Embryogenesis begins with fertilisation, and defective activation of the egg by the sperm is implicated in human infertility. Today’s paper, published in the most recent issue of Development, investigates the role of the sperm protein PLCζ in egg activation and the calcium oscillations that accompany it. We caught up with co-first author Alaa Hachem and his PI John Parrington, Associate Professor in Cellular and Molecular Pharmacology at the University of Oxford and Fellow of Worcester College, to hear more.

John, can you give us your scientific biography and the main questions your lab is trying to answer?

JP I studied at the University of Cambridge where I took Natural Sciences.  An important inspiration for me was doing my third year studies at the Department of Zoology where scientists like John Gurdon, Ron Laskey, Michael Bate, and Alfonso Martinez-Arias, were harnessing the power of molecular biology to address some key questions in developmental biology. I then did my PhD at the Imperial Cancer Research Fund with Ian Kerr investigating how transcription factors regulate the activities of cells in the immune system.

My first post-doc saw a change of direction. I worked under the supervision of Karl Swann and Tony Lai at the National Institute of Medical Research trying to identify the molecular basis of the process whereby an egg is induced to develop into an embryo. These studies continued at University College London where I was able to continue my research into egg activation thanks to an MRC Career Development Award and an MRC Senior Research Fellowship.  These studies culminated in the discovery – jointly with Karl Swann, Tony Lai, and Keith Jones – that the physiological agent of egg activation appears to be a novel sperm-specific protein called PLCζ. Subsequently, at the University of Oxford, where I moved to begin a lectureship at the Department of Pharmacology, my group were the first to show that mutations in PLCζ are associated with certain types of human infertility. PLCζ triggers egg activation by inducing Ca²⁺ signals in the egg.

Over the last decade, my group have been studying more generally the role of calcium signals in a variety of important pathophysiological processes

Over the last decade, my group have been studying more generally the role of calcium signals in a variety of important pathophysiological processes, in particular the role of the intracellular signalling molecule NAADP as a calcium mobilising messenger. These studies culminated in my discovery, in collaboration with Antony Galione, that two-pore channel, or TPC, proteins, are endolysosomal calcium channels regulated by NAADP. To study the mechanism of action and role of NAADP and TPC proteins, we generated knockout mice with loss of expression of TPC1 and/or TPC2. Our studies of these mice have identified important roles for TPCs in processes as diverse as smooth muscle and cardiac contraction, insulin secretion and sensing, autophagy and skeletal muscle function, intracellular trafficking, secretion of enzymes by the pancreas, brown adipose tissue thermogenesis, muscle development, and neo-angiogenesis.

Most recently, I became interested in using the new CRISPR/Cas9 gene editing technology to create a PLCζ knockout mouse. The reason for this was that despite numerous studies pointing to an important role for PLCζ in the egg activation process, no one had provided the definitive evidence in the form of gene knockout that PLCζ was the physiological agent of egg activation. In fact, we had previously tried using the standard embryonic stem cell approach to make a PLCζ knockout but for some reason we never managed to get germline transmission. With only a limited budget for the project, when we heard about CRISPR/Cas9 gene editing, this seemed to offer a rapid and economic way to make a PLCζ knockout. And indeed we did, in a single generation, although from this we then generated two knockout lines, our studies of which are described in the Development paper.

As well as running a lab, you have been involved in public science communication and writing books on the genome and gene editing. How did you get in to this side of science, and how important is the public understanding of science to you?

JP I’ve been interested in the communication of science to the public ever since the start of my career as a PhD student. I’ve written about new scientific discoveries and the ethics and politics of science for various publications since that time and also taken part in a variety of science communication initiatives with young people. I have debated the science and ethics of cloning and assisted reproductive technologies with sixth form students, and even took part in a project with the Northern Ballet to teach cosmology to 14-15 year olds using dance and drama. For several years now, my wife Margarida Ruas – who is also a joint first author on the Development paper – and I have taken over the classroom of a year 6 (10-11 year old) primary school classroom, to give a lesson on ‘Sex, Reproduction, and DNA’. As well as a short Powerpoint presentation, this involves us showing the children fertilization of a sea urchin egg under the microscope, freezing objects in liquid nitrogen to mimic the freezing of human embryos in clinical ART labs, and the children extracting DNA from a strawberry. The popularity of this lesson was demonstrated by comments from the children such as ‘science is wicked!’ and ‘this is the best lesson I’ve ever had!’

You can learn more about the British Science Assication Media Fellows in this video.

To further my skills in science communication, I have also completed part-time diploma courses in science communication at Birkbeck College, London, and in creative writing at the University of Oxford. Although I’ve been involved in many science communication initiatives over the years, I think a crucial moment was being awarded a British Science Association Media Fellowship. This involved me working as a science journalist at The Times in London for six weeks in the summer of 2012. I really enjoyed covering all manner of different science topics during this period, and I was very pleased that 22 of my articles were published. This gave me confidence and helped develop my writing and communication skills.

One of the stories I published concerned ENCODE – the project whose aim is to map all the functional activity in the genome. This later became the subject of my first book – The Deeper Genome, published by Oxford University Press in 2015 – which tackles the controversial question of how much of the genome is junk and how much is important. My second book – Redesigning Life, published by OUP in 2016 – is about gene editing, but also other new approaches such as optogenetics and stem cell technology. I have just finished writing a book about genetics for secondary school students, and I’m working on one about the biology of human consciousness for OUP. So you could say I have caught the book-writing bug. It’s definitely a juggling act finding the time to write popular science books as well as doing my university teaching, research and writing papers, reviews, and grants. But I have really enjoyed being able to tackle broader scientific topics than I can in my research, and the extra readership that this has brought. It has also been fun to talk about the subjects covered by the books at science and literary festivals and have a debate with the audience, who can be of a variety of ages and backgrounds. All of which should make it clear that I take communication of science to the public, including the debate about its direction and ethics, very seriously.

Alaa – how did you come to join John’s lab? I understand your PhD is funded by the Iraqi government?

AH The first time I met John was during a fertility conference in Yazd, Iran, where he presented his latest studies of PLCζ’s role during fertilization and its link with human infertility. His talk caught my attention and I decided to approach him to ask him more about his future projects, and also about the chance of doing a DPhil in Oxford under his supervision. I found him very keen in extending my knowledge of the process of mammalian egg activation, and he was also very happy to help me with my application to Oxford. In 2013, I joined his lab after successfully securing a generous grant from the Higher Committee For Education Development in Iraq (HCED-Iraq). The prime minister’s initiative provided me with sufficient funds for covering the cost of college and university fees, accommodation and living expenses, as well as some money for the research itself.

Can you give us key results of the paper in a paragraph?

AH&JP Activation of the egg by the sperm is the first, vital stage of embryogenesis. The sperm protein PLCζ has been proposed as the physiological agent that triggers the Ca²⁺ oscillations that normally initiate embryogenesis. However, there has been no evidence that gene knockout of PLCζ abolishes the ability of sperm to induce Ca²⁺ oscillations in eggs. We used CRISPR/Cas9 gene editing to generate PLCζ knockout mice. Our studies of sperm from PLCζ knockout males showed that these fail to trigger Ca²⁺ oscillations in eggs. This therefore provides the first definitive evidence that PLCζ is the physiological trigger of these Ca²⁺ oscillations. Remarkably, however, some eggs fertilized by PLCζ-null sperm can develop, albeit at greatly reduced efficiency, and after a significant time-delay. In addition, PLCζ knockout males are subfertile but not sterile, suggesting that in PLCζ’s absence, eventually egg activation can occur via an alternative, although much less efficient route. This is the first demonstration that in vivo fertilization without the normal physiological trigger of egg activation can result in offspring.

In the absence of Ca²⁺ oscillations, how do you think egg activation occurs?

AH&JP The unfertilized egg is kept in a state of meiotic arrest by the maturation promotion factor, or MPF. A later decline in MAPK activity correlates with the formation of pronuclei and entry into interphase of the first cell cycle. Previous studies have shown that MPF levels can be reduced artificially in various ways, even without a Ca²⁺ stimulus, triggering egg activation and development to blastocyst in vitro. This mode of egg activation, without Ca²⁺ release, may explain the spontaneous activation observed by previous studies in ovulated, hamster and mouse eggs left to reside in the oviduct for extended periods of time in the absence of fertilization. Moreover, eggs from the C57BL/6 strain, the strain used in our studies, have been shown to have a particularly high susceptibility to spontaneous activation during in vitro maturation. Therefore a related mechanism might be responsible for the activation we observe, with the self-activation that aging, unfertilized eggs experience that normally results in fragmentation or embryonic arrest, being rescued by the PLCζ knockout sperm. A more intriguing possibility is that some stimulus supplied by the sperm, either a soluble factor, or the product of sperm-egg binding, is triggering egg activation in the absence of PLCζ. Although we failed to observe the Ca²⁺ oscillations typical of fertilization following ICSI or IVF with PLCζ knockout sperm, it remains possible that PLCζ knockout sperm are inducing an atypical Ca²⁺ signal in the egg that somehow we failed to detect in the current study. Examining this possibility will be an important focus for future studies.

How might your PLCζ mice be useful for studying male infertility and its treatment?

AH&JP Currently infertility in men whose sperm fail to induce egg activation, can be treated by inducing egg activation artificially with mechanical, electrical, or chemical stimuli. However, the long term effects of such treatments on human development remain far from clear, an issue of some concern given that the Ca²⁺ signals induced by these treatments are highly non-physiological. The availability of PLCζ knockout sperm now makes it possible to assess in a mouse model, how artificial egg activation stimuli, used in the clinic, might affect embryonic gene expression and offspring growth, metabolism and behaviour. Importantly, it will provide a way to test the efficacy and safety of recombinant PLCζ protein as an alternative therapeutic agent to treat infertility caused by egg activation deficiency.

When doing the research, did you have any particular result or eureka moment that has stuck with you? And what about the flipside: any moments of frustration or despair?

AH Eureka moments are always a joy to share with friends and colleagues. However, such moments don’t come easily as they are usually preceded by great amounts of frustration and despair. I remember the moment when we observed the genotyping results of the mice produced after performing the CRISPR-Cas9 gene targeting of the PLCζ gene. The results showed us for the first time that mutation by out-of-frame deletion had occurred, and all the guide-RNAs had worked successfully with both wildtype Cas9 and Cas9 nickase. That was a big moment of relief because previously we tried to mutate the coding sequence of the PLCζgene in mouse zygotes, however we weren’t successful due to a one nucleotide difference between the guide-RNA we had designed based on the standard mouse genome sequence and the genomic DNA of the strain of mice used for the gene targeting. At the time, I felt extremely unlucky for this rare mismatch to have occurred, as it ruined five months of my work. However, a friend told me that it doesn’t show how unlucky I am, but rather how ‘special’ I am for this rare event to occur during my scientific career.

Another moment that really pleased me was when I collected the epididymis from the first homozygous knockout male at the age of 8 weeks. A previous unpublished conference report had claimed that knockout of the PLCζ gene was detrimental to the spermatogenic process, leading to no sperm being produced, which would have made it impossible to use gene knockout to test the physiological importance of PLCζ in the egg activation process. Thankfully, these claims didn’t hold true as I discovered that male PLCζ knockout mice were able to produce sperm with normal parameters of viability, motility, and capacity to undergo capacitation and the acrosome reaction. This allowed us to confirm PLCζ as the source of the  Ca²⁺ oscillations at fertilization which otherwise would not have been possible.

The PLCζ knockout mice allowed us to begin unravelling long-awaited questions regarding the identity of the ‘sperm factor’ that triggers embryogenesis and its role in the egg activation process. Initially, I assumed that  the males we had created would be totally infertile, given that their sperm lacked the capacity to trigger the normal signal that induces embryogenesis. Yet, one day we received an email from our co-author Jonathan Godwin saying that some of the mutant males had managed to impregnate a few of the wildtype females. This was puzzling, and I initially assumed it was due to a mistake in labelling the homozygous mutants. However, the genotyping results of the new offspring confirmed they were heterozygous animals and that the knockout males had not been mislabelled. Therefore, in order to address this unexpected finding, we cultured zygotes generated with KO sperm by in vitro fertilisation and in vivo mating, which confirmed occurrence of activation in mice eggs in the absence of the normal Ca²⁺ oscillations, albeit with greatly reduced efficiency and after a significant delay. Importantly, the findings showed for the first time birth of individuals of a mammalian species in the absence of the normal physiological stimulus, and we have also witnessed the importance of PLCζ for achieving the normal physiological levels of fertility in mammals.

What are your career plans following this work?

AH I am an aspiring academic and so inherently I am drawn to teaching and research. Therefore, I plan to go back to Iraq where I hope to teach university students in embryology along with setting up my own lab where I will continue to study aspects of mammalian reproduction and infertility. My goal is to help develop the scientific community in Iraq. I believe that my studies at the University of Oxford has equipped me with the knowledge and skills to have a positive impact on both students and research. In the future, I hope to translate the findings clinically by working with infertility clinics in Iraq.

My goal is to help develop the scientific community in Iraq

And what next for the Parrington lab?

JP Providing the definitive evidence by gene knockout that PLCζ is the physiological trigger of the Ca²⁺ oscillations that initiate embryogenesis has been very exciting for me, since I have been on the search for the identity of the ‘sperm factor’ that kick-starts life ever since my first post-doctoral post. However, our findings raise as many questions as they answer, and I’m particularly keen now to study how egg activation and even development to term can occur in the absence of the physiological agent of egg activation. Given that some previous studies have shown that embryo development is sensitive to differences in the pattern of Ca²⁺ signals in the egg, there is also the interesting question of whether the offspring conceived by PLCζ knockout males differ in important respects, e.g. in their growth, metabolism, behaviour, lifespan, etc. compared to offspring conceived in the normal fashion. This issue also has clinical importance given that artificial egg activation stimuli are now being used to treat certain types of human infertility in which egg activation fails to take place without assistance. Given that such stimuli generate a Ca²⁺ signal that is quite different from the normal sperm-induced Ca²⁺ oscillations, we would like to use the PLCζ knockout sperm as a null background to test the efficacy and safety of these clinically used artificial egg activation stimuli in an animal model. In addition to these PLCζ-focused studies that I would like to carry out in the future, I am also very interested to continue my studies into the mechanism of action and pathophysiological role of the TPC endolysosomal Ca²⁺ channels that we are also investigating using gene knockout approaches.

Finally, what do you like to do when you are not in the lab?

AH I usually enjoy watching sci-fi movies and some sport activities when I have time.

JP As well as writing popular science articles and books, I read a lot of popular science. I also read books about history, politics, and a fair number of novels. I also like to travel and I usually try and learn a bit of the language of the country I’m visiting. I’m a political activist, and I’m particularly interested in the ethics and politics of science. For relaxation I like walking, running, exercising at the gym, cooking, listening to music, and watching the occasional film. Otherwise, my two children keep me pretty busy!

Hachem, A., Godwin, J., Ruas, M., Lee, H. C., Ferrer Buitrago, M., Ardestani, G., Bassett, A., Fox, S., Navarrete, F., de Sutter, P., Heindryckx, B., Fissore, R.
and Parrington, J. 2017. PLCζ is the physiological trigger of the Ca2+ oscillations that induce embryogenesis in mammals but conception can occur in its absence. Development 144:2914-2924.

This is #26 of our People Behind the Papers series; the full archive can be viewed here

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The full programme and booking information for the On Growth and Form 100 conference are now online at https://www.ongrowthandform.org/conference/

The three-day conference takes place in the University of Dundee and the University of St Andrews on 13-15 October and features speakers from around the world, exploring the many aspects and influences of D’Arcy Thompson’s landmark book – including presentations of fascinating developmental biology research following in D’Arcy’s footsteps. Don’t miss it!

https://www.ongrowthandform.org/conference/

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Here are the highlights from the current issue of Development:

PLCζ ‘waves in’ mammalian oocyte activation

At fertilisation, fusion of the sperm with the oocyte activates a slew of downstream processes to kick-start embryogenesis. This ‘oocyte activation’ event induces the cortical reaction to prevent polyspermy, triggers oocyte metabolic and DNA synthesis pathways, and reactivates meiosis. In mammals, there is evidence to suggest that a phospholipase C isoform, PLCζ, initiates calcium oscillations associated with oocyte activation when delivered from sperm to egg. Hence, PLCζ is thought to be crucial for the activation process. Here, on p. 2914, John Parrington and colleagues directly test this hypothesis using CRISPR/Cas9 to generate a PLCζ knockout mouse. They report that while the production and quality of sperm are unaffected in knockouts, the sperm fail to induce calcium waves at fertilisation, confirming that PLCζ is required for this event. Intriguingly, although the majority of eggs fertilised by the knockout sperm do not develop, some are able to initiate embryogenesis, albeit in a delayed manner. Remarkably, PLCζ knockout mice could father a small number of offspring that develop to term, suggesting that oocyte activation can occur via an alternative route when PLCζ-triggered calcium oscillations fail. These findings will provoke further investigation into what this alternative mechanism might be, and the PLCζ knockout will be a useful tool to study infertility in mammals.

HIFs help make two halves

Just as in adulthood, an organism must respond to changes in its external environment during embryogenesis. Oxygen levels can fluctuate within a tissue, and animals have evolved a conserved signalling pathway to orchestrate a cell’s response to low oxygen levels (hypoxia). Critical to this pathway is the transcription factor hypoxia-inducible factor α (HIFα), which is degraded in conditions of normoxia. However, in low oxygen, it binds with its partner HIFβ to hypoxia-response elements and activates downstream genes that are important for a cell to cope with oxygen depletion. On p. 2940, Yi-Hsien Su and colleagues demonstrate that the hypoxia signalling pathway is active in the early sea urchin embryo, in a graded manner that mirrors the emerging dorsoventral axis. They report that while hifα mRNA is distributed uniformly throughout the embryo, the protein is stabilised at the dorsal side, and degraded more ventrally. They found that HIFα protein restricts nodaltranscripts to the ventral ectoderm only, and that the dorsoventral axis is affected by artificial perturbation of HIFα levels. Interestingly, they also found evidence for an intrinsic hypoxia gradient in embryos, which may be a forerunner to dorsoventral patterning. Together, these results provide a fascinating insight into the question of how environmental signals can impact early development.

A fishy response of transposons to demethylation

DNA methylation is an epigenetic mechanism that promotes heterochromatin formation, silences imprinted loci, the X chromosome, repeats and transposable elements. The idea that DNA methylation also represses differentially expressed genes has been challenged by experiments showing that loss of genomic methylation, either during early embryonic development or in mutants, does not result in a burst of gene activation. On p. 2925, work by Kirsten Sadler and co-workers confirms and extends the model that DNA methylation functions primarily as a gatekeeper for transposons. They report that mutants with a hypomethylated genome upregulate interferons, leading to the recruitment and expansion of immune cells in the developing larva. Rather than being directly due to derepression of interferon genes in the demethylated state, interferon production is stimulated by the detection of nucleic acids in the cytosol of a cell. This normally indicates the presence of a virus, but the mutants used in the study were not infected. So, what elicits this response? The authors reveal that the aberrant transcription of transposable elements caused by demethylation results in the production of cytosolic DNA that triggers the antiviral response. This mechanism could act as an early-warning system, allowing cells in the developing embryo with widespread epigenetic abnormalities to be put under immune surveillance so they can be rapidly eliminated if necessary.

PLUS…

An interview with Jenny Nichols

The 2017 BSDB Cheryll Tickle Award winner talks to us about her career in science, the importance of collaboration, and the similarities between playing musical instruments and manipulating embryos.

Wilms’ tumour 1 (WT1) in development, homeostasis and disease

This Primer summarises our current understanding of the diversity of WT1 functions in mammalian tissues and organs and how WT1 mutations can lead to disease.

Cellular and molecular mechanisms coordinating pancreas development

This Review describes the gene regulatory networks, signaling pathways, morphogenetic movements and cell dynamics underlying organogenesis of the mammalian pancreas.

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I joined Dr. Craig Albertson’s lab as a graduate student in 2009, where I quickly became fascinated by these cute cichlid fishes. They’re colorful, they breed their young in the mouth, and some of them have funny looking faces like this blue mbuna (Labeotropheus fuelleborni):

My research started on the genetic control of bone development in these cichlids, trying to understand the development and evolution of their facial shape. We soon discovered that the ptch1 gene, a member of the Hedgehog signaling pathway, plays a major role in shaping multiple bones in the cichlid head. In brief, some cichlid species, such as the blue mbuna above, have one version of the ptch1 gene that leads to a facial skeleton suitable for scraping algae off the surface of rocks; some other cichlid species have a different version of the ptch1 gene which leads to a slightly different skeletal shape that make them good at capturing evasive prey via suction feeding. These findings add up to a fast-growing body of knowledge about the genotype-phenotype map, which has been of great interest to biologists for decades.

While I’m proud of my contribution as a young scientist, one thought often haunts me: although I call it a “major role”, the genetic difference in ptch1 only accounts for 10-20% of the variation in bone shape. The vast majority of the variation remains to be explained, what’s going on there?

From a geneticist’s point of view, this is exactly what you would expect to find in a trait as complex as the shape of facial skeleton: there’re probably many genes working together to produce the final phenotype. Some genes, like ptch1, can have a relatively large effect and will pop out in our analysis. But most of genes will have such a small contribution that could be easily swamped by all kinds of background noise and therefore, would be very difficult to detect. Considering the size and life history of cichlids, it’s logistically impossible for me to trace down those genes.

How about environmental factors? Genetics is certainly not the only player, I should take a detour.

I was really intrigued by a plasticity experiment in the lab, where we fed the cichlids with different food and induced skeletal changes in the face. And I started wondering, is there any other behavior that can also change their bones?

Then, the gaping behavior of cichlid larvae quickly caught my eyes.

6 day old cichlid larvae gaping

The cichlids we study are mouth brooders. Mom usually keeps all her eggs in the mouth for several weeks. But in order to study their development, we will extract the embryos from the female’s mouth and raise them in a flask. This is one of the first skills I learned when I joined the lab, and I noticed this gaping behavior right away – well, dozens of cute little baby fish gaping their mouths is kind of hard to miss.  I didn’t pay much attention to this behavior at that time. And I think probably most people would have thought the same thing when they first see the video above – these larvae are just doing their normal fishy thing, they’re just breathing. It looks cute, but that’s pretty much it.

However, when I looked at this behavior with the new perspective, I soon realized its bizarreness: they were gaping really fast. Some of them would gape more than 250 times in 1 minute, so fast that I could barely count it in real time. And I had to count in Chinese, English numbers were just too long for me. That felt like a lot of gaping, were they really just breathing? A quick literature search said no: fish larvae at such a young age rely mostly on their skin for breathing. I was excited to find this out. Initially, I thought maybe this larval breathing behavior could influence bone development as a side effect. Now I got a new idea: perhaps it’s not about breathing at all. They might actually be working out their mouths to stimulate bone development.

As a graduate student, the first thing you need to do when you have an idea like this is to convince your adviser that you’re not crazy, that it’s a legit scientific hypothesis. So, I started to collect some preliminary data, which turned out to be encouraging: I found that the larvae of the blue mbuna, who has a longer jaw bone called the retroarticular process (RA), gaped at a higher frequency than another species with short RA. This is exactly what I expected to see, more gapes more bones.

At this point, both Craig and I became very interested in this hypothesis, and started to brainstorm ways to test it. We thought about all kinds of ways to manipulate the gaping behavior, from varying environmental factors like oxygen and temperature, to chemical treatment like caffeine and tricaine (commonly used for anesthesia in fish), to more goofy ideas like botox and gag. Most of them were rejected either because they would cause an overall impact on development, or simply not feasible in our fish. In the end, I decided to take a rather adventurous route, that was to surgically cut the ligament attached to the RA. We liked this approach because it’s a local and targeted experiment: the surgery would only attenuate the mechanical stimuli being applied directly to the RA without affecting the other bones and muscles that participates in the gaping behavior. The surgery worked beautifully: when I cut the ligament in the larvae of the fast gaping species, they developed a shorter RA.

The surgery was sort of a loss-of-function experiment, and we also did a gain-of-function experiment that originated from what I call a happy accident in the lab. One day I ran out of standard containers for the larvae when I was monitoring their gaping behavior, so I had to substitute with small beakers. Then I found that when restricted to a smaller environment, the larvae will gape at a higher frequency. Although it ruined my gape counts for that brood, I took advantage of this phenomenon and tried to induce gaping behavior in the larvae of slow-gaping species with smaller container. Just as I expected, they developed longer RAs.

These two experiments suggested that the seemingly trivial gaping behavior of cichlid larvae affected the development of their RA development, and the effect size was, interestingly, on par with what we found with genetic difference in the ptch1 gene. But what’s more interesting, is that we found larvae that gaped more is expressing more ptch1.

Taken together with our previous studies, we think the Hedgehog signaling pathway plays a dual role in shaping the facial skeleton. On one hand, genetic differences in the ptch1 gene leads to differential bone growth in the facial skeleton; on the other hand, mechanical stimuli also induce bone growth via Hedgehog. At this point, we’re curious about how these mechanisms evolved. It will be super cool if the epigenetic (or plastic, whichever way you prefer to call it) route came in place first, and genetic mutations came later to fix the difference – genes as followers.

And for me personally, I really want to learn the larval gaping behavior in nature. My observation of the gaping is done in beakers and Petri dishes after all. And I just can’t stop thinking what the larvae are doing in their mother’s mouth, which is a much smaller environment than my restriction experiment. Do larvae gape even faster? Does the mom modulate their gaping? Or perhaps they don’t gape at all? These questions won’t help us cure cancer. They’re not major challenges with great impact in academia or industry. I’m just curious, simple as that, which is exactly why I love doing science.

Baby fish working out: an epigenetic source of adaptive variation in the cichlid jaw. Yinan Hu, R. Craig Albertson. 2017. Proceedings of the Royal Society B: Biological Sciences. 

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Curator position at Xenbase, the Xenopus Model Organism database

Xenbase (www.xenbase.org) is the Xenopus bioinformatics and genomics resource. Xenopus is a major model for fundamental cell and developmental biology and a model for human disease. Xenbase is a totally free, and globally accessible database, used by Xenopus researchers worldwide, and is funded by the National Institute of Child Health and Human Development. Xenbase has two performance sites: the curation team is based in Cincinnati, OH (PI: Dr. Aaron Zorn) and the developer/database management team is based at the University of Calgary in Canada (PI Dr. Peter Vize).

Xenbase is seeking to fill 1 full time Curation position to join the curation team at the Division of Developmental Biology, Cincinnati Children’s Hospital, Cincinnati, OH, USA. Curation positions offer a challenging job away from the wet-lab and research bench, where interpreting, annotating and displaying complex data is our main task. Curators also develop strategies to improve data curation; work to improve data display/querying on the website; interact with our user community at research conferences; develop programming skills; and contribute to Xenbase publications.

Job Description:
• Curation and annotation of published Xenopus scientific literature, focusing on gene expression and the extraction of other research data: genes, transgenic constructs, antibodies, morpholinos, phenotypes, genetic interactions, gene product functions and models of human disease.
• Import and annotate data from large-scale screens (e.g., loss-of-function morpholino screens, gain-of-function mRNA screens).
• Help develop new features: curation and processing of public and directly submitted RNA-seq and ChIP-seq NGS data from Xenopus experiments, curation of mutant phenotypes and transgenics; implementing GO annotations.
• Co-author reports and publications, and give presentations at national and international meetings/workshops.

Qualifications:
• MSc or PhD degree in bioinformatics and/or developmental biology, genomics, genetics, molecular biology, zoology, anatomy or related field.
• Demonstrated ability to produce scientific papers, reports and presentations
• Demonstrated ability to work in a team as well as independently, efficiently (i.e., both quickly and accurately) and be self-motivated
• Strong interpersonal and communication skills, including excellent written and spoken English.

Preference will be given to an applicant with:
• Experience with a bioinformatics, genomics or model organism database
• Experience in data annotation/biocuration, knowledge of relational databases, and familiarity with ontologies.
• Experience in Xenopus or other vertebrate (mouse, zebrafish or chick) developmental biology.
• Experience in analyzing genomics data, using GRN software, genome browsers and common bioinformatics tools.

How to Apply: 
Please submit your application to: aaron.zorn@cchmc.org
with the following information:
• A cover letter, including a statement of interest/purpose
• CV/Resume.
• Copy of your degree(s).
• List 3 references/referees whom we may contact (please include their postal address, email and phone number).

Salary and Start Date:
Salary will be commensurate with qualifications and experience. Start date is negotiable, but expected to be in late 2017.

The successful applicants will be employees of Cincinnati Children’s Hospital and will undergo background checks, orientation and a 6-month probationary period. Employees are required to receive an annual flu vaccination.  
More information about working at Cincinnati Children’s Hospital, and living in Cincinnati, can be found here: http://www.cincinnatichildrens.org/careers/working/default/

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A position as postdoctoral research associate is available in the Ear Repair group within the NIHR Biomedical Research Centre at Guy’s and St. Thomas’ NHS Foundation Trust and King’s College London. The Ear Repair group is led by Professors Andrea Streit, Karen Steel and Abigail Tucker, and is part of the BRC Oral Health Theme based in the Centre for Craniofacial and Regenerative Biology at King’s College London. The Centre is one of the leading centres for Craniofacial Biology and offers a vibrant research environment in the heart of London.
We are looking for an enthusiastic and highly motivated candidate holding a PhD in biomedical science or a related field with a background in molecular biology, mouse genetics, neuroscience and/or developmental neurobiology. Understanding of and expertise in ear development and physiology will be an advantage, as will experience in drug delivery strategies. The project investigates the mechanisms underlying ear homeostasis and hearing loss, looking at new ways of restoring function, with the long term aim to design new strategies to treat hearing loss. The successful candidate will have training and expertise in one or more of the above areas, an excellent academic track record, be committed to a scientific career and will be keen to work in an interdisciplinary team. The project itself offers many training opportunities in cutting-edge biomedical science, as well as in different transferrable skills.

To apply for this post go to https://www.hirewire.co.uk/HE/1061247/MS_JobDetails.aspx?JobID=77419

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Looking back on the journey: Intracellular uptake of macromolecules by brain lymphatic endothelial cells during zebrafish embryonic development eLife van Lessen et al., 2017

Just over two years ago, while I was a Masters of Neuroscience student at University College London, I became interested in the emerging concepts of brain lymphatics and sleep dependent macromolecule clearance within the brain. Until quite recently, the brain was considered to be without lymphatic vasculature or dedicated mechanisms of clearance of its own. Then in 2013, Lulu Xie of the Nedergaard lab published a blockbuster paper in Science that suggested that sleep facilitates fluid and metabolite clearance from the brain (Xie et al., 2013). Following in 2015, a double header in Nature and The Journal of Experimental Medicine (Louveau et. al. 2015, Aspenlund et al. 2015) demonstrated that lymphatic vessels exist within the dura mater of mouse and human meninges. Together these papers opened an entirely new outlook for how our energy intensive brain might maintain homeostasis, with potential implications for neuropathology and degenerative disease. I was attracted to the interdisciplinary aspects of this budding field, and the topic became a fundamental obsession for me after several involved discussions with Dr. Jeff Illif who had written the original brain paravascular clearance paper (Illif et al., 2012) and moved from the Nedergaard lab to head his own at Oregon Health and Science University in 2013. I was deeply encouraged by the time he took to act as a sounding board and to offer mentorship and direction as I decided how to pursue this field for my PhD.

I had a potential idea for how to tackle this field from a new angle after Dr. Jason Rihel from UCL’s Department of Cell and Developmental Biology gave a lecture for the Master’s program, during which I discovered that his lab uses molecular and genetic techniques to study sleep using zebrafish. I found his approach and style as a creative, big-picture scientist appealing. Zebrafish also made sense to me as a model through which to investigate this complex clearance mechanism, as they are small and optically transparent throughout larval stages, allowing for accessible imaging and high throughput studies. However, when I approached Dr. Rihel (Rihel et al. 2010) with a PhD proposal to investigate these processes in zebrafish, he warned me that not only would this be a new area for his lab, requiring new methods, but we also might find that zebrafish simply lack brain lymphatics/clearance. To finally convince Dr. Rihel that it was worth investigating, I poured over these new papers along with zebrafish papers describing lymphatic development, many of which came from a well-known lymph and vascular lab in Muenster, Germany led by the seasoned scientist, Professor Stefan Schulte-Merker.

About the same time, in Professor Schulte-Merker’s lab, a postdoctoral researcher by the name of Max van Lessen had also caught the brain lymphatics ‘bug’. Digging through the same literature, Max had his own “eureka” moment when he realized that the transgenic vascular and lymphatic lines, including a few created by his colleague Andreas van Impel, might have labelled the brain. Such transgenic tools would allow for dynamic investigation of the origins and molecular properties of brain lymphatics, if they were present and conserved in bony fish.

Serendipitous timing brought Professor Schulte-Merker to our doorstep last March for an unrelated meeting with our colleague and neighbour, Professor Steve Wilson. When I heard from my colleague Dr. Eirinn Mackay that he was down the hall, I jumped up and caught him as he was heading out the door. Taking advantage of the brief moment, I thrust my hand at him for a hearty shake and I began excitedly outlining that I believed brain lymphatics found in mice and humans would be conserved in zebrafish. I told him that in some of the images from papers, I had seen evidence of lymphatic markers present in the head of the zebrafish and wanted to know his opinion. Professor Schulte-Merker smiled and nodded. “Indeed, we have noted this and are looking into it.“ I asked out of curiosity why had everyone in the zebrafish vascular community focused exclusively on the trunk of the fish? Most images of zebrafish lymphatics I found in the literature had frustratingly cropped the top of the head from full view. Professor Schulte-Merker smiled wryly and stated that “everyone was so engrossed with the peripheral system in the past that no one ever bothered to look up.” It struck me that so much of discovery is about simply “looking up”. Not being afraid to challenge long standing norms and established or popular avenues of thought to follow instincts that bring us to look with new eyes upon something we have seen before a billion times.

I also discovered that science is, more often than not, luck, happenstance, and the willingness to cooperate to get more done. We can work alone and hoard, or we can branch out and be, as my friend Seanne in California always likes to put it, “better together”. My PhD supervisor Dr. Rihel cultivates an atmosphere of collaborative research, and his fearlessness in sharing ideas and taking chances paved the way. The Schulte-Merker lab didn’t hesitate to reciprocate in the same spirit, and immediately a cooperation was forged. To begin parallel investigations into brain lymphatics, the Schulte-Merker lab sent us a combined transgenic line with kdr-l expression labelling all vasculature in mCherry (Hogan et al., 2009) and flt4 (Vegfr3), an established venous-lymphatic marker in mCitrine (van Impel et al., 2014).

We at the Rihel Lab have an interest in the conservation of these systems and mechanisms to ultimately elucidate the impact on potential clearance through sleep, and the Schulte-Merker lab thinks about this problem through the angle of vascular and lymphatic origin and development. We have complementary skill sets that allowed the work to begin at an incredible pace, building a whirlwind storyline. My colleagues often remark that this project made them feel like they had been strapped to a rocket ship.

The Mystery Cells

To help with the anatomical search for brain lymphatics, I recruited my remarkably talented second supervisor as a co-investigator (Dr. Tom Hawkins), as he is one of the most highly inquisitive people I have met, with a wide breadth and depth of technical expertise which would clearly be needed. We began by looking at adult transgenic brains, carefully dissecting and removing them using confocal microscopy to examine both the surface of the brain as well as the interior portions of the skull. What we saw initially was somewhat astonishing. We had imagined we might find lumenized vessels; however, along much of the surface of the brain were what looked to be flt4 positive cells of some sort, glowing brightly in mCitrine, clearly differentiated from the kdr-l blood vascular marker in brilliant mcherry. While kdr-l expression labels both arteries and veins, flt4 expression is an established marker of venous and and lympahtic endothelium. Importantly, our cells were entirely negative of kdr-l expression, which in the trunk can only be found in lymphatic vessels.

They possessed large inclusions and spindly processes which appeared to be connecting each to the other in close proximity to surface vasculature. A Sir Henry Welcome Fellow in our lab, Sabine Reichert, took one look at the images and without blinking stated, “they look like some sort of endothelial cell type.” It quickly became clear that she was right.

We dove in with immunohistochemistry using anti GFP and RFP antibodies followed by cryosections in adult brains to investigate carefully where these cells were positioned. Our cryosections revealed that these cells populated the meninges surrounding the entire brain exclusively and were associated with particular vessels diving into the parenchyma from the meningeal layer. This was an exciting moment for us as we recognized that the work done in mice and in humans had placed brain lymphatics in the meninges and our cells were positioned in a similar location. Although much work remained, we in London felt we were on track and may have identified a brain lymphatic system in the fish, suggesting evolutionary conservation within the vertebrate lineage.

Origins

Around the same time Professor Schulte-Merker and I had met, Max and the Schulte-Merker lab in Germany had found beautiful evidence of head lymphatics in the embryonic brain. Max had been capturing live images from the earliest stages of development onwards, patiently waiting to see when, how, and from where expression of these flt4 positive structures would emerge to get a hint of their developmental origin. They reasoned that if peripheral lymphatics sprout from venous structures early in development, perhaps the same would occur in the brain. While training the confocal microscope on the choroidal vascular plexus (CVP) and mesencephalic vein (MsV) which loops around the optic tectum of the zebrafish brain, they saw a clear sprout comprised of flt4 positive cells emerging around 56 hours post fertilization (hpf) from the CVP that then grew in an arc along the MsV at the edges of the tectum in parallel to the vasculature. Residual kdr-l expression in these flt4 positive cells suggested differentiation from venous endothelium.

Over the course of three days, this flt4 positive ring from what we began calling the stereotypical tectal “loop”, a cohort/string of loosely connected single cells which aligns closely to the MsV. In the peripheral nervous system of zebrafish, angiogenesis begins with the sprouting and development of arteries, followed by the concurrent development of veins and lymphatic vessels with both requiring three key genes: ccbe1, vegfc and flt4. Both lymphatic sprouts and venous sprouts initially originate from the posterior cardinal vein and ultimately form their own independent structures as they grow towards the myoseptum (van Impel et al., 2014). Since these novel brain cells sprouted from a vein and expressed flt4, Professor Schulte-Merker remarked that they were most likely lymphatic in origin.

Macromolecule Uptake

If these novel cells were in fact functionally similar to mammalian lymphatic structures, Max hypothesised that these cells may have function in the uptake and clearance of macromolecules, similar to what has been reported for murine brain lympahtic vessels. Incredibly, tracers injected either into the tectal neuropil or into the hindbrain ventricle accumulated at flt4 positive cells forming the tectal loop within minutes of injection. This uptake is so rapid and specific that when I first showed Dr. Rihel a similar dye injection experiments that my colleague Dr. Chintan Trivedi and I had performed on the two-photon microscope in a wild type fish, he thought I was showing him an image of the lymph transgenic line, as the dye took on the precise location and shape of the flt4 positive cells! With so much activity going on in both of our labs, we realized that it was time to have an in-person meeting to facilitate discussion and avoid excessive experimental overlap.

I made a pre-Christmas journey to Muenster to mutually share our findings along with new ideas and techniques. During my time there, I was also introduced to Andreas van Impel, another postdoctoral researcher in the Schulte-Merker lab, with a humble and steadfast demeanour who had created a number of the stunning complex lymphatic lines we were currently using. He brought a great deal of experience and insight to our discussions and grounded us with his thoughtful interjections. When Max presented his work, we both were so excited that we were wildly gesticulating and scribbling notes. At one point, one of us climbed atop of the table to point out a key piece of research from an old paper from the 1930’s, with Andreas calmly leaning in with an amused smile on his face. It was one of the most vibrant scientific visits I have had, and it solidified a strong sense of teamwork. Face to face contact I found is the cornerstone of collaboration. Trust must be earned and built, while looking one another in the eye, so colleagues can both sense and see our intentions, and know we are all labouring for the same goal.

Shortly after my visit, Max shared he had mused that the cells had macrophage or dendritic-like characteristics, even though their lymphatic origin suggested a novel cell type. This had Max hunting after shared receptors or other common characteristics, which may offer further clues about our cells. In this reading, Max discovered a universal pattern recognition receptor called the mannose receptor found in both macrophages and dendritic cells and was a major way in which cargo was endocytosed into these cell types. Thus, he decided to apply both Pyrimidyn-7, a quintessential blocker of dynamin-dependent endocytosis, and mannan, a bacterial polysaccharide that binds to and saturates the mannose receptor in independent experiments to see if he could stop tracer uptake. He shared on a call that he was left deeply puzzled as it looked like the tracers had still localized to the flt4 positive cells anyway, even in the presence of the endocytic blocker.

Fortunately, Max had also injected the pH sensitive dye (pHrodo-advin) which would only fluoresce when it encountered an acidic environment inside of a cell. In previous experiments, he had seen both the tracers and the pHrodo fluoresce at the flt4 positive cells together, demonstrating for the first time lymphatic uptake of macromolecules at the single cell level. Being in the habit of injecting pHrodo along with his usual tracer, he also used this dye cocktail in his search for an effective concentration of mannan or Pyrimidyn-7, which revealed that the usual fluorescent tracer still accumulated at the cells. However the injection of either blocker effectively stopped the internalization of pHrodo. It didn’t make sense! How could the fluorescent tracer be taken up, but the pHrodo not? Suspicious, he took a closer look, and realized that the usual fluorescent tracer had merely localized to the membrane of the cell and was not actually being internalized. 3D reconstructions revealed that the fluorescent tracers were lodged into the membrane. If pHrodo was injected before the blocker it still happily fluoresced in the acidic lysosomal compartments within the cell but never co-localized with the fluorescent tracers lodged in the membrane after endocytic blockage.

Thus, it seemed that one process “docks” the tracer to the cell membrane while subsequent cargo internalization into acidic compartment is likely mannose receptor dependent. If it hadn’t been for his habit of using the pHrodo he may just have given up thinking the blockers hadn’t worked. I learned a few valuable lessons from Max’s results. First, I learned that experiments don’t have to be complicated and showy. They can often be incredibly straightforward. The other lesson was to always look closer and deeper before walking away from an experiment in frustration. So much of science emerges from happy accidents. We hear it again and again, and that is because it’s just so true.

Max and his colleagues expanded the analysis to additional lymph markers as well as markers for macrophages, etc. For example, they examined transgenic lines labelling classic lymphatic markers such as Lyve1 (Okuda et al., 2012) and Prox1 (van Impel et al., 2014) to further confirm these cell’s lymphatic identity. Much to our combined excitement, each lymphatic line labelled the quintessential tectal loop. Finally, to rule out that these flt4, prox1, lyve1 positive cells were macrophages, Professor Schulte-Merker suggested breeding the lyve1 line in DsRed (Okuda et al., 2012) with the mpeg1 macrophage line in GFP (Ellett et al., 2011). This showed clear differences between the cell types based on location, morphology, and expression. To completely eliminate the possibility, they also examined whether the morpholino knockdown of the macrophage transcription factor pu.1 had any effect on the lymphatic tectal loop. Andreas injected morpholinos, and after a tense few days of waiting for the results, the experiment revealed that our flt4 positive cells remained undisturbed, even as all macrophages were eliminated. We were elated, and we all finally allowed ourselves to believe that we had a novel cell-type on our hands.

Back in London, Tom and I began our hunt for the identity of these cells from a very different angle, using electron microscopy to obtain a definitive view of the location and ultrastructural features of these cells in relation to blood vasculature. Tom has had decades of experience with transmission EM, so he warned me not to get too excited, as disappointment was likely. Using the enormous TEM microscope with the assistance of Mark Turmaine: the quiet EM master, we happily navigated our way over the ultrathin slices in what, to me, was reminiscent of the broadcasts from the first moon landing. To our delight, and despite the forewarning, our first EM run was crystal clear with excellent structural resolution. The cellular landscape was wild and extraordinary, full of intricate interdigitations of membranes and strangeness all around. Guided by venerable EM studies on goldfish meninges, we could clearly identify a distinct meningeal tri-layer, inside which the cells that contained these large inclusions were exclusively found. We were able to immediately identify structures with multiple large inclusions in close proximity to surface brain vasculature with the size and distribution shown in our confocal images. They were clearly separable from perycites, vascular endothelial cells, and their basement membranes.

We combed over mammalian and teleost EM images of the meninges to try to identify these cells. One candidate was the Fluorescent Granular Perithelial Cells (FGP), which were discovered and characterized in rodents by Maso Mato in the 1980’s. Mato cells were known to reside in the meninges and contained numerous, heterogeneous inclusions (Mato et al., 1986, 2002), and we had several hours of heated debates in Dr. Rihel’s office as we stared at side-by-side comparisons of classic EM images and our new data. Ultimately, we decided that our cells were most probably distinct from Mato cells, or at least they represented a specific subtype, as our new cells’ inclusions were less heterogeneous than expected for Mato cells, and they had morphologically distinct shapes.

The ”Competition”

As the first part of our story of these cells was beginning to wrap up, Max and I were made aware that two other labs had come to similar conclusions to us. Suddenly, we were plunged into a race in which we weren’t precisely clear who had what, and when, in entirety.

Moments like these, when you find out other labs are working on similar avenues, can be stressful at best, and to be frank, Max and I had moments of panic together. The prospect of potentially being “scooped” looms heavy above all of our heads at any given moment. Each one of us is deeply connected to our research and have spent countless hours sweating and toiling over our projects. They become a part of us. And hearing that others have found something similar can be particularly disorienting as discovery, though made for the common good, can feel so deeply personal. Professor Schulte-Merker and Dr. Rihel kept insisting that each lab would be contributing to the scientific enterprise and bring novel insights into the nature of these cells.

This also meant that the community would be excited about this discovery. In retrospect, with the three respective papers now published, (Galanternik et al., 2017, Bower et al., 2017, van Lessen et al., 2017), it is clear that each lab approached these cells from unique perspectives. Galanternik et al. discovered these cells using a transgenic mannose receptor line and indicated that they believed these to be fluorescent granular peritheliaI cells (FGP or Mato Cells) (Galanternik et al., 2017). Bower et al. utilized the transgenic lyve1 lymphatic line to reach their discovery, and amongst other interesting findings they ablated these cells to find that the ablations impacted the integrity and development/regeneration of meningeal vasculature (Bower et al., 2017). Both those papers also performed valuable RNAseq analysis using differential gene expression analysis to rule out the cell’s having blood endothelial, smooth muscle, macrophage, or pericyte identity. This exciting combined evidence opens up an entirely new avenue of research into brain lymphatics in zebrafish.

Arachnocyte, Kumocyte, BLEC

Finally, as we debated what to name these cells, the other labs disclosed the names with which they were identifying the cells in their papers, with Galanternik et. al. calling them FGPs and Bower et al. settling on perivascular Lymphatic Endothelial Cells (pLECs). We ultimately decided on Brain Lymphatic Endothelial Cells (BLECs) to be precisely descriptive of their properties; however, internally we all enjoyed the name we had additionally selected which was Kumocyte. Kumo means spider in Japanese, describing their spidery appearance, they had previously been colloquialised as arachnocytes for the same reason. I think they will always remain Kumocytes to us somehow, as it speaks to their unique morphology and “personality” and we have taken to fondly calling them that within the Rihel lab.

We are grateful for this opportunity to share our experience as we are so often confined by formality, word limits, and jargon that we miss the opportunity to tell “the story”. The story which discusses the more personal aspects of the craft of discovery, especially the journey behind the scenes that keeps all of us all coming back again and again despite the tough days, which are many. After all, in biology and science, we are all just witnesses to nature, merely reporting what we see with awe and wonder. It’s always been there, but there’s that kid in all of us that gets our mind-blown every time we get to be the ones to see something with human eyes for the “first time.“ I also never want to forget on this journey, not just in science, but in life, that we truly are only as good as those we surround ourselves with. Each and every colleague on my floor has contributed, and given their time and expertise without hesitation. They say it takes a village to raise a child…..and so it also goes with raising a PhD student.

We can’t wait to see what this field of brain lymphatics and clearance holds. Certainly, dogma that the brain is without dedicated lymph structures of its own is being rewritten, even in fishes. As we start at the drawing board and re-examine all of our old assumptions, what else will we discover around the corner?

Shannon Shibata-Germanos and Max van Lessen

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Cluster Hire in Quantitative and Computational Developmental Biology, North Carolina State University

As part of the Chancellor’s Faculty Excellence Program, NC State University seeks three outstanding faculty at any rank to expand the new interdisciplinary faculty cluster on Quantitative and Computational Developmental Biology. Together with NC State’s existing strength in quantitative/computational sciences and engineering, this cluster will propel NC State to the forefront of efforts to define new principles in developing biological systems.

Successful candidates will engage in transformative research which integrates expertise in biological, biophysical, computational, and/or mathematical methods to investigate dynamic events in human, animal or plant development on any level, from the nanoscale to the whole organism. The cluster seeks individuals with expertise in using state-of-the-art quantitative methodologies and/or computational modeling to address fundamental aspects of Developmental Biology such as:

• the mechanisms that regulate individual cell behavior, proliferation, differentiation, potency, and reprogramming,
• how material properties and dynamic physical / mechanical forces influence collective embryonic cell behavior and tissue shape,
• the formation, maintenance and appropriate scaling of the geometric configurations of cells and tissues that comprise organs and organisms, and
• the mechanisms that underlie the evolution of new biological forms and facilitate morphological plasticity in response to stress or environmental change.

Although we are interested in individuals who can bridge experiment and theory and have the ability to translate research outcomes to address biomedical or agricultural challenges, applicants whose expertise is mainly in the computational realm are also strongly encouraged to apply. Demonstrated collaboration as a member of a multi-disciplinary team is essential.

Hiring may occur at the level of Assistant, Associate, or Full Professor. The home department is anticipated to be in one of the four participating colleges (Engineering, Sciences, Veterinary Medicine and Agriculture and Life Sciences) and will be determined based on credentials and research fit. Hires will be expected to provide key leadership in quantitative and/or computational biology across the university, teach in existing courses and develop specialized coursework in their area of expertise.

Minimum requirements include a PhD in a relevant field from an accredited institution. Interested candidates should submit: a CV, a 2-3 page research plan, a cover letter describing prior multi-disciplinary research efforts and how their research prospectus addresses the goals of the cluster (see https://facultyclusters.ncsu.edu/clusters/modeling-the-living-embryo/), and
contact information for 3 references. Materials for consideration will be accepted electronically via http://jobs.ncsu.edu/postings/88538. Review of applications will begin immediately and continue until the position is filled. Questions about the position may be directed to Dr. Nanette Nascone-Yoder (nmnascon@ncsu.edu).

NC State University provides a vibrant environment for research, teaching and mentoring across disciplines; ample opportunities will be available for collaborations with existing faculty and other newly hired colleagues in the cluster. Our location in the Research Triangle also facilitates interaction with faculty at Duke University and the University of North Carolina at Chapel Hill, as well as with industry and government agencies.

The Chancellor’s Faculty Excellence Program is bringing some of the best and brightest minds to join NC State University’s interdisciplinary efforts to solve some of the globe’s most significant problems. Guided by a strong strategic plan and an aggressive vision, the cluster hiring program is adding new faculty members in select fields to add more breadth and depth to NC State’s already strong efforts. The Chancellor’s Faculty Excellence Program marks a major initiative of the university’s strategic plan, “The Pathway to the Future”   We invite you to explore more information about the Chancellor’s Faculty Excellence Program and this cluster at http://workthatmatters.ncsu.edu/.

NC State University is an equal opportunity and affirmative action employer.All qualified applicants will receive consideration for employment without regard to race, color, national origin, religion, sex, gender identity, age, sexual orientation, genetic information, status as an individual with a disability, or status as a protected veteran. Persons with disabilities requiring accommodations in the application process please call (919) 515-3148.

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The Mokalled laboratory is seeking applicants for a Research Technician II position in the Department of Developmental Biology at Washington University School of Medicine.  Our lab uses the zebrafish model system to study neural regeneration after spinal cord injury or disease.  We welcome ambitious applicants with enthusiasm for neuroscience, regenerative biology, and zebrafish research. Training will be provided. This position offers opportunities for co-authorships on published manuscripts.

Duties:

• Assists with research studies, experiments and assays including collection of data, preparation of solutions and set-up and maintenance of equipment. Main experimental duties include molecular biology, zebrafish husbandry and microinjection.
• Performs data entry and maintains data files on research.
• Complies with established safety procedures and maintains required documentation on laboratory and specimen conditions.
• Ensures lab conditions and equipment are properly cleaned and maintained in accordance with established procedures.
• Assists with general lab maintenance and cleaning.

Required qualifications:

Bachelor’s degree and up to 1 year of experience in lab setting or equivalent combination of education and experience equaling 4 years required.  Please send a cover letter, CV, and list of 3 or more references to mmokalled@wustl.edu.

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