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Amniote gastrulation without a streak

Posted by , on 12 June 2013

The three principal germ layers of the vertebrate embryo, ectoderm, mesoderm and endoderm, emerge from the pluripotent epiblast during the process of gastrulation. Being especially interested in the molecular and cellular mechanisms underlying the emergence of mesoderm, the source of a diverse set of tissues and cells including blood, muscle and bone, we decided to take a closer look at the primitive streak, the anatomical correlate of gastrulation in birds and mammals.

 

We initially set out to analyse the molecular mechanisms and signalling processes associated with formation and patterning of mesoderm in the primitive streak, reporting our findings in a previous Development paper (Alev et al., 2010). We then went on to tackle the long debated question about the evolutionary relationship between the primitive streak of amniotes and the blastopore of anamniotes (Shook and Keller, 2008), and asked whether it was possible to uncouple mesoderm induction and primitive streak formation. In order to efficiently address this question we needed a novel, simple and reproducible way to manipulate mesoderm differentiation and primitive streak formation, preferably in vivo, finally settling for in ovo subgerminal cavity injection. The subgerminal cavity is an ideal “reaction vessel” that can be used to assess the in vivo effects of growth factors and small molecules on the pre-gastrulation epiblast, as well as the differentiation of these cells giving rise to the germ-layers during gastrulation.

 

We found that subgerminal cavity injection of FGF can potently induce a ring of mesoderm in the marginal zone with more than half of the treated embryos having no primitive streak, which is generally assumed to be essential for gastrulation and mesoderm formation in birds. Further analysis of this unexpected “circumblastoporal” mode of mesoderm formation in chick revealed that the induced ring of mesoderm even had anamniote-type dorso-ventral polarity. Looking for an explanation as to why the induced mesoderm forms only in the outer margin of the epiblast (the marginal zone) while the center of the epiblast can not be turned into mesodermal precursors despite the uniform presence of FGF, we found that Wnt signalling, which is present in the marginal zone, is required not only for the circumblastoporal mode of mesoderm induction, but in concert with FGF can even turn the entire epiblast into mesodermal precursor fate. We also showed that TGFβ signalling contributes mainly to epithelial mesenchymal transition (EMT) and dorsalisation of the induced mesodermal precursors (Alev et al., 2013).

Brachyury WISH of FGF4 injected chick embryo 36h post-injection

To further explore the possibilty that this hidden capacity for anamniote-type mesoderm formation might be also present in other amniotes, we tested the effects of subgerminal cavity injection of FGF in two other bird species: the quail, a close relative of chick; and emu, a basal ratite. We could induce circumblastoporal mesoderm in not only these bird species but could also generate a mesoderm ring in FGF-injected embryos of the Chinese soft-shelled turtle. Our observation that even a reptile, which does not possess a primitive streak to begin with, still maintains the ability for anamniote-type circumblastoporal mesoderm formation highlights the evolutionarily conserved nature of this phenomenon, supporting our hypothesis that the evolutionary emergence of amniotes was characterized by the restriction of a mesoderm inducing signal, likely FGF, to one side of the epiblast, while the overall capacity for anamniote-like circumblastoporal mesoderm formation was retained. Further support of our hypothesis may arise from studies of mesoderm induction in additional non-model organisms such as urodele and cacecilian amphibians and prototherian mammals.

 

Current model organisms are often selected out of experimental convenience rather than their necessarily being “model” representatives of the lineages they belong to, in contrast to the sheer variety of organisms studied during the height of comparative anatomy and embryology over a hundred years ago. It therefore doesn’t come as a surprise that even though a recent examination of reptilian gastrulation (Bertocchini et al., 2013) strongly suggests that the primitive streak is not a conserved feature among the amniotes, contrary statements still permeate vertebrate embryology textbooks. A revival of the utilisation of non-model organisms such as reptiles could thus help bypass the limitations of current models, especially in light of the recent advances made in the field of genomics. The usage of non-model-organisms in combination with comparative genomics and comparative embryology may thus lead to unexpected novel insights into questions old and new.

 

 

Alev, C., Wu, Y., Kasukawa, T., Jakt, L.M., Ueda, H.R., Sheng, G., 2010. Transcriptomic landscape of the primitive streak. Development 137, 2863-2874.

 

Alev, C., Wu, Y., Nakaya, Y., Sheng, G., 2013. Decoupling of amniote gastrulation and streak formation reveals a morphogenetic unity in vertebrate mesoderm induction. Development.

 

Bertocchini, F., Alev, C., Nakaya, Y., Sheng, G., 2013. A little winning streak: the reptilian-eye view of gastrulation in birds. Dev Growth Differ 55, 52-59.

 

Shook, D.R., Keller, R., 2008. Epithelial type, ingression, blastopore architecture and the evolution of chordate mesoderm morphogenesis. Journal of experimental zoology. Part B, Molecular and developmental evolution 310, 85-110.

 

 

 

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ISSCR Annual Meeting 2013

Posted by , on 11 June 2013

Hi there,

My name’s Harry and i’m going to be blogging from the ISSCR annual meeting in Boston, starting tomorrow. I’ll try and add daily updates to let you know what’s new in the stem cell field and give an overall impression of the ISSCR experience. Hopefully if you click on the ISSCR tags below this will link to all my posts.

You can also follow me on Twitter (@HGLeitch) if you want more regular updates. Opinions are my own(!).

 

 

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Red fish, blue fish, Brainbow fish!

Posted by , on 11 June 2013

Here is a little backstory to our zebrafish Brainbow (Zebrabow) paper published in Development. After finishing up my graduate work in Josh Sanes’s lab at Harvard, I decided to join Alex Schier’s lab to work on zebrafish neural development. Ironically, Alex soon moved from NYU School of Medicine to Harvard and I ended up being just across the street from Josh’s lab. The close proximity sparked the collaboration (also with Jeff Lichtman’s lab) to adapt the mouse Brainbow multicolor fluorescent labeling technique to zebrafish.

One fun aspect of this project is that many beautiful images are produced that capture people’s imagination. Our colorful zebrafish images have reached far and wide over the years, gracing the covers of many posters, meeting booklets, and conference websites. One image was even made into a mouse pad by Olympus, the microscope manufacturer.

photo

To make Zebrabow more than a tool to take pretty pictures, we addressed several key technical issues about this technology, as reported in our Development paper. We showed that Zebrabow labeling is broadly applicable to many tissues in embryonic, larval, and adult animals. Furthermore, the diverse fluorescent colors in Zebrabow animals are stable and faithfully inherited after cell division, making it an ideal tool for lineage-tracing analysis. Our work is just the tip of the iceberg of what Zebrabow can do. Last summer we distributed our Zebrabow lines to more than 100 labs with the hope for many interesting uses for this technology.

twophotonhighresslice

Unexpectedly, there are also some caveats to having pretty Zebrabow pictures around for all to see. My daughter visited the lab a while ago, and I wanted to impress her with beautiful GFP-labeled axons. She took a look in the microscope and asked, “Where are all the other colors?” The bar has officially been raised.

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In Development this week (Vol. 140, Issue 13)

Posted by , on 11 June 2013

Here are the highlights from the current issue of Development:

 

Centrosomes and cell fate: a Notch ahead

F1.smallAsymmetric cell divisions (ACDs) play a crucial role in controlling cell fate and generating cell diversity during development. The centrosome is known to be involved in ACD, and recent studies have shown that centrosomes exhibit dynamic and asymmetric movements that regulate orientation of the mitotic spindle. Here, Yohanns Bellaiche and co-workers identify a novel type of centrosome movement during cytokinesis (p. 2657). The authors demonstrate that centrosome movements in Drosophila sensory organ precursors are regulated by the cell fate determinant Numb; the asymmetric localisation of Numb regulates asymmetric centrosome movements. Moreover, they report, Numb acts via the microtubule-binding protein CRMP rather than via its classical effectors. Finally, the researchers show that CRMP in turn participates in the regulation of endosome dynamics and thus likely the recycling of the Notch receptor Delta. They thereby establish a functional link between centrosome dynamics, Notch signalling and cell fate. These findings suggest a model in which asymmetric centrosome movements participate in differential Notch activation to regulate cell fate.

 

Fishing out maternal and zygotic transcriptomes

F1.small-1Early embryonic development occurs in the absence of transcription; instead, it relies on maternal mRNAs and proteins present within the egg. It is believed that this period of transcriptional quiescence is maintained by factors that eventually become titrated out during early cleavages, thus leading to zygotic genome activation. How exactly this transition occurs, however, is unclear. Here, Jim Smith, Steven Harvey and co-workers use exome sequencing and RNA-seq to distinguish between maternal and zygotic transcriptomes in early zebrafish embryos (p. 2703). Using single nucleotide polymorphisms to identify maternal and paternal transcriptomes, and using the appearance of paternal mRNAs as an indicator of zygotic transcription, the researchers identify the first zygotic genes to be expressed in the embryo. Zygotic transcription, they report, begins after ten cycles. Prior to this, changes in mRNA levels are observed but these are due to post-transcriptional regulation of maternal mRNAs and not due to transcription. Finally, the researchers demonstrate that different modes of regulation are required for zygotic transcription initiation.

 

Imaging the neurogenic niche

F1.small-2Neural stem/progenitor cells in the mammalian hippocampus generate new neurons throughout life. But how do these integrate into a mature and functional neural circuitry? Here, Sebastian Jessberger and colleagues address this question by using a new imaging approach to analyse neurite growth from newborn granular cells (p. 2823). Using a novel system for culturing sections of mouse hippocampus, combined with retroviral labelling to mark newborn neurons and their progeny, the researchers visualised neurite growth over several days using confocal imaging. Dendritic processes, they report, extended in different directions, with all neurons showing a clear apical extension at ∼4 days. Moreover, the dendrites in such slice cultures follow a linear growth pattern that is characteristic of the growth patterns observed in the intact brain, as assessed by snapshot-based analyses, thus validating their approach. This approach for visualising the adult neurogenic niche opens up the possibility of investigating the dynamic events that occur during adult neurogenesis in both physiological and diseased states.

 

A new vein of lumen formation

F1.small-3The correct formation of blood vessels is essential for the development of a functional vasculature. Various mechanisms of vascular lumen formation have been described to date but, now, Wiebke Herzog and colleagues examine the development of common cardinal veins (CCVs) in zebrafish and show that these form via a previously undescribed mode of lumen formation (p. 2776). The researchers use in vivo time-lapse studies together with lineage tracing approaches to show that the angioblasts that form CCVs are specified as a population that is distinct from arterial-fated angioblasts. Once specified, these then form CCVs by a novel mechanism, which the authors term ‘lumen ensheathment’: endothelial cells (ECs) delaminate and align along an existing luminal space, extend via migration and eventually enclose the lumen. The delamination and migration events, they report, require cadherin 5, while EC proliferation within developing CCVs requires erythrocyte-derived Vegfc. These findings uncover a new mode of vessel formation, as well as highlighting important crosstalk between the haematopoietic and EC lineages.

 

Specifying hepatopancreas progenitors

F1.small-4The liver and ventral pancreas are thought to develop from a common pool of multipotent progenitors. Although a number of studies have identified factors required for either pancreas or liver specification, factors that are distinctly required to specify the entire hepatopancreas system have not yet been reported. Now, Joseph Lancman and co-workers uncover a common genetic program, involving hnf1ba and wnt2bb, that specifies progenitors of the liver, ventral pancreas, gall bladder and associated ducts in zebrafish (p. 2669). By characterising a new hnf1ba hypomorphic mutant that phenocopies pancreatic defects found in people with HNF1B monogenic diabetes, the researchers show that hnf1ba regulates pancreas specification and β-cell numbers. Furthermore, they report, the combination of Hnf1ba partial loss with conditional loss of Wnt signalling reveals that these pathways synergize during a narrow developmental window to specify hepatopancreas progenitors; Hnf1ba acts to generate a Wnt permissive domain in the foregut that in turn adopts a hepatopancreatic fate. In summary, these findings highlight a new model for hepatopancreas specification and provide important insights into pancreas and β-cell development.

 

A lnc between coding and non-coding RNAs

F1.small-5Long non-coding RNAs (lncRNAs) have recently emerged as key regulators of gene expression in embryos and in embryonic stem cells (ESCs). Recent large-scale genomics approaches have identified thousands of putative lncRNAs but are these all truly non-coding RNAs? Here, on p. 2828, Alex Schier, Eivind Valen and colleagues set out to answer this question. The researchers use ribosome profiling to identify translated transcripts, combined with a machine-learning approach to classify open reading frames (ORFs) and to validate zebrafish lncRNAs. They find that many proposed lncRNAs are in fact protein-coding contaminants. Moreover, their study reveals that many zebrafish and ESC lncRNAs resemble the 5’ leaders of coding RNAs, suggesting a novel mechanism for lncRNA regulation. Overall, the findings presented here clarify the annotation of lncRNAs, as well as offering a valuable resource that can be used for identifying translated ORFs and hence novel protein-coding genes that function during zebrafish development.

 

Plus…

 

Lineage-dependent circuit assembly in the neocortex

F3crop.largeSong-Hai Shi and colleagues review recent findings on the generation, migration and organization of excitatory and inhibitory neurons in the neocortex, and discuss how the lineage history of neurons influences the assembly of functional circuits.

See the Review article on p. 2645

 

 

The San Francisco Declaration on Research Assessment

DORARead the Editorial by our Editor-in-Chief, Olivier Pourquié on p.2643

See also the earlier Node post (and some feedback from the community) about this declaration.

 

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Woods Hole Images round 3 – vote for a Development cover

Posted by , on 7 June 2013

It is time for round 3 of last years’ Woods Hole embryology course images! The regenerated planarian won the last round , but who will you vote for this time? Below are 4 great images, and you can decide which one will feature in the cover of Development. To see bigger versions, just click on the image.
 
Voting will close noon GMT on June 27th.

 
 

1. Ventral view of a Drosophila melanogaster embryo (stage 12/13) fluorescently stained for Repo (yellow; nuclei of glial cells), axons (red, anti-HRP), Hedgehog (green; Hh-GFP), Elav (pink, nuclei of neurons), and nuclei (blue, DAPI). The different maximum projection images show different combinations of the color channels. This image was taken by Davon Callander (Oregon State University).

 
 

2. Color inverted image of a skeleton preparation of a pig (Sus scrofa domesticus) embryo. This image was taken by Marina Venero Galanternik (University of Utah), Rodrigo G. Arzate-Mejía (Universidad Nacional Autonoma de Mexico), Jennifer McKey (Universite Montpellier) and William Munoz (The University of Texas MD Anderson Cancer Center).

 
 

3. Male stolon (ventral view, anterior up) of the annelid, Proceraea sp., fluorescently stained for acetylated tubulin (green), serotonin (yellow), F-actin (red; phalloidin), and nuclei (blue; DAPI). Confocal z-stacks were viewed as maximum projections and tiled together to cover the entirety of the animal (body length approximately 7.5 mm).This image was taken by Eduardo Zattara (University of Maryland, College Park).

 
 

4. Generation of a secondary body axis resulting from an organizer graft in Xenopus laevis. The graft was performed at stage 10 and the tadpole was photographed at stage 41. This image was taken by Elsie Place (MRC National Institute of Medical Research).

 


 

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We’re celebrating!

Posted by , on 3 June 2013

Today we’ve added the 1 millionth antibody to CiteAb, making us the world’s biggest citation based antibody search engine!

www.citeab.com

Despite reaching a million antibodies we are still very new and would appreciate feedback from users of the Node.

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Postdoctoral Position in laboratory of Olivier Pourquié (IGBMC, Illkirch, France)

Posted by , on 3 June 2013

Closing Date: 15 March 2021

A postdoctoral position is open in the laboratory of Pr Olivier Pourquié at the IGBMC.

 

Project:

The project will focus on establishing differentiation protocols to produce cells of the muscle lineage from mouse and human pluripotent stem cells (ES and iPS). Talented and motivated scientists with a strong background in mouse and human pluripotent stem cells research (ES/iPS) are encouraged to apply.

 

IGBMC:

The Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC) is an international institute with a strong focus on cell and developmental biology, and core mechanisms involved in gene expression, where the common language is English. It offers cutting edge imaging platforms (light and confocal microscopy, electron microscopy), plus many common facilities including proteomics, and sequencing. It is located within a biking distance from the lively city of Strasbourg in a nice area of France near the Vosges mountains.

 

Job Requirements:

–       PhD degree in molecular biology, developmental biology, stem cell biology, or related fields

–       Strong background in mouse/human pluripotent stem cell research (ES and iPS cells), molecular biology and microscopy absolutely required

–       Expertise in muscle tissue development / physiology appreciated

–       Organization skills

–       Excellent oral and written communication skills (English language) to contribute to scientific reporting

–       Management skills and aptitude in leading and working with research teams (the candidate will supervise a group of 2 technicians)

 

Candidate should submit a CV and a cover letter outlining relevant experience and reasons for interest in this position. Names of 2-3 references should also be provided by email to pourquie@igbmc.fr

 

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EMBO practical course “Imaging of Neural Development in Zebrafish”

Posted by , on 3 June 2013

8-15 September 2013, Karlsruhe, Germany

We would like to welcome applications to the EMBO zebrafish workshop that will take place at KIT, Karlsruhe (Germany) in the fall of 2013.

The objective of this EMBO Workshop is to provide theoretical and practical background on the zebrafish model system for direct assessment of open questions using modern microscopy techniques.

We encourage you to share the information on the EMBO course among potential applicants, aiming at experiencing the latest microscopy techniques to study development of nervous system using zebrafish.

For more information on the workshops please visit following webpage:

http://events.embo.org/13-neural-zebrafish/

We look forward to meeting you during the course.

Steffen Scholpp, Lucia Poggi, Mihaela Zigman, Uwe Strähle, Robert Geisler

The Organizers

Key date:

Registration Submission Deadline: 15th June 2013.

13-neural-zebrafish9-1

 

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Categories: Events

This month on the Node: May

Posted by , on 31 May 2013

May was another busy month on the Node, with a combination of research, publishing and Node news, in addition to several job posts. One very exciting news was that the Node has a new community manager! You can learn more about Cat on her introductory post and you can always contact her with suggestions and comments.

2012Round2_C_small

Woods Hole Image Competition- round 2

Round 2 of the ever popular cover competition of pictures taken by last year’s Woods Hole course students was underway this month, with a last minute victory by the two headed regenerated planarian! Congratulations to Chang Liu of the Shanghai Institute of Biochemistry and Cell Biology for the winning image, which will feature in the cover of Development  in the next few weeks.

Research highlights

The hair follicle bulbWe had three very interesting posts this month about recent research papers:

– Kif Liakath-Ali posted about a recent paper on lingual epithelium stem cells

– Chris Dooley focused on two recent papers on the sequencing of the zebrafish genome and the associated gene knockout project

– And Bruce Morgan commented on his recent paper describing how changes in the hair follicle niche cells can lead to hair loss

Publishing news

DORAThis month saw a group of editors and publishers signing the San Francisco Declaration on Research Assessment (DORA), a set of recommendations trying to address some of the issues with the current citation and impact factor system. Katherine posted about DORA, so if you have a comment or view on it, please join the discussion!

A recent post on the Node also announced a new Stem Cells & Regeneration website within Development, highlighting stem cell-related content in the journal, alongside a special issue on this area.

Interviews:

This month featured two very interesting interviews on The Node. The first interview was with biochemist-turned-sculptor Briony Marshall, and focused on some of the beautiful developmental biology-inspired pieces in her current exhibition in London. The Node also interviewed Aditya Saxena, a PhD student in Helen Skaer’s lab who won the poster prize at the last BSDB meeting.

Carnegie stages 6 - 10

Also on the Node:

– EuroStemCell announced a Stem Cell-themed non-fiction writing competition, alongside other news

– PostPostDoc discussed bullying and intimidation in the lab

– The Node followed the #LiteraryPaperOpenings hashtag on twitter and collated some of the best tweets in a storify

Happy Reading!

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COB Workshop: Evolution of the human neocortex

Posted by , on 30 May 2013

The Company of Biologists is hosting a workshop entitled “Evolution of the neocortex: how unique are we?“.

This  promises to be an exciting event, discussing brain evolution and the unique features of the human cortex. The 3-day workshop is part of a successful series of small, intimate meetings hosted by the Company. Each event has only around 30 attendees: 20 invited invited speakers, plus 10 places for early career scientists. This is a unique opportunity to interact informally with leaders in the field!

We still have a couple of places free for early career scientists, so there is still a chance to attend! There are no registration or accommodation costs. For more information, please see the Workshop website, where you will find the application form.*

*We are accepting late registrations, so please don’t worry about the deadline on the form!

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