Welcome to the Eublephosphere!
Eublephosphere (noun) – A place in which those who study the leopard gecko (Eublepharis macularius) reside.

Our names are Noeline Subramaniam and Kathy Jacyniak and we are Master’s students at the University of Guelph in Ontario, Canada. Under the supervision of Dr. Matthew Vickaryous, we have become budding regenerative biologists! Our research laboratory investigates the most fundamental question in biology: why are some tissues and organisms able to regenerate, whereas others cannot? Our model for these investigations is the leopard gecko (Eublepharis macularius), a popular reptile in the pet trade.

Research Focus

Figure 1: Members of the Vickaryous lab from left to right: Dr. Matt Vickaryous, Kathy Jacyniak, Noeline Subramaniam and Emily Gilbert

Ongoing research in our lab aims to identify and understand the biological mechanisms that permit and promote scar-free wound healing and regeneration.Emily, a PhD candidate, is examining the role of neural stem/progenitor cells in the spinal cord and the brain in response to regeneration, Noeline is investigating the role of angiogenesis in scar formation, and Kathy is exploring the role of cardiac stem/progenitor cells in the heart. If you would like to learn more about the research conducted by the Vickaryous lab, check out our website: http://www.vickaryouslab.com/. You can also follow us on Twitter @VickaryousLab, including our infamous #FluorescentFridays.

Figure 2: Dorsal view of a gecko with a beautiful original tail. This gecko was an environmental control for one of our previous studies.

Why lizards?

Many species of lizards are able to self-detach a portion of their tail to avoid predation, and then regenerate a replacement. Tail detachment, or caudal autotomy (‘self-cutting’), is a dramatic – and somewhat deceptive – phenomenon. For most species (including leopard geckos), the tail is autotomized within a vertebra at a location known as a fracture plane (essentially a non-mineralized gap in the vertebral centrum). In addition, the main arterial supply to the tail (the caudal artery) develops a regular series of muscular sphincters, with one sphincter located in advance of each fracture plane. Therefore, the site of tail loss rarely bleeds more than a couple of drops. Tail regeneration then begins spontaneously following caudal autotomy.

Why leopard geckos?

Figure 3: The leopard gecko (Eublepharis macularius) is the model we use in our lab. They can spontaneously regenerate their tail following tail loss.

Although many lizards can regenerate their tails, the husbandry requirements for some species can be challenging (e.g., the lizards are aggressive or shy, have narrow ranges of preferred temperature and humidity, particular and unusual dietary and habitat preferences, and/or are intolerant of handling). As a result, early work on lizard tail regeneration tended to focus on a few types of locally available (and wild caught) species kept over short periods of time. However, with the increasing popularity of reptiles a pets, some lizards – including leopard geckos – are now captive bred and (at least in North America) widely available. Leopard geckos are hardy, accept being handled, and have relatively simple husbandry requirements: a plastic enclosure similar in size to those used for rats and mice; a water dish; a couple of hide boxes; a heat pad under one end (to establish a thermal gradient – leopard geckos are ectotherms); and escape-proof lid (see below).

Leopard gecko fun facts:

– Caudal autotomy is not necessary for regeneration. Tails surgically amputated outside the fracture plane still regenerates
– They are members of the Eublepharidae – the ‘true eyelid’ geckos. Unlike other geckos, they have movable eyelids
– They are native to parts of Pakistan, Afghanistan, India and possibly Iran
– They have temperature-dependant sex determination – the temperature of the environment during the embryonic period helps determine the sex of the offspring

A typical day in the Vickaryous lab:

We typically start the day with a cup of coffee/tea and a discussion about our experimental plans for the day. We manage our own leopard gecko colony, and everyone has to pitch in with the daily feedings and weekly weigh and measure adventures. After the geckos have been attended to there is a good chance someone in the lab will be serially sectioning and staining tissue. Not only do we love our histology, but learning to section seems to be a rite of passage in our lab…

Figure 4: Undergraduate students Rebecca McDonald (left) and Alaina Macdonald (right) working in our histology prep lab. The majority of our time spent here includes processing, embedding, sectioning and staining tissues we use to study regeneration

Feeding, housing and maintaining leopard geckos:

Our gecko colony is maintained in an environmental chamber (about 28˚C) here at the University of Guelph. Leopard geckos are not terribly social, so we house each one separately – this also makes keeping track of who’s who much easier. Our gecko enclosures are roughly 36cm x 22cm x 22cm in size, and topped by a perforated lid. Although leopard geckos lack subdigital adhesive pads, they have small claws and are capable climbers (and aspiring escape artists). Fortunately, they seem to prefer showing off their talents rather than following through with the getaway.

Leopard geckos eat live prey, and we feed ours mealworms (larval Tenebrio molitor, the mealworm beetle) dusted with a calcium and vitamin D3 (cholecalciferol) powder. The use of calcium and vitamin D3 is necessary in that it helps prevent metabolic bone disease, a nasty group of disorders that can lead to brittle or distorted limb elements and jaws, paralysis and reduced growth.

Weekly duties include cleaning and changes cages, and weighing and measuring geckos. Both require a certain degree of gecko wrangling skills, and tolerance of gecko urine. Although leopard geckos are typically docile and easy to handle, they will occasionally let loose a stream – especially if you forget to wear a lab coat.

Figure 5: Aerial view of a standard gecko enclosure. Features include: two small huts and a bowl of water.

Impact of ongoing research:

Tail replacement by leopard geckos is a striking example of a naturally evolved mechanism of multi-tissue regeneration. As one of the closest living relatives to mammals, leopard geckos provide a powerful platform to study the biology of regeneration, with numerous biomedical implications. Unlike other species, leopard geckos (and other lizards) can voluntarily self-detach the tail and have a variety of structural adaptations to minimize (or at least localize) tissue damage. Thus, autotomy-mediated tail loss is arguably a less-invasive alternative to amputation to initiate the regeneration program.

Follow us on Twitter: @VickaryousLab

Website: http://www.vickaryouslab.com/

Figure 6: Tail regeneration (in dorsal view). Left to right: the original tail, two regenerating tails and variation in two complete regenerate tails.

This post is part of a series on a day in the life of developmental biology labs working on different model organisms. You can read the introduction to the series here and read other posts in this series here.

(10 votes)

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This cartoon was first published in the Journal of Cell Science. Read other articles and cartoons of Mole & Friends here.

To read part I- ‘The imposter’ click here. To read part II- ‘The teaching monster’ click here.

(1 votes)

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Pint of Science is a science outreach organization that holds an annual festival across 9 countries (UK, Ireland, France, Italy, USA, Australia, Spain, Germany and Brazil) in 6 major themes, to bring the deeper, ground-breaking questions of science and the researchers working on them in contact with public at the local watering holes. The 2015 festival was from May 18^th to 20^th. I was fortunate enough to be invited to talk in the Pint of Science USA – Boston/Cambridge on the theme of Brain Games: From Development to Empathy gaps. It was a fantastic and engaging evening. I had the distinct pleasure of sharing the microphone with a brilliant fellow researcher Dr. Emile Bruneau from MIT who works on using neuroscience tools to discover new ways of understanding and solving conflict resolution issues across different tense areas in the world. I was given the opportunity to go first and talked about “Coding in Biological Systems: Bioelectrical Control of Tissue Identity and Anatomy” for about 10-15 mins. The transcript of my talk can be found below in this post. This was followed by a barrage of very interesting and engaging questions from the audience. Then it was Dr. Bruneau’s turn to speak and I had the distinct pleasure of being his guinea pig for an onsite experiment/demonstrations. I thoroughly enjoyed it and I believe everyone had a blast.  Many deep questions and discussions followed his talk. I had the good fortune of discussing science questions, thoughts and ideas with Dr. Bruneau and some of the audience members after the event. A wonderful effort and event organization by the Pint of Science USA – Boston/Cambridge coordinators Eleana Manousiouthakis, Daniel Whittet, and Shannon Spreen and the Pint of Science community all together in making this happen. The events’ major sponsors were elife, AHA consulting engineers, USGBC Massachusetts, BGlo and D!A.

Dr.Emile and myself have a nice discussion after our talks

Title: Coding in Biological Systems: Bioelectrical Control of Tissue Identity and Anatomy

“How many of you know who a potter is? Ok for those who may not know, potter is a person who creates these beautiful pots and structures out of clay/mud. And the way that this person does this is; they have a spinning wheel on which they place a limp of moist clay and give it beautiful shape by applying forces with their hands.

Now in nature you see all kinds of beautiful shapes and structures, from that of butterflies to various birds all the way to humans. How are these shapes in nature formed? There is no external force shaping them as they develop. So where is the information for shape structure and organs stored? All animals develop from an embryo. An embryo is a single cell with one piece of genetic information. So is the information for shape and structure stored in the genetics? To find out lets do a thought experiment. Say an alien comes down to earth and hands us humans a piece of genetic information of an animal that we have never seen before. Will we be able to predict this animal’s shape, structure, organs and functions of those organs based only on the genetics of it? The answer is no! This does not mean genetics is not important. Genetics does carry important information but not all the information, only a fraction of this information. Where else then could the information for shape and structure be present? Every cell, including the developing embryo sits in a multidimensional environment where it is sensing or gathering information from all these dimensions. One of these dimensions is genetics, others including mechanical forces, chemical signals and biophysical or Bioelectric signals. We work on understanding what information is stored in bioelectrical signals and how powerful this information is.

What are bioelectrical signals? They are not externally applied electrical currents, but are endogenous (from within) currents. They are also not very fast and transient currents like one sees in the neurons. They are rather very slow currents changing over very long periods of time like hours to days! Now take any cell. It has a membrane that separates its inside from its outside. In this membrane there are proteins that act as pumps and channels. These proteins use a lot of energy to shuttle charged molecules like sodium, potassium, chloride etc, in such a way that the inside of the cell is negative and the outside positive; thus converting every cell into a battery. This voltage difference across the membrane is called membrane voltage (Vmem) and for each cell it is about 10s of mV. Now if you take a sheet of cells and look at the Vmem of cells you don’t see uniform Vmem but rather beautiful patterns of Vmem. This is highly evident in developing embryos where one sees these beautiful patterns of Vmem that occur as the embryo develops. So what information do these bioelectrical patterns contain?

To study this we use frog embryos which are experimentally practical for various reasons. We have identified one of the patterns that is critically involved in eye development. What is remarkable is that if we imprint that eye pattern elsewhere of the embryo say for example on the gut, we end up forming a whole eye on the gut of the frog tadpole. Similarly we are able to form eyes on the tail and even butt of the tadpole! What is even more remarkable is that the tadpoles are able to see through these extra eyes and their brains are able to process the visual information coming from them no matter where they physically might be present! This is done using a robot which not only records the behavior of these animals but is able to teach them and test them how much they have learned. We have now done similar experiments for bioelectric control of brain tissues as well, and are able to induce brain tissues even in the tail of the tadpole.

So what is the purpose of all this? This will help us know where and how the information for shape and structure is stored within bioelectrical signals and how it is read and implemented. This is very important for purposes of regenerative medicine, where if there is any traumatic injury we can use this to regenerate the tissues or organs. Another use is once we know this information encoding we can detect birth defects very early on before they manifest and may even be able to add that information back and correct the defect. Lastly cancers can be viewed as lump of cells that have lost the information of structure, shape and function. If we can implant the right information into the tumors we might be able to have them differentiate and incorporate into normal tissues and perform normal functions. Ok at this point I will stop and take any questions you all may have for me.  “

(1 votes)

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From Maps to Circuits:
Models and Mechanisms for Generating Neural Connections

http://maps2015.org

7-9 December 2015

Strasbourg, France

Understanding the development of the nervous system is a key challenge
that has been approached by both experimental and theoretical
neuroscientists. In recent years there has been a gradual move towards
the two groups working more with each other. The idea of this workshop
is to bring key people together who have shown an interest at
combining theoretical and experimental techniques to discuss current
problems in neuronal development, and plan future collaborative
efforts. Time at the end of each day will be devoted to a group
discussion about questions that have been raised during the day to
identify possible research directions and people willing to pursue
them.

This meeting follows on from our first meeting on this theme held in
Edinburgh, July 2014 (details at http://maps2014.org); a special issue
of Developmental Neurobiology was devoted to papers related to work
presented at this meeting, see
http://onlinelibrary.wiley.com/doi/10.1002/dneu.v75.6/issuetoc

Confirmed speakers

Jianhua Cang (Northwestern University)
Claudia Clopath (Imperial College London),
Robert Datta (Harvard Medical School)
Stephen Eglen (University of Cambridge)
Marla Feller (University California Berkeley)
Patricia Gaspar (École des Neurosciences de Paris)
David Holcman (École Normale Supérieure Paris)
Andrew Huberman (University California San Diego)
Siegrid Löwel (Georg-August-Universität Göttingen)
Christian Lohmann (Netherlands Institute for Neuroscience)
Till Marquardt (European Neuroscience Institute, Göttingen)
Filippo Rijli (Friedrich Miescher Institute for Biomedical Research, Basel)
Jennifer Rodger (University of Western Australia)
Charles Stevens (Salk Institute)

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I’m conducting a study on biologists’ use of math; in order to identify the main mathematical and statistical tools currently used in the field. The results of the study would help to inform the graduate and undergraduate curriculum in Biology and Mathematics–as well as the curriculum of related training programs outside of academia–so that they would better align to student needs at their future workplace.

The first part of the study consists of an online survey designed for biologists from the areas of “developmental biology” and “ecology, evolution, and behavior”. It is been however quite difficult for me to find participants in the area of developmental biology. I was wondering if some of you would be willing to participate of the survey or/and help me to find participants, passing around the link below.

The ideal participant would be someone who majored in biology and is now working in developmental biology. This survey was piloted by faculty members and students at UT Austin, with an average completion time of 9 minutes. I can offer no compensation but I would be happy to share the results of the study, and make you eligible to win one of four $25 Amazon gift cards—I know it is not really not much, but I am limited to a very small budget. The online survey is completely anonymous; you will just need to click on the link below to take it.

https://utexas.qualtrics.com/SE/?SID=SV_3sAsOBtAhu4R8BD

Thank you so much for taking the time to consider my request, and please let me know if you have any further questions.

Pablo Duran (pduran@utexas.edu)
Doctoral candidate in Mathematics Education
The University of Texas at Austin

(5 votes)

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Having just begun my new role at the Society of Biology five weeks ago, I’m embarking on the exciting prospect of travelling around the country to meet potential members and find out about the important work they are doing. The mood is very high at the Society as we were granted Royal status only last week, honouring the prominence of biology and the vital work of our Society as the voice of life sciences across the country. Today, I’m at the Young Embryologist Network Annual Meeting held at King’s College London, where 200 young researchers will be presenting their work, updating their knowledge and mingling with peers.

Research topic areas include early embryonic development, stem cells and differentiation and forces in morphogenesis, but as my knowledge doesn’t extend further than my BSc in Human Sciences, it’s way beyond my expertise. However, what I learnt the most from and can definitely share with you were the wise words of Professor Jon Clarke, Head of Department of Anatomy at KCL, about advancing a career in academia:
‘Identify a niche and an important question that you can contribute your skills to. I wasn’t at the top of my class or the best in my school: I moved from working in the CNS in amphibians, to teaching anatomy, so don’t be afraid to do several postdocs before you go for a job. There’s no single way to get a good job, but the keys are: work hard, publish well, become excellent communicators of your research and most of all, work in a good lab that’s best for your research area. If you’re going to stay in academia, you need to know that this is the right job for you – if outside reading and going to seminars seems like a chore, get out now.

‘Most permanent research jobs are in Universities and an important part of these jobs is likely to be teaching undergraduates: so start teaching now and make sure it’s on your CV. Be prepared to get good at teaching something unusual or out of your comfort zone to students; you’ll learn a huge amount from it, it will improve your science and will give you ‘the edge’ for job applications. Have enthusiasm and curiosity: in every lab meeting, be exceptional and ask good questions – remember you are always on show. Be able to identify important areas of need and ask the right questions. This will help you to get an outstanding letter of recommendation, one that is better than anyone else in the interview. And obviously, publications: A good teaching profile will really help your career prospects, but essentially won’t trump your research profile.’

It seems from talking to delegates that the atmosphere is competitive, as with all research activities, and they are looking for ‘the edge’ that will get them their next post. Professor Clarke gave suggestions on how to diversify your skill set to raise you above your peers, including teaching skills and finding an effective niche. But essentially, he suggested that regardless of the decisions you make in your career, what matters most is your ability to present your case to the decision making body, and build a cohesive and persuasive argument of how you came to your current post and why you want to move to your next one. It’s advice like this that translates across all professions.

I previously worked for a professional conference organiser for medical and scientific associations and would be simultaneously organising multiple large conferences. I commend the organisers, all volunteers, for their excellent work and the smooth running of the conference. The feedback I received from talking to delegates in the exhibition area was brilliant, with specific comments noting the rising standard of the talks given. Thank you to Amanda Patist and Claire Bromley for their assistance: I personally really enjoyed the event, meeting the delegates and exhibitors, and wish YEN all the best for future meetings.

Written by Alexandra Spencer, Marketing & Membership Coordinator at the Society of Biology. The Society of Biology is the UK’s leading professional association for the life sciences: representing a single unified voice for biology: advising Government and influencing policy; advancing education and professional development; supporting our members, and engaging and encouraging public interest in the life sciences. 

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Here is some developmental biology related content from other journals published by The Company of Biologists.

Xenopus as a developmental model of neuroblastoma

Neuroblastoma (NB) is a paediatric form of cancer derived from the sympathetic nervous system. Recent genome-wide sequencing data suggest that often NB does not have a clear genetic cause, leading the authors to hypothesize that NB results from aberrations of normal development. To test this hypothesis, Anna Philpott’s group used a population of anteroventral noradrenergic (AVNA) cells from Xenopus embryos. These cells share several features with mammalian sympathetic neurons, including the expression of noradrenergic-associated genetic markers such as the achaete-scute complex-like 1 (Ascl1) gene, which encodes a transcriptional driver of neurogenesis. By comparing AVNA and NB cells, the authors found that, whereas Ascl1 is only transiently expressed in AVNA cells, it is aberrantly maintained in NB, where it is phosphorylated on multiple serine-proline sites. The authors then show that differentiation of AVNA cells is enhanced by dephosphorylated Ascl1. Moreover, this process is inhibited by experimental manipulations of NB-associated genes, but, interestingly, dephosphorylation of Ascl1 is able to overcome this inhibition. This work demonstrates that Xenopus AVNA cells represent a unique system to study sympathetic nervous system development and its relationship to NB. Moreover, it suggests that Asc11 phosphorylation might promote stalled differentiation leading to NB, thus identifying a potential target for therapeutic purposes. Read the paper here  (Open Access) and the authors’ Node post here.

Modelling DMD-associated cardiomyopathy in patient-derived iPSCs

Duchenne muscular dystrophy (DMD) is a genetic muscular disorder characterised by progressive muscular weakness and wasting, with cardiac complications (such as dilated cardiomyopathy) and respiratory muscle failure arising in the late stage. To investigate mechanisms of dilated cardiomyopathy in DMD, Lei Yang and collaborators derived cardiomyocytes (CMs) from DMD-patient-specific induced pluripotent stem cells (iPSCs). Compared to control cells, DMD iPSC-CMs exhibited elevated cytosolic calcium, mitochondrial damage and increased cell apoptosis. To further dissect the mechanisms underlying these alterations, the authors performed transcriptional and translational analyses and identified a mitochondrially initiated molecular cascade – which involves CASPASE3 (CASP3) activation – as being responsible for the increased apoptosis in DMD iPSC-CMs. Notably, the application of the membrane sealant Poloxamer 188 could prevent calcium overload and CASP3 activation, significantly reducing apoptosis in these cells. Thus, the authors established a useful in vitro system to disclose mechanisms of cardiomyopathy in DMD and to identify molecular targets that could be pharmacologically manipulated. Read the paper here (Open Access).

 
Endocytosis and micropinocytosis internalise cadherin-6B in EMT

Epithelial-to-mesenchymal transition (EMT) is an integral developmental and physiological process, but can also be utilised by cancer cells at the initiation of metastasis. A requirement for EMT is the post-translational removal of adhesion proteins from the plasma membrane. Here (p. [164426]), Lisa Taneyhill and Rangarajan Padmanabhan study cadherin-6B (Cad6B) internalisation to elucidate the mechanisms of EMT in chick cranial neural crest cells. The authors found that in neural crest cells that are initiating EMT, Cad6B was detected in cytosolic puncta that were endocytic, rather than exocytic, in nature. They then identified two intracellular motifs that were potentially important for regulating Cad6B internalisation. Mutating the p120-catenin-binding (EED) motif, but not the dileucine (LI) motif, significantly increased Cad6B internalisation, supporting the idea that Cad6B is removed from the plasma membrane through endocytosis. However, although Cad6B colocalised with clathrin, the colocalisation was not exhaustive, suggesting that an additional mechanism is involved in Cad6B internalisation. Therefore, the authors used an array of pharmacological treatments to show that Cad6B was removed from the plasma membrane through both endocytosis and macropinocytosis, and that both of these processes depended on dynamin. This study demonstrates that EMT and neural crest migration require Cad6B internalisation through endocytosis and macropinocytosis. Read the paper here.

Generating an in vitro 3D cell culture model from zebrafish larvae for heart research

Spontaneously beating 3D ‘heart’ structures can be developedin vitro from larval zebrafish using a novel, fast and inexpensive method that could be employed in ecotoxicology and biomedical safety testing. Read the paper here.

(2 votes)

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My review of the Young Embryologist Network London Meeting 2015

As I write this, I am currently listening to the third very impressive senior PI talk in a row addressing ‘how to succeed in science’. This one is from an incredible woman who came through the German system, did a PhD at the EMBL in Heidelberg, did a postdoc there for about 10 minutes where she developed an incredible new model system, won loads of prizes, and is now running a large and successful lab at Cambridge basically solving every medical problem in pregnancy. In Cambridge she has started a family and been incredibly successful. She looks about 32.

So, I have decided to stop listening (and stop crying inside) and to start a review of the research talks. Hilariously, I am actually a judge of the talks this year (I have taken off marks for every time someone says ‘’so we took a system approach’’, and then speaks about a phenomenon/gene/conceptual framework that was entirely known or in place before people said this in talks. It’s not their fault of course – we are all prisoners of fashion. But here I am, I can be no other, as somebody once said). As ever, I have just picked out a few highlights. This is not because the other talks were bad, but because I have a very fat son who is 7 weeks old. So I have been getting up approximately every 2 hours for about 90 minutes every night for the last 7 weeks to feed an apparently insatiable baby who has supplanted me as the most important man in my wife’s life. I have slept through some talks. Sorry.

Vanessa Chong (Oxford)

This talk was awesome. This girl is at the end of her 2^nd year (remember that). She is a student of Tatjiana Sauka-Spengler at the Wetherall Institute of Molecular Medicine. This is an institute that reverses the reprehensible modern trend, and has a translational name but does incredible basic research. She has developed a method for purifying nuclei from genetically targeted cells in zebrafish neural crest, and then conducted large-scale transcriptomics. She identified very counter-intuitive expression of neural crest developmental transcripts in non-neural crest tissues. What? But, she also found opposite strand transcription of these same genes in only the neural crest lineage. Antisense non-coding transcription is correlated with gene expression but not transcription (of neural crest genes). She is a 2^nd year. Don’t know if I mentioned that. Impressive stuff.

Lizzy Ward (UCL)

A wonderfully old-fashioned bit of developmental biology examining the signalling of the notochord to the forming somites and ultimately vertebrae. She showed really nicely and elegantly that the notochord does signal to the somites, and that this signal is generic i.e. doesn’t vary in nature along the A/P axis of the notochord. It might only end up in a smaller journal (thought the last such study from the Stern lab ended up in Science), but it was beautifully presented and very elegant.

Rebecca McIntosh (KCL/UCL)

A talk on a favourite topic of mine: basal progenitors. These are proliferative cells in the CNS that unlike most CNS progenitors maintain a connection not to the apical surface, but the basal one. Such cells have expanded enormously in number in the primate neocortex and are suggested to underlie the evolution of large neocortices; they have expanded enormously in the cerebella of amniotes and are suggested to underlie the evolution of large cerebella (some wonderful work from an extremely talented and very good looking young lecturer in East London). Rebecca’s talk examined their biology in a much more simple and elegant system: the zebrafish spinal cord and hindbrain. This talk was a really nice combination of sophisticated genetic labelling and high-end microscopy, combined with really sensible and simple questions. The work quantified basal divisions in zebrafish (i.e. in a very small brain) and showed that elaborate cellular behaviours accompany their specification and patterns of division. As ever with good research, it generated more questions than it answered.

John Robert-Davis (KCL/Crick Institute)

John is a great bloke who is incredibly charismatic and depressingly bright. His thesis examined the molecular and mathematical basis of contact inhibition in Drosophila hemocytes and won the Beddington Prize for the best thesis produced in the UK last year in developmental or cellular biology. He is now a postdoc at the Crick Institute and I suspect a shoe-in for some incredible fellowship – he published his PhD work in Development and Cell. His talk was crap. That is all you need to know.

Okay, okay, it wasn’t crap. It was, in fact, utterly brilliant. And entirely predictably so. He won the prize for the best talk. And he used zorb ball (look it up) as a genuine scientific metaphor. Apparently he’s also a standup comedian. And he’s a great bloke. Urgh.

Alexander Fletcher (Oxford)

I should preface this review by saying that I am generally quite unimpressed by a lot (though certainly not all) of the modelling that is done in relation to developmental biology. I am sure that I am quite possibly completely mistaken and very very happy indeed to be corrected and saved from my own ignorance by the armies of physical scientists now being recruited into biology. Nevertheless, insert Martin Luther quote here.

I recently sat through a talk that had the sentence: ‘’of course without any inertia, you generate chaotic Turing signalling patterns.’’ And people say that recruiting physicists into developmental biology is confusing the field…

With my very poor mathematical ability (it is actually not that poor), I took the following from this other talk, as from so many others: we made a model using real data and we know how to do this (at this point the vast majority of the audience who do things like experiments become very scared – there is usually a slide with lots of maths on). We tested the model using real data. The real data supported the model. The model generated zero (this is the point of distinction between the good modelling studies and the majority) novel hypotheses. We concluded that the laws of physics apply to living things. A room full of biologists (or possibly just those who make funding decisions?) was incredibly impressed.

The final talk thus came as a wonderful surprise. It was brilliantly cynical, entertaining and I think important. It discussed how the process of writing code for models for scientific applications like developmental biology is hugely ineffective and should be assessed and rigorously examined by people who know how to write good code, before being foisted upon those who don’t. An idea whose time I hope has come. www.cs.ox.ac.uk/chaste.

(3 votes)

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Department/Location: Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute, University of Cambridge

Salary: £38,511-£48,743

Reference: PS05620

Closing date: 14 June 2015

Limited funding: The funds for this post are available until 31 January 2018 in the first instance.

Three UK Research Councils; Biotechnology and Biological Sciences Research Council, Engineering and Physical Sciences Research Council and the Medical Research Council have invested £25m in research and equipment to develop the UK Regenerative Medicine Platform (UKRMP www.ukrmp.org.uk). The Pluripotent Stem Cell Platform (PSCP) is one of five UKRMP Hubs formed to tackle critical challenges in developing regenerative treatments from laboratory discoveries. PSCP brings together researchers from several institutions to develop the processes for taking pluripotent stem cells and their derivatives from laboratory research to the manufacture of safe, consistent and effective products suitable for clinical applications.

PSCP now has an exciting opportunity for a Project Manager with experience in management and administration at the interface between academia and industry.

We are looking for a proactive and motivated individual to manage the PSCP portfolio according to industry best practice; determining and delivering to agreed scope, quality, budget and deadlines. The Project Manager will manage a number of projects concurrently and will maintain accurate project plans, work schedules, issue and risk logs to enable projects to be delivered successfully. You will coordinate regular meetings at the different partner sites. You will develop summary documents and reports, will organise meetings and will lead the daily administration of the project. You will be a confident and articulate communicator who delivers information concisely and effectively. You will have the experience and interpersonal skills to build effective relationships with funders and a range of stakeholders including industry and regulators.

Experience of finance management, budgeting, and administration in a research-led environment is essential. Experience of leading, or working as part of, a management team with accountability for group and individual performance is highly desirable.

You will be educated to at least degree level and have a further qualification (MSc, PhD or MBA).

Once an offer of employment has been accepted, the successful candidate will be required to undergo a health assessment.

To apply online for this vacancy and to view further information about the role, please visit: http://www.jobs.cam.ac.uk/job/6475. This will take you to the role on the University’s Job Opportunities pages. There you will need to click on the ‘Apply online’ button and register an account with the University’s Web Recruitment System (if you have not already) and log in before completing the online application form.

The closing date for all applications is Sunday 14th June 2015.

Please upload your Curriculum Vitae (CV) and a covering letter in the Upload section of the online application to supplement your application. If you upload any additional documents which have not been requested, we will not be able to consider these as part of your application.

Informal enquiries about the post are also welcome via email on cscrjobs@cscr.cam.ac.uk.

Interviews will be held on the afternoon of Wednesday 1st July 2015. If you have not been invited for interview by 24th June 2015, you have not been successful on this occasion.

Please quote reference PS05620 on your application and in any correspondence about this vacancy.

The University values diversity and is committed to equality of opportunity.

The University has a responsibility to ensure that all employees are eligible to live and work in the UK.

(1 votes)

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Figure 1. “Cytoplasm”, illustration by David S. Goodsell, the Scripps Research Institute.

When contemplating the illustrations by David S. Goodsell (Figure 1), the first thing that stands out is how cells are packed full with those wonderful little machines we call proteins. They move, interact and change shape to produce cellular functions, so our ability to observe where they localize and monitor their behaviour is extraordinarily important in biology. In general, immunocytochemistry is preferred for static protein detection, while the dynamics of protein localization can be highlighted by transgenic constructs including the protein of interest fused to a fluorescent tag. Importantly, whereas tagged proteins are irreplaceable tools for live imaging at high resolution, immunocytochemistry is far from dead. Both antibodies and fluorescent tags are so widespread in use that sometimes we assume that the results we obtain must reflect the endogenous condition. As a matter of fact, when comparing data from static and dynamic protein detection techniques, inconsistencies can emerge. On one hand artifacts can derive from handling of the tissue, for example by fixation and permeabilization (Schnell et al., 2012). On the other hand, direct tagging of a protein or perturbations in its expression levels can interfere with the way proteins fold, interact and therefore localize. In a notable example, Swulius and Jensen (2012) found that YFP-tagged MreB (a bacterial actin homolog) artifactually nucleates to form long helical structures. The description of a helical cytoskeleton in rod-shaped bacteria heavily influenced this field of research for over 10 years. However, the aggregates were not found in microstructural analyses of bacteria with untagged MreB, demonstrating that the filaments were a byproduct of protein tagging.

In the current issue of Development, we contributed a report investigating a potential alternative for protein detection in living organisms, which unifies the advantages of antibodies and live imaging.

Figure 2. Schematic structure of camelid heavy-chain antibodies and chromobodies, compared to conventional IgGs.

In the late 1980s, a serendipitous discovery led to the description of heavy-chain antibodies in the plasma of camelids (Hamers-Casterman et al., 1993). Similar molecules are also found in sharks, however their advantage for animal immunity is not clear. These atypical IgGs are devoid of light chains, such that their heavy-chain variable portion (termed VHH or nanobody) can alone recapitulate antigen binding (Figure 2). This convenient property introduces the possibility of cloning entire repertoires of nanobodies and screening for binding to an antigen of interest in vitro, for example by phage display.

Nanobodies are already important reagents utilized in X-ray crystallography. They preferably bind convex surfaces, thereby helping the stabilization of protein conformations or by providing additional contact surface for protein aggregates (De Meyer et al., 2014). Their small size (15 KDa, one tenth of a conventional antibody) and high affinity are valued properties. In addition, with more and more monoclonal antibodies currently being evaluated for therapeutic application, nanobodies are becoming increasingly popular. They offer a variety of advantages, among which high tissue penetration, blood clearance, stability and solubility (Holliger and Hudson, 2005).

In a most innovative adaptation of nanobodies, Rothbauer, Zolghadr et al. (2006) fused the VHH binding moiety to a fluorescent protein and tested the resulting “chromobody” in mammalian cells. In this context, chromobodies can recapitulate the localization and original behaviour of endogenous proteins, with minimal functional interference, presumably because of their high binding turnover. Remarkably, chromobodies appear to fold correctly by establishing their only disulphide bond even in the reducing intracellular environment.

Video 1. 5 hpf zebrafish embryo injected with actin chromobody mRNA, showing localization to the plasma membrane and to the perinuclear actin cap. Centrosomes can be identified during cell division, after which the nuclear compartment and its actinic cap reassemble (arrowheads). Scale bar: 50 µm.

Inspired by these results, Ulrich Rothbauer (now at the Universität Tübingen) initiated a collaboration with our group at the Max Planck Institute for Developmental Biology, aimed at testing chromobody reagents in zebrafish. Would antigen binding be maintained in an entire organism? Would it be stable over consecutive developmental stages? We decided to focus on chromobodies binding to two proteins with well-known localization patterns, F-actin and PCNA. This choice was also motivated by the fact that interfering with actin or PCNA function would probably lead to visible phenotypes, conveniently showing side effects of chromobody expression. After injecting chromobody mRNA in zebrafish zygotes we could observe localized fluorescence at the expected sites where the respective antigens normally accumulate (Video 1). However, because of the expression variability and aspecific death in many embryos, we decided to switch to genetically-encoded expression systems. Therefore, by combining chromobodies and heat-shock or Gal4-dependent expression, we collected live imaging data supporting the actual binding of chromobodies to endogenous intracellular antigens in zebrafish, as confirmed by costaining with antibodies. With to the newly generated heat-shock and UAS transgenic lines we encountered some photobleaching problems, however we could detect actin and PCNA dynamics with high fidelity in different tissues or in the entire embryo, throughout embryonic development.

A very important question regards the impact of chromobodies in the context of a living embryo. Will they interfere with endogenous protein function? Strikingly, we observed that transgenic zebrafish embryos and larvae were morphologically normal throughout development and adult stages. They did not show aberrations in cell cycle progression (when monitoring the PCNA chromobody), nor they could alter the migration of lateral line primordium cells (in the case of the actin chromobody), even though it is known that interfering with actin dynamics at the leading edge of the primordium will perturb its migration (Xu et al., 2014). We sought a possible explanation for these observations. We detected fast recovery after photobleaching in HeLa cells stably expressing chromobodies. Importantly, the original localization of the antigen was immediately recapitulated by unbleached chromobodies, indicating that these molecules have elevated k_off and k_on, which imply a very transient antigen binding mode. We propose that the short binding half-life displayed by chromobodies prevents the emergence of global functional defects in living embryos.

In our work we show that chromobodies can be used as research reagents in living vertebrates, and substantially behave as already described in cultured mammalian cells. We introduce chromobodies as a complementary technique to more standard immunocytochemical and fluorescent-tag-based approaches. Chromobodies are advantageous because they allow live imaging without the need to directly modify or overexpress the protein of interest, approaches that can potentially produce artifacts. From the application side, chromobodies can be used as readout for phenotyping purposes or for high-throughput drug screening. As an example, the PCNA chromobody reports cell cycle progression just by displaying the localization dynamics of PCNA, which allows to differentiate single cell cycle stages and confers better resolution during S phase and S/G2 transition compared to FUCCI (Sakaue-Sawano et al., 2008).

Recent publications reported improved methods to clone and isolate nanobodies (Fridy et al., 2014), hence we hope that the high-throughput identification of novel binders will boost the availability of these reagents for basic research community. Although our results are preliminary because they focus only on two commercial chromobodies, we anticipate a widespread adoption of these promising reagents.

Featured article

Panza, P., Maier, J., Schmees, C., Rothbauer, U., & Sollner, C. (2015). Live imaging of endogenous protein dynamics in zebrafish using chromobodies Development, 142 (10), 1879-1884 DOI: 10.1242/dev.118943

References

De Meyer, T., Muyldermans, S. and Depicker, A. (2014). Nanobody-based products as research and diagnostic tools. Trends Biotechnol. 32, 263–270. DOI: 10.1016/j.tibtech.2014.03.001

Fridy, P. C., Li, Y., Keegan, S., Thompson, M. K., Nudelman, I., Scheid, J. F., Oeffinger, M., Nussenzweig, M. C., Fenyö, D., Chait, B. T., et al. (2014). A robust pipeline for rapid production of versatile nanobody repertoires. Nat. Methods 11, 1253–1260. DOI: 10.1038/nmeth.3170

Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hammers, C., Songa, E. B., Bendahman, N. and Hammers, R. (1993). Naturally occurring antibodies devoid of light chains. Nature 363, 446–448. DOI: 10.1038/363446a0

Holliger, P. and Hudson, P. J. (2005). Engineered antibody fragments and the rise of single domains. Nat. Biotechnol. 23, 1126–1136. DOI: 10.1038/nbt1142

Rothbauer, U., Zolghadr, K., Tillib, S., Nowak, D., Schermelleh, L., Gahl, A., Backmann, N., Conrath, K., Muyldermans, S., Cardoso, M. C., et al. (2006). Targeting and tracing antigens in live cells with fluorescent nanobodies. Nat. Methods 3, 887–889. DOI: 10.1038/nmeth953

Sakaue-Sawano, A., Kurokawa, H., Morimura, T., Hanyu, A., Hama, H., Osawa, H., Kashiwagi, S., Fukami, K., Miyata, T., Miyoshi, H., et al. (2008). Visualizing Spatiotemporal Dynamics of Multicellular Cell-Cycle Progression. Cell 132, 487–498. DOI: 10.1016/j.cell.2007.12.033

Schnell, U., Dijk, F., Sjollema, K. A. and Giepmans, B. N. G. (2012). Immunolabeling artifacts and the need for live-cell imaging. Nat. Methods 9, 152–158. DOI: 10.1038/nmeth.1855

Swulius, M. T. and Jensen, G. J. (2012). The Helical MreB Cytoskeleton in Escherichia coli MC1000/pLE7 Is an Artifact of the N-Terminal Yellow Fluorescent Protein Tag. J. Bacteriol. 194, 6382–6386. DOI: 10.1128/JB.00505-12

Xu, H., Ye, D., Behra, M., Burgess, S., Chen, S. and Lin, F. (2014). Gβ1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium. Dev. Biol. 385, 316–327. DOI: 10.1016/j.ydbio.2013.10.027

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