Many salamanders can regenerate limbs, and even a seven-year-old child appreciates exactly the reasons why this feat is so remarkable.  How can an animal that has been living its life, using its leg full of muscles and bones and tendons and nerves every single day, suddenly grow a new one at some random time?  When the leg first grew on the same salamander when it was just a tiny animal, we can imagine it followed a prescribed developmental program for its instructions and used a prescribed pool of progenitor cells for its construction.  Once the limb tissues are already working, the cells are differentiated and doing their jobs, so how do they go back in time and create tissues anew?  Scientific understanding of how limbs develop has progressed immensely over the last several decades, but understanding of how limbs can regenerate and why some animals do it better than others has remained elusive.  This lag in understanding vertebrate limb regeneration on a molecular level stems from the fact that the two model systems with the most sophisticated tools for studying limb development—chick and mouse—do not naturally regenerate entire limbs as adults.  Figuring out what the regenerative roadblocks are in chick, mouse, and human will be imperative for improving regeneration in species that do not do it well.  However, it will be equally important—and perhaps fundamental for elucidating these roadblocks—to understand how animals that regenerate limbs remarkably well do it.  This is why we are developing tools that allow for more precise molecular genetic and cell biological inquiry into axolotl limb regeneration.

EGFP expression in a live animal. This axolotl hindlimb was amputated mid-femur. The blastema that formed was infected with EGFP-encoding retrovirus two weeks post-amputation, and the limb was allowed to fully regenerate. Cells descended from infected blastema cells express EGFP. Since the injection was limited to the blastema, cells proximal to the amputation plane do not express EGFP.

Two things a researcher might want to do when studying how salamanders regenerate limbs are tracking cells during regeneration and expressing introduced genetic elements to analyze their effects.  In the past, labeling methods such as heavy isotopes, dyes, and electroporation of plasmid DNA have been used in regenerating salamander limbs.  Electroporation of plasmid DNA has also been used to mis-express genetic elements.  While studies using these methods provided key insights into, for example, which cells become mitotically active following amputation, all three of these methods suffer a similar drawback:  the element gets diluted with each successive cell division, and in a regenerating limb, lots of cell division occurs.  It was impossible to follow cells from the stump, through the blastema (group of relatively dedifferentiated cells that forms at the tip of the stump and will give rise to internal limb tissues in the regenerate), and into the new limb.  Transplantation studies using tissues from permanently marked donors, such as animals induced to have a different ploidy or transgenic animals, have aimed to bypass these caveats, but transplantation is not ideal in many situations (for example, some tissues cannot be cleanly separated, and the procedure itself is obviously invasive and best done well before limb regeneration will be studied).  Furthermore, donor tissue is relatively limited at this point to just a few genetic lines of axolotls constitutively expressing a fluorophore, and generation of novel animals by transgenesis is a lengthy and labor-intensive process.  Expression studies using electroporated plasmid DNA were limited because even good electroporations may not lead to enough sustained expression to detect an effect.  In our Development paper, we showed that retroviruses can infect regenerating axolotl limbs.  These retroviruses are simply injected into limb tissue, and they can infect any mitotically active cell they encounter.  Since the retroviral genomes integrate into host cells, they can be used to permanently express a label such as GFP, which allows for tracking cells during regeneration, opening the door to many future studies.  Expression from the retroviral vectors is robust and using retroviruses might be a powerful way to finally address the consequences of misexpressing candidate genes during limb regeneration.  Additionally, we showed that retroviral infections can in principle be targeted to specific cell types by targeting infections to vascular endothelial cells.  This works by borrowing technology exploited by other researchers in the mouse.  Axolotl and mouse cells do not make the receptor for a particular coat protein found on the surface of certain bird viruses.  However, if axolotls or mice are made to express that receptor, they can become infected by viruses with these coats, and expression of the receptor can be confined to particular cell types by the researcher provided genetic elements for doing so (for example, a cell-type-specific promoter) exist.  Hence, the retroviruses can also someday be targeted to a whole battery of different specific cell types in regenerating axolotl limbs once cell-type-specific promoters are found.  These tools will allow for more precise control of labels and introduced gene expression during regeneration.

Figuring out how salamanders regenerate limbs won’t just satisfy the curiosity of the seven-year-old in us all, it also stands to someday dramatically improve the lives of the millions of people living with the consequences of limb amputation.  With key risk factors such as diabetes and peripheral artery disease on the rise, limb loss is unfortunately becoming an even more common problem in places like the United States, giving scientists a call to arms when it comes to unraveling the mystery of limb regeneration.

(10 votes)

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Let’s start this monthly summary with two fantastic posts that were left out of last month’s roundup due to the holiday period (and scheduled posts).

Idoia Quintana studies shark brain development in Spain, and travelled to Scotland recently to learn how to work with mouse brains, so she could do some cross-species comparison.

“At the beginning was tricky, working with mice was a big challenge for me; but at the same time was amazing to do research in a species in which several optimized techniques are available. Particularly I enjoyed learning slice culture techniques and I hope to have time to implement them in shark embryos and perform some axon guidance experiments upon my return.”

Benjamin Coyac attended the UPMC/Curie Institute International Course in Developmental Biology, and described what it was like to study in Paris for several weeks with students from around the world.

“The intensity of the program enabled us to get to know each other very quickly. Little talks about science or our lives as international students began to build a common experience in our shared interest in developmental biology. By the end of the program, not only we had acquired scientific skills in theory, methods and practicals, but also we became friends and started to build a strong network of future developmental scientists.”

And now on to the January posts.

Mouse development
Stephanie Vanderweide travelled from California to Montreal in the middle of the Canadian winter to learn how to microinject 2-cell and 8-cell mouse embryos in Yojiro Yamanaka’s lab, as part of a project to study the molecular mechanisms involved in the first lineage decision of the developing mouse embryo.

Heather writes about a technique developed in the Niswander lab, where she’s a student. The lab set up a confocal-based live imaging system to visualize mouse embryo development in real time.

“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.”

Woods Hole Embryology course
The application period for the 2013 Woods Hole Embryology Course has been extended to February 8. If you’re accepted to the course, we may see your images on the Node in the future: this is the course that produces the gorgeous images that Node readers have selected for Development covers. Speaking of which, the first round of images from the 2012 course are now up!

Publishing discussions
Finally, we’ve covered two very different discussions related to scientific publishing: peer review and the secrets behind methods sections.
-Katherine Brown wrote a post responding to an earlier discussion, about the future of peer review – both in general and in the specific case of Development.
-Eva followed the #overlyhonestmethods hashtag on Twitter, where within a few days hundreds of scientists shared the true stories behind their methods sections.

Also on the Node
-Lots of new job ads
–Hope for Huntington’s
–Top posts of 2012

(1 votes)

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A two-year post-doctoral positionis available in the group of Guillaume Balavoine and Michel Vervoort (http://www.ijm.fr/en/ijm/research/research-groups/metazoaires/) at the Institut Jacques Monod (IJM) in Paris (France). The IJM is a leading French biological research institute, comprising about 25 interactive research groups and high-quality technological facilities, including a cutting-edge imaging platform.

The primary research focus of the group is to reconstruct the early stages of animal evolution, by comparing the genetic networks that regulate the developmental patterning of key aspects of the body plan across metazoans. The main model studied by the group is the annelid worm Platynereis dumerilii, an emerging model species. Platynereis is a member of the Spiralian/Lophotrochozoan branch of the bilaterian tree and is hypothesized to be as close to a “bilaterian living fossil” as a bilaterian can be, both in terms of genome organization and body plan.

The post-doc project aims at understanding and modelling cell movements and cell shape changes that direct CNS and segment morphogenesis in Platynereis, as well as determining the roles of the Planar Cell Polarity (PCP) and Rho/ROCK/MyoII pathways in these behaviours. The project will be centered on the use of live imaging, molecular and modelling tools.

Candidates should have a strong background in developmental and/or evolutionary biology. Expertise in live imaging would also be welcome. Candidates must hold a Ph.D. degree in developmental or evolutionary biology and have at least one first author publication in a peer-reviewed journal.

Potential candidates should send their application by mail to Guillaume Balavoine (balavoine.guillaume[at]ijm.univ-paris-diderot.fr) and Michel Vervoort (vervoort.michel[at]ijm.univ-paris-diderot.fr) with a statement of interest, a Curriculum Vitae and contact informations for two referees.

The position will remain open until filled; however applications received by March 15^th 2013 will be given priority. The starting date is flexible (in 2013), with an early date preferred.

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Each year, students of the Woods Hole Embryology course produce some amazing images. Last year, readers of the Node selected four images from the 2011 course to appear on the cover of Development.

Now it’s time to do the same with the images from the 2012 course. Here’s the first batch of four images. Please vote in the poll below the images for the one you would like to see on the cover of Development. (Click any of the images to see a bigger version.) Poll closes on February 19, noon GMT.

1. Chick ectopic limb. An FGF-4-soaked bead was implanted at stage 14. The embryo was fixed four days later, and stained with alcian blue to reveal the developing cartilage of the skeleton. An ectopic limb can be seen developing next to the normal forelimb, and the bead is still present in the body wall. This image was taken by Elsie Place (MRC National Institute of Medical Research).

2. Two day old Xenopus embryo epidermis, highlighting multiciliated cells. The embryo had been injected with mRNAs encoding membrane blue fluorescent protein, Centrin GFP, and Clamp RFP at the 4-cell stage and imaged as a live mount by confocal microscopy. This image was taken by Andrew Mathewson (Fred Hutchinson Cancer Research Center).

3. Confocal image (extended focus Z stack) of an E10.5 day mouse embryo (lateral view; thorax) immunostained with antibodies against PECAM (endothelial factor present in the vasculature; red), beta-III-tubulin (neurons; green) and DAPI (cell nuclei; blue). This image was taken by Joyce Pieretti (University of Chicago), Manuela Truebano (Plymouth University), Saori Tani (Kobe University) and Daniela Di Bella (Fundacion Instituto Leloir)

4. Mouse embryo, day E9.5. Widefield fluorescence image showing immunostaining with anti-Tuj1 (orange) and anti-glucagon (green), counterstained with DAPI (cyan). This image was taken by Eduardo Zattara (University of Maryland, College Park).

(7 votes)

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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.

(2 votes)

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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…

(1 votes)

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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!

(4 votes)

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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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