For the last round of Woods Hole images this year we have an exciting development- the last round is a movie round! Below are 4 great movies from last year’s Woods Hole embryology course, and you can vote for your favourite. The most voted movie will be featured in the homepage of Development and a still or collection of stills from the movie will be the cover of a coming issue of the journal. You can see what the cover will look like by clicking on the link below each image.
 
 
Voting will close on noon GMT on the 30th of September.
 
 
 
 

1. Drosophila embryogenesis.  Lateral view of a Drosophila melanogaster embryo with anterior to the left and ventral down.  The embryo carries a Histone 2A-RFP transgene that allows visualization of all nuclei and was imaged by confocal microscopy (maximum intensity projections created from each timepoint).  During the approximately eight hours of development the embryo goes from stage 6 to stage 13, during which time the embryo undergoes gastrulation, germband extension, germband shortening, and the appearance of morphological segmentation. This movie 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). Cover image.
 
 
 
 
 

2. Ascidian metamorphosis – extension of ampullae.  Metamorphosis of the colonial ascidian, Botrylloides violaceus, imaged by widefield microscopy.  During the three-hour period the ampullae extend out over the substrate, and eventually this individual will bud off additional colonies. This movie was taken by Matthew Clark (University of Oregon). Cover image.
 
 
 
 
 

3. C. elegans early cell divisions. The embryo contains both GFP:Histone H2B and GFP:gamma tubulin allowing for visualizaion of both the chromosomes and the centrosomes respectively. At the 8 cells stage, the embryo contains the AB.a1, AB.ar, AB.p1, AB.pr, MS, E, C and P3 cells. The 2 cells that divide at the bottom near the end of the movie are the MS and E cells (with E dividing first). Imaged by confocal microscopy (maximum intensity projections created from each timepoint). Movie covers approximately 17.5 minutes of development. This movie was taken by Daniela Di Bella (Fundacion Instituto Leloir), Joyce Pieretti (University of Chicago), Saori Tani (Kobe University) and Manuela Truebano (Plymouth University). Cover image.
 
 
 
 
 

4. Zebrafish lateral line migration.  Zebrafish lateral line primordium migration leaves behind clusters of cells that will form neuromasts, which are mechanoreceptive organs that allow the fish to detect water movement.  Here the cells of the primordium and neuromast clusters are visualized in the Tg(-8.0cldnb:lynEGFP)zf106 transgenic line in which claudin B-GFP fusion protein highlights the cell membranes. Each frame of the movie is a maximum intensity projection of a confocal Z-stack.  Lateral view with anterior to the left and ventral down.  Movie covers approximately 8 hrs of development. This movie was taken by Eduardo Zattara (University of Maryland, College Park ). Cover image.
 
 
 

If you are interested in using any of the images or movies in this post, please contact the Node to request permission

After fertilization, embryos undergo rapid, synchronous cell divisions until the point of the midblastula transition (MBT) where the cell cycle lengthens.  This transition is also known as the maternal-zygotic transition (MZT), as the embryo switches from relying on maternally-deposited mRNAs to undergoing its own transcription.

The trigger for this transition was previously unknown but thought to be related to the increasing ratio of nuclear material to cytoplasm.  The embryo does not grow in these early stages, but keeps dividing its cytoplasm between more and more cells.  So as the amount of cytoplasm decreases relative to the nuclear component of the cells, it was proposed that at a critical ratio the embryo undergoes a transition where the cell cycles lengthen and become asynchronous, and embryos begin transcribing mRNA.

Now a recent paper in Science has proposed a mechanism for this observation.  Four DNA replication factors – Cut5, RecQ4, Treslin and Drf1 – are implicated in the cell cycle elongation, which occurs in concert with a decrease in the density and synchrony of DNA replication initiation events, directly related to a decrease in the abundance of mRNA and protein of these replication factors.

This was demonstrated in vitro using Xenopus egg extracts containing sperm nuclei, by addition of in vitro translated protein of the replication factors.  There was an increase in DNA synthesis compared to extract without the addition of protein.  This was also carried out with addition of more nuclei to extract to artificially increase the nucleus: cytoplasm ratio, which reduced the rate of synthesis; addition of proteins to this extract increased the rate of synthesis again.

mRNA microinjection, to direct protein overexpression in vivo in Xenopus embryos, of the replication factors demonstrated no slowdown or loss of synchrony in the cell cycle at the MBT and an increase in the number of cells and DNA content compared to controls.  Animal caps – the top portion of blastulae that later forms ectoderm – were dissected out of injected embryos and dissociated, then used for pulse-chase labelling of replication forks, showing that overexpression of the replication factors resulted in an increased rate of DNA replication.  This was achieved not by affecting elongation, but rather increasing the number of origins of replication.

The authors also found that in embryos overexpressing the four replication factors, there was an earlier activation of the cell cycle checkpoint kinase Chk1, normally activated during the MBT, that had been linked previously to the depletion of the nucleotides required for DNA synthesis.

Whilst overexpressing the four replication factors resulted in increased cell number and no cell cycle lengthening, these embryos were also severely restricted in their ability to undergo gastrulation and form closed blastopores, with high rates of embryo death by the stage of neurulation.  This phenotype was rescued partially by co-injection of morpholinos, small synthetic oligonucleotides that prevent mRNA translation, targeting Cdc6, a protein involved in prereplicative complex formation.  The idea behind targeting this protein was to reduce the extent of origin licensing, illustrating that developmental problems caused by overexpression of the replication factors were indeed caused by increased rates of replication initiation.  The rescued embryos also showed normal lengthening of the cell cycle at the MBT as well as normal activation of Chk1.

Overall, the paper has illustrated the role of replication initiation rates in regulating normal embryonic development, tying in nicely to the observation that an increasing nuclear: cytoplasmic ratio correlates with events at the MBT.  Furthermore the authors speculate that this may be an important mechanism in regulating the length of S-phase across development, and across eukaryotes.

Beware of the Frog

References:

Collart, C. et al. Titration of four replication factors is essential for the Xenopus laevis midblastula transition.  Science 341, 893-896 (2013).

“If you’re looking for some really high quality career workshops specifically designed for doctoral and postdoctoral bioscientists then the Society for Experimental Biology’s new Bioscience Futures series could be just what you need!”

I must admit to being somewhat biased when I write the above advertising strapline, since I am not only organsing the initiative but also contributing to it! However, I am proud of the six one-day workshops we have to offer this year. With only 35 participants per workshop there is going to be plenty of interaction and opportunities to really delve into each career subject. The workshops on offer are as follows and include world recognised experts in their field:

1. Planning your career – how to find and keep your perfect job (11th October) – Sarah Blackford (www.biosciencecareers.org)
2. Writing funding proposals (25th October) – Carmen Gervaise (http://www.hfsp.org/)
3. Publishing your research – Beginner level (5th November) – Margaret Cargill (http://www.adelaide.edu.au/directory/margaret.cargill)
4. Successful Applications and interviews (8th November) – Sarah Blackford (www.biosciencecareers.org)
5. Publishing your research – Intermediate/advanced level (26th November) – Irene Hames @irenehames
6. Using social media to promote and enhance your career (25th November) – Anne Osterrieder @AnneOsterrieder

Go to the Society for Experimental Biology website to see an overview of the programme, as well as biographies of the tutors, more information on the workshops and registration (buttons on the left hand side). Note that a fee is levied for each workshop at a break-even cost. If you have difficulties with the finance you can ask your PI or head of department if they have funds available for contract staff or students to attend external professional development courses.
http://www.sebiology.org/meetings/bioscience_futures/Overview.html

I hope you like what you see and maybe I’ll see you in London in the Autumn. If you want to ask me anything in the meantime contact me via twitter @Bioscicareer or email s.blackford@lancaster.ac.uk

August might be the month when most people go on holidays, but the Node was still full of activity! Here are some of the highlights:

Research

– Stefano de Renzis wrote about his recent paper using powerful microscopy techniques to study the role of endocytosis in morphogenesis.

– Tatsuya posted on what is like working with turtles and the background to his paper on the evolution of turtle carapaces.

– Gary discussed a new paper using X-ray tomography and synchrotron radiation to study gastrulation in frogs.

– and Christele started her ‘Stem Cell Beauty’ blog, in collaboration with EuroStemCell, by choosing a great image from a paper describing how to make hepatocytes in vitro.

Interviews

This month saw two new interviews on the Node:

– An interview with NIMR developmental neurobiologist and new editor of Development, François Guillemot, focused on his career, research and his lab’s future move to the Crick Institute.

– while the Node interviewed Marianne Bronner at the ISDB meeting, and asked her about her fascination for the neural crest and  her passion for mentoring.

Resources:

– Mario introduced his new software silicoCROSS, which may make your genetic cross writing that little bit easier.

– while Florian described E-CRISP, a software that allows the design of CRISPR constructs in 8 different organisms.

Also on the Node:

– Patricia chose her favourite Biology TED talks– a great selection!

– Caroline examined how ‘alternative’ careers are perceived in science, by discussing the reactions of people around her to her decision to leave the bench for a career as a reviews editor at Development.

– and check out the jobs page for the several new positions advertised this month.

Node news:

– The Company of Biologists, the non-for-profit company behind Development and the Node, launched its YouTube channel– check it out for some amazing research movies!

– the Node summarised the best (developmental) biology content spotted on the internet this month- look out for more great internet content on the Node in the future!

– and we revealed the new Node postcards, which you will be able to collect in future conferences!

Happy reading!

E-CRISP is now available under: www.e-crisp.org

Genome editing by applying the CRISPR/Cas system has been shown to be a promising new tool in genetics. CRISPR/Cas works by guided DNA double strand breaks (DSB) at specific loci in the genomic or exogenic DNA, where various kinds of sequence alterations can be introduced (exploiting the cellular DSB-repair machinery). As the CRISPR/Cas system is rapidly developing, it remains important to systematically assess which design parameters/properties of CRISPR/Cas constructs influence experimental outcome.

Bioinformatics methods can be used to find suitable target sites for the DSB in a systematic manner.  We developed E-CRISP to design CRISPR constructs and provide the possibility to alter various design parameters systematically. A fast nucleotide indexing approach and the application of a binary interval tree for construct annotation make E-CRISP very fast.

Until now E-CRISP is available for eight different organisms, including mouse, rat, fish, fly, worm, Arabidopsis, yeast and human, providing the possibility to use it for different model organisms. This list is easily extendable upon request.

The only information needed to design a CRISPR with this application, is the organism of interest and the target gene-symbol or sequence. Default parameters can be used or adapted to suit the particular needs. E-CRISP identifies and evaluates potential CRISPR targets by a combination of options including specificity (should target one locus only), nucleotide composition, experimental purpose and genomic context (if it targets a gene, which exon, lies in CpG island) among others.

Additionally, E-CRISP offers a re-evaluation tool to identify and annotate targets of CRISPR constructs designed by other sources (or designed by E-CRISP itself).

Image analysis is powerful and essential in modern biology. However, many people working on image analysis might be struggling with following problems:

• Which tool is appropriate to address my question?
• Who should I ask my very specific question about image analysis?
• I want to increase my skills, but I do not know how.
• I became an image analysis specialist. Is there a community for me?

To deal with these problems, we decided to organize a quite unique type of meeting in Barcelona to strengthen the network among those involved in bioimage analysis and to provide direct solutions:

1. Open Community Meeting (Oct. 7 – 8, 2013): Top-developers, leading analysts and biologists sit together to share current status of each to exchange information and share the problem for a more effective, direct and efficient solutions. Anyone could join to acquire solid information on the front-line of bioimage analysis and to share your views.

2. The open community meeting is followed by a course targeting microscopy facility staffs  (Oct. 9 – 12, 2013) to propagate the knowledge and techniques of image analysts to the scientific community (For a course targeting biologists, we are applying for an EMBO practical course in 2014, the second round after the BIAS2013).

3. In parallel with the course, we startup to build a public webtool that is expected to evolve into a practical solution for building image analysis pipeline. The participants will discuss freely over various tools, manually annotate and added tags to all available image analysis tools. For this symposium participation to this activity will be invitation-based since it is still in an early phase of its development but annotations/taggings will be open to public in near future. We call this precursory trial as “Taggathon”  (Oct. 9 – 11, 2013).

Please visit the website below for more details and for your registration:

http://eubias2013.irbbarcelona.org

This event was conceptualized at the last European Light Microscopy Initiative meeting 2013 in Arcachon, is mainly sponsored by EuroBioImaging (www.eurobioimaging.eu) and OME, is open to further sponsor contributions and participation from the private sector.

EuBIAS2013 is hosted by IRB Barcelona and is organized by many people from the University of Dundee, EMBL (Heidelberg), IRB Barcelona, CRG (Barcelona), EPFL(Lausanne), ETH (Zuerich), DZNE (Bonn), Institut Curie (Paris).

If BioImage Analysis is key to your research, do not miss this unique event to commit yourself in boosting the accessibility to BioImage Analysis tools and strengthening the community. If you know anyone who might be interested in this meeting, please let them know and pass this information.

Organizers:

• Kota Miura, EMBL Heidelberg
• Julien Colombelli, IRB Barcelona
• Sébastien Tosi, IRB Barcelona
• Jason Swedlow, Univ. of Dundee

Here at the Node we are very excited about our new conference giveaway- the Node postcards! We have selected a set of 4 beautiful images that have featured in the Woods Hole image competitions in the last few years: the dwarf cuttlefish, the E10.5 mouse embryo, the bat skeleton preparation and the set of Drosophila embryos. And they are not just pretty pictures- the postcards have space at the back where you can write your message, although you might want to follow the lead of the Node team and use them to decorate your desk!

We hope that you like our selection, and that you will collect the postcards at the Company of Biologists stand in your next conference! And don’t forget to also collect the Node tea bags, and have a tea break on us!

POSTDOCTORAL POSITION IN DEVELOPMENTAL NEUROSCIENCE is immediately available to study signaling factors that regulate cortical development using in vivo mouse models.  In this NIH-funded project, we explore signaling factors that influence the restricted proliferation of intermediate neural progenitors in the developing cerebral cortex.  We focus on regulators of small RhoGTPases, comprised of guanine-nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), that control the GTP-loading state and activity of RhoGTPases. Highly motivated candidates who have recently received a PhD or MD (<4 years from completion of degree) with a strong cellular and molecular biology, biochemistry and/or neuroscience background and familiarity with molecular techniques are encouraged to forward their CV, three references, and a brief statement of research interest by email to:

Jill Weimer, PhD
Sanford Research/USD
Children’s Health Research Center
2301 E 60th St. N
Sioux Falls, SD 57104
Phone: 605-312-6407
Email: Jill.Weimer@sanfordhealth.org
website: http://www.sanfordresearch.org/researchcenters/childrenshealth/weimerlab/

PhD student position at the IBMB-CSIC, Barcelona

Laboratory of Developmental Neurobiology

 http://www.ibmb.csic.es/index.php?pg=laboratorio&idLaboratorio=18&tab=lab_home

We are looking for: Enthusiastic researchers with a BSc or Masters Degree in biomedical sciences with interest in Developmental Neurobiology Good academic records are required Good spoken and written command of English

We offer: A highly multidisciplinary and competitive training programme in biomedical research. Access to state-of-the-art infrastructures.

The selected candidate will investigate the role of extracellular signals and the genetic networks that control cell numbers, cell identity and cell shape changes during the embryonic development of the neural tube, using live-imaging, cell- and molecular biology in two animal model chick and zebrafish embryos

Those interested please send CV, a cover letter justifying the interest of the applicant in the project to emgbmc@ibmb.csic.es

Application deadline on September 30th, 2013  

Here are the highlights from the new issue of Development:

FGF10 function in the lung branches off

Lung development in mice involves specification of the primary lung field followed by the formation of lung buds, which subsequently undergo outgrowth and branching morphogenesis to form the stereotypic bronchial tree. Localised expression of Fgf10 in the distal mesenchyme adjacent to the sites of lung bud formation has long been thought to drive branching morphogenesis in the lung but now, on p. 3731, Stijn De Langhe and colleagues challenge this model. They show that lung agenesis in Fgf10 knockout mice can be rescued by ubiquitous overexpression of Fgf10, demonstrating that localised Fgf10 expression is not required for lung branching morphogenesis in vivo. Instead, they report, localised Fgf10 prevents the differentiation of distal epithelial progenitors into Sox2-expressing airway epithelial cells, thus suggesting that Fgf10 plays a role in proximal-distal patterning. Furthermore, they show that, later in development, Fgf10 can promote the differentiation of airway epithelial cells to basal cells, a finding that has important implications for understanding and improving lung injury and repair.

Stem cell quiescence outFoxed

Hair follicles cyclically degenerate and regenerate through adult life: after an initial growth phase, hair follicles enter a destructive phase and then go through a quiescent stage before re-entering the next growth phase. This cycling involves hair follicle stem cells (HFSCs) but how these cells transition between the phases of the hair follicle cycle is unclear. Here, Hoang Nguyen and colleagues report that the forkhead transcription factor Foxp1 is crucial for maintaining HFSC quiescence (p. 3809). The authors show that Foxp1 is expressed in adult mouse HFSCs and that ablation of Foxp1 in skin epithelial cells shortens the quiescent phase of the hair cycle and causes precocious HFSC activation. Furthermore, they report that overexpression of Foxp1 in keratinocytes leads to cell cycle arrest as well as to upregulation of Fgf18, which has been previously implicated in controlling HFSC quiescence. Finally, the researchers demonstrate that exogenously delivered FGF18 can prevent the HFSCs of Foxp1-null mice from being prematurely activated, confirming that FGF18 acts downstream of Foxp1 to regulate stem cell quiescence.

Fasci(cli)nating link between signal transduction and morphogenesis

The molecular mechanisms that link intracellular signalling pathways to changes in tissue morphology are unclear. Using the Drosophila embryonic hindgut as a model, Martin Zeidler and co-workers demonstrate that the transmembrane protein Fasciclin III (FasIII) regulates intracellular adhesion and links signal transduction to morphogenesis (p. 3858). The researchers show that normal hindgut curvature is dependent on JAK/STAT signalling, and that JAK/STAT pathway activity asymmetrically localises to the inside curve of the developing hindgut, where it drives FasIII lateralisation. In addition, they demonstrate that FasIII promotes intracellular adhesion both in vivo and in cells in vitro. Based on these findings and the differential interfacial tension hypothesis, the researchers establish a mathematical model of the developing hindgut, which suggests that intracellular adhesion mediated by FasIII is sufficient to explain the curvature observed in the hindgut. These findings, together with additional studies of tissue folding in the Drosophila wing disc, suggest that FasIII-dependent modulation of intracellular adhesion might be a general mechanism by which organs are shaped during development.

Dlk1 muscles out of regeneration

Muscle development is driven by a set of myogenic factors, but how these are regulated during normal development and during regeneration is unclear. Here (p. 3743), Charlotte Harken Jensen and colleagues show that delta-like 1 homolog (Dlk1), an imprinted gene, is a crucial regulator of the myogenic program in mice. They report that Dlk1-null mice exhibit impaired muscle development due to a defective myogenic transcriptional program: the myogenic genes Mef2c, Meis1 and Myod1 are suppressed in these mice. Surprisingly, however, they find that depletion of Dlk1, which is known to be re-expressed in regenerating muscle, in fact enhances muscle regeneration both in vitro and in vivo. This improved regenerative capacity in the absence of Dlk1 is associated with an enhanced myogenic program, and is not due to altered adipogenic-myogenic commitment. Together, these findings highlight a dual function for Dlk1 – as an enhancer of muscle development but as an inhibitor of muscle regeneration – and may open up new possibilities for improving muscle regeneration in human disease.

A new cloud on the horizon of mouse ooocytes

The piRNA pathway silences retrotransposons and hence maintains genome integrity in the germline. Several components of the piRNA pathway localise to a structure called the nuage, which has been detected in many animal germlines, including mouse testes and Drosophila oocytes. Now, Ai Khim Lim, Barbara Knowles and colleagues show that a nuage-like structure can be found in mouse oocytes (p. 3819). They report that the nuage proteins mouse vasa homologue (MVH), Piwi-like 2 (PIWIL2/MILI) and tudor domain-containing 9 (TDRD9) transiently colocalise to a nuage-like structure in mouse oocytes shortly after birth. Furthermore, they report, the nuage protein GASZ, which is functionally but not structurally linked to the nuage in testes, is also present in cytoplasmic granules in oocytes. Using mutant mice, the authors demonstrate that the nuage genes Mvh, Mili and Gasz control retrotransposon repression through the piRNA pathway. Importantly, however, they find that these null-mutant females, unlike their male counterparts, are fertile, thus highlighting that retrotransposon activation and sterility are uncoupled in female mice.

A novel role for TGFβ in lymphangiogenesis

Lymphangiogenesis, the formation of lymphatic vessels, involves multiple growth factors and receptors, including vascular endothelial growth factor C (VEGFC) and its receptor VEGFR3. Here, on p. 3903, Yoh-suke Mukouyama and co-workers uncover a role for TGFβ signalling during lymphatic network development in mice. The researchers first develop a novel, whole-mount imaging technique to visualise lymphatic vessels in the anterior dorsal skin of mouse embryos. Using this approach, combined with conditional knockout of TGFβ receptors (Tgfbr1 or Tgfbr2) in lymphatic endothelial cells (LECs), they show that a loss of TGFβ signalling in LECs leads to reduced vessel sprouting and hence a global decrease in lymphatic network complexity. Furthermore, they report, LEC proliferation is increased following TGFβ receptor depletion. Finally, they demonstrate that TGFβ signalling in a dermal lymphatic cell line can upregulate the expression of VEGFR3 and the VEGFC co-receptor neuropilin 2. These studies, together with other findings, suggest that TGFβ plays a dual role during lymphangiogenesis, both enhancing LEC sprouting while decreasing LEC proliferation.

PLUS…

Cohesin in development and disease

Recent studies have shown that cohesin, which was named for its ability to mediate sister chromatid cohesion, can influence gene expression during development. Here, Ana Losada and colleagues provide an overview of how cohesin functions in development and disease. See the Development at a Glance poster article on p. 3715

Molecular causes of aneuploidy in mammalian eggs

Mammalian oocytes are particularly error prone in segregating their chromosomes during their two meiotic divisions, resulting in the creation of an embryo that has inherited the wrong number of chromosomes: it is aneuploid. Here, Keith Jones and Simon Lane review recent data on factors that determine successful segregation in female meiosis and explain how this might be related to an age-related decline in female segregation accuracy. See the Primer article on p. 3719