details and important hints of how Wingless works during wing development. It also shows that this might be a theme for the way Wingless works in Drosophila. Using elegant novel engineering technology they substitute the wildtype copy of Wingless for a membrane tethered form, and show that a fly with this genotype is viable. From the point of view of morphology it is a very good looking fly, though it has some small defects on growth, physiology and reproduction; but, for all practical purposes -certainly those that concern Drosophila pattern formation buffs- it is good. The defects should make people think and opened many interesting questions about Wnt signalling. Significantly, the authors go on to show that most of the growth of the wing disc is associated with ubiquitous low levels of Wingless expression in all the cells of the disc during the early/mid part of imaginal development (second and early third larval instars to the experts) and that therefore all cells have access to the levels of Wingless that they need, when they need it. This is consistent with previous suggestions that to understand Wingless in the wing disc one has to take into consideration its different patterns of expression and focus on early events. Its extension to the rest of development and other tissues of the fly is extremely important and invites much thought.

These observations are, indeed, surprising. However, rather than rejecting them and looking for small holes in the experiment (there is no perfect experiment in Biology) maybe we should simply use them to.reconsider our views of Wnt signalling. After all, none of the people who are raising caveats about this work –and there are a few- fluttered an eye brow when thinking about Wingless as a Morphogen, disregarding experiments to the contrary and boosting weak results in journals of wide readership, to favour an idea which was only an idea (see http://amapress.gen.cam.ac.uk/?p=1191) . The new work is not without issues but they are minor and do not have to do with the techniques, the membrane tetheres Wingless or the experimental design (all of them fine), but rather with what the results tell us about Wingless and Wnt signalling. Importantly: the work does not imply that Wingless does not diffuse in Drosophila; it does!. The work does not say that diffusible Wingless might not play a role, in Drosophila or other organisms; it might!. All that the work says is that in Drosophila, during normal development, there is not a major need for Wingless to diffuse a long range to perform its function. One of the reasons why it is important to take stock of this, is because there are few experiments (any?) in vertebrates that show a requirement for ling range diffusion of Wnt proteins in pattern formation. The accolade of Wingless as a Morphogen in vertebrates is a shallow extrapolation of the statements about Wingless in Drosophila, mostly in NCS but also, by mimicry, in other journals. Let us hope that this work leads to a more careful and rigorous analysis of the function of these molecules (NB this does not mean that reviewers should ask for endless lists of experiments, it simply says that we should be a bit more critical of what we have and a bit more thoughtful in the interpretation of the results).

One of the reasons for the use of Wingless in Drosophila might have to do with the rapid and robust development of the organism. Computation of signals take time and the wing disc (as all development of the fruit fly) is under extreme temporal constrains that have led it to evolve rapid mechanisms built around deeply interlocked gene regulatory networks (behold the early segmentation cascade). Thus, in evolutionary time it might be easier to reengineer the system, on the basis of the Gene Regulatory Networks, than to change the properties of the molecules (in the case in hand here, the diffusibility of Wingless). Thus the situation in the wing.

The twilight of the Morphogen?

The notion of a Morphogen is very tightly linked to pattern formation and signalling and in the light of the XXI century, it might be good to look at its roots and maybe return to the Turing version instead of the wolpertian PI version. Many people would be happier. While it is becoming increasingly clear that signalling molecules can elicit concentration dependent responses, it is not at all clear what the role of these responses are in vivo. We find correlations between concentrations and differential patterning but as one often gets in reviewers comments: correlation is not causation. In these considerations we should not forget the issue of time integrals and their relationship to spatial concentration. It seems that we need to divorce ourselves from simple naïve notions based on qualitative models. The Wnt version might just be one.

In the end rather than hanging on to notions for which, let us remember, there was precious little evidence for, we should look at the new results and think about what they tell us about the system rather than about a molecule. There is much in the work of Alexandre et al about this and we should take it beyond the simple ‘Morphogen or no Morphogen’. A twist to some famous words of Jean Rostand come to mind: theories pass, the wing remains.

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I am a former diploma student in Emmanuel Farge’s team “Mechanics and genetics of embryonic and tumoral development” (Paris). Watching embryos could only convince me of Lewis Wolpert’s famous claim that “gastrulation is the most important time in your life” – but I also think we should keep in mind Theodosius Dobzhansky’s statement that “nothing in biology makes sense except in the light of evolution”.  Hence, the evolution of gastrulation should be an especially important topic. In this post, I hope to share the excitement we got at trying to untangle some of its mysteries.

According to Plato, the universe was necessarily a sphere – as this was the most perfect of all possible shapes. When it came to creating people, Plato’s Creator was similarly well-intended, and first made us ball-shaped; but it was quickly obvious that spherical people were suboptimal, as they wouldn’t stop tumbling around. In an unusually self-critical move for a divine being, Plato’s creator added legs and arms on second thought, so that early humans would not be bound to passively follow gravity but could “get over high places and get out of deep ones”.

We share with Plato’s people the fact that we started as spheres. One of the first achievements of developmental biology was to demonstrate that all animals come from ball-shaped eggs (either spherical or, in the fanciest cases, looking like roughly rugby ball-shaped ellipsoids). The first cell divisions cleave the egg into multiple cells, but don’t alter the overall form. This ball shape is only lost when the first cell movements start deforming the embryo, and this is called gastrulation. While universally recognized as a developmental step in animal embryos, gastrulation comes in a broad diversity of shapes and flavors, and precisely defining it can be a tricky task. The simplest definition that still captures the essence of the phenomenon is probably the following: gastrulation is what happens when you stop being a ball.

Why should we gastrulate?

Gastrulation is so familiar to us that we easily forget that its very existence is mysterious. Why do cells need to move at all? Why should the making of an animal require elaborate, origami-like folding, complex migrations and relocations? Why can’t embryos directly make the cells in their final needed place? This would certainly be possible: all major independently evolved multicellular groups (plants, fungi, and brown algae) managed to reach high levels of organization only by the use of oriented cell divisions, differential growth, and a little bit of trimming by programmed cell death. No cell migration involved, no massive tissue folding needed. Yet why does the making of all animals, from jellyfish to cockroaches to zebras, require this beautifully absurd, but universal, dance?

The historically first solution to this riddle came from the man who first recognized gastrulation (and who coined the word), the German biologist Ernst Haeckel. While Haeckel’s figure recently attracted a lot of fierce (and maybe excessive) criticisms for the inaccuracies he introduced in his drawings of embryos, he also made many seminal contributions to biology – one of which was to identify, and tentatively explain, the universal occurrence of gastrulation. His hypothesis was rooted in the early idea that embryonic development recapitulates evolution: early animals, the proposal goes, were indeed shaped as balls, even as adults, but they had the possibility to form a transient cavity by folding inwards when they touched the substrate or encountered a piece of food. The simple, temporary gut thus created allowed to locally concentrate digestive enzymes, and maybe also to trap preys. This structure was later stabilized during evolution as a permanent digestive cavity. Our own cells, Haeckel further claimed, still have to move inside during embryonic development because they recapitulate this ancestral response. By analogy to the embryonic stage (gastrula), the hypothetical ancestor was called Gastraea.

The Gastrae hypothesis for early animal evolution. Here, early animals are depicted as flattened balls, that could transiently form a cavity by invagination when they were encountering food. This transient cavity would have then changed into a permanent structure, with cells on the inner and the outer side acquiring different identities (ectoderm and endoderm, respectively). A similar change of shape, with external cells having to move inside, would be recapitulated, in a modified form, during gastrulation in all animal embryos. After http://scienceblogs.com/pharyngula/2007/02/21/basics-gastrulation-invertebra/

Still now, the Gastraea hypothesis has its supporters and its critics. Whether one loves it or hates it, it probably remains the only attempt so far at solving the riddle of gastrulation. Yet one can still wonder: even if the Gastraea hypothesis explains how gastrulation appeared, why has it been universally maintained since then? Why could animal embryos deeply modify gastrulation movements in evolution (one would hardly recognize Haeckel’s Gastraea in a human or a nematode gastrula) but never completely abolish them? Could movements perform a so far unrecognized function, one that would provide a selective pressure to maintain them?

Gastrulation movements provide ancient mechanical signals to determine cell identity

We set to investigate this question in a project carried out in Emmanuel Farge’s lab (Institut Curie, Paris). The activity of the lab has concentrated, over the past 10 years, on demonstrating that morphogenetic movements during embryonic development perform another function besides the establishment of shape: they are also signals. Cells can perceive when the movements of neighboring cells are deforming them, and react by turning on or off the expression of certain developmental genes. Thus, mechanical strains that accompany morphogenetic movements during development are not only an output of the regulatory activity of the genome, but, in turn, provide inputs that feed back on gene expression. However, examples of this phenomenon remained few (mechanical induction of the transcription factor Twist in the anterior midgut of drosophila embryos for example, or mechanical induction of joint fate at the articulation of two bones during early mouse development). Moreover, these examples were all disconnected – each case study on a model organism revealed a completely different picture from previously investigated species, providing little support for any evolutionarily ancient function for mechanotransduction in development.

We set out to test one precise hypothesis: do mechanotransduction pathways respond to gastrulation movements? Are mechanical signals necessary to induce the correct molecular cell identity at the correct position when the animal stops being a ball and first starts acquiring its shape? If the answer were yes, and if this phenomenon were of general importance in animal embryos (with the same transduction pathways responding to deformations and activating the same downstream cassette in the gastrulas of several, distantly related species), this could contribute to explain why gastrulation movements were conserved in evolution: the mechanical cues they provide are a pre-requirement for cell fate specification.

We started by comparing zebrafish and drosophila, that belong to the two main branches of the bilaterian evolutionary tree (Deuterostomia and Protostomia). In both species, we used assays that first blocked gastrulation movements, and then performed exogenous deformations to artificially rescue mechanical strains. Interestingly, in both species, we found that blocking gastrulation movements blocked the expression of certain developmental genes, and that rescuing movements reestablished it. Moreover, both the identity of the responding cells and the identity of the responding genes turned out to be similar in both species: in both cases, the responsive cells were the presumptive mesoderm, and mechanical signals activated key early transcription factors for early mesoderm specification – notail in zebrafish (a brachyury orthologue) and twist in drosophila. Finally, in both species, the mechanotransduction pathway turned out to be the same: cell deformation in the presumptive mesoderm promotes phosphorylation of β–catenin at a conserved site (tyrosine 654 in zebrafish and 667 in drosophila), which in turns promotes its translocation into the nucleus, where it acts as a transcription factor and turns on mesoderm genes.

Mechanical induction of mesoderm identity in flies

In Drosophila, the study was performed on snail-/- mutants. These mutants lack invagination of the ventral furrow, which gives rise to the bulk of the trunk mesoderm in the fly. Previous research had demonstrated that gently poking the ventral side of a drosophila snail mutant rescues invagination with 70% of success. In snail mutants lacking invagination, twist expression fades prematurely in the mesoderm (though it is correctly initiated at earlier stages). Rescuing invagination rescues a high level of twist expression in the ventral furrow. Strikingly, even in the 30% of cases when poking does not rescue invagination, a small increase of twist expression was observed in response to cell deformation. Altogether, these results indicated that the mechanical constraints linked to ventral furrow invagination maintain a high level of Twist expression in the drosophila mesoderm.

Twist immunostaining in the ventral furrow (future mesoderm) of Drosophila embryos. (a) is a wild-type individual, (b) a sna mutant with reduced Twist expression and (c) a sna mutant where invagination has been rescued by indentation. This also rescues high levels of Twist. (d) levels of Twist measured in sna mutants that were poked, but failed to respond by invaginating; the quantification shows that poking alone partially rescued Twist expression.

Mechanical induction of mesoderm identity in fish

In zebrafish, the first morphogenetic movement that happens is epiboly: cleavage produces a small mass of cells on top of a ball of nutritive yolk. The first task of the cells is to enclose the yolk, and to achieve this, they start spreading over it and engulfing it. At the beginning of this spreading movement, the cells of the presumptive mesoderm – which form a marginal ring around the bottom of the mass of cells – undergo a specific deformation: they are stretched and dilated. Shortly after being deformed, these cells show nuclear translocation of β–catenin and, under the control of β–catenin transcription activity, start expressing notail, thus acquiring the first molecular signs of mesoderm identity. Could cell deformations due to the first gastrulation movements be required to induce this identity, as they are in drosophila? We demonstrated, by blocking gastrulation movements using specific inhibitors of non-muscle myosin II (blebbistatin) or microtubules (nocodazole), that deformation is indeed required for β–catenin to move into the nuclei and for notail expression to be initiated. Moreover, rescuing movements by artificial bulk compression of the embryo, by washing away blebbistatin, or by injecting magnetic particles in the embryos and using a magnetic ring to pull the marginal cells, resulted in a rescue of β–catenin nuclear translocation and notail expression.

Zebrafish embryos at the dome stage (beginning of gastrulation). (a) controls, (b) embryos with movements blocked by blebbistatin treatment, (c) PIV (Particle Image Velocimetry) quantification of cell deformations at the margin of artificially compressed blebbistatin-treated embryos (blue is dilation and red is compression), (d) PIV quantification of cell deformations after washing blebbistatin, which prompts resumal of wild-type movements and (e) schematic drawing of magnetically deformed embryos. (f-j) β–catenin immunostaining of embryos in each experimental condition, showing that cell movements are necessary and sufficient for nuclear β–catenin translocation in marginal cells. (k-o) brachyury in situ hybridization of embryos in each condition, showing that cell movements are necessary and sufficient for brachyury expression around the margin (future mesoderm).

An ancient function for mechanotransduction in mesoderm induction?

The drosophila/zebrafish comparison suggested one tempting conclusion: that mesodermal identity was under the control of mechanical signals already in the last common bilaterian ancestor, more than 570 million years ago, and that mechanical signals still induce mesoderm identity in the embryos of most, and maybe even all, bilaterian animals. Indeed, one strikingly recurrent theme in bilaterian development is the expression of brachyury along the blastopore – an observation that could be elegantly explained if brachyury were under the control of conserved mechanical strains.

If so, one might need to consider a revised status for embryonic geometry in developmental biology – not only as an output of gene expression, but as an integral part of the regulatory networks that control development. By doing so, we might get insights into how the evolution of embryonic shape and the evolution of differentiated cell identity (often considered as largely independent issues) actually constrain each other. We might also come closer to understanding why, in the individual development of every single animal of our planet, the dance of gastrulation needs to reenact, in an unbroken chain since our Precambrian ancestors, the way the first animals stopped looking like balls, and paved the way for the diversity of animal shapes we see today.

References

Brunet, T., Bouclet, A., Ahmadi, P., Mitrossilis, D., Driquez, B., Brunet, A.-C., Henry, L., Serman, F., Béalle, G., Ménager, C., et al. (2013). Evolutionary conservation of early mesoderm specification by mechanotransduction in Bilateria. Nat. Commun. 4. (open access)

Piccolo, S. (2013). Developmental biology: Mechanics in the embryo. Nature 504, 223–225.

On the Gastraea theory

Wolpert, L. (1992). Gastrulation and the evolution of development. Dev. Camb. Engl. Suppl. 7–13.

On Haeckel’s drawings

the case for the fraud: Richardson, M.K., Hanken, J., Selwood, L., Wright, G.M., Richards, R.J., Pieau, C., and Raynaud, A. (1998). Haeckel, embryos, and evolution. Science 280, 983, 985–986.

the case against the fraud: Richards, R.J. (2009). Haeckel’s embryos: fraud not proven. Biol. Philos. 24, 147–154.

(4 votes)

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This Wellcome Trust-funded post-doc position is available to carry out computational modelling of coordination of planar polarity as part of an interdisciplinary team using Drosophila epithelial development as a model system. The Strutt lab (MRC Centre for Developmental and Biomedical Genetics, University of Sheffield) studies  the planar polarity signalling pathways that control coordinated cell polarisation in animal tissues. In collaboration with Prof. Nick Monk (University of Sheffield) we are now seeking to combine our biological expertise with computational modelling approaches, to build an integrated understanding of coordinated cell polarisation.

The post-doc will be involved in developing quantitative models to understand the effects of integrating collective protein behaviour and different regulatory mechanisms in the coordination of cell polarity, and to devise and assist in the experimental testing of such models. The work will build on existing theoretical models for coordination of cell polarity and incorporate quantitative data producedthrough analysis of conventional and super-resolution images of developing tissues in both the wildtype state and following experimental manipulation.

Informal enquiries may be directed to David Strutt (d.strutt@sheffield.ac.uk) or Nick Monk (n.monk@sheffield.ac.uk). Formal applications should be made directly to the University of Sheffield (http://www.sheffield.ac.uk/jobs Job Ref: UOS007732) by no later than the 16^th January 2014.

Recent relevant publications:

Brittle, Thomas & Strutt (2012) Planar Polarity Specification through Asymmetric Subcellular Localization of Fat and Dachsous. Curr Biol 22: 907-914

Strutt, Warrington & Strutt (2011) Dynamics of core planar polarity protein turnover and stable assembly into discrete membrane subdomains. Dev Cell 20: 511-525

Strutt & Strutt (2009) Asymmetric localisation of planar polarity proteins: Mechanisms and consequences. Semin Cell Dev Biol 20: 957-963

Fischer, Houston, Monk & Owen (2013) Is a persistent global bias necessary for the establishment of planar cell polarity? PLoS ONE 8: e60064

Schamberg, Houston, Monk & Owen (2010) Modelling and analysis of planar cell polarity. Bull. Math. Biol. 72: 645-980

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The Royal Institution Christmas Lectures are an annual event where science is celebrated and young people are inspired. This year’s lectures celebrate the Life Fantastic, and will showcase the excitement, beauty and medical potential of developmental biology.

If you were brought up in the UK, the Royal Institution (RI) Christmas Lectures will have been almost as much part of Christmas as Christmas pudding or the Queen’s speech. This annual event started in 1825, instigated by Michael Faraday, and has been broadcasted on the BBC in the festive season since 1966. From the very beginning that the Christmas Lectures were aimed primarily at a young audience, something very rare in Victorian times. The lectures aim to dazzle and inspire young people in equal measure, and often include fantastic demonstrations. The list of speakers over the years is impressive: from Michael Faraday himself to Sir David Attenborough.

Following in the footsteps of such distinguished scientists, this year’s speaker is developmental biologist Dr Alison Woollard. The Woollard lab is based in the Biochemistry Department at the University of Oxford, and focuses on the molecular mechanisms of cell fate determination during C.elegans development. Her series of talks, entitled Life Fantastic, aims to excite and inspire young people, bringing developmental biology to a wider audience. In Alison’s own words: ‘I’m incredibly excited and proud to present this year’s Christmas Lectures. This is partly because my area of science, developmental biology, tends to be under-represented in the media and in science communication, but mainly because Life Fantastic is such an interesting story to share.’

Life Fantastic will be divided into three lectures. The first lecture will focus on how all organisms start as a single cell and develop into a complex beings. The second lecture will explore the concept of mutations, how small changes in the DNA can have evolutionary consequences, and how our knowledge of mutations can be important to understand disease. The final lecture focuses on ageing, and how developmental biology and genetics may potentially help us to live longer. True to the spirit of the Christmas Lectures we have been promised an action-packed series of lectures, where Alison’s model organism, C.elegans, will take central stage, and also featuring lobsters, mussels, Chihuahuas, goose and naked moles!

So if you ever wanted to explain to your family what developmental biology is and why it is important, or just want to see developmental biology presented in all its glory, why not watch this year’s Christmas Lectures? We are certainly in for a (Christmas) treat!

Watching in the UK:

The RI Christmas lectures will be broadcasted on BBC4 in the following days:

–       Lecture 1: Where do we come from? – 28^th December (8 p.m.)

–       Lecture 2: Am I a mutant?- 29^th December (8 p.m.)

–       Lecture 3: Could I live forever?- 30^th December (8 p.m.)

Watching outside the UK:

The RI Christmas Lectures will be available to stream on the RI website in early 2014.

Special Christmas Lectures I’m a scientist, get me out of here!:

The Royal Institution  is collaborating with ‘I’m a scientist’ in a special I’m a scientist, get me out of here! RI Christmas Lectures Q&A with Alison and other developmental biologists. Question submission is now open, and answers will start being published with the broadcast of the first lecture. You can read more about this project here.

The RI advent calendar:

The RI is also running a science advent calendar this year, exploring all 23 chromosomes and mitochondrial DNA. These outreach videos explore different science concepts, including developmental biology, such as the movie below explaining the concept of stem cell. You can watch all the movies released so far here.

Images credit: Paul Wilkinson

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With the pre-festive season and the long winter darkness that accompanies it, it is appropriate to wonder how daylight/darkness cycles affect our biology. Regular daily variations such as daylight/darkness cycles are called circadian rhythms. For example, human skin needs to respond to harmful UV radiation generated by sunlight in a circadian manner. Amazingly, our cells have developed an inherent and self-sustained clock in order to adapt their behavior to these daily fluctuations.

In a recent study published in Cell Stem Cell, Janich and colleagues tried to understand how circadian rhythms could modulate self-renewal and differentiation of human skin (epidermal) stem cells. Part of their approach consisted in using genetic engineering to increase and sustain the expression of the core clock genes Per1 and Per2, core clock genes being required for regulation of circadian rhythms in cells. They show that this over-expression of Per1 and Per2 results in spontaneous stem cell differentiation.

In this picture, one can observe skin that is obtained from the transplantation of a mixture of human skin stem cells (in red and in green, transplanted at 1:1 ratio in the 3 panels) into recipient mice. In all three panels, the red cells are “normal” cells. In the control left panel, the green cells are also “normal” (EV). One can see that the bottom (basal) layer, the one in which the stem cells reside, is composed of green and red cells. In the middle panel, the green cells have been engineered to over-express Per1. In the right panel, the green cells over-express Per2. In contrast to the left panel, the green cells that over-express Per1 or Per2 are found in the upper skin layers (differentiated cells) and not in the basal layer containing the stem cells. From this observation, the authors conclude that over-expression of the core clock genes Per1 or Per2 triggers epidermal stem cell differentiation.

This example shows that circadian rhythms play important roles in stem cell decisions. Interestingly, it has also been shown in another study that strong disturbance of circadian rhythms can lead to premature ageing. So while you enjoy your Christmas holidays, make sure you get your beauty sleep, your stem cells need it!

Janich, P., Toufighi, K., Solanas, G., Luis, N. M., Minkwitz, S., Serrano, L., Lehner, B. and Benitah, S. A. (2013) ‘Human epidermal stem cell function is regulated by circadian oscillations’, Cell Stem Cell 13(6): 745-53.

doi: 10.1016/j.stem.2013.09.004.

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DNA extraction from fruit is an easy experiment that makes a great demonstration for kids’ science fairs. I ran a DNA extraction stall at Oxford’s Wow!How? family science fair a few years back. Unfortunately I didn’t take any photos at the time but I had a lot of fun this weekend recreating the experiment in my kitchen!

The experiment is hands on and messy, which kids tend to love, and there’s plenty of opportunity to explain why DNA is important in telling the cells in our body what to do. You could even go into more detail and explain some of the concepts of genetics to older children.

During a busy science fair there might not be time to wait 20 minutes for the extraction solution to work. To avoid having to wait you could set up stations for each of the steps below and every half an hour or so prepare a handful of fruit/extraction buffer solutions and also some alcohol/purified DNA mixtures.

Click here for a downloadable instruction sheet that can be printed off for children/parents to take home.

Here’s what to do:

1) Prepare your equipment

You will need:

– Two kiwis

– Pineapple juice

– Table salt

– Washing up liquid

– Cold alcohol – put in the freezer before you start the experiment (I used surgical spirit but strong rum also works well)

–  Two small glass beakers (or plastic cups)

– Sieve

– Bowl

– Tall glass/measuring cylinder

– Kitchen Roll

– Stirring rod (or chopstick)

– Knife

– Fork

– Chopping board

2) Make the extraction solution

The DNA is tightly packaged inside the nucleus of cells. The membranes of the cell and of the nucleus  are rich in fats so we can break them down using a detergent. The salt helps to get rid of the proteins that package the DNA tightly inside the nucleus.

– In one of your beakers measure out about 80mls water

– Add half a teaspoon of salt and stir until dissolved

– Add two teaspoons of washing up liquid and stir gently avoiding making too many bubbles

3) Prepare your fruit mush

DNA can be extracted from anything living. You could also try this experiment with strawberries or bananas. Make sure you remove the fruit skins as they are mostly dead and don’t contain DNA. The kiwi needs to be broken up so the extraction solution can get to the cells.

– Peel your kiwis and chop into small pieces

– Add the chopped up kiwi to the second small beaker and use the fork to mush it up

4) Add the extraction solution to the fruit mush

In this step the detergent breaks down the cell membranes so the DNA can be released. The salt removes proteins that are bound to the DNA.

– Add your extraction solution to the kiwi mush

– Leave at room temperature for about 20 minutes

5) Filter the solution

This gets rid of the fruit pulp and seeds and should leave a pure solution of DNA

– Put your sieve over a clean bowl and line the sieve with a few sheets of damp kitchen roll

– Pour your green mush into the sieve carefully, being careful not to break the kitchen roll

– Use a fork to gently push the mixture through the sieve.

– The pulp and seeds should be left in the sieve and there should be a greenish liquid in the bowl. Transfer this to a tall glass or measuring cylinder.

6) Purifying the DNA

If you want an even purer solution of DNA then we need to remove proteins that are bound to the DNA. Pineapple juice contains an enzyme that breaks down proteins. If you haven’t got any pineapple juice then contact lens cleaning solution can also be used.

– Add pineapple juice to the green liquid. You will need about 1ml of pineapple juice to 5mls of the green DNA solution.

– Leave at room temperature for about 5 minutes

7) Precipitating the DNA

DNA dissolves in water so will not be visible. However, it does not dissolve in alcohol so if we add surgical spirit then the DNA will collect as a white mass at the top of the tube.

– Remove the alcohol from the freezer

– Carefully pour the alcohol down the side of the glass

– You need about equal volumes of DNA solution to alcohol

8) Visualise the DNA sample

After about 10 minutes you should be able to see a mass of white stringy stuff at the top of the tube (see right hand photo). This is the kiwi DNA! You can fish this out using the chopstick and place it onto a piece of card to take home.

Sources

This protocol is adapted from the following sources:

http://www.funsci.com/fun3_en/dna/dnaen.htm

http://www.thenakedscientists.com/HTML/content/kitchenscience/exp/how-to-extract-dna-from-a-kiwi-fruit/

http://www.nuffieldfoundation.org/practical-biology/extracting-dna-living-things

This post is part of a series on science outreach. You can read the introduction to the series here and read other posts in this series here.

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Organisation: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Studentship starting: October 2014

Application Deadline: 10th January 2014

Interviews: 14th February 2014

We invite applications from committed and creative candidates for a 4 year MRC DTA PhD studentship. This studentship is aimed at individuals who have a clear idea of their preferred research topic and supervisor and will commence their PhD project in Year 1.

Stem Cell Biology

Stem cells are defined by the dual capacity to self-renew and to differentiate. In adult tissues stem cells sustain homeostatic cell turnover and enable repair and regeneration throughout the life time of the organism. In contrast, pluripotent stem cells are generated in the laboratory from early embryos or by molecular reprogramming. They have the capacity to make any somatic cell type, including tissue stem cells.

Stem cell research aims to identify and characterise which cells are true stem cells, and to elucidate the physiological, cellular and molecular mechanisms that govern self-renewal, fate specification and differentiation. This research provides new foundations for biomedical discovery, biotechnological and biopharmaceutical exploitation, and clinical applications in regenerative medicine.

Cambridge Stem Cell Community

The Cambridge Stem Cell Institute is exceptional in the depth and diversity of research in both fundamental and translational Stem Cell Biology. The Institute provides a dynamic and interactive research community with over 50 PhD students. Choose from over 30 participating host laboratories using a range of experimental approaches in different tissues and diseases http://www.stemcells.cam.ac.uk/researchers/.

Programme Outline

During the first year students will enter their host lab to commence their PhD research and in addition will join with other PhD students to:

i) study fundamental aspects of Stem Cell Research through a series of group discussions led by leaders in the field;

ii) learn a variety of techniques, such as advanced imaging, flow cytometry, and analysis of complex data sets.

Students will complete their PhD research and thesis submission over years 2-4.

Please note applicants must meet the MRC funding eligibility requirements – please check the eligibility requirements at http://www.mrc.ac.uk/Fundingopportunities/Applicanthandbook/Studentships/Eligibility/index.htm 

To Apply: visit http://www.stemcells.cam.ac.uk/careers-study/otherstudentships/ for full details.

Any questions? Email: cscr-phd@cscr.cam.ac.uk

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Organisation: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Studentship starting: 01 October 2014

Application Deadline: 1st April 2014

Interview Date: 29th April 2014

Programme Overview: This studentship is targeted to applicants with a Physical Sciences, Mathematical or Computational Sciences background, who are interested in applying their training to aspects of stem cell biology.

This programme provides students with an opportunity to spend time in three different labs during their first ‘rotation’ year, before deciding where to undertake their thesis work for years 2-4.

Physical Biology of Stem Cells: Stem cells are defined by their dual capacity to self-renew and differentiate into somatic cells. Great inroads have been made towards understanding how stem cells generate tissue and sustain cell turnover in tissue. At this time most of the inroads have been made by studying the individual biochemistry of the stem cell; much less progress has been made in understanding their function across scales – from molecules to tissue – or how they interact with their physical environment.

In studying the physical biology of stem cells, the aim is to identify and characterise the importance of physical, chemical, mathematical, and engineering considerations in the function of stem cells. This could include mathematical modelling of homeostasis in tissues, engineering controlled environments to control stem cell function, imaging and biotechnology, using single molecule approaches to study molecular interactions, systems biology, or investigating the importance of the stem cell’s response to forces in its environment.

The research generated by the MRC studentships should provide new foundations for biomedical discovery, biotechnological and biopharmaceutical exploitation, and clinical applications in regenerative medicine.

Qualification Eligibility: We welcome applications from those who hold (or expect to be awarded) a relevant first degree at the highest level. You should have a passion for scientific research, specifically with a Physical Sciences, Mathematical or Computational Sciences background.

Financial Support: All applicants must meet the MRC funding eligibility requirements outlined at http://www.mrc.ac.uk/Fundingopportunities/Applicanthandbook/Studentships/Eligibility/index.htm

To Apply: Please visit http://www.stemcells.cam.ac.uk/studentships/phy-biol/ for full details. Please note you will be required to complete and submit a departmental application form, a copy of current CV, provide two references and upload a copy of your transcripts as part of the application process.

Visit http://www.physbio.group.cam.ac.uk/ for details of the current Cambridge Physical Biology network.

Any questions? Email: cscr-phd@cscr.cam.ac.uk

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Organisation: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute

Studentship starting: October 2014

Application Deadline: 10th January 2014

Interviews: 30th & 31st January 2014

Stem cells are defined by the dual capacity to self-renew and to differentiate. These properties sustain homeostatic cell turnover in adult tissues and enable repair and regeneration throughout the lifetime of the organism. In contrast, pluripotent stem cells are generated in the laboratory from early embryos or by molecular reprogramming. They have the capacity to make any somatic cell type, including tissue stem cells.

Stem cell biology aims to identify and characterise which cells are true stem cells, and to elucidate the physiological, cellular and molecular mechanisms that govern self-renewal, fate specification and differentiation. This research should provide new foundations for biomedical discovery, biotechnological and biopharmaceutical exploitation, and clinical applications in regenerative medicine.

Cambridge Stem Cell Community
The University of Cambridge is exceptional in the depth and diversity of its research in Stem Cell Biology, and has a dynamic and interactive research community that is ranked amongst the foremost in the world. By bringing together members of both the Schools of Biology and Medicine, this four year PhD programme will enable you to take advantage of the strength and breadth of stem cell research available in Cambridge. Choose from over 30 participating host laboratories using a range of experimental approaches and organisms.

Programme Outline
During the first year students will:

i) perform laboratory rotations in three different participating groups working on both basic and translational stem cell biology;

ii) study fundamental aspects of Stem Cell Biology through a series of teaching modules led by leaders in the field;

iii) learn a variety of techniques, such as advanced imaging, flow cytometry, and management of complex data sets.

Students are expected to choose a laboratory for their thesis research by June 2015, and will then write a research proposal to be assessed for the MRes Degree in Stem Cell Biology. Students will then commence a 3 year PhD. 

Visit http://www.stemcells.cam.ac.uk/careers-study/studentships/ for full details

Any questions? Email: cscr-phd@cscr.cam.ac.uk

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My name is Rob Fordham and I’ve just finished my PhD at the Wellcome Trust/MRC Cambridge Stem Cell Institute, University of Cambridge, supervised by Dr Kim Jensen (now Associate Professor at the Biotech Research and Innovation Center (BRIC), University of Copenhagen), and Professor Roger Pedersen.* During my PhD I was fascinated with intestinal development- and here I hope to convince you why this is of interest not just to developmental biologists!

The mature mammalian intestinal epithelium is comprised of villi, lined with absorptive enterocytes, protruding into the lumen, and proliferative crypt compartments invaginated into the underlying mesenchyme. The mature intestinal epithelium is the most rapidly self-renewing tissue in adult mammals, with a turnover time in the mouse of 3-4 days. We now know that this high rate of cell replenishment is driven by the division of intestinal stem cells sitting at the crypt base, intercalated between Paneth cells. This has made the intestine an excellent model system for studying tissue stem cell biology, in both homeostasis and disease. However, the emergence of this state, with functionally mature tissue stem cells, from its foetal progenitors during development was relatively poorly understood when I started my PhD.

Morphologically, we do know that by E9.0 of mouse development, a sealed gut tube has formed, which progressively lengthens over the subsequent four days. Around E14.5, the pseudostratified epithelium transitions into a simple columnar epithelium, followed by mesenchymal invagination around E15.5 to form nascent villi. Thus, in mice at birth, the intestine is developmentally immature, consisting simply of a series of undulating villi and proliferative intervillus regions. During the first two postnatal weeks, the intervillus regions lengthen into nascent crypts, coincident with the emergence of Paneth cells. Development of the human intestine follows broadly the same sequence of events as in the mouse, except that crypts start to form in utero, during the second and third trimesters. The early postnatal mouse intestine thus provides an interrogatable model system to understand the events that occur in humans during late gestation.

In 2009, methods were devised for the long-term three-dimensional culture of adult intestinal epithelium as organoids. These organoids require Wnt signalling and contain all the major cell types of the adult intestinal epithelium in vivo, including stem cells and Paneth cells (Sato et al., 2009).

By adapting these culture conditions for foetal material, we were able to demonstrate the existence of a rapidly proliferating population of intestinal progenitors that exists prior to the establishment of mature intestinal stem cells. These cells, present in the foetal intestine from E14.5, grow in vitro as cystic Foetal Enterospheres (FEnS). Through subsequent postnatal development, FEnS are replaced by organoids, and beyond around P15 only organoids can be grown. We also found that a similar population of proliferative progenitors exists in the human intestine towards the end of the first trimester (Fordham et al., 2013).

Mouse Foetal Enterospheres (mFEnS), expressing GFP, in 3D culture. From Fordham et al., Cell Stem Cell (2013)

Thus, an otherwise developmentally transient population can be expanded in vitro, allowing direct comparison between foetal and adult intestinal stem cell populations for the first time. One important finding was that FEnS could grow without the Wnt-augmenting factor R-spondin, and even in the presence of Wnt signalling inhibitors, in stark contrast to adult organoids, which require Wnt signalling. We further demonstrated that canonical Wnt signalling is sufficient to induce the maturation of foetal intestinal progenitors into adult-like organoids (Fordham et al., 2013). Another group, from Brussels, independently described the same population of immature mouse intestinal progenitors. Mustata et al., (2013) showed that the foetal-to-adult conversion could be induced by inhibiting Notch signalling. Together these two findings suggest that the maturation of foetal progenitors proceeds in concert with establishment of the stem cell niche and that intestinal epithelial maturation requires modulation of Wnt and Notch signalling.

Of course this is all very interesting for developmental biologists, and has raised new questions for the intestine field to now address, such as the extent and relevance of heterogeneity within the pool of intervillus cells.

But I believe this also has important implications for the more translational arm of stem cell research. In particular, it is well known that most of the current protocols aiming to generate various tissue types from pluripotent cells tend to result in immature foetal-like populations, rather than functionally mature adult tissues. Indeed, we were able to generate intestinal cells from the directed differentiation of human iPS cells, but these cells displayed more foetal than adult characteristics (Hannan et al., 2013; Fordham et al., 2013). Clearly the conversion of these immature cells into an adult state in vitro will require a much better understanding of tissue stem cell specification during normal development in vivo.

Since the overarching aim of regenerative therapy is replacement of damaged tissue, we were interested in assessing the in vivo potential of immature intestinal cells. Importantly we now had a system, in the form of mouse FEnS, to sufficiently expand immature cells for transplantation experiments. Previous work from Tokyo Medical and Dental University had shown that adult colonic organoids, transplanted into mice with damaged colons, could contribute to regeneration of the colonic epithelium (Yui et al., 2012). Working with the group in Tokyo, we were able to demonstrate that mouse FEnS could similarly contribute to colonic epithelial regeneration following transplantation (Fordham et al., 2013). This proof-of-principle experiment revealed that foetal cells cultured for prolonged periods in 3D culture retain the potential for differentiation into more-mature cell types, perhaps with greater lineage plasticity than adult-derived cells.

Therefore this work should highlight why translational stem cell research should not lose sight of basic developmental biology- and that developmental biology is perhaps not so far removed from translational impact as people think!

GFP-expressing mouse FEnS, transplanted into a damaged colon, contribute to epithelial regeneration

Summary of the major findings presented in Fordham et al., Cell Stem Cell (2013)

 References

Fordham, R.P. and Yui, S. et al., Transplantation of Expanded Fetal Intestinal Progenitors Contributes to Colon Regeneration after Injury. Cell Stem Cell (2013) Dec 5; 13 (6) 734-744

Hannan, N.R.F., Fordham, R.P. et al., Generation of multipotent foregut stem cells from human pluripotent stem cells. Stem Cell Reports (2013) Oct 10; 1 (4); 293-306

Miyoshi, H. and Stappenbeck, T.S. The Young and the Wnt-less: Transplantable Fetal Intestinal Spheroids without Wnts. Cell Stem Cell (2013) Dec 5; 13 (6) 637-638

Mustata, R.C. et al., Identification of Lgr5-independent spheroid-generating progenitors of the mouse fetal intestinal epithelium. Cell Rep. (2013) Oct 31;5(2):421-32

Sato, T. et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature (2009) May 14;459(7244):262-5

Yui, S. et al., Functional engraftment of colon epithelium expanded in vitro from a single adult Lgr5⁺ stem cell. Nat. Med. (2012) Mar 11;18(4):618-23

This work was funded by the Medical Research Council and Wellcome Trust

*from January 2014, I will be a Postdoctoral Research Fellow in Professor Owen Sansom’s group at The Beatson Institute for Cancer Research, Glasgow

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