I’ve been asked to present the back-story behind our recently published manuscript in Development “Transcription precedes loss of Xist coating and depletion of H3K27me3 during X-chromosome reprogramming in the mouse inner cell mass.”

Mammalian dosage compensation occurs by silencing one X-chromosome in female cells, termed X-chromosome Inactivation (XCI).  Balancing X-linked gene transcription is critical for female development, and in most cells, the absence of XCI is lethal.  Interestingly, female mouse germ cells and inner cell masses (ICMs) appear to be an exception, capable of handling imbalances in X-linked gene dosage and exhibiting activation of both X-chromosomes.  These uncommitted cells have the unique ability to reset the epigenetic profile of inactive X-chromosomes.  As a graduate student, I was drawn to investigate the mechanism of X-chromosome reactivation because I wanted to understand how epigenetic reprogramming occurs naturally in the embryo.

Most of what is known about the molecular events that initiate XCI has been extrapolated from observations of XCI in mouse cells.  In the mouse embryo, the choice of which X-chromosome to be silenced is made stochastically; maternal and paternal X-chromosomes have an equal chance of being silenced and the end result is a random pattern of XCI.   In contrast, extraembryonic tissues exhibit an imprinted form of XCI, and the paternal X-chromosome appears to be predetermined for silencing.  It has been the prevailing opinion in the field that a master controller, the Xist non-coding RNA, regulates XCI.  Xist RNA has been linked to almost every step in the XCI mechanism, including choice, initiation, and maintenance; however, its exact role is still unknown.  What is clear is that Xist transcripts coat the X-chromosome in cis, recruiting epigenetic modifying proteins that are responsible for forming inactive-X heterochromatin.

It had previously been shown that in all cells of the female preimplantation mouse embryo, Xist transcripts coat and recruit the repressive histone modification H3K27me3 to the Xp.  However, X-linked gene expression at single cell resolution had not been directly analyzed, relative to cell fate in the embryo.  In our manuscript, we definitively show that all cells, irrespective of lineage, establish imprinted XCI by the early blastocyst stage.   This sets up a situation in the ICM; epiblast cells must undergo a reactivation/inactivation cycle.  During ICM maturation, epiblast cells exhibit progressive upregulation of Nanog expression, coincident with their transition to ground-state pluripotency.  Curiously, Xist is a target of OCT4, SOX2, and NANOG.  Based on the concept that Xist is the master regulator of XCI, it had been predicted that progressive upregulation of NANOG in the ICM leads to Xist repression, resulting in loss of Xist coating and triggering reactivation of Xi genes.

We used a knockout of Grb2 to upregulate Nanog expression in the ICM.  As might be predicted, increased NANOG dosage led to Xist repression, indicated by loss of Xist coating.  However, X-linked gene silencing is maintained in cells that lack Xist coating, perhaps suggesting that induced epigenetic reprogramming is incomplete in these cells.  These provocative results point to other factors, in addition to Xist, that are needed initiate Xi gene silencing.  Recent results from Kalantry et al. Nature 2009, Namekawa et al. Mol. Cell Biol. 2010, and Okamoto et al. Nature 2011 support these conclusions and the need for the field to reconsider mechanisms regulating XCI.
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Last month, the advocate-general of the European Court of Justice gave his opinion on a long-running legal debate about a patent filed several years ago in Germany. If the Court follows his recommendation, patenting of applications using embryonic stem cells will be prohibited on moral grounds.

13 leaders of major stem cell projects in Europe responded to the advocate-general’s statement with an open letter published in Nature this week. They express serious concerns about the impact of a patenting ban on European research.

EuroStemCell, Europe’s stem cell hub, has collected comments and information about this case on its Stem cell patents topic page. Visit the site to read the open letter and find out more about the case.

Post a comment on the site, or add your signature to the letter if you wish: http://www.eurostemcell.org/stem-cell-patents

(2 votes)

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Physiologically speaking of course…

As mammals we have biological clocks, or circadian rhythms that respond to light. Dark or blue light set off signalling mechanisms, which ultimately regulate the melatonin levels in our system. Melatonin is a hormone which can induce sleep, if at sufficiently high levels in our system. (Hence your doctor prescribing you melatonin if you have trouble sleeping at night. Suffice to say countless grad students are probably on this stuff right now). (Photo: Festival Colours by jmtimages, Flickr CC)

Plants also have a light-sensing system, which can respond to different times of day and seasonal changes. This can determine  fruit maturation and changing colour in the leaves etc. For plants, blue light has a revitalizing (instead of drowsy) effect. If you’ve ever left your plants without light for too long, they go yellow. Irradiate them with a little blue, and the green will return within a couple of days. (Photo: Autumn Colours in Australia’s National Capital, personal collection).

Interestingly, plants and animals have the same type of photoreceptors to blue light, called Cryptochromes (CRYs), even though they elicit a different response. (Photoreceptors = light absorbing molecules or pigments.)

CRYs initiate the light response, analogous to a molecular domino effect. The pigments bind specialized proteins that in turn, regulate transcription factors that can switch on the light response genes involved in development. Termed as photomorphogenesis, light dependent changes in development can include initiation of flowering, or extension of roots in germinating seeds.

Currently, characterizing the molecular interactions in the plant blue-light/CRY1 response seems to be a hot topic. Two highly similar articles were just published in Genes & Development, which was drawn to my attn by Eva (cheers). They were produced by two different research crews, using virtually the same methods to similar results. It’s not by total coincidence either. Characterizing molecular interactions in plants involve the same gold standard biochemical and genetic assays (i.e. transgenic plants, yeast hybrid systems, loss-of-function mutants etc.)

In summary: the blue-light/CRY1 pathway doesn’t directly switch genes on. instead, it switches off the default dark condition mechanism. In the dark, SPA1 and COP1 proteins negatively regulate the light signalling pathway. Active COP1 normally marks transcription factor HY5 for degradation. SPA1 binds COP1 to enhance this activity. If HY5 were allowed to accumulate, it promotes the expression of multiple genes involved in photomorphogenesis, such as flowering, or germination.

What the groups found: Blue-light triggers CRY1 to bind SPA1. This in turn inhibits SPA1 binding to COP1. In 2001, another group found that CRY1 can also interact directly with COP1 to inhibit its activity in a 2nd way. Result: no COP1 activity, HY5 accumulates. Photomorphogenesis is observed. (CRY1 could not bind SPA1 in the dark, or under red light).

(photo: blue light, by psychiks, Flickr CC. does this make you feel sleepy?)

Some discordance over the CRY1-SPA1-COP1 interaction: One group claims that CRY1 causes SPA1 to dissociate from COP1. (exact mechanism unclear, allosteric binding = conformational change?) the other group suggests that CRY1 impedes COP1 and SPA1 binding, possibly by sandwiching itself between them. Refer to sandwich model in Liu et al. 2011 Figure 4D, or kit-kat dissociation model in Lian et al. 2011 Figure 5G. Devil’s in the details: both groups developed their models based on two biochem assays ~ yeast two/three hybrid and co-immunoprecipitation. However, they only tell you if things bind or not. Interestingly, blue-light and CRY1 were required to prevent SPA1 and COP1 binding. Under blue light, CRY1 could bind both COP1 and SPA1, just not sure how.

The interaction hasn’t been characterized in animals, although they do contain the CRYs. Mammals even have COP1, which can also mark other transcription factors for degradation. Inexplicably, breast cancers were found to have over-expressed levels of COP1. Not sure if CRY also inactivates mammalian COP1, although, one group did stick the mammalian COP1 into Arabidopsis, and found it reacts to blue-light. Interestingly, photoreceptors and COP1 were initially found in Arabidopsis. (score 1 for the plant model of genetics).

Plants:

Liu B, Zuo Z, Liu H, Liu X, & Lin C (2011). Arabidopsis cryptochrome 1 interacts with SPA1 to suppress COP1 activity in response to blue light. Genes & development PMID: 21511871

Lian HL, He SB, Zhang YC, Zhu DM, Zhang JY, Jia KP, Sun SX, Li L, & Yang HQ (2011). Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism. Genes & development PMID: 21511872

Both research groups hail from China and the US. They’re also based in some of the top universities in the world (Peking Uni, UCLA etc.), which makes for some hefty competition, among neighbours no less.

Human Photoreceptors and COP1:
Yi C, & Deng XW (2005). COP1 – from plant photomorphogenesis to mammalian tumorigenesis. Trends in cell biology, 15 (11), 618-25 PMID: 16198569

Yi C, Wang H, Wei N, & Deng XW (2002). An initial biochemical and cell biological characterization of the mammalian homologue of a central plant developmental switch, COP1. BMC cell biology, 3 PMID: 12466024

Cashmore AR (2003). Cryptochromes: enabling plants and animals to determine circadian time. Cell, 114 (5), 537-43 PMID: 13678578

(2 votes)

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We’ve had to find a new location for Thursday’s (today’s) Node meetup at the BSDB meeting.

Find us Thursday April 28 at 8PM in the Gulbenkian Cinema Cafe/Bar.

This is the cafe with the big windows that you pass when you follow the footpath from Eliot Dining Hall to the Woolf College building. This happens to be *exactly* where you will be walking around that time when you go from dinner to poster viewing, so you might as well pop in!

Addendum: We have just found out that there will be a theatre production at Gulbenkian which has intermission from 8:15 to 8:35. This means that there will also be about 200 theatre-goers in the bar at that time. We have the Node banner with us, so you will be able to spot us. However, if it gets too noisy, we might move to the Woolf building earlier, and use one of their upstairs seminar rooms. But just look for the banner: if it’s not in the cinema bar, we’re in the Woolf building.

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Two postdoctoral positions are available immediately at the University of Pennsylvania: (1) to analyze the gene regulatory network underlying neural crest formation (Hong & Saint-Jeannet, 2007. Mol Biol Cell 18, 2192-2202; Hong et al., 2008. Development 135, 3903-3910); and (2) to characterize the molecular regulators of cardiac neural crest migration (Lee & Saint-Jeannet, 2011. Development 138, 2025-2034). Interested candidates must have a PhD, MD or VMD degree and a strong background in developmental biology and/or molecular biology.

Please submit your CV, and contact information for 2 references to Dr. Jean-Pierre Saint-Jeannet, University of Pennsylvania, School of Veterinary Medicine, Department of Animal Biology, 3800 Spruce Street, Philadelphia, PA 19104, USA. e-mail: saintj@vet.upenn.edu

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The Node’s staff asked me to write a short “behind the scenes” on our paper just released in the May 15 issue of Development, “Cardiac neural crest is dispensable for outflow tract septation in Xenopus” http://dev.biologists.org/lookup/doi/10.1242/dev.061614

In the summer of 2008 when Dr. Young-Hoon Lee joined my laboratory from Chonbuk National University for a sabbatical we discussed a number of potential projects, and very quickly decided to analyze the contribution of the neural crest to the cardiovascular system in Xenopus laevis. This was a project that we considered several years earlier when Dr. Lee was a postdoc in my lab, but at the time Young-Hoon opted for a different line of research. To address this question we started using the lipophilic dye DiI to label the putative cardiac neural crest in neurula stage embryos. We found that the label cells were consistently excluded from the outflow tract septum. We were a little skeptical about this result, because of what we know of the cardiac neural crest in other species, but mostly because only a small number of neural crest cells is being labeled by DiI injection, and we could not exclude the possibility that unlabelled neural crest cells were contributing to the outflow tract septum. We then decided to move to a tissue transplantation system, where entire segments of the neural crest from RFP-labeled embryos were grafted onto an unlabeled host embryo. These experiments are not trivial and I have to give credit to Young-Hoon for his persistence and exceptional technical skills. Again in these experiments RFP-labeled neural crest cells were exclusively confined to the aortic sac and arch arteries and never populated the outflow tract cushions, confirming our initial observations using DiI. Consistent with these observations, upon cardiac neural crest ablation the outflow tract and the spiral septum developed normally and expressed the molecular markers specific to these lineages. This was a surprise. In chick and mouse the cardiac neural crest provide the separation for the systemic and pulmonary circulations at the arterial pole, remodeling the outflow tract into two vessels by forming the aorticopulmonary septum. In zebrafish where there is no separation between both circulations cardiac neural crest cells contributes myocardial cells to all regions of the heart. In Xenopus we were expecting to find something that was in between fish and amniotes. This is not the case – in frogs cardiac neural crest cells stop their migration before entering the outflow tract. Our next challenge was to determine the embryonic origin of Xenopus outflow tract septum. We were greatly helped in this quest by a recent study from the group of Michael Kuhl at Ulm University (Germany) that carefully mapped the cardiogenic lineages in Xenopus (Gessert and Kuhl. 2009. Dev Biol 334: 395-408). By transplantation of GFP-labeled regions of the cardiogenic mesoderm we found that the septum was derived from the second heart field, presumably by epithelial-to-mesenchymal transformation of the endocardial cells lining the cardiac cushions.

The picture below shows a lateral view of a host Xenopus laevis embryo after transplantation of both an RFP-labeled neural crest graft (red) and a GFP-labeled second heart field graft (green).

How to explain these differences in the deployment of cardiac neural crest across species? With a single ventricular chamber the separation of the pulmocutaneous and systemic blood is incomplete in frogs. Therefore a possible explanation is that in species that have an evolutionary need for a fully divided circulation the neural crest was recruited into the outflow tract septum to help complete the separation of both circulations at the arterial pole of the heart.
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Here are the research highlights from the current issue of Development:

FatJ keeps neural progenitor pools in shape

The correct development and functioning of the spinal cord depends on the patterning, proliferation and differentiation of neural progenitor cell cohorts along the dorsoventral axis of the neural tube. But how are the numbers of these cells controlled? On p. 1893, Stuart Wilson and colleagues demonstrate that the cadherin FatJ acts via the Hippo pathway to regulate the size of neural progenitor pools in the chick neural tube. Using a large-scale RNAi screen of all cadherin genes expressed in the neural tube, the researchers identified a role for FatJ in neural tube patterning. FatJ, they report, is expressed in intermediate regions of the neural tube, and a loss of FatJ leads to an increase in the size of the progenitor pool in this region. This effect is mediated via the Hippo pathway component Yap1. Thus, this first report of a large-scale RNAi screen in a whole vertebrate organism reveals an important role for FatJ in controlling the proliferation and differentiation of specific interneuron classes.

Endoderm-mesoderm signalling controls pancreatic fate

Inductive interactions between endodermal and mesodermal cells play a role in organogenesis, but the molecular nature of these interactions are unknown. Keiichi Katsumoto and Shoen Kume (p. 1947) now demonstrate that reciprocal signalling mediated by the chemokine ligand CXCL12 and its ligand CXCR4 can regulate pancreatic fate in chick embryos. The researchers show that, prior to blood vessel formation, angioblasts reside in close proximity to the somatic mesoderm and the gut endoderm, tissues from which pancreatic precursors arise. Importantly, they demonstrate that CXCL12 expressed in the gut endoderm functions to attract these angioblasts, which express its receptor CXCR4 and in turn signal back to the gut endoderm to induce pancreatic cell fate; ectopic expression of Cxcl12 attracted angioblasts and resulted in an expanded pancreatic bud, whereas a CXCR4 inhibitor prevented angioblast migration towards the gut endoderm and perturbed vessel formation. The authors propose that reciprocal CXCL12-CXCR4 signalling can spatiotemporally regulate angioblast migration and pancreas induction, shedding light on the early mechanisms that establish pancreatic fate.

Myosin flies in to control oocyte polarisation

During Drosophila oogenesis, interactions between somatic cells and germline cells are crucial for oocyte polarisation and the subsequent formation of the body axes, but the molecular nature of the signals that regulate these interactions is not known. Here, on p. 1991, Trudi Schüpbach and co-workers show that, by regulating myosin activity, protein phosphatase 1β (PP1β) in the posterior follicle cells (PFCs) of Drosophila ovaries controls the generation of an oocyte polarising signal. The researchers first identified a loss-of-function mutation of flapwing, which encodes the catalytic subunit of PP1β, that disrupts oocyte polarisation. They show that excessive mysosin activity in PFCs caused by PP1β disruption leads to defective PFC Notch signalling and endocytosis. This sensitivity to defective Notch signalling, they report, requires JAK/STAT and EGFR signalling in the PFCs. Importantly, the researchers also identify a Notch-independent role for myosin activity in controlling oocyte polarisation, highlighting an additional link between the mechanical force-generating machinery of the cell and the ooctye polarisation pathway.

Xenopus loses heart in neural crest cells

In amniotes, cardiac neural crest (NC) cells contribute to the septum of the heart outflow tract to allow for separation of the systemic and pulmonary circulations. It is not known, however, whether cardiac NC cells are also involved in outflow tract septation in amphibians. Here, Lee and Saint-Jeannet show that cardiac NC cells are dispensable for outflow tract septation in Xenopus (p. 2025). Using a combination of tissue transplantations and molecular analyses, they show that the amphibian outflow tract is derived from precursor cells in the second heart field, as is observed in birds and mammals. In contrast to the situation in amniotes, however, the second heart field, and not the cardiac NC, gives rise to the septum of the Xenopus outflow tract. The researchers propose that this significant difference is an amniote-specific novelty that has been acquired evolutionarily to increase the mass of the outflow tract septum with the need for a fully divided circulation.

X inactivation: novel mechanisms eXist

X-chromosome inactivation (XCI), which is achieved by chromosome coating with Xist RNA and repressive chromatin modifications, results in the silencing of one of the two X chromosomes in female cells. In early mouse XX embryos, the paternal X chromosome is silenced during imprinted XCI. However, in pre-implantation mouse embryos, inner cell mass (ICM) cells reverse this imprinted XCI in order to initiate random XCI later in development. Now, Terry Magnuson and colleagues show that, during this reversal process, gene transcription from the inactive-X precedes the loss of Xist coating (p. 2049). By analysing the kinetics of X-chromosome activity in developing ICMs, the researchers show that reactivation of gene expression from the inactive-X can occur in the presence of both Xist coating and repressive histone H3K27me3 marks, suggesting that X-linked gene transcription and Xist coating are uncoupled during this period. This finding, they propose, alters our perception of the reversal process and supports the existence of Xist-independent silencing mechanisms in the mouse embryo.

NPH3-like proteins PIN down auxin transport

PIN-FORMED (PIN)-dependent auxin transport is essential for many aspects of plant development, and PIN proteins show a polar localisation within cells that determines the direction of auxin flow. Here, Masao Tasaka and co-workers show that Arabidopsis MACCHI-BOU 4 (MAB4)-like proteins can specifically retain polarised PIN proteins at the plasma membrane of cells and, hence, can control directional auxin transport and plant development (p. 2069). Using genetic anaylsis, the researchers show that combined mutations of MAB4 subfamily members result in auxin-related phenotypes and severe reductions in PIN polarisation. Furthermore, they report, mutations of MAB4 genes result in increased PIN internalisation from the plasma membrane but have no effect on intracellular trafficking. Notably, they show that the MAB4 subfamily proteins are localised to the cell periphery in a polarised manner almost identical to that of PIN localisation. These findings suggest that MAB4 proteins, which are related to NON-PHOTOTROPHIC HYPOCOTYL 3 (NPH3) proteins, may regulate the specific localisation of PIN proteins to the plasma membrane.

Plus…

Review: Principles of planar polarity in animal development

Planar polarity refers to the coordinated polarisation of cells or structures within the plane of a tissue. Here, Goodrich and Strutt review the molecular mechanisms and cellular consequences of planar polarization, identifying common principles of planar polarity in animal development. See the review article on p. 1877

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Axons have such important jobs to do that they require their own support staff.  Schwann cells are responsible for ensheathing axons of the peripheral nervous system with myelin, which allows rapid conduction of action potentials.  The process by which Schwann cells do this was understood to involve cytoskeletal regulators, and a recent paper in Development describes how N-WASp plays a role.

Schwann cells begin the myelination process by spiraling lamellipodia-like processes around an axon.  Actin is known to be important for Schwann cell myelination, yet an understanding of the actin effectors involved is incomplete.  In a recent paper, Jin and colleagues demonstrate the role of N-WASp, a protein that links extracellular signals with actin polymerization, in Schwann cell maturation and myelination.  Schwann cells in N-WASp deletion mutants were unable to extend lamellipodia-like processes normally, which likely contributes to the myelination problems also seen in these mutants.  Although these mutant mice were viable through old age, they did display some motor defects.  Images above show axons in normal (top) and N-WASp mutants (bottom).  Without N-WASp, axons (green) remain without myelin, as seen as low levels of the myelin basic protein (MBP, red).

Jin, F., Dong, B., Georgiou, J., Jiang, Q., Zhang, J., Bharioke, A., Qiu, F., Lommel, S., Feltri, M., Wrabetz, L., Roder, J., Eyer, J., Chen, X., Peterson, A., & Siminovitch, K. (2011). N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination Development, 138 (7), 1329-1337 DOI: 10.1242/dev.058677

(1 votes)

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A few weeks ago, you selected this sea urchin image from the Woods Hole embryology course as a cover for Development. We have many more images from this course, and more opportunities to choose a journal cover. Here is the second batch.

Which of these images would you like to see on the cover of Development? Please vote in the poll below the images. (Click any image to see a larger version.) You can vote until May 3, 12:00 (noon) GMT

1. Developing pharyngeal arch region of mouse embryo showing immunofluorescence detection of neurofilaments (green), DiI labeling of trigeminal ganglion (red), and nuclei detected with DAPI (blue). This image was taken by Hozana Andrade Castillo (University of Sao Paulo).

2. Crepidula fornicata (slipper limpet) embryo undergoing cleavage from 4 to 8 cells. Confocal image shows DNA in blue (DAPI) and microtubules in yellow (alpha tubulin). This image was taken by Anna Franz (University of Oxford).

3. Confocal image of a squid embryo. All nuclei are stained with DAPI (blue). Phalloidin staining reveals neural structures (red), while cilia on the surface of the embryo are highlighted by acetylated tubulin staining (green). This image was taken by Davalyn Powell (University of Colorado Denver).

4. Muscle of the ascidian, Ciona intestinalis, visualized by phalloidin staining (red). Nuclei (blue) stained with DAPI. This image was taken by Christine Carag Krieger (University of Pennsylvania).

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The next deadline for Development’s travelling fellowships is coming up on April 30, and Development would like to encourage you to apply if you are a graduate student or postdoc planning to work for a few months in a distant lab. Have a look at the fellowship site for the full requirements, and read these stories from previous recipients on the Node:

Tetyana (from the Ukraine) went to India:
Research Snippets from the Land of the Tiger
The Maggot Meeting 2010

Cristian (from Chile) went to Germany:
Developing Science in a Far Country: The Paradoxes of Life

Shreeharsha (from India) went to Japan:
Research in the Land of the Rising Sun

Dávid (from Hungary) went to Japan:
Nippon

Terry (from the US) went to Israel:
International Experience

Giovanni (from Italy) is currently in the Netherlands:
A nice Lab Experience in Amsterdam

If you’re planning a lab exchange as part of your research, don’t forget to apply for travel funding before April 30. Good luck!

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