Here are the highlights from the current issue of Development:

Pancreatic injury unlocks cell potential

Identifying methods by which pancreatic β-cells can be produced is of major therapeutic importance. Whether there are adult pancreatic cells with the potential to make new β-cells is a matter of much debate. During embryonic development, the transcription factor Ptf1a initially marks multipotent progenitors, before becoming restricted to acinar cells. Here (p. 751), Christopher Wright and colleagues test whether mature Ptf1a-expressing cells can regain multipotentiality upon injury by labelling Ptf1a-positive acinar cells in mice and following their fate after pancreatic duct ligation. Remarkably, not only do new duct cells arise from the labelled cells, but some labelled cells start to express endocrine markers and display the hallmarks of mature β-cells, suggesting transdifferentiation of acinar cells into β-cells. This process is inefficient and slow, but can be enhanced by prior ablation of endogenous β-cells. Thus, pancreatic injury appears to induce reactivation of a more embryonic-like multipotent state in Ptf1a-expressing cells, from which endocrine cells can differentiate, possibly opening up new avenues for generating β-cells.

Lipid leads the way in wound healing

During epithelial wound healing, actin assembles at the leading edge of cells that border the wound, forming dynamic protrusions and, in some cases, an actomyosin cable. Together, these actin-rich structures are essential for wound closure. The process of dorsal closure in Drosophila shares many characteristics with wound healing and is a convenient system for cell biological analysis. Building on earlier results showing that the apical polarity determinant Par3/Bazooka (Baz) is lost from the leading edge of cells during dorsal closure, Tom Millard and colleagues (p. 800) now uncover a molecular mechanism by which Baz localisation regulates actin dynamics. Baz is known to bind the lipid phosphatase Pten, and the authors find that loss of Baz from the leading edge causes Pten redistribution. This, in turn, leads to an accumulation of the lipid PIP3 at the leading edge, which promotes formation of actin protrusions that are required for closure. This pathway is conserved during both dorsal closure and wound healing, offering a mechanistic basis for actin assembly during epithelial closure.

Mapping the neural crest

Neural crest (NC) cells arise in the neural tube (NT), undergo an epithelial-mesenchymal transition, and migrate away along defined routes, differentiating into multiple lineages. Precisely how NC cells exit the NT, and whether their fate is predetermined by their initial position within the NT, has been controversial. To address these issues, the Kulesa and Bronner laboratories performed a collaborative study (p. 820). Using a combination of photoactivation and two-photon time-lapse microscopy, they precisely marked individual or small groups of NC precursors in vivo in the chick embryonic NT and followed their fate. They found that most NC cells exit the NT at the dorsal midline, and that some precursors remain resident in the dorsal midline, producing an unordered emigration of cells. Moreover, they showed that differentiation potential is not defined by initial position within the NT, as has previously been suggested, although time of NT exit did influence fate. Together, these results suggest a more plastic and dynamic behaviour for NC cell emigration than previously appreciated.

X inactivation: the great escape

X-chromosome inactivation (XCI) enables dosage compensation between XX females and XY males, and its absence causes lethality, owing to defects in extra-embryonic tissues. However, it has also been shown that some genes are able to escape XCI in these tissues. Here, Catherine Corbel, Edith Heard and colleagues reconcile these findings and show that the inactive X (Xi) in one particular extra-embryonic cell type – trophoblast giant cells (TGCs) – has an unusual chromatin status (p. 861). Using RNA FISH on sections of postimplantation mouse embryos, they show that XCI is maintained in embryonic lineages, whereas TGCs show a high level of escape from XCI. Partial re-expression of most X-linked genes analysed, with the exception of the G6pd housekeeping gene, was observed in TGCs. In addition, the Xi in TGCs possesses an unusual organization and chromatin status, exhibiting both active and inactive chromatin marks. The authors propose that this apparent ‘bivalence’ of the Xi might account for its instability in TGCs and suggest that additional mechanisms maintain silencing at key loci.

HNF1β controls nephron development

The nephron is a highly specialised unit of the kidney. It arises by mesenchymal-to-epithelium transitions. After epithelialization, a polarized renal vesicle forms, and this further differentiates into a comma-shaped body and a S-shaped body (SSB), in which the future nephron segments are mapped into proximal, intermediate and distal domains. How SSBs are patterned and subsequently differentiate during kidney morphogenesis is poorly defined. Here, two papers use complementary approaches to show that hepatocyte nuclear factor 1β (HNF1β), which is known to be required for the earliest steps of metanephric kidney development and is implicated in developmental renal pathologies, controls this early patterning.

On p. 873, Silvia Cereghini and co-workers show that conditional inactivation of Hnf1b in murine nephron progenitors causes abnormal SSB regionalisation and morphology. In particular, Hnf1b deficiency leads to the absence of a proximal-medial SSB subdomain. This defect correlates with a downregulation of Notch pathway components and of Iroquois transcription factors, and perturbs the subsequent differentiation and morphogenesis of SSBs. Using parallel studies in Xenopus embryos, the researchers show that Hnf1b is required for the acquisition of proximal and intermediate tubule fate, acting again through the Notch pathway and Iroqouis genes. Together, these results show that HNF1B is required for the acquisition of a proximal-medial segment fate in vertebrates and uncover a previously unappreciated function of a novel SSB subdomain.
Using a similar gene targeting approach, Evelyne Fischer and colleagues (p. 886) demonstrate that Hnf1b inactivation in the murine metanephric mesenchyme (MM), which gives rise to nephron progenitors, leads to drastic tubular defects. The researchers report that mutant embryos show significant alterations to SSB structure: the typical bulge of epithelial cells between the intermediate and distal SSB segments is absent in mutant embryos. The lack of Hnf1b correlates with decreased expression of several genes, including the Notch ligand Delta-like 1, and results in impaired tubular expansion and differentiation. Finally, the researchers show that the nephron defects observed in Hnf1b-deficient mice resemble those observed in human foetuses carrying HNF1B mutations. The authors conclude that HNF1β plays an essential role in controlling the formation of a specific SSB sub-compartment by activating a set of crucial kidney development genes.

PLUS…

Stem cells living with a Notch

Freddy Radtke and colleagues review the role of Notch signaling in stem cells, comparing insights from flies, fish and mice to highlight similarities, as well as differences, between species, tissues and stem cell compartments. See the Review article on p. 689

Human pluripotent stem cells: an emerging model in developmental biology

Zhu and Huangfu discuss how studies of human pluripotent stem cells (hPSCs) can complement classic approaches using model organisms, and how hPSCs can be used to recapitulate aspects of human embryonic development ‘in a dish’. See the Review on p. 705

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Happy 2013 everyone! I hope you’re all settling into the year nicely.We sent out our EuroStemCell January newsletter last week and I thought some of you might be interested in our latest schools activities and fact sheets on stem cell research.

Highlights this month include a new lesson for 12-14 year olds on Stem cell treatments and ethics and a blog from Cambridge Stem Cell Institute researchers about their successful school visit using our CSI: Cell science investigators lesson.

Our collection of fact sheets is always growing: the latest additions are on (1) Umbilical cord blood and stem cells and (2) the role of commercial organisations in developing stem cell treatments. We’ve also added more fact sheet translations – most recently into French, Spanish and Italian.

Remember: you can stay in touch inbetween newsletters by following @eurostemcell on Twitter or liking us on Facebook. Your feedback is always very welcome – via these channels or use our contact form to get in touch.

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The Woods Hole image voting posts are some of the most popular posts on the Node (and yes, there will be a new one up VERY soon!). These images are all made by students of the Woods Hole Embryology course, and you still have a chance to be part of the 2013 class!

The application deadline for all Woods Hole summer courses, including this one, has been extended to February 8th. The course itself runs from June 1 to July 14, and is open to graduate students, postdocs, and junior faculty.

Scholarships are available for accepted students, so don’t let money be an issue in your decision to apply.

For more information, see the course website. Good luck! We hope to see some of your images and posts on the Node in the coming year…

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Hello, my name is Stephanie and I’m a graduate student in Dr. Amy Ralston’s lab at the University of California Santa Cruz.  I just returned from a trip to Dr. Yojiro Yamanaka’s lab at McGill University in Montreal, Quebec.  This trip was funded by the Development Travelling Fellowship from Company of Biologists.  I highly recommend checking it out, receiving this grant was a great, hassle-free experience!

In my time at Dr. Yamanaka’s lab I learned how to synthesize and  inject mRNA constructs into 2 and 8-cell mouse embryos.  I also learned how to live image the embryos as they develop and analyze the data gathered from the imaging.  During my visit I injected GFP and RFP mRNA for easy visualization of my injection success.  I will be using these techniques back at UCSC to study the molecular regulation of the first lineage decision in the mouse embryo.  The molecular mechanisms underlying the first asymmetries and subsequent lineage decisions in the mouse embryo are only beginning to be understood.  I will be using microinjection to over-express a variety of intra-cellular signaling molecules and transcription factors and then assessing the fate of the injected cells.

My time in Montreal was very cold!  Coming from California I’m not adapted to living in subzero temperatures, and most days were below zero (Celsius).  In fact, on the coldest day the high was -22 C, and felt even colder because of the windchill.  It was great to visit French Canada though, very different from other regions in Canada.  McGill University was very international and I met scientists from all over the world who are working there now.  I’m glad to be back in California though, where the high today in Santa Cruz was 15C, well above zero.  I’m also excited to get our injection system up and running and start collecting data.  I included a picture of a 16 cell mouse embryo which I injected at the 8 cell stage.  One cell was injected with H2B conjugated to RFP, marking the DNA, and that cell has subsequently divided.  This was fun and very technically challenging to learn!

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O-GlcNAc signalling during embryonic stem cell differentiation

My lab is studying the signalling mechanisms governing the onset of differentiation of pluripotent embryonic stem (ES) cells. Work from this and other labs (e.g. 1, 2) has revealed a critical role for autocrine FGF signalling and consequent sustained phosphorylation of the kinase Erk during the differentiation process. Although essential to the differentiation process, Erk activation is not the only signalling event regulating the decision between self-renewal and differentiation of these cells (3). Cellular signalling is most commonly associated with post-translational modification of proteins by addition of a phosphate to serine, threonine and tyrosine residues by the large family of kinase enzymes. However, there are other protein post-translational modifications with increasingly recognised very important roles in protein control. One of these is the addition of β-O-linked N-acetylglucosamine (O-GlcNAc) to serine or threonine residues, first described over 25 years ago. This modification (O-GlcNAcylation) involves the covalent addition of a single sugar to aminoacids via O-glycosidic linkage and occurs with similar time scales, dynamics and stoichiometry as protein phosphorylation.

Compared to the body of work accumulated around the study of phosphorylation, O-GlcNAcylation is much less understood, and relatively little is known about the types of extracellular signals controlling it. However, as these two modifications can occur (mutually exclusively) on the same residue or (in an antagonistic or synergistic fashion) on neighbouring ones, it is easy to see how O-GlcNAcylation can modulate the phosphorylation downstream of a large number of signal transduction cascades (4).

In recent years evidence has been accumulating for a critical role played by O-GlcNAcylation in ES cells, although its precise function(s) and the mechanisms operating are still poorly defined. This project aims to study in detail the role of O-GlcNAcylation on cell signalling, using ES cells as a model system. ES cell differentiation is a complex process, governed by the interaction of multiple signalling pathways (e.g. ERK, Gsk3/Wnt, BMPs etc.) Work in our lab has identified an important and novel role for O-GlcNAc during mouse ESC differentiation, and we have generated preliminary data showing how alteration of O-GlcNAc levels (using a specific inhibitor of the O-GlcNAc hydrolase, GlcNAcstatin, abbreviated GNS) affects cell signalling, gene expression and the self-renewal/differentiation balance. Other labs have recently reported that O-GlcNAc modification of the ESC transcription factors Oct4 and Sox2 controls their function suggesting further mechanisms by which O-GlcNAc profoundly affects cell behaviour (5,6)

This project will build on these preliminary findings and define the mechanisms by which O-GlcNAc affects ES cell function using cell biological, biochemical and proteomic approaches.

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PhD project title:

Non-genetic mechanisms of inheritance influenced by the maternal environment

Project description:

In the last few decades, it has been becoming increasingly clear that organisms can express phenotypes that are inherited in a non-Mendelian fashion. Studies in humans, for example, show that grandfathers who experienced dramatic diet fluctuations as children influenced the incidence of heart disease in their grandchildren. The mechanisms for transgenerational effects, however, are poorly known.

The aim of this project is to study the effect of maternal environment on sex determination of a species of nematode. In this nematode, if the mother smells a specific chemical, she will give rise mostly to hermaphrodites. Otherwise, the mother will produce mostly females. The student will investigate the mechanisms by which an odorant signal can change the epigenetic status of the germline, thereby influencing sex determination. This project will involve RNA sequencing of the germline of mothers that experience odorant stimuli. Mechanistic tests will be performed by genetic manipulation of the nematodes either by mutational analysis or RNA interference.

Key experimental skills involved:

The student will gain experience in analyzing large datasets derived from next-generation RNA sequencing, as well as standard methods in molecular biology and model systems, such as generation of transgenics and RNA interference.

References:

Kaati, G., Bygren, L. O., Pembrey, M. and Sjostrom, M. (2007). Transgenerational response to nutrition, early life circumstances and longevity. Eur J Hum Genet 15, 784-790.

Jablonka, E. (2012). Epigenetic inheritance and plasticity: The responsive germline. Prog Biophys Mol Biol. xxx 1-0 (advanced online).

Chaudhuri, J., Kache, V. and Pires-daSilva, A. (2011). Regulation of sexual plasticity in a nematode that produces males, females, and hermaphrodites. Curr Biol 21, 1548-1551.

Shakes, D. C., Neva, B. J., Huynh, H., Chaudhuri, J. and Pires-daSilva, A. (2011). Asymmetric spermatocyte division as a mechanism for controlling sex ratios. Nat Commun 2, 157.

Contact details for application enquiries:

http://www2.warwick.ac.uk/fac/sci/lifesci/study/pg/research/phd/studentships/#SLS_PhD_Pires

http://www2.warwick.ac.uk/study/postgraduate/apply/

andre.pires@warwick.ac.uk

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Developmental biologists have long relied on the power of observation to understand how embryos develop. In addition, pharmacologic and genetic manipulation of embryos gives us clues as to the mechanisms involved in proper developmental processes. The ability to combine embryo manipulation with observation of embryonic development in real time has been possible for quite some time when using model organisms that develop externally, such as chicks, frogs and zebrafish. However, for those of us that use a mammalian model system, the technology to observe development in real time has lagged way behind. How we long for a way to watch organ systems develop and cell populations migrate and differentiate in the early embryo. While we have many sophisticated genetic tools to study these types of processes, we are limited to looking at simple “snapshots” of time based on when the embryos are dissected and fixed. The task of generating a robust confocal microscopy-based live imaging platform for early mouse embryos was taken on by R’ada Massarwa, a post-doc in the Niswander lab and the culmination of this work was recently published in Development (2013 Jan;140(1):226-36).

During the creation of this live imaging system, she chose to observe the process of neural tube closure, which occurs between days 8.5 and 10.0 of embryonic growth (E8.5-E10.0) in the mouse. Following dissection and experimental setup, the embryos are able to survive up to 16 hours of live imaging. Thus, by performing a series of experiments in which embryos were dissected at increasing somite stages, the entire process of neural tube closure was observed. This type of careful sequential experimentation also showed that the culture and imaging system did not interfere with the proper growth and movement of the tissues. The result: beautiful movies that not only show us how the neural tube develops, but also highlight all the exciting possibilities that this system brings to the study of early mammalian development.

We have been using this system to study neural tube closure, but there are many other tissues and organs that develop during these time periods (E8.5-E10.5) that are amenable to imaging including the heart, face, limbs and neural crest. By using tissue-specific Cre- recombinase reporter strains, the behavior of individual cell types can now be observed in real time in the early mammalian embryo. Also, combining fluorescent reporter strains with genetic knock-out strains and imaging the mutant embryos as the phenotype begins and progresses can provide a much better understanding of how the loss of gene function affects a developmental process. This system also provides access to the embryo itself for pharmacologic and physical manipulation. Overall, the potential for what can be learned using this live embryo imaging system is incredible. We are excited to share this technology with the scientific community and we look forward to seeing how all of you are able to use it to your advantage. Happy imaging!

Massarwa R. & Niswander L. (2012). In toto live imaging of mouse morphogenesis and new insights into neural tube closure, Development, 140 (1) 226-236. DOI: 10.1242/dev.085001

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Every time a biologist drives pluripotent cells to differentiate into a specialized cell type, patients of all sorts of diseases, disorders, and injuries allow their hope to grow.  A research group recently reported how to drive differentiation of human pluripotent stem cells into medium-sized spiny neurons, the neurons that are some of the first to undergo degeneration in Huntington’s Disease.

hPS (human pluripotent stem) cells have the ability to differentiate into countless specific cell types, and can be either human embryonic stem cells or induced pluripotent cells.  hPS cells can generate various neuronal cell types, so their use in studying neurological diseases and regenerative therapies for such diseases is notable.  Huntington’s disease is an untreatable genetic neurodegenerative disease that typically begins with the degeneration of medium-sized spiny neurons (MSNs), neurons found in the basal ganglia region of the brain.  A recent paper in Development describes how hPS cells can be driven to an MSN fate.  Carri and colleagues began a combinatorial modulation of the pathways involved, beginning with BMP/TGFβ pathway inhibition.  About 20% of the neurons differentiated from hPS cells in these experiments are DARPP-32+/CTIP2+ MSNs also containing dopamine D2 and A2a receptors.  These resulting MSNs showed a firing pattern and neuromodulation identical to mature, authentic MSNs.  Carri and colleagues transplanted the hPS cell-induced neurons into the striatum of acid-lesioned rats, leading to their in vivo survival and differentiation towards an MSN fate.  In the images above, hPS cells were differentiated into neurons that contained DARPP-32 (green), a marker for MSN identity.

For a more general description of this image, see my imaging blog within EuroStemCell, the European stem cell portal.

Carri, A., Onorati, M., Lelos, M., Castiglioni, V., Faedo, A., Menon, R., Camnasio, S., Vuono, R., Spaiardi, P., Talpo, F., Toselli, M., Martino, G., Barker, R., Dunnett, S., Biella, G., & Cattaneo, E. (2012). Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32+ medium-sized spiny neurons Development, 140 (2), 301-312 DOI: 10.1242/dev.084608

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We haven’t posted one of these in a while, but there are quite a few things coming up. Remember to also keep an eye on the calendar, and add events there.

Conference and course deadlines in the next few weeks:
January 14 – abstract submission deadline for the annual meeting of the Dutch Society for Developmental Biology
The meeting is January 30 in Utrecht.

January 18 – abstract submission deadline for the joint meeting of the British Societies for Cell and Developmental Biology
March 17-20 Warwick University
Early registration discount ends February 15.

January 31 – abstract submission deadline for the International joint meeting of the German Society for Cell Biology and the German Society for Developmental Biology
March 20-23 Heidelberg
Early registration discount ends February 15

February 1 – application deadline for the Woods Hole Embryology Course
(This is the course that produces the wonderful images that you’ve seen on the Node and on Development covers.)
June 1 – July 14 2013

Other deadlines:
The deadline to apply for the job of Community Manager for the Node is on January 20.

Less urgent, but worth noting:
Registration will soon* open for the 17th International Congress of Developmental Biology. This meeting is only held every four years, and this year it’s in Cancun, so you won’t want to miss this!

(* I first wrote “January 21st”, but had it mixed up with the JSDB meeting )

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Abcam is a leading web based business supplying research tools to life scientists worldwide. Our head office is in Cambridge (UK), we also have offices in Bristol (UK), Cambridge, Eugene and San Francisco, (USA), Tokyo (Japan), Hong Kong and Hangzhou (China).

A position has become available to develop the product portfolio in the Epigenetics and Nuclear Signalling research area.

You will be part of a larger team responsible for identifying and prioritising targets for antibody production at Abcam. You will be key in ensuring the antibodies produced receive the highest level of validation and data using both internal and external resources. The role will focus on investigating key topics within the field of Epigenetics and Nuclear Siganalling and driving antibody production to meet customer needs. The ideal candidate will be flexible, work well in a team, be comfortable working to deadlines, prioritising different tasks and have good attention to details. Excellent communication skills are essential. You will have contacts within the scientific community and be confident networking to establish new links. Research experience in the field of Epigenetics, Chromatin or Nuclear Signalling is essential.

This position will provide an exciting opportunity for a motivated individual with a PhD or significant research experience who is looking to make the step into a more commercial environment. All relevant training will be provided.

Key Responsibilities:
i) Develop a strategy to identify new targets for antibody production in the field of Epigenetics and Nuclear Signalling. Prioritise production of the most commercially and scientifically valuable antibodies
ii) Post-production validation and characterization of existing antibodies using both internal and external resources.
iii) Networking with the scientific community to identify unmet needs and new product opportunities for Abcam
iv) Providing scientific guidance and input to troubleshoot production or QC issues for key products, working closely with our laboratory and other members of the team
v) Identify current and upcoming scientific topics in order to provide data/information to the Marketing department, including but not limited to, topics for marketing literature, scientific meetings, top selling products.

Our culture is one that empowers individuals, with responsibility given at an early stage. We place great emphasis on knowledge and experience. The working environment is fun and fast-paced, with everybody working together as a team to deliver great service and the best products to our customers. In addition to competitive salaries we can offer an attractive flexible benefits package which includes a profit-share scheme and share options.

To apply, or for more information please follow this link and submit your CV and cover letter: http://hire.jobvite.com/j/?cj=ozd0Wfws&s=The_node

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