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

(2 votes)

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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!

(4 votes)

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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/

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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  

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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

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POSTDOCTORAL POSITION IN VASCULAR BIOLOGY is available to study the cellular and molecular processes regulating the development of the lymphatic vasculature using in vivo mouse models.
Highly motivated individuals who recently obtained a PhD or MD degree and have a strong background in developmental and vascular biology are encouraged to apply. Interested individuals should send their curriculum vitae, a brief description of their research interests, and the names of three references to:

Guillermo Oliver, Ph.D
Dept. of Genetics
St. Jude Children’s Research Hospital
262 Danny Thomas Place,
Memphis, TN 38105-3678
Email: guillermo.oliver@stjude.org
Phone: 901-5952697
Fax: 901-5956035

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Marianne Bronner is a developmental biologist at the California Institute of Technology. At the International Society of Developmental Biology (ISDB) meeting in 2013 she was awarded the prestigious Conklin medal for her work on the cells of the neural crest. The Node interviewed Marianne at the ISDB meeting and asked her about her fascination for the neural crest and her passion for mentoring.

You initially trained as a biophysicist. How did you first become interested in developmental biology?

I really liked physics, chemistry and maths and only took one undergraduate course in biology. I majored in biophysics because that was the only option that incorporated all the things I wanted to do. After my undergraduate I wanted to a PhD, but didn’t know on what. I applied for programmes in biophysics, thinking I wanted to be a structural biologist. Because I had done so little biology, I had to take many biology courses when I got to graduate school. One of the courses I took was developmental biology and I learned about the work of Nicole LeDouarin and her beautiful quail-chick chimera experiments. I found it all fascinating, but I particularly gravitated towards the neural crest. There are times in your life when you go from being incredibly naïve to suddenly saying: ‘that’s it’. That was my moment, and I have worked on it ever since.
 

Your lab has worked over the years on different aspects of the neural crest. What do you find fascinating about it?

Everything! Initially I was mostly fascinated about how these cells could give rise to so many different derivatives, their multipotency. It relates back to the central question in developmental biology – how do you generate a complex organism from just a single cell – but I viewed it as a simpler system than the embryo as a whole. My initial experiments aimed to find out if single neural crest cells were multipotent or whether there was a mix of determined and undetermined cells in the neural crest.

However, there are certain questions that you really want to address, but the technologies to address them are not available. I worked on the lineage questions as far as I could but then realized I was stuck and couldn’t go much further. I got interested in migration, which is also fascinating- how the cells move to particular locations, and how their fate is linked to where they go. I started working on the interactions between neural crest cells and the extracellular matrix, analyzing pathways of migration. Later on, when more tools were available, I went back to the lineage question.

You have used a variety of model organisms to study the neural crest: from more standard models like Xenopus and zebrafish to lampreys and amphioxus. Why do you use such a range of models?

I started most of my work in chick, and my initial work on the neural crest was very vertebrate specific. I used Xenopus and chick because they were easy to manipulate. Around 1990 I started teaching at the embryology course at Woods Hole, and as I sat through the lectures of other people I realized that my focus had been quite narrow. This course looks at organisms ranging from simple marine species to mice and it got me thinking about evolutionary questions. The fascinating thing about the neural crest is that it is a vertebrate specific cell type. Why did these cells suddenly arise in the vertebrate lineage? To address this question I had to look across chordates, so I decided to work on a basal chordate and a closely related, non-vertebrate chordate.

At Woods Hole I met David McCauley, who was very interested in evo-devo and came to work for me. We decided to start working with lamprey, but this was not easy: lampreys are not genetically-tractable organisms, live in large deep lakes, and like salmon they swim into the streams where they were born, lay their eggs and die. You can’t exactly grow them in labs! David went up to the Great Lakes every year to collect embryos and did some basic embryology. Then we discovered FedEx, and we started setting up the lamprey system in our own lab. Another postdoc came to work on this project, Tatjana Sauka-Spengler, and she really took the lamprey into the genomic age: making cDNA libraries, BAC libraries and so on.

By this point were looking at the gene regulatory networks that define neural crest and we wanted to know how the gene regulatory networks in lampreys compared with those in other vertebrates. We found that most of these networks were already conserved all the way down to lamprey. But when we looked at the non-vertebrate chordate, the amphioxus (which does not have neural crest), the group of genes that were important for neural crest specification were present in the genome, but were not expressed in the presumptive neural crest region. We concluded that this is where the transition occurred.

What are the scientific questions that you are excited about? What directions do you want to go in with the neural crest?

I feel like I am asking the same questions I always have, but the way we can approach them now is much more sophisticated. For the last decade I have been trying to understand, from a gene regulatory perspective, how you make a neural crest cell: how a cell is first formed at the neural plate border, why it comes to reside within the dorsal neural tube and why it then migrates out of the neural tube. Now I want to try to understand how the cells decide whether they should become cranial facial cartilage, or neurons, or something else. I would like to do that by analysing the gene regulatory circuits that act during late cell migration and as cells terminally differentiate. People have looked at the very end point in the differentiation networks, and I have looked a lot at the beginning points, but that middle territory is still unexplored. We are already doing a lot of transcriptome analysis to identify all the players that come on during those times, and we now need to figure out what their function is and how that they fit into a circuit.

You have said before that your achievements in mentoring are those of which you are most proud. Why is that?

My concept of how to run a lab has been based on how to raise my children. I think you get a lot more out of people by loving them. I also feel like I owe the people who work for me a debt of gratitude, for all their hard work, and I try to help them becoming the best kind of scientist they can be. Everybody has different abilities: some people are very independent right from the start and can go off and build their careers, and others need a lot of guidance and help. I feel like I am a good mentor and I’m able to take people at many different levels and help them along the right pathway. In some cases that is just giving someone a nice environment where they can work in and do whatever they want. In other cases I really try to guide people and say: ‘at this point in your career you should do this’. When I look back at the people that I’ve trained, I see that some are doing similar things to what I do, while others have gone in different directions. I feel that I helped them getting where they are and that is extremely gratifying.

You seem to enjoy the mentoring process. Did you have a particularly inspiring mentor?

Surprisingly no- I was anti mentored! I think there are two different ways to learn how to be a good mentor: one is to have had good mentoring, and the other is to not. It is not that I didn’t have good mentoring- I just came out of nowhere. I would not recommend any career decisions that I have made.

I applied to grad schools together with this boy I was dating at the time, and I decided where to go based on where he wanted to go. We broke up within a year, and the school I went to was terrible for me: it was extremely sexist at that time, and I was one of the very few women in the biophysics programme. I then went on to work in a lab where the PI was horrible and very sexist too. I almost dropped out of research: I applied for a teaching job but did not get it. I was so disappointed that I decided instead to change labs.

I discovered I wanted to work on the neural crest and I moved to the lab of Alan Cohen. He was a very nice guy, but he had already decided he didn’t want to do science anymore and was going to med school. He was not around, but it was a permissive environment- I got on with my work and learnt most of what I needed from other people in the lab. I got my PhD fairly quickly, but since I didn’t have any mentors, I didn’t have people to write job recommendation letters for me. Malcolm Steinberg, who was at Princeton, was probably the closest thing I had to a mentor. He was a really good scientist and took a liking to me, so he wrote my job letters.

I got my job at UC Irvine not because of anything I had done but because they wanted to hire my husband. I took a non-tenured track job there, which really wasn’t a smart move, because it was very hard to convert it to a tenure track position (although luckily some great colleagues at Irvine helped me to do that later). I was right out of grad school and I had no postdoctoral experience. I didn’t really have anyone to rely on, which is maybe why it is so important to me to be a good mentor to others. I have learnt so much by trying different things and making mistakes that now I have a rather large body of knowledge about what not to do.

You say that you would not advise anyone to make the same decisions that you have, but do you have any advice for young scientists? 

You have to be happy. So when picking a lab, either for graduate school or for a postdoc, make sure that you can get along with the lab head. Make sure they are a strong mentor who supports people, not only when they are in their lab but also after they have left. Secondly, look at the environment in general, and make sure that you like the other people in the lab, not only the PI. You are going to be spending 4 years or more at this place, and you want be happy there. Choosing the right place is really important.

Choosing the right question is equally important. You want to find something that grabs you and that you will be happy working on for quite a long time, but it should also be something tractable. There are some questions out there that are extremely interesting but so difficult that they can discourage you.

Finally, make a network. Find people that can help you in addition to your mentor: it could be your peers or other faculty members. Getting lots of feedback on your work, especially from people that can give you a big picture view when you are in the middle of your experiments and really detailed oriented, is very helpful and it can help you correct your course and save time.

In the last year you had your work in cell biology recognised by the ASCB, and now, here at the ISDB, you won the Conklin medal. What do these prizes mean to you?

I am so thrilled- I have never won anything before! I’m particularly grateful because I know that one of the reasons I am getting recognition is thanks to the people I have trained. They are starting to move up in the faculty ranks and as they appreciate what I did for them, they are helping me get these awards. I am really happy, very grateful, and very touched. It is a lot of work to put together these nomination packages and it means a lot to me, especially because it has come from them.

What would people be surprised to find out about you?

I was born in Europe and I escaped from Hungary when I was 4 years old. My parents are holocaust survivors, and probably a big reason why I like mentoring is because I feel like I have to give back.

(8 votes)

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Can you imagine a supermarket where doctors could just go and pick the cells they need to cure their patients? Just like a pharmacy…or a blood bank?

Well, this is the dream most stem cell biologists are working towards. In order to make this dream a reality, scientists are trying (with more or less success!) to develop protocols with which they could use a stock of stem cells and differentiate them towards a cell fate of interest in order to obtain functional specialized cells…which ideally could then be used for cell replacement therapies and drug screenings.

In a recent study published in Development by S. Ogawa and colleagues, human pluripotent stem cells (hPSCs; ie: stem cells that can become any tissue of the body) were successfully differentiated into mature liver cells. To this end, the authors developed an elaborate in vitro liver differentiation protocol that consists of successive steps that recapitulate liver development. First, hPSC are differentiated into definitive endoderm and then to hepatoblasts (immature liver cells), a process dependent on activin/nodal signalling.  Subsequently, 3D cell aggregation and cAMP signalling are required to obtain hepatocyte-like cells, hepatocytes being mature liver cells.

In this picture, one can observe 3D cell aggregates the scientists produced from hPSCs. Cells express the proteins ASGR1 (in green in the picture) and ALBUMIN (ALB, in red in the picture), a combination that is specific to hepatocytes.

So in the future, if these cells are proven to function properly, they could potentially be used in therapies against all sorts of liver diseases, in drug screening studies or to produce bio-artificial liver devices!

S. Ogawa et al., Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes. Development 140, 3285 (August 2013). doi: 10.1242/dev.090266

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I have recently returned from a spate of international conferences that afforded me the opportunity to experience meetings for the first time as an editor, instead of as an active researcher. It was also the first time I had seen my former lab mates since leaving the bench, and the reunion was a very happy one indeed. I was surprised, however, by the avid interest in my new career, and even moreso by the hushed tones in which these conversations took place. It seemed that numerous people I spoke with harboured some interest in leaving the bench, although this had never been spoken of when we worked side-by-side. Even people I had never met before confided in me that they, too, were considering a career beyond the lab, although they could scarcely admit this to their colleagues. So why the secrecy?

Upon reflection, I can sympathise. The response to my own decision to leave the bench was mixed. My closest colleagues were happy for me, including my principle investigator (PI) at the time, but there were some who expressed great surprise and even disappointment. Notably, this seemed to come from higher up: senior postdocs and other PIs who had always imagined that I would stay on. “It’s a shame, you could have made it” was one such response. Though undoubtedly intended as a compliment, I was nonetheless bothered by this. Surely what constitutes “making it” is a personal decision.

It is often said that parents only want what is best for their children, and yet often parents will push their children in a particular direction. The same may also be true for PIs and their students. It is certainly important to encourage students to fulfill their potential, but care must be taken when deciding where that potential lies. The PI should not assume that the student has a burning desire to run their own lab. For many, this is undesirable, and when we consider the number of PhD being produced, it is certainly unrealistic (see Nature article “Education: the PhD factory”). Instead, careful consideration must be given to understanding the various strengths and weaknesses of each person, as well as their interests, motivations and goals: not just for their career but also for their life. These are big questions, and neglecting to address them may often lead to frustration and disappointment for both PI and student.

Most students these days are reasonably well aware of “alternative careers in science” (see careers post on the Node). Almost every major conference has the obligatory session, and some institutes and universities have taken an active approach to promoting this. This is an excellent first step, however I fear that it will not be enough. Instead, we need to change the attitude from within and remove the stigma associated with leaving the bench. A PhD provides training in much more than the specific subject at hand: there is an entire smorgasbord of critical skills that are learnt during the process: project management, conflict resolution, lateral thinking, public speaking, writing, teaching, and team-building are just some of these assets. And assets they are: there is a reason why the big banks and consulting companies will in theory accept candidates with a PhD in any discipline. The mere process of completing a PhD bestows these valuable skills upon the graduate.

There are a number of different approaches that can be taken to breakdown the barriers to career diversity in science. As a minimum, students should be educated about and gain exposure to a wider range of career paths as part of their studies. I’m not just talking about “tech transfer” but the whole gamut: teaching, journalism, publishing, politics, law, consulting, science outreach, and (shock horror) administration. The prevailing dogma that the very best PhD students should remain in academia must be challenged, and the challenge must come from within. Simply being good at something doesn’t mean one should be fated to it.

(9 votes)

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A major challenge when studying the cell biological bases underlying morphogenesis is represented by the gap between the sub-cellular and the tissue-organ scale (Figure 1). Ideally, one would like to follow tissue dynamics with sub-cellular resolution and obtain ultra-structural details about the organization of specific plasma membrane domains, cytoskeletal structures, intracellular compartments and vesicular carriers involved in signaling and adhesion. It is, indeed, the spatio-temporal modulation of membrane and cytoskeletal dynamics in individual cells or group of cells, which ultimately drive tissue remodeling during organismal development. Cell shape changes play, indeed, a fundamental role during embryonic development and are often initiated by expansion or contraction of specific plasma membrane domains. While the role of the cytoskeleton in driving plasma membrane remodeling is well established, the contribution of membrane trafficking remains an open question.

Figure 1. Scales in developmental biology: from organisms (left panel, cross section of a Drosophila m. embryo ~0.5 mm) to organelles (right panel, endosome ~0.5 microns). Tissue and organ morphogenesis depends on the modulation of intracellular machines operating in the nanometer scale.

In my laboratory, for example, we are interested in understanding how endocytosis contributes to shaping cells and tissues during morphogenesis.  Although, the role of endocytosis in signaling and cell polarity is well-established, to what extent endocytosis is directly involved in shaping cells is less understood. The cellularization of the early Drosophila embryo provides an ideal system for studying the mechanisms driving plasma membrane remodeling during morphogenesis. Over the course of one hour a syncitium of 6000 nuclei is divided into an equal number of polarized epithelial cells by invagination and growth of the apical plasma membrane (for video see, http://youtu.be/kwN1aNAvTwk). Scanning electron microscopy studies revealed that during this process the apical plasma membrane undergoes a dramatic morphological re-organization characterized by the retraction of villous protrusions and flattening of the apical surface. To date, however, the apical surface of the embryo has proven extremely difficult to visualize in real time using traditional live imaging techniques.

In our recently published paper (Fabrowski P. et al 2013) we adapted Total Internal Reflection Microscopy (TIFR-M) to visualize endocytic dynamics during surface flattening in live Drosophila embryos. This microscopy technique relies upon an evanescent wave that exclusively illuminates a region in close proximity (10-200 nm) to the coverslip. In cell culture, TIRF-M has been successfully employed to visualize vesicle fission and fusion events with the plasma membrane (Toomre D. et al 2000). Furthermore, because of its high sensitivity and low signal to noise ratio, TIRF-M can be applied also to image single molecules dynamics (Jain A. et al 2012).

Imaging living organisms is not as simple as imaging single molecules in solution or cells grown on glass coverslips. In living organisms, such as developing embryos, cells move, are not adhering to coverslips and autofluorescence sometime poses a serious limit to the imaging techniques that can be employed. In the Drosophila embryo, cells are enclosed by the vitelline membrane and are surrounded by peri-vitelline fluid. The vitelline membrane is a proteinaceous waxy layer that tends to be autofluorescent when illuminated with blue (488) light. It seemed therefore unlikely that TIRF-M could be employed to visualize plasma membrane dynamics in live embryos.  However, after several failed attempts, we realized that it was possible to direct the laser light with such an angle that an evanescent wave could be generated at the interface between the vitelline membrane and the peri-vitelline fluid. This approach allowed us to visualize, for the first time, the morphogenetic remodeling of the apical surface over the entire course of cellularization (see movie below and  also movie S1 in Fabrowski P et al. 2013)

Quantification of endosome dynamics, marked by the small GTPase Rab5 tagged with GFP at its endogenous locus, revealed a massive increase in apical endocytosis that correlates with changes in apical morphology (Figure 2, for video see, http://youtu.be/9X5uM85lBBM).

Figure 2. TIRF-M imaging of apical endocytic dynamics during plasma membrane remodeling in live Drosophila embryo. From left to right, thee snapshots of early, middle and late stages of cellularization showing the up-regulation of endocytic vesicles marked by Rab5 (green). The plasma membrane is labeled by Gap-43 and it is shown in white. Scale bar, 10 microns.

In a series of experiments, which I will not describe here in detail, we demonstrated that endocytosis is required for surface flattening. We, therefore, decided to investigate the endocytic mechanisms driving this morphogenetic process. The large quantities of plasma membrane contained in protrusions together with the relative fast kinetics of flattening (~10 minutes) raised the question of whether clatrhrin coated vesicles mediated endocytosis could, by itself, be sufficient to drive membrane remodeling. To address this question, we employed Correlative Light-Electron Microscopy (CLEM), a powerful, but time-consuming technique, that allows one to correlate fluorescently labeled particles onto a corresponding electron microscope image. By combining CLEM with electron tomography we reconstructed the 3D organization of GFP labeled endocytic membranes. In summary, the results of this analysis revealed that surface flattening is driven by the activation of a prominent tubular endocytic pathway characterized by the formation of tubular plasma membrane invaginations that serve as platforms for the de novo generation of vacuolar Rab5-positive endosomes. Thus, surface flattening is an endocytosis dependent morphogenetic process during which endosomes form directly at the plasma membrane rather than by fusion of incoming clathrin coated vesicles.

In conclusions, the application of these powerful microscopy techniques to multicellular systems will undoubtedly help bridging the gap between the sub-cellular and the tissue-organ scale. The possibility, for example, of visualizing endocytic events during tissue differentiation should help characterizing the molecular regulation and spatial organization of signaling systems in an in vivo context with an unprecedented level of resolution.

References

Jain A, Liu R, Xiang YK, Ha T. (2012) Single-molecule pull-down for studying protein interactions Nat Protoc. 7(3), 445-52. doi: 10.1038/nprot.2011.452.

Toomre D, Steyer JA, Keller P, Almers W, Simons K. (2000) Fusion of constitutive membrane traffic with the cell surface observed by evanescent wave microscopy. J Cell Biol. 149 (1), 33-40.

Fabrowski P, Necakov AS, Mumbauer S, Loeser E, Reversi A, Streichan S, Briggs JA, De Renzis S. (2013) Tubular endocytosis drives remodelling of the apical surface during epithelial morphogenesis in Drosophila. Nat Commun.4:2244. doi: 10.1038/ncomms3244.

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