The School of Natural Sciences invites applications for three full-time, permanent Lectureships in the broad area of Biology encompassing the full spectrum of genes, organisms and ecosystems. As part of the School of Natural Sciences, the biology and zoology subject areas are dynamic and growing elements with strong research records and successful recruitment onto our undergraduate and postgraduate degrees. Currently we are looking to expand our research base and our teaching and outreach capabilities. Bangor University is committed to excellence in research, teaching & scholarship and offers attractive career prospects for staff with such expertise.
Details of current staff research interests are available online. The School benefits from modern research laboratories and microscopy suites, a Natural History Museum, freshwater and marine aquaria, fly room, reptile and rodent facilities, plus a number of temperature controlled rooms, a botanical garden located on the fringes of the Menai Strait, a herbarium, and avian housing facility. Further research opportunities are facilitated by close links to the Centre for Ecology and Hydrology (CEH), the Centre for Environmental Biotechnology (CEB), the Schools of Ocean Sciences, Computer Sciences, and Psychology and access to the Supercomputing Wales High Performance Computing cluster.
Candidates should have a PhD in biological science, be able to develop research programmes of their own, contribute to the School’s Research Excellence Framework (REF) submission, show an enthusiasm for teaching, be willing to engage with outreach activities and also have excellent interpersonal skills.
Interviews will be held in the week commencing 3rd September. The successful candidates will be expected to start on 1st October 2018 or as soon as possible thereafter.
Informal enquiries may be made to Professor Chris Freeman, email c.freeman@bangor.ac.uk; and copied to Dr Nia Whiteley, email n.m.whiteley@bangor.ac.uk.
The transience of flowers is proverbial. Degeneration of flowers is elicited after successful pollination by the onset of seed and fruit development. However, also unpollinated flowers do not last forever – on the contrary, the life span of unpollinated flowers is a tightly regulated trait that differs greatly among plant species. Some plant species like orchids of the genus Phalenopsis have very long-lived flowers that can stay receptive for weeks, even months. Most plant species, however, sustain unpollinated flowers for much shorter time spans. In the most extreme cases mere hours pass between flower opening and withering.
In the Programmed Cell Death lab at the VIB Center for Plant Systems Biologywe are using the model plant Arabidopsis thaliana to study the longevity and senescence of unpollinated flowers. Although Arabidopsis is the most advanced model species for plant developmental biology, it is actually not particularly convenient to investigate the biology of flower senescence. Arabidopsis flowers are tiny, and moreover they are self-pollinated, so that the male floral parts (anthers) have to be either removed manually by emasculation, or male-sterile mutants have to be used.
A typical Arabidopsis flower aligned next to a pin. The floral stigma is at the level of the pointed end of the needle.
Every Arabidopsis researcher knows how to perform emasculation and pollination in order to cross different mutants, marker lines, or accessions. And many of us know that a flower emasculated on Friday might already have started to senesce by Monday, hence making successful pollination and seed set inefficient or impossible.
In order to observe and stage flower withering in Arabidopsis, we set up a macroscopic imaging system using a single lens reflex camera equipped with a macro objective. Taking images every 10 minutes under constant light enabled us to monitor flower senescence in detail. We could observe that concomitant with the withering of petals and sepals, also the floral stigma was degenerating.
The stigma of flowers is the primary receptive surface for pollen grains. In Arabidopsis the stigma consists of over 200 elongated fingerlike cells, the so-called stigmatic papillae, specialized epidermal cells that serve to intercept the pollen. In Arabidopsis, hundreds of pollen grains compete to fertilize about 50 ovules located inside the pistil of each flower. Once in contact with the stigma cells, pollen grains will hydrate, and germinate to form a single tip growing cell, the pollen tube. The pollen tube penetrates the papilla cell wall, growing in between the cell wall and the plasma membrane to the base of the papilla cell. From there on, guided by chemical cues, the pollen tube grows through the style and the transmitting tract to the ovules. There, it will release its two sperm cells to fertilize the egg cell and the central cell of the female gametophyte, thus initiating embryo and seed development.
The journey of the pollen tube during plant reproduction in Arabidopsis. Pollen grains (orange) are shed from the anthers and adhere on the stigmatic papilla cells. During the preovular guidance phase, pollen grains hydrate and germinate, and pollen tubes (red) penetrate in the papilla cells and grow through the style and transmitting tract. After that, pollen tubes are attracted by the ovules, enter the ovules with a near one-to-one relationship during the ovular guidance phase. Finally, pollen tube growth stops, and the sperm cells are released into the female gametophyte to perform double fertilization. Picture is a courtesy of Zhen Gao.
The structure of the intact Arabidopsis stigma appears a bit like a little hedgehog due to the hundreds of erect elongated papilla cells. Once flower senescence sets in, individual papilla cells start to break down, leading first to a ragged, and finally to a completely collapsed appearance of the stigma.
Pollination assays at different time points during this process showed that stigma degeneration coincided with a sharp decline in seed set, suggesting that viable papilla cells are necessary for successful reproduction. To test this hypothesis, we specifically ablated papilla cells by expressing diphtheria toxin chain A (DT-A) under a stigma-specific promoter. As we had assumed, DT-A induced stigma degeneration likewise caused a strong reduction of seed set.
In order to quantify stigma longevity, we faced a considerable problem – the macro setup was only able to follow a single flower at a time, and a price tag of about 1400 Euro per unit made the parallel acquisition of time courses impracticable. Coincidence came to our aid: Setting up a skype conference call we noticed that a cheap webcam without autofocus can be brought very close to an object (in that particular case a certain PhD student’s uvula) and amazing magnification can be achieved. After some experimentation with different webcams, we rigged up a phenotyping system with 20 webcams. We used a custom-made script to operate the open source camera surveillance software so that each camera acquires one picture frame every 10 minutes. Although the image quality was of course not comparable to the conventional macro lens setup, in the end of the day we had established a phenotyping platform with 20 imaging units for mere 250 Euros.
Webcam imaging platform to follow flower senescence in Arabidopsis
Analysis of the time lapse images revealed a remarkable reproducibility of stigma collapse on average around 56 hours after emasculation of a flower at the developmental stage 12c1.
The apparently precisely timed collapse of papilla cells suggested that an active cell death process might be occurring in Arabidopsis stigmata. Based on a large-scale meta-analysis of mRNA transcriptome profiles, our lab has established promoter-reporter lines of genes transcriptionally upregulated prior to a number of developmentally regulated programmed cell death (PCD) events2. Microscopic analyses revealed that most promoter-reporters come up during stigma senescence, thus linking stigma degeneration with established forms of developmentally controlled PCD processes3. Microscopic imaging of papilla cells is not trivial; although the individual cells are thankful objects for cell biological analyses, the stigma as a whole is a rather large and very sensitive three-dimensional structure. In order to investigate cell biological hallmarks of PCD in living stigmata, we could not use conventional slide-and-coverslip setups lest we create injuries and mounting artefacts in papilla cells. After testing diverse setups, we found that mounting the entire flower in an agar block and viewing the stigma from top with a long working-distance water dipping lens on an upright Zeiss 710 confocal microscope allowed a minimally invasive way to perform live-cell imaging of papilla cell death. In order to visualize cell death, we used a live/death stain in which fluorescein-diacetate (FDA) fluorescence indicates living cells, while nuclear staining of the membrane impermeable stain propidium iodide (PI) reveals plasma-membrane permeation as a committing step of cell death.
Cell death/ viability staining of stigmatic papilla cells. Fluorescein-diacetate (FDA) fluorescence indicates living cells, while nuclear staining of the membrane impermeable stain propidium iodide (PI) reveals plasma-membrane permeation as a committing step of cell death. Picture is a courtesy of Yulia Salanenka.
Alternatively, we used a tool developed in our lab, a tonoplast integrity marker (ToIM) line4. This plant line expresses a cytoplasmic green fluorescent protein (cGFP) and a polycistronically produced vacuolar red fluorescent protein (vRFP). Living cells display clearly separated GFP and RFP domains indicating an intact vacuolar membrane (tonoplast). Collapse of the large central vacuole in mature plant cells, another hallmark of plant programmed cell death5, is visible as merging of red and green fluorescence signals.
A confocal maximum intensity projection of a representative stigma from a Tonoplast Integrity Marker line (ToIM). Green labels cytosolic GFP, red shows RFP localized to the vacuole. The fusion of both signals appears yellow and indicates a loss of cellular compartmentalization symptomatic of cell death.
Our analyses revealed that as in the macroscopic setup, stigma degeneration occurred over a time span of 12 hours. Fascinatingly, the death of individual papilla cells followed a pattern from the periphery towards the center of the stigma, and often small clusters of individual cells underwent vacuolar collapse almost synchronously. On the level of individual cells, the PCD program occurred surprisingly fast; vacuolar collapse, plasma membrane permeation, nuclear fragmentation, and finally cellular collapse occurred within about one hour. The reproducibility of these successive events confirmed our hypothesis of a tightly regulated cell death program.
In order to discover regulators of this cell death process, we set out to perform an RNA-seq analysis monitoring the transcriptional changes occurring in the stigma over time. We manually dissected the stigmata from flowers at 1, 2, and 3 days after emasculation, corresponding to young, mature, and senescent flower stages. In total we had to stage, emasculate, and dissect way over individual 2000 flowers, a feat that we only managed with lots of training and a great team effort.
Emasculation session in the PCD lab. The combined efforts of at least three people were required during the laborious process of Arabidopsis flower emasculation. The stigmata of about 2000 emasculated flowers was collected and subjected to RNA-Seq analysis.
Illumina RNA-seq revealed a large number of differentiallty regulated genes (1180 genes out of more than 25 000 predicted genes in Arabidopsis) over the course of stigma senescence. Interestingly far more genes were upregulated (897) than downregulated (283), again indicating an actively controlled program controlling stigma senescence and papilla cell death.
RNA sequencing also confirmed the strong upregulation of developmental PCD-associated genes. In order to identify key regulators of this process, we investigated the expression profiles of transcription factors (TFs). Interestingly, the plant specific NAC TF family was strongly overrepresented in senescence-associated TFs. Among the most strongly upregulated NAC TFs was the well-established leaf senescence regulator ORESARA1/ANAC092 (ORE1, Korean for “long-living”), and the flower-specific ANAC074, a previously uncharacterized NAC TF that we dubbed KIRA1 (KIR1) after the killer “Kira” in the Japanese manga “Death Note”.
Using our webcam-phenotyping platform, we investigated an established knock-out mutant of ORE1 and found a slight, but not significant delay of stigma collapse. Interestingly, a newly established KIR1 knock-out mutant showed modest, but significant extension of stigma life span. We crossed the mutants and the resulting kir1 ore1 double mutant showed a clear synergistic effect which led to a doubled life span in comparison to the wild type. We also generated lines overexpressing dominant repressive mutant versions of KIR1 and ORE1, and some of these lines had an even stronger effect with some stigmata only collapsing at 11 days after emasculation. Intriguingly, both recessive kir1 ore1 loss-of-function mutations, as well as expression of dominant repressive alleles showed an uncoupling of stigma life span from floral organ longevity. Although the stigma life span was considerably increased, sepals and petals senesced similar to the wild type.
In a complementary approach, we investigated KIR1 gain-of-function mutants. Estradiol-inducible systemic misexpression of KIR1 in seedlings caused a rapid growth arrest caused by a widespread ectopic cell death. This demonstrates that the transcriptional program controlled by KIR1 is sufficient to elicit cell death outside of the stigma context. When inducing precipitate KIR1 expression specifically in the stigma, the papilla cell death and loss of receptivity occurred significantly earlier than in estradiol treated wild types or mock treated mutant lines. These results demonstrated that KIR1 functions to actively terminate the receptive life span of the flower by promoting a cell death program in the stigma.
This picture shows an emasculated Arabidopsis flower undergoing localized estradiol treatment in order to induce KIR1-GFP overexpression in the stigma. The floral stigma is imbibed into an estradiol-containing droplet for 6 hours and pollinated after that.
A central question arising from this investigation was of course whether aged but viable kir1 ore1 loss-of-function mutants could be successfully pollinated. When performing a pollination time series, we discovered that in dominant repressive KIR1 and ORE1 mutants, there was as significant, but rather modest extension of flower receptivity. In the recessive kir1 ore1 double mutant, there was no extension of floral receptivity at all. We used pollen from a transgenic Arabidopsis line that expresses β-glucuronidase (GUS) under a pollen-tube specific promoter to visualize pollen tube growth on pollinated stigmata after addition of the GUS substrate X-Gluc6. Microscopic analyses showed that pollen hydrated and germinated, but that pollen tube growth on aged mutant stigmata was strongly reduced in comparison to younger wild type or mutant stigmata. No pollen tubes could be detected to enter the style or the transmitting tract of the aged mutant flowers, suggesting that KIR1 / ORE1 loss of function is sufficient to suppress age-induced stigma PCD, but not sufficient to maintain stigma function in a corresponding fashion.
In summary, our research on stigma senescence suggests that a KIR1-ORE1 dependent cell death program actively terminates stigma and flower receptivity. However, as suppression of this cell death program is not sufficient to extend stigma function, we assume there must be additional, KIR1 – ORE1 independent pathways that either passively or actively terminate stigma function in the absence of cell death. With research going on in our lab we attempt to research these pathways in order to effectively modulate flower receptivity.
While Arabidopsis serves as a model system to discover these pathways, our research might also be applicable to outcrossing crop species. An extension of floral receptivity, especially under spells of environmental stresses, which are deleterious to plant reproduction, might be a key strategy to stabilize the yield of seed and fruit bearing crops7.
I recently attended the biennial Development, Cell Biology and Gene Expression C. elegans Meeting, this time in combination with the 2018 European Worm Meeting, in Barcelona. C. elegans meetings are always pretty special, defined more than anything else by the strong sense of community between researchers, or, as well like to call ourselves, ‘Worm People’. Worm culture has extended from a magazine (The Worm Breeder’s Gazette) to comedy shows (The Worm Show), and worm-inspired art collections (The Worm Art Show) curated by Ahna Skop, designer of this year’s meeting logo. This shared love of the worm translates into an atmosphere of supportiveness and openness within the community, making worm meetings ideal places to share new data and ideas – and at the same time being a huge amount of fun.
The meeting logo was designed by Ahna Skop to represent the extensive work that has been carried out investigating the neurobiology of the worm. C. elegans remains the only animal to have its entire connectome mapped.
A person who exemplified the ethos of the worm research community, and the memory of whom permeated the meeting, was John Sulston, who sadly passed away earlier this year. John Sulston was one of the first scientists working on C. elegans with Sydney Brenner at the LMB in Cambridge during the ’70s. He is known most famously within the worm field as the person who gave us the complete C. elegans cell lineage, a fundamental cornerstone of C. elegans research. This lineage represents years of work, as he monitored in real time every cell division of the larval – and following this, embryonic – worm, in order to build a remarkably accurate map of wild-type development from zygote to the adult. It is only very recently (40 years later) that any additional cell divisions have been identified. The concept of lineage is so fundamental to the ‘worm world’ that lab members also identify themselves according to their ‘lineage’ – tracing themselves back to that first ‘zygotic’ lab in Cambridge through a network of supervisors – in fact, many of the conference speakers introduced themselves in this way at the meeting.
However, John Sulston’s scientific legacy extends beyond the relatively small sphere of C. elegans biologists, as he was also the scientist who lead the British side of the Human Genome Sequencing Project. A first consequence of this was that the C. elegans genome was actually completed first, cementing it’s place in history as the first sequenced genome of a multicellular organism (John was apparently just as excited – if not more so – to resolve the worm genome as to complete the human genome). But more importantly, John Sulston was also responsible for fighting to make the human genome sequence freely accessible to all, not patented and the property of a company. The contributions that John Sulston made to the scientific community during his time with the Human Genome Sequencing project, and how his work with C. elegans shaped his scientific ideology, can be read in a book called The Common Thread, which he wrote with the science writer Georgina Ferry. His attitude towards open-access science and the free sharing of data and resources remains a defining property of the C. elegans field.
The meeting: attendees and talks
The meeting, held at the World Trade Centre on the waterfront in Barcelona and organised by Sander van den Heuval, Sophie Jarriault and Alex Hajnal, opened to an audience of 465 people, representing research from 33 different countries. As well as the talks, the meeting also involved two lively poster sessions, and a fabulous evening of food, wine and dancing by the sea – and where better to party than in Spain? I certainly came away from the meeting with more worm-sympathetic friends than I’d started with.
As there were unfortunately too many presentations to talk about them all here, I have summarised a selection of my favourites. However, a complete list of the abstracts can be found here.
Meeting attendees, representing 33 countries.
The World Trade Centre (arrow) in Barcelona, where the meeting was held. Photo by the author.
Worm behaviour and neurogenesis
When Sydney Brenner first selected Caenorhabditis for research, his ultimate goal was to solve the genetics and cell biology of behaviour. To this end, the electron microscopist Nichol Thompson created serial sections (up to 5000, uninterrupted) of the worm in order to map each synapse, and subsequently a draft of ‘the Mind of a Worm’ was published in 1986. Given that the worm is the only organism to date to have it’s entire connectome mapped, the field of neurobiology – and more recently behaviour – is an intensely studied area of C. elegans research.
Upon completion of the C. elegans post-embryonic lineage, it was established that the adult hermaphrodite worm had 302 neurones, and the male 385. However, as I have already mentioned, the first post-Sulston cells to be identified added two neurones to the male count, discovered by Arantza Barrios of UCL, London, and published in 2015. At this meeting, Arantza Barrios revealed how her lab, together with the lab of Richard Poole, also at UCL, have since gone on to update the number of male neurones yet again (to 389) although this time the cells in question were always known to be there. However, they have now been identified as undergoing a fate and morphology change during larval development, producing a pair of putative proprioceptive neurones, the PHDs, from a pair of glial cells. The Barrios and Poole labs, with the help of Scott Emmons and Steven Cook at the Albert Einstein College of Medicine in New York, who through electron microscopy established the connectivity of the newly discovered neurones, have identified that the PHDs are essential for efficient male mating behaviour. As Arantza described, males are usually lead by the tail (in which their hermphrodite-sensing organ and spicule for sperm-delivery are located) during mating. In contrast to this known mating behaviour, a newly-described behaviour (termed the Molina Manoeuvre after the post-doc who discovered it) involves an unusual forward sweep along the hermaphrodite followed by a reversal in direction in order to relocate the vulva. This manoeuvre is only performed in the case of the male being unsuccessful in its first attempt to mate. Without the PHDs, the Molina Manoeuvre cannot take place successfully and the male is likely to lose its potential mate.
Wiring diagram of the newly-discovered PHD neurones, shown to be involved in male mating behaviour. L. Molina-Garcia et. al. “A direct glia-to-neuron natural transdifferentiation ensures nimble sensory-motor coordination of male mating behaviour” BioRxiv https://doi.org/10.1101/285320
Many mutant animals, or wild-type animals subjected to different environments, exhibit changed behavioural patterns. Systematic quantification of this behaviour can allow both detection of the behavioural differences and analysis of the specific change. Andre Brown of Imperial College London spoke about how his lab have broken down the 2-dimensional behaviour of C. elegans in a petri dish into a series of distinct positions, and uses the analogy of language to compare these positions to letters in an alphabet. He showed, using automated imaging of free-moving worm populations followed by extraction of relevant features, how wild-type worms are more likely to use a certain subset of ‘letters’ compared to mutant animals. His newly developed algorithms and imaging systems are likely to be useful for a wide range of applications, from behavioural genetics to agrochemical analysis.
Oliver Hobert from Columbia University in New York spoke about his lab’s recent feat of creating a comprehensive neurotransmitter map which overlays the anatomical map of the worm connectome, completed for both hermaphrodites and males. Most neurones in the worm fall into one of the main neurotransmitter classes: glutamatergic, GABAergic, or cholinergic. Upon completion of the neurotransmitter map, the Hobert lab has been able to identify 86 different classes of neurone within these classes, and through mining of the Wormbase database of gene expression, further show how a group of only 33 different transcription factors are able control these identities, acting as ‘terminal selectors’. (Remarkably, Oliver points out, the Wormbase collection of C. elegans gene expression patterns allows between 5 and 141 cell markers to be identified for each neurone – that’s an average of 32 markers per cell.) The Hobert lab deduced that there must, therefore, be a combinational code for neurone identity, and Oliver described experiments where the loss of one of a pair of transcription factors can have varying consequences on the identity of differing neurones. A second observation was that these terminal selector transcription factors are predominantly homeobox genes (c.f. only 10% of all transcription factors in the worm are homeobox genes). The lab went on to develop a worm strain that could be used to identify gene expression in any neurone based on co-expression (NeuroPal – Neuronal Polychromatic Atlas of Landmarks) and characterised gene expression of all homeobox genes in all neurones. They found that every neurone expresses at least one homeobox gene, with 113 out of 118 neurone classes expressing a unique homeobox gene code. This means the same homeobox gene gets used again and again to specify different neurones. A striking observation was made when looking for commonalities between the neurones that express the same transcription factor: they are all synaptically connected. This has lead to speculation in the lab of how evolution may first have resulted in individual networks of neurones, each based on the presence of a founding homeobox gene specifying a single neurone that subsequently duplicated, before acquiring additional specialisation across the network to encode multiple neuronal types. These networks, Oliver proposes, may have later linked up to form the connectome as we know it.
Tissue-specific expression analyses
Tissue-specific RNA-seq in the worm has provided a challenge for the field, especially for those interested in understanding the factors that drive differentiation. Current analyses include using known gene expression profiles coupled with machine learning algorithms to sort sequencing profiles obtained from FACS-sorted single cells back to their tissue of origin, such as the efforts of John Murray’s laboratory at the University of Pennsylvania. John presented his lab’s work on dissecting the cell-specific embryonic expression profile over time, using a ‘whole-organism shotgun’ approach. This approach involves using the RNA-seq data obtained from individual, separated cells from mixed stage embryos. He then profiles each cell in order to map it back to the cell lineage, using known gene expression ‘hallmarks’ obtained from expression patterns of individual genes. Using this data, it is possible to start to answer questions such as which genes are required for acquiring cell identity versus those that are required for maintaining cell identity. An interactive database of this data is under development and should be available soon (keep an eye on BioRxiv). In contrast, Annabel Ebbing, from Hendrik Korswagen’s lab at the Hubrecht Institute in the Netherlands, described how RNA tomography can contribute to our understanding of spatially-resolved gene expression patterns without any prior knowledge of gene expression – rather, this approach relies on the highly invariable anatomy of the worm. RNA tomography involves cryo-sectioning young adult animals at 20µm intervals along the anteroposterior axis, before using CEL-seq to sequence each section. The data were aligned to provide a comprehensive map of gene expression along the anteroposterior axis of the worm, at single cell resolution. This map has already revealed sex-specific differences in the expression of previously-unidentified genes required for male fertility in the male reproductive tract, and has been made available here.
Outline of the RNA tomography method. Worms are sectioned at 20µm intervals before RNA from individual sections is sequenced. The relatively simple anatomy of the worm allows for single-cell and tissue resolution of sequencing data using this section width. Ebbing, A.*, Vertesy, A.*, Betist, M., Spanjaard, B., Junker, J.P., Berezikov, E., van Oudenaarden, A., and Korswagen, H.C. Spatially-resolved transcriptomics of C. elegans males and hermaphrodites identifies novel fertility genes. Submitted.
Cell fate and plasticity
The understanding of cell fate determination, and its opposite, fate plasticity, is important due to the potential implications of reprogramming in cancers and regenerative medicine. C. elegans provides two models with which to study in vivo reprogramming: cells which undergo natural transdifferentiation, and induced conversion of cells by ectopically expressed transcription factors.
The most famous natural transdifferentiation event in the worm is the Y to PDA transition in the tail. Y is a fully differentiated rectal epithelial cell that, partway through larval development, detaches from the rectum, migrates and converts into the PDA neurone. This process was first detected by John White and noted in his lineage, and later confirmed to be a bona fide transdifferentiation event by Sophie Jarriault. Sophie’s lab have continued to contribute to the field of reprogramming, and more recently her work has expanded to encompass other natural fate changes in C. elegans. Claudia Riva from the Jarriault lab talked about her work on these other cells. Although direct conversion of a cell from one fate to another is rare (the two known examples are the changing fate of Y, and also the phasmid socket PHso1, which produces the PHD neurone required for the Molina Manoeuvre), there are also cases of seemingly differentiated cells dividing to produce a cell of a different fate. These include the K cell in the rectum, which divides to produce the DVB neurone, and the G1 excretory pore cell, which gives rise to two RMH neurones. Claudia showed that even though these cells undergo a cell division, the ‘plasticity cassette’ comprising SEM-4, EGL-27, SOX-2 and CEH-6, identified to be required for the Y to PDA transition, is also required for the production of DVB from K. For example, in sem-4 mutant animals K goes on to divide but K.p remains epithelial. However, upon halting the cell cycle using a temperature sensitive allele of lin-5, she demonstrated that cell division is also essential for the production of DVB. The asymmetric division of K requires Wnt signalling, like many other asymmetric pathways in C. elegans, leading the lab to investigate the interplay of Wnt (during asymmetric cell division) and plasticity reprogramming factors (for the acquisition of neuronal cell fate) in the process of K dividing to produce the DVB.
Anna Reid, who works in the Tursun lab at the Berlin Institute for Medical Systems Biology in Germany, talked about her work on the forced reprogramming of mesodermal coelomocytes. There are three pairs of coelomocytes located in the pseudocoelom of the worm. The lab has found that the ectopic expression of the GATA factor ELT-2 is sufficient to convert these cells to the intestinal fate, and upon closer inspection found that they even produced an intracellular lumen lined with microvilli. Testing the limits of forced reprogramming, she found that the zinc transcription factor CHE-1, which specifies the fate of the ASE neurones, was also found to be able to reprogram the coelomocytes, with a 70% success rate for one of the three pairs of cells. Intriguingly, since the coelomocyte fate is lost at the time of conversion, so too is the expression of the coelomocyte-specific transgene inducing the forced transition event (in this case, the unc-22 promoter is used to drive ectopic che-1 expression in the coelomocytes). Thus only a pulse of expression must be sufficient to convert fate. Anna finds that this pulse of ectopic CHE-1 expression leads to the endogenous che-1 locus to be switched on in these cells; the fate-converting experiment in a che-1 mutant background is much less efficient. Lastly, Anna explained how the coelomocytes, now shown to be amenable to either intestinal or neuronal reprogramming, are much more resistant to a muscle fate change. Anna suggests that this is surprising, since the muscle lineage is much closer to that of the coelomocytes than the neuronal lineages. This raises the question of how lineage proximity can affect fate plasticity: if two lineages are closer, does this make it easier or harder to reprogram between them?
Transcriptional metabolic network rewiring
Marian Walhout, from the University of Massachusetts Medical School, USA, delivered a very elegant talk on the work her lab are carrying out in the field of systems biology. She explained how the worm has two metabolic pathways for breaking down proprionate, one of which is dependent on vitamin B12, and the other not. The B12-dependent pathway is preferable to the worm as it does not risk the build up of toxic intermediates, but the pathways are interchangeable in the event of no B12 being available and subsequent build up of proprionate. (Marian also points out that the standard laboratory culture conditions of growing C. elegans on the OP50 strain of E. coli means that the worms are always B12-deficient in the lab.) To convert from a B12-dependent pathway to a B12-independent pathway requires the transcriptional machinery to switch to expressing the enzymes required for the new pathway. However, in order to achieve this only when there is a sustained deficiency of B12, and not just in the case of a temporary peak in proprionate, requires a delay to be built into the switch. Marian explains how this delay has been modelled in bacterial systems by a coherent type 1 feed-forward loop with an AND-logic gate, providing persistence detection in response to a stimulus. However, this type of transcriptional circuitry has never been observed in an animal system, until now. To turn on the B12-independent pathway, Marian’s lab have found that two genes, nhr-10 and nhr-68, together act in such a persistence switch to turn on five genes of the B12-independent metabolic pathway, and in this way prevent this potentially hazardous pathway being turned on during a transient peak in proprionate.
Model for a persistence detector in the form of a coherent type 1 feed-forward loop with an AND-logic gate. Transcription factors A and B are both required to turn on their targets. In the case of a transient stimulus, however, gene A will not be expressed long enough to make sufficient amounts of gene B, in order to turn on the switch. Yet if the stimulus is sustained, transcription factor A will activate the expression of gene B, and the target genes will be transcribed.
Meiosis
Abby Dernburg from the University of California, Berkeley shared her work on how crossovers are regulated during meiosis. In C. elegans, each pair of homologous chromosomes undergoes just one crossover event, and all other double-stranded breaks are repaired without the transfer of genetic information. Little is known about how chromosomes convey this crossover information along their lengths. Through fluorescent analysis, allowing direct visualisation of the synaptonemal complex assembly which forms between homologous chromosomes, the Dernburg lab have been able to quantify the kinetics of synapsis. This has lead to the conclusion that the synaptonemal complex in fact acts as a liquid crystal – an ordered assembly of molecules that show liquid-like mobility – rather than a static complex. This rapid diffusion within the complex could be the mechanism by which information is rapidly transported down the length of the homologous chromosomes. The lab is now testing how each of the previously-identified factors for regulating crossover events, including the RING finger proteins ZHP-1-4, CDK-2 and COSA-1, can act with the synaptonemal complex to regulate and limit the number of crossovers to one per chromosome pair.
Membranes in biology
David Sherwood of Duke University, USA, delivered a wonderful talk on his work studying basement membranes in C. elegans. I had first come across David’s work as a Masters student, reading about how the anchor cell invades through the basement membrane that separates the gonad from the uterine seam during vulval formation. During his talk, David described how his lab have created a toolkit of fluorescently labelled endogenous basement membrane components (17 components and 13 receptors) in order to study the composition and dynamics of this ancient extracellular matrix. One of the striking results from this work has been the discovery of the huge range in the rates of turnover and mobility between these tagged components: FRAP and FLIP experiments show that whereas some components take hours to recover, others recover in a matter of milliseconds.
Meera Sundaram from the University of Pennsylvania, USA, is more interested in the apical membranes of cells – during this meeting she shared some of the work from her lab on how the worm builds tiny, seamless tubes from a single cell. There are three possible ways for a single cell to form a tube: through endocytosis and internal vesicle fusion, through membrane invagination followed by exocytosis, or through auto-fusion after the cell has rolled around the lumen. In the excretory system of C. elegans, there are two cells which each form a tube. These are the duct cell, which forms a seamless tube, and the pore cell, which remains seamed. Both tubes are formed by an initial rolling of the cell, but the duct cell then auto-fuses in an EGF-Ras-ERK dependent manner. When looking for targets of this pathway, Meera’s lab discovered that this fusion is dependent on the activation of the fusogen aff-1, which has already been shown to drive fusion in the uterine seam and between seam cells. Auto-fusion of the duct cell occurs early in development, and is followed by morphogenesis and elongation of the tubular cell into its final shape. Using the degron system to deplete aff-1 after the fusion event but before remodelling, Meera showed that surprisingly, aff-1 was also required for the elongation of the duct cell. In the absence of aff-1, she showed using STED microscopy that the cell accumulates vesicles containing apical cargoes, and also had nucleus-sized ‘hair-balls’ of membrane attached to the basement membrane, suggesting that AFF-1 is required for apically-directed trafficking. AFF-1 is present at endocytosing membranes, so may be required for scission. Vesicle trafficking is known to be required for remodelling of the duct cell, as Rab11, required for the recycling of endocytic vesicles, is essential for duct cell elongation. Thus Meera’s lab has discovered a novel role for fusogens during vesicular trafficking.
Model of the role of AFF-1 during endocytic scission in the duct cell of the excretory system.Soulavie F, Hall DH, Sundaram MV. The AFF-1 exoplasmic fusogen is required for endocytic scission and seamless tube elongation. Nature Communications. 2018;9:1741. doi:10.1038/s41467-018-04091-1
This was not the only talk at the meeting that presented a novel role for fusogens, however. Piya Ghose from the Shaham laboratory in the Rockefeller University, New York presented her work on how the fusogen EFF-1 was required for phagosome sealing during the programmed cell death of the tail spike cell. The role of fusogens in cell biology appears to be broader than was previously thought.
The ageing worm
The striking genetic control of worm lifespan has been demonstrated many times over the last few decades, and this together with the worm’s relatively short natural lifespan makes it an attractive model for ageing research. Adam Antebi of the Max Plank Institute for Biology of Ageing in Cologne, Germany, presented his lab’s work on how nucleolar size in the young worm can be used as a predictor of the lifespan of that worm – with worms with smaller nucleoli living the longest. Not only is this trend true for worms, but he showed how it also holds true for fruit flies, mice and humans, suggesting that the pathways of ageing that converge on nucleolar size are conserved. Adam’s lab found that nucleolar size is controlled by the NCL-1 tumour suppressor, with long-lived animals representing different longevity pathways exhibiting small nucleoli, decreased rRNA and ribosomal protein expression, and decreased fibrillarin, a nucleolar protein. Indeed, knockdown of fibrillarin is sufficient to reduce nucleoli size and increase lifespan in worms.
Nucleolar size inversely correlates with lifespan, and as such is a cellular hallmark of longevity. Tiku V, Jain C, Raz Y, et al. Small nucleoli are a cellular hallmark of longevity. Nature Communications. 2017;8:16083. doi:10.1038/ncomms16083.
Wild C. elegans
C. elegans research has primarily been performed on a single, Bristol isolate of C. elegans, called ‘N2’. However, there are myriad natural isolates of C. elegans around the globe. Resources for wild isolate research include those of Mark Blaxters lab in Edinburgh, and Erik Anderson, who manages the association mapping resource CeNDR. Jan Kemmenga has also produced a library of sequenced RILs between two highly polymorphic strains, the Bristol and Hawaiian isolates, that can be used to map differences in any phenotypic differences between these strains.
Marianne Félix, of the Marie Curie Institute in Paris, has been sampling wild C. elegans for many years, studying natural variation and using polymorphic genomes for quantitive genetic approaches to C. elegans research. Marianne described at the meeting a recently accepted (Lise Frézal et. al., Current Biology, in press) story investigating germline mortality in a C. elegans wild isolate. She described how the project started by leaving wild worms in a petri dish on her bench – resulting after a few generations in a sterile population. Luckily the lab was able to recover the population before it was too late, and realised they were observing a temperature-sensitive Mrt phenotype (mortal germline). Mrt phenotypes had already been observed in screens by Jonathan Hodgkin, which isolated mutants in DNA repair. The lab found that many of the wild isolates exhibited Mrt phenotypes to differing degrees of severity – the strain QX1211, for example, exhibits sterility in one generation. Many of these strains were isolated from hot countries, raising the question of how these strain survive in their natural habitats. Marianne observed that infection of the worms by natural pathogens was able to suppress the Mrt phenotype, indicating that the phenotype was in fact an artefact of the worms’ sterile environment in the lab petri dish. In order to understand the genetics behind why some strains exhibit the Mrt phenotype and not others, RILs were created between two strains, one Mrt and one not, JU1395 and MY10. Through phenotyping followed by whole genome sequencing, the Mrt phenotype was mapped to the set-24 locus on chromosome II. However, upon inspection of set-24 alleles across various wild isolates, Marianne’s lab found that the set-24 Mrt allele was, in fact, a rare allele in the wild. Thus to understand the common factor shared between the Mrt wild isolates, GWAS (genome-wide association mapping) was performed across 95 wild isolate genomes. This approach identified the more commonly present Mrt factor as morc-1, on chromosome III, a gene which helps to silence repetitive elements. Thus two different quantitive genetic approaches to find the cause of the same phenotype resulted in the identification of two different Mrt alleles, one of which was relevant for a particular strain (set-24), and one of which helped describe the differences between many wild isolates (morc-1).
Family, friends and enemies of Caenorhabditis
Although Caenorhabditis elegans took centre stage at the meeting, there were a few friends and even enemies of our favourite worm present. Marie Delattre from the Université de Lyon in France told us about one such worm, Mesorhabditis belari, which has a curious mechanism of genetic reproduction. For comparison, C. elegans has two sexes: hermaphrodite (of genotype XX) and male (of genotype X0). Populations are predominantly hermaphrodite, although the loss of one X chromosome at a rate of 1:1000 means that males do occur at a very low incidence. If a male mates with a hermaphrodite, mendelian genetics dictates that 50% of the resulting cross progeny, fertilised by sperm not carrying an X chromosome, are male. By contrast, Mesorhabditis belari is present in populations comprised of female and male individuals (so far so good). However, although the male is required by the female to fertilise her oocytes and to produce viable offspring, the male animal only rarely passes any of its DNA to the next generation, and progeny are predominantly clones of the female parent. So what is the point of producing males at all? Marie discovered that females produce two different kinds of embryos upon oocyte fertilisation. To understand the difference between them, it should first be mentioned that C. elegans oocytes, as well as those of Mesorhabditis belari, undergo only meiosis I before fertilisation by the sperm; meiosis II occurs shortly after fertilisation and it is only after expulsion of the sister gamete that the male pronucleus joins the female pronucleus to form the zygote. So, to go back to the oocytes of Mesorhabditis belari, it was found that 10% of oocytes carry out meiosis II after fertilisation, as expected, followed by fusion of the male and female pronuclei and subsequent development of the embryo – into male worms only. The remaining 90% of fertilised oocytes of Mesorhabditis belari do not complete meiosis II at all. Instead, the male pronucleus is prevented from expanding and fusing with the female nucleus, which enters mitosis to produce a female embryo – containing only the DNA of the female parent. In this scenario, it would seem that there is no evolutionary advantage to producing males. Marie turned to evolutionary game theory to provide some answers. She modelled two genetically distinct populations of Mesorhabditis belari, using factors to describe population size (i.e. how many progeny are produced) and how many worms will migrate away and towards each population. Using this model, she was able to identify a scenario in which males of Mesorhabditis belari presented an advantage to the population: if the males were to preferentially mate with their sisters. Indeed, Marie realised that the very low mating efficiency of these worms in the lab may also be explained by this preference, and upon performing the experiment, she found this to be the case. Thus, males provide their genetically similar sisters with sperm for the activation of oocytes, initiation of polarisation and for providing centrosomes – just not for their DNA.
A more nefarious creature (at least from the point of view of C. elegans researchers) is Pristionchus Pacificus. This is a dimorphous worm that in one of its forms is carnivorous – enjoying munching on C. elegans, amongst its other prey.
If one can overcome the macabre, however, an interesting question presents itself: how does Pristionchus Pacificus avoid becoming cannibalistic? James Lightfoot, from the lab of Ralf Sommer at the Max Planck Institute for Developmental Biology in Germany, explained how self-recognition (and thus avoidance of eating its own species) was achieved. In order to test the limits of self-recognition, James mixed populations of four different carnivorous species (including the sister species of Pristionchus pacificus) together on a plate, and observed that all four species recognised and avoided eating only themselves. If larvae from their own species were allowed to populate a plate and were later removed and replaced by the larvae of an alternative species, the new larvae were not protected from the predatory worms. Together these experiments suggested that self-recognition involved a surface-bound molecule. During his investigation, James found that individual isolates of Pristionchus pacificus, such as those isolated from Washington and those from California, will happily eat each other (even though they are members of the same species) but avoid worms from their own genetic pool. This observation allowed him to cross together the two strains to produce recombinant inbred lines (RILs), followed by phenotyping and sequencing the resulting strains. Impressively, the lab had to overcome a significant incompatibility region that obscured the data, and eventually managed this by inducing double stranded breaks along the chromosomes using CRISPR. This resulted in the identification of the locus ‘self-1’. Deletion of this locus resulted in a loss of self-recognition, and it was found to encode a 63 amino acid short peptide. An alignment of 38 strains revealed that although most of the self-1 sequence was highly conserved, it contained a highly variable terminal sequence. In addition, the copy number of this gene varied extensively between strains. James and his colleagues found that changing this variable region between strains was enough to protect the opposite strain, and that even a single amino acid change in this region was enough to affect self-recognition. However, he has identified some strains that carry identical self-1 sequences, but do not eat each other, suggesting that there are as yet still undiscovered mechanisms that regulate self-recognition in Pristionchus pacificus.
EMBO Young Investigator lecture
Luisa Cochella of the Research Institute of Molecular Pathology (IMP) in Vienna, delivered the EMBO Young Investigator lecture. Luisa studies the role of miRNAs (short, approximately 22nt long non-coding RNAs that repress gene expression) during development, and started her own lab after working as a postdoc in Oliver Hobert’s lab at Columbia University.
Luisa Cochella presents the EMBO Young Investigator lecture at the European Worm Meeting. Her slide is showing John Sulston’s lineage of the worm.
Luisa described how it has been previously proposed that miRNAs can act as ‘de-noisers’ of transcription, expressed in domains where their target genes are not normally transcribed, to reinforce the transcriptional control of the gene. Alternatively (and she suggests, perhaps more interestingly), miRNAs can act on their own to change cell identity. miRNAs begin their existence as miRNA precursor molecules (a self-annealing loop of RNA) which must be processed by first the RNase III Drosha, together with Pasha (partner of Drosha), and then again by Dicer to produce the mature miRNA which can be recognised by Argonaute. Embryos of a strain containing a temperature sensitive allele of Pasha, pash-1(mj100), fail to elongate during embryogenesis at the restrictive temperature, showing that miRNAs are essential for embryogenesis in the worm. Individual knockouts of 90 of a total 150 miRNAs, performed by Eric Miska and Bob Horwitz, showed no developmental arrest. However, upon deletion of entire miRNA families, they found that two families in particular were important for development. These were the mir-35 family (comprising 8 members) and the mir-51 family (6 members). Deletion of the mir-35 family phenocopies the pash-1 allele, whereas deletion of the mir-51 family resulted in animals that arrested slightly later during embryogenesis. Rescue of a miRNA gene family knockout strain can be achieved by any one of the family members.
By sequencing miRNA from embryos, Luisa revealed that 75% of all miRNA molecules belonged to one of the mir-35 or mir-51 families. Using a micro-processor by-pass strategy, which uses splicing machinery rather than Dicer to process synthetic ‘mirtrons’, the lab was able to rescue pash-1 mutant embryos with only two mirtrons, representing each of the mir-35 or mir-51 families. However, these animals still arrested during larval development, suggesting a role for the remaining 25% (by expression) of miRNAs. Despite some of these 25% of miRNAs being expressed at very low levels, Luisa hypothesised that they may be essential for very specific developmental events, and could be expressed in as little as one cell during development. She tested the expression patterns of these lowly-expressed miRNAs and found that they were, indeed, expressed in very restricted domains. One such miRNA, mir-791, was found to be expressed in a subset of neurones: BAG, AFD and ASE. These are all CO2-sensing neurones. In behavioural assays, loss of mir-791 resulted in a reduced avoidance response, and could be rescued by reintroduction of mir-791. This indicated that mir-791 has a specific role in conferring fate of these neurones, and is not merely involved in the robustness of the expression of target genes. Luisa found that the target genes for mir-791 were two ubiquitously expressed genes, akap-1 and cah-3. The only cells where the expression of these genes is repressed is in the CO2-sensing neurones. In this way, she has found that miRNAs may act in parallel to transcription factor control of gene expression to confer specific cell fates.
Keynote talk
Paul Sternberg from CalTech in California delivered the keynote talk at the meeting. The worm community owes Paul a huge debt for the many research resources that he has cofounded in his time working on C. elegans – including Wormbook, Textpresso and Wormbase. He began his career with C. elegans with the Horwitz lab at MIT at about the same time as John Suston’s lineage was published, at the start of what would become a hat tick for C. elegans research: the lineage, the connectome and the genome. Using this information, he set out to ‘solve the worm’. His lab maintain a very broad spectrum of research interests, and here he highlighted their recent work on the dauer life-form of the worm. C. elegans larvae enter an alternative lifecycle during conditions of starvation or overcrowding, in which they may remain as a dauer animal for up to three months. Indeed, in the wild this is the most commonly-found life stage. Upon finding food, the dauer animals re-enter the life cycle as L4 larvae, and progress to adulthood. Paul suggests that the dauer stage might also serve other functions in the worm, however, than just a means to survive starvation. He notes that animals of the genus Steinernema are able to jump significant distances as dauers, due to the energy stored in their cuticle. Many genes are shared between Steinernema and Caenorhabditis, and his lab are now investigating the differences in gene expression between dauers and other larvae in C. elegans.
During his talk Paul highlighted the many resources available for C. elegans researchers, including the CRISPR-based knock-out by knock-in (using the hygromycin resistance cassette) library being created to complement the knockout consortium database of null alleles. He also mentioned the cGAL bipartite expression system that his lab has recently adapted for the worm, and finally his initiatives in micropublication. Micropublication is available through Wormbase and allows concise (one figure) reports to be made and referenced within the scientific community. As he reminds us at the end, the 1953 DNA double helix paper also had just one figure…
Keynote address: Worm Tales by John White
One of the meeting highlights was the keynote address delivered by John White, FRS. John was a graduate student in the lab of Sydney Brenner in the 1970s, and is now Professor Emeritus at the University of Wisconsin-Madison. During his time at the LMB, John co-developed confocal microscopy and published ‘The Mind of a Worm’, and in so doing founded the field of connectomics. His address focused on the historical perspective of modern Caenorhabditis research, from its origin as a brain child of Sydney Brenner to its establishment as a major genetic model organism. (Wormbook has a wonderful memoir describing John White’s experience as part of the first C. elegans research lab that is definitely worth a read.)
In his talk, John explained how Sydney would holiday at the laboratory of marine biology in Woods Hole, contemplating his next scientific adventure (he had already been involved with solving the genetic code, in the process discovering a STOP codon). He was particularly interested in a molecular solution to behaviour, and had the foresight to understand that this would involve a precise understanding of the research organism’s anatomy. However, current model organisms were either too complex (Drosophila has approximately 135,000 neurones) or genetically intractable for this purpose. Sydney came across Bovari’s lineages of the nematode Ascaris, which suggested that these worms were likely to have stereotyped lineages, and also Goldschmidt’s work, which showed Ascaris to have a very simple nervous system. A 1948 paper published in Nature by Dougherty and Calhoun had also suggested the possible significance of nematodes as model organisms. Thus, after some experimentation which showed the nematode nervous system could be visualised with electron microscopy, Sydney settled on the worm as his model. The first strain of Caenorhabditis elegans was isolated from mushrooms in Bristol in England, becoming the ‘canonical’ research strain, and 55 years ago Sydney isolated the first mutant worm.
According to John White, Sydney undertook a very successful PR campaign in the early ’70s to garner support for his new research model. He travelled around America, speaking at many events in order to attract the best and most adventurous postdocs to his laboratory – who also came with funding. People who worked at the LMB with Sydney included many current lab heads and notable researchers: Bob Horwitz, Marty Chalfie, Judith Kimble, Cynthia Kenyon, Barbara Meyer, Jim Preiss, Iva Greenwald, Andy Fire, Julie Ahringer, Benjamin Podbilewitz, John Sulston, Bob Edgar and Ed Hedgecock, amongst others. Graduate students included John White, Jonathan Hodgkin, Mario de Bono, Richard Durbin and Tony Hyman. John explained how Sydney’s approach to supervision could be described as ‘benign neglect’, centring around 10am coffee break discussions that may or may not be related to the lab’s current research. John attributes much of the success of early worm research to this strategy, which forged strong communication and support links between researchers.
Sydney’s first task was to get the genetics of the worm up to the standard of Drosophila, generating mutants and tools such as balancers, and also mapping the C. elegans genome. This work culminated in the publication of a single author paper in 1974. At the same time, the neuroanatomy of the worm was being dissected using electron microscopy. However, Sydney soon moved onto other scientific projects and left C. elegans in the hands of his coworkers. Jonathan Hodgkin took over the construction of the genetic map, and John Sulston decided to help with the effort of White and colleagues to reconstruct the neural network by completing the post-embryonic lineage of the worm. After finding out that those in charge of constructing the embryonic cell lineage were ‘doing it wrong’, Sulston apparently locked himself away for 18 months and produced the accurate map we have today. This work paved the way for the identification of lineage mutants, and the work on cell-cell communication and programmed cell death that was pioneered in the worm. An obituary for John Sulston written by John White can be found here.
John Sulston holding his cell lineage of the nematode C. elegans.
The whole meeting represented a wonderful collection of the continuation of the work initiated by John Sulston, during which he was remembered and missed by the C. elegans community. The historical perspective of John White was a good reminder of how creativity is fostered by the combination of curiosity, tenacity and communication between researchers, values that I believe are central components of the C. elegans research community and make it such a pleasure to be part of.
In our recent paper published in Nature, we unravel a new mechanism of an extracellular matrix protein secreted by muscle satellite (stem) cells, thereby playing the unusual role of acting as a signaling molecule to maintain the stem cell population. Here, I share the story behind this discovery and discuss the questions related to niche regulation.
In a ChIP-sequencing screen in myogenic cells performed by the Tajbakhsh and Stunnenberg labs (Castel et al., 2013) to identify direct targets of nuclear NOTCH (NICD) and its downstream effector RBPJ, we found an enrichment of extracellular matrix proteins, among which were specific collagens genes including Collagen 5 (Col5a1 and Col5a3) and Collagen 6 (Col6a1 and Col6a2). I was in the first year of my PhD when Dr. Philippos Mourikis, an ex post-doc in the Tajbakhsh lab, asked me to help with some collagen immunostainings on embryonic muscles over-expressing NICD (E17.5 Myf5Cre; R26NICD) (Mourikis et al., 2012). We nicely showed that both COLV and COLVI secretion were upregulated upon Notch induction.
As Col5a3 gene was the most responsive gene to Notch activity, both in vitro and in vivo, we thought that Col5a3 KO mice might present a muscle phenotype (Huang et al., 2011). In collaboration with Dr Guorui Huang from the Greenspan lab, we analyzed the number of satellite cells (PAX7+) in adult and postnatal P8 pups muscle sections. Unfortunately, we could not observe any alterations in stem cell number nor proliferation capacity (BrdU pulse).
PAX7 (satellite cells), BrdU (proliferation) and Laminin immunostaining on transverse sections of Tibialis Anterior muscle of Control (WT) and Col5a3 KO P8 (8 days after birth) juvenile mice. No difference in satellite cells number and proliferation capacity between control and mutant pups.
As my initial PhD project was on hold due to mice breeding issues, we decided that I could perform a simple culture experiment to close up the collagen story. We performed two sets of experiments: in the first, we added different collagens in soluble form on cells plated on gelatin-coated dishes; in the second, cells were cultured directly on collagen-coated dishes. Surprisingly, only COLV in its soluble form retained cells in a more stem-like state delaying their proliferation and differentiation. With these unexpected results, we realized that COLV might be acting as a signaling molecule rather than a biomechanical cue. As collagen molecules are known to bind to specific receptors such as Integrins or Discoidin Receptors (Leitinger, 2011), we tested whether their inhibition could abrogate COLV anti-myogenic effect. To our disappointment, COLV was still able to delay myoblast differentiation even when these receptors were inhibited.
After looking at the results from several angles, we came to the conclusion that we lacked a mechanism and we should probably wrap up the story with what we had, so that I could go back to my initial PhD project. With a sense of unfinished business, we started gathering the data into figure formats. We were then struck by an old RT-qPCR assay on satellite cells that were treated with the different collagens, where Calcitonin receptor (CalcR), a known satellite cell quiescence marker, was upregulated more than 20-fold. We remembered that CALCR is a G-protein couple receptor (GPCR) and found in vitro evidence that some collagens could indeed bind to GPCRs (Luo et al., 2011; Paavola et al., 2014). This was a major turning point in the project, and the path was wide open for detailed mechanistic studies.
In parallel on the other side of the world, our collaborator Dr. So-Ichiro Fukada in Osaka had just a few weeks earlier published a very nice study showing that specific ablation of Calcr in muscle stem cells led to their spontaneous exit from the quiescent niche (Yamaguchi et al., 2015). Using their retroviral tools to overexpress CALCR and Calcr null muscle stem cells, we were able to demonstrate that CALCR was necessary to respond to COLV. This was too exciting, my other project had to wait!
EdU (2h pulse) and CALCR staining of GFP+ C2C12 cells isolated by FACS and transduced with Calcr-GFP or mock-GFP retrovirus and cultured for 24 h with COLI (top) or COLV (bottom). Quantification of EdU+ Calcr-transduced C2C12 cells or mock-GFP cells treated for 24h with COLV or with the controls, COLI and HOAc. There was no significant difference between HOAc and COLI treated samples.
On the other hand, these results were puzzling…CalcR already has its ligand: Calcitonin hormone produced by the parathyroid gland, suggesting that satellite cell quiescence is under systemic control. Why would the regulation of muscle stem cells that are dispersed throughout muscle masses in the body – and that would be solicited with different kinetics – be under systemic control? Can COLV act as the local ligand of CalcR for muscle stem cells?
Meanwhile, our first priority was to assess whether the loss of function of COLV specifically in satellite cells using Tamoxifen-inducible Tg:Pax7-CreERT2; Col5a1flox mice could affect their behavior. Another unexpected surprise was the observation that only 18 days following recombination, satellite cells escaped the quiescent niche, differentiated and fused to the pre-existing fibres.
Another consideration is the source of collagen in skeletal muscle – being predominantly from interstitial myofibroblasts, which deposit collagen between myofibres (Zou et al., 2008). We also found expression of Col5a-1, -2, and -3 in other cell types present in the muscle; namely Fibro-Adipogenic Progenitor (FAPs) and resident macrophages.
So why does this source of COLV not compensate for the loss of satellite cells-produced COLV?
Possible explanations include the basal membrane (BM) forming a physical diffusion barrier between the satellite cells and the interstitial space – rendering them blind this source of COLV. Although the myofibre that is in intimate contact with satellite cells under the BM could be a provider, we showed that myofibres do not express detectable COLV to sustain satellite cell quiescence.
Another possibility is that COLV produced by satellite cells has a specific configuration. In our siRNAs experiments on isolated myofibers, we observed that targeting of the Col5a3 isoform phenocopied siRNA against Col5a1, strongly suggesting that the active triple helix is the a3-COLV isoform composed of both a1 and a3 chains as an [α1(V)α2(V)α3(V)] heterotrimer. Although the presence of soluble COLV is difficult to demonstrate in vivo, we favoured the possibility that COLV produced by satellite cells rapidly binds to CALCR upon secretion, rather than being integrated in the collagen network. This would then explain why loss of COLV function exhibited a phenotypic loss of satellite cells within such a short period.
We then needed to demonstrate that the newly discovered Notch/COLV/CALCR axis is functional for the maintenance of quiescent cells in vivo. To do so, we injected Col5a1 cKO (Tg:Pax7-CT2; Col5a1flox) mice subcutaneously with a derivative of the known CALCR ligand calcitonin (Elcatonin). Interestingly, exogenous Elcatonin administration compensated for the lack of COLV and prevented their exit from quiescence, thereby validating our results linking COLV and CALCR. It also told us that endogenous calcitonin was not bioavailable in the muscle to rescue the loss of COLV. Interestingly, thyroidectomy in adult individuals does not show any striking overall pathological consequences (Russell et al., 2014); it would be interesting to study satellite cell dynamics in mice suffering from hyper/hypothyroidism.
An interesting outcome of the rescue experiments was that Elcatonin administration in control mice also showed an improvement in self-renewal capacity, suggesting that this could be a good therapeutical candidate for myopathies.
Rescue of loss of COLV by Elcatonin in an ex vivo self-renewal reserve-cell model, where PAX7+ non-proliferative cells return to quiescence. MyHC and PAX7 staining of Control (Ctr: Tg:Pax7-CT2; Col5a1+/+; R26mTmG) and Col5a1 null (Tg:Pax7-CT2; Col5a1flox/flox; R26mTmG) non-treated (NT) or treated with Elcatonin (Elcat). No GFP+/EdU+ cell (12h pulse) could be detected in any of the conditions indicating GFP+ cells are quiescent (data not shown).
In addition, it has been shown that the satellite cell population is heterogenous according to the muscle anatomical location and even within a single muscle (Rocheteau et al., 2012; Sambasivan and Tajbakhsh, 2015). Therefore, we cannot exclude the possibility that some satellites cells do indeed respond to calcitonin stimulation. Our results showing that Elcatonin administration induces a deeper quiescent state could support this notion. In addition, CALCR is expressed in about 80% of satellite cells, suggesting that 20% of them are most likely not under direct Notch/COLV/CALCR control.
While exploring the quiescence in muscle stem cells, we learned that when studying the role of collagens one should consider not only the traditional mechanotransduction role of these molecules, but also their potential role as signaling agents. Given that neural and intestinal, and perhaps other stem cells, are maintained by Notch signaling, it is possible that this model operates in stem cells located in other tissues and organs.
The Echeverri lab at the MBL seeks a highly motivated individual to join the Eugene Bell Center for Regenerative Biology and Tissue Engineering as a Postdoctoral Researcher. The successful candidate will work on the molecular mechanisms of scar free skin regeneration in axolotls.
The specific goal of the project is to examine the role of different cell types in responding to the injury cue and in later remodeling collagen.
Basic Qualifications:
Applicants should have a Ph.D. in a biology related field. Must have prior experience working in the field of cell and developmental biology, as well as experience with molecular biology. Must be independent, enthusiastic, self-motivated, productive, and enjoy working in a highly collaborative environment.
Preferred Qualifications:
The ideal candidate will have direct experience with working in vivo in an animal model. Previous experience with cell culture, molecular biology and imaging would be a plus.
Required documents:
Cover letter explaining specifically why you are interested in joining our lab to work on this project and what positive qualities you would bring to our team.
Curriculum vitae.
List of 3 references (Please do not have letters sent at this time. Letter writers will be contacted directly by the PI)
Please e-mail your application to Dr. Echeverri: echev020@umn.edu
The Echeverri lab at the MBL seeks a highly motivated individual to join the Eugene Bell Center for Regenerative Biology and Tissue Engineering as a Postdoctoral Researcher. The successful candidate will work on the comparative evolution of molecular regeneration in various aquatic research organisms. The specific goal of the project is to examine how pathways that are essential for regeneration have evolved in different species with different regenerative capacity.
Basic Qualifications:
Applicants should have a Ph.D. in a biology related field. Must have prior experience working in the field of cell and developmental biology, as well as experience with molecular biology. Must be independent, enthusiastic, self-motivated, productive, and enjoy working in a highly collaborative environment.
Preferred Qualifications:
The ideal candidate will have direct experience with working in vivo in an animal model. Previous experience with cell culture, molecular biology and imaging would be a plus.
Required documents:
Cover letter explaining specifically why you are interested in joining our lab to work on this project and what positive qualities you would bring to our team.
Curriculum vitae.
List of 3 references (Please do not have letters sent at this time. Letter writers will be contacted directly by the PI)
Please e-mail your application to Dr. Echeverri: echev020@umn.edu
Contact inhibition of locomotion is a widespread phenomenon in migrating cells. However, cells often migrate collectively as a sheet, raising the question of how contact inhibition is overcome in these scenarios. A new paper in Development addresses this problem by studying the signals that regulate collective migration in Xenopus leading edge mesendoderm (LEM) cells. We caught up with the paper’s two authors Martina Nagel and Rudolf Winklbauer, Professor in the Department of Cell and Systems Biology at the University of Toronto, to find out more about the story.
Rudolf and Martina
Rudolf, can you give us your scientific biography and the questions your lab is trying to answer?
RW I did my PhD work at the MPI for Virus Research in Tübingen, now MPI for Developmental Biology, in the lab of Peter Hausen. I was the first graduate student in his lab who used Xenopus to study embryonic development, and I remained with the frog embryo ever since. As a postdoc in Ray Keller’s lab at Berkeley, I settled on my present research agenda: cell migration in the gastrula. Back in Germany, I led a small research group in Hausen’s Department at the MPI in Tübingen. From 1993 to 1999, I held an Assistant Professor-like position at the University of Cologne. It was non-tenure-track, and to keep me off the street afterwards, Peter Hausen offered me a Senior Researcher position at his Department. Since 2001, I am at home at the Cell and Systems Biology Department at the University of Toronto.
As it turned out, almost every gastrulation movement in the frog embryo can be considered a cell migration process. The basic question is: how do cells migrate across and between other cells all the while they adhere firmly to each other to keep the embryo together? Naturally, with this question in mind, one becomes also interested in problems of cell adhesion and tissue mechanics.
Martina, PubMed tells me you first published with Rudolf in 1991 – how did you initially come to work together, and what questions have driven your research since then?
I was Rudi’s first graduate student, and my task was to find out how prospective head mesoderm cells find their target region as they migrate through the frog gastrula. Subsequently I studied also other aspects of mesoderm cell motility, but this primordial question is still my main interest.
In that 1991 paper, you described how LEM aggregates formed oriented lamellipodia during their migration. What initially drew you to this phenomenon?
MN That lamellipodia all pointed in the direction of overall mesoderm movement had been observed in the intact embryo. It had been the first indication that the cells could somehow sense the direction of their target at the animal pole of the embryo. We reproduced this orientation process in vitro to study its mechanism.
RW Tina politely disregarded my enthusiastic attempts to supervise my first graduate student and, against my nicely laid-out plans, performed an elegant experiment which demonstrated that mesoderm cells are guided by cues in the extracellular matrix of the ectoderm.
MN A major step in our understanding of the cell guidance mechanism was the identification of PDGF-A signalling as an essential factor in the process. We did this in collaboration with Karen Symes, Boston University. Our subsequent attempts to understand how PDGF-A actually functioned as a guidance cue led us to the current work.
Protrusion formation and behavior of single mesoderm cells on fibronectin. Movie 1 from the paper
Can you give us the key results of the paper in a paragraph?
MN Outside of the embryo, mesoderm cells show contact inhibition of migration: if the lamellipodium of a cell contacts another mesoderm cell, it collapses, and movement ceases. On the endogenous substratum, however, PDGF-A bound to the extracellular matrix suppresses contact inhibition, which allows cells to form long-lasting lamellipodia. In a way that we do not yet understand, this suppression of lamellipodia collapse is spatially biased, leading to the preferential survival of protrusions that point into the predetermined direction of migration. This potentially generates the uniform orientation of lamellipodia that was the starting point of our investigation.
SEM images of the substratum-facing surface of the LEM, from Fig. 8 in the paper
So you have pathway involving a series of inhibitory interactions linking Pak1 to ephrin: do you have any idea of the molecular basis of any of these interactions?
MN Ideas, yes; but no data yet. We find it an attractive hypothesis that direct interaction between factors enables or prevents signalling between cells, depending on the size of these factors. For example, Eph receptor/ephrin signalling requires close cell-cell contact, but patches of syndecan 4 in the cell membrane would locally preclude such close contact due to the size of the syndecan molecules. Another possibility is the exchange of cytoplasmic signals between factors, for example between the PDGF receptor and integrin. But so many putative downstream signalling pathways exist for all the factors involved…
When doing the research, did you have any particular result or eureka moment that has stuck with you?
RW Many eureka moments; too many!
MN Ever so often, we thought we finally understood how the whole thing worked, until the next experiment gave us exactly the opposite result from what we had predicted.
RW Back to the drawing board. But the nice thing was: after a while…
MN … a long while…
RW … after a while, this pattern changed, we more and more often predicted correctly what would happen, and gradually most of our observations found their place in the puzzle.
A mesoderm aggregate on a fibronectin-PDGF substratum. Movie 9 in the paper
And what about the flipside: any moments of frustration or despair?
MN As just mentioned: whenever a really cool idea went to dust after an experiment.
RW Cell and developmental biologists have done an enormous job over the last decades, and so many facts and concepts are known now that are relevant to our work. This makes adding new results to the existing body of knowledge so complicated. Where we had to align two little known interacting factors for a paper thirty years ago, we now have to juggle a handful of them. And they all are known to do multiple things and to interact with multiple other factors.
And where will this work take you?
MN We hope that we will better understand how migration is guided by the substratum in systems where cells move as parts of a large, tightly coherent cell mass.
RW We also fancy that we might identify a core pathway regulating contact inhibition of migration. By determining the position in the pathway of other factors that we and others have found to be involved in the process. We wonder whether such a pathway exists, or whether lamellipodia collapse is easy to get and regulated in different ways in different contexts.
When you think about it more generally, cells being able to freely crawl around in the organism, to exchange places and to re-group is an essential part of what makes an animal an animal at the cell level. Green plants don’t do it, red or brown algae won’t, and so on. Fungi seem to try. And after all: it is animals who found out all this stuff about cell migration that we were just talking about.
Finally, let’s move outside the lab – what do you like to do in your spare time in Toronto?
RW Watching how our little backyard garden turns slowly into a tiny patch of forest in the middle of the city, where the squirrels put on a show, the sparrows ask for bread crumbs, and…
MN Half the year it is winter in Toronto!
RW Well, say a third of the year. And there are always books.
Iain Martyn & Tatiane Kanno share their experiences of the discovery of the human organizer
“It’s alive!” Iain’s first impressions
“Hybrid human chicken embryos: HALF HUMAN – HALF CHICKEN abomination created in US lab” was my favourite headline reporting on our work1. While the headline and accompanying article managed to miss the science completely, the author may have been surprised to know how close he or she came to capturing mood of that first, Frankenstein-like moment of discovery of the “abomination”.
For starters, it really was a dark and stormy night. The lab, high on the seventh floor of a sheer, grey, impenetrable tower, was deserted and silent. Only the intermittent odd hum and hiss of an incubator or the sound of rain and wind lashing on the windows broke the stillness and betrayed the presence of something living and growing in its confines. Far below, next to the seething, storm-overloaded river, a hunched figure made its way hurriedly across a narrow bridge towards the tower.
That would of course be me, making the dash from my apartment to the lab foolishly without a rain-jacket and trying not to get soaked. Almost exactly 24 hours previously I had grafted human embryonic stem cells into a developing chicken embryo, a long-shot search for the never-before-seen human organizer, and now it was time to check the result. What exactly was I going to see? What would it look like? A monster? Some sort of bird-human chimera? Despite the gothic atmosphere I thought to myself it was more likely that all I was going to see was a mess of dead or dying cells. This was after all my first attempt and I was a novice at chick embryology. The only reason I was here at this midnight hour in the first place was because I had run late the night before, taking over six hours to set up what any half-competent chick embryologist could do in two. Still, as I made my way into the darkened lab, took the grafts from the incubator, and loaded them onto the microscope, I could not help the apprehension rise within me.
In the first dish the cells were indeed dead or dying, torn apart by a clumsy error made during the grafting. In the second dish the graft was alive, but relatively unchanged from last night, undisturbed by the developing host chick and not disturbing it in turn. In the third dish…well three really is a lucky number: in the third dish was a little “abomination”. There, besides the normally developing host chick, the fluorescently tagged human cells had grown, expanded their area, and fused with the host tissue. More dramatically, they had also coalesced and grown into a long thin rod-like structure, emanating from the center of the graft and pointing like a dismembered finger towards the host. This is the point where lightning should have struck, thunder should have boomed, and I should have stood up and shouted “it’s alive!”, but I was more concerned with gathering evidence and recording what I saw. In fact, I think I only released the breath I’d been holding when I was sure I had taken two good pictures with the microscope’s camera and saw that they were each safely stored on the computer.
Good thing that I did as well, for none of the remaining grafts showed anything so remotely as dramatic. And when I returned to the successful graft the following morning to see if it had grown into anything even more remarkable I found only dead or dying cells. Those pictures and the memory of the previous night were all that remained, and if it were not for them, and not for that one successful graft, I might have given up and gone back to my co-PIs Ericand Alito report that it was a total failure. As it were, I became convinced that if it happened once it would happen again. The way forward to fully studying and proving the existence of the human organizer was still long and difficult, and it required teaming up with a bona fide chick embryologist, but after that night I was sure we could get there.
Iain and Tati conducting graft experiments
“…but what is it?” The striking moment for Tati
My story in the Brivanlou lab begins before I join the team as a postdoc, not so very long ago. At that time, I was a PhD student visiting the lab to learn and perform some experiments with embryonic stem cells. When I finished my internship, I returned to Brazil to defend my thesis. Few months went by and there I was, coming back to New York.
It was my first day back as an official lab member when I first came across this project. I remember being in the conference room, feeling that mixture of excitement and anxiety for starting a new chapter in my career when I heard “Hey, welcome back! Can I show you something cool?” That was the moment when I was introduced to lucky embryo number 3. As an embryology enthusiast, I got thrilled with those pictures! Some ideas had already started to pop up in my mind. We teamed up to optimize the chick experiments and that was just the beginning of our long journey in search for the human organizer.
The first set of grafting took longer than I expected: even being very familiar with chick embryo manipulation, it was my first time trying to generate a chimera. It was late, I was exhausted and hungry crossing the narrow bridge back home, but I was also feeling an excitement and eagerness for the daybreak to see the results. As it turned out, our first grafted embryos looked more like a Picasso painting. I still think MoMa museum would love to exhibit our nightmarish sci-fi art. But a tweak here and there and we managed to keep the embryos alive and looking more… normal-ish! After that, it was a marathon. Besides the long hours in lab grafting, swayed by Brazilian forró songs and replenishing ATP with Iain’s hidden snacks, we also had to go through endless washing steps for in situ and long confocal imaging sessions.
And then, finally there it was! In the elongated structure emanating from the human cells we found expression of SOX2!! How awesome that could be?! To me, that was the mind-blowing moment, but of course I still had to hold my horses and wait for the in situ results of SOX3 probe to confirm our findings. SOX2 and SOX 3 were ectopically induced in chick cells that surrounded the human cells!! We had generated our very first chick-human chimera, our “Chuman”! Our results bring valuable insights into early human development.
This work was one of those “high risk, high reward” kinds of project. It could lead to an amazing discovery or could give us nothing. Gladly, with a wonderful teamwork, we got the reward!
Welcome to our monthly trawl for developmental biology (and other related) preprints!
This month we found a tranche of preprints getting deep into the mechanics of fly development, a clutch on organoids (retinal, cerebral, cortical!), an investigation into the role of gender in scientific collaboration, and a veritable zoo in our evo-devo section – from ladybirds to placozoans via pufferfish, hydra and choanoflagellates.
The preprints were hosted on bioRxiv, PeerJ, andarXiv. Let us know if we missed anything, and use these links to get to the section you want:
Amot regulates neuronal dendritic tree through Yap1
Katarzyna O. Rojek, Joanna Krzemien, Hubert Dolezyczek, Pawel M. Boguszewski, Leszek Kaczmarek, Witold Konopka, Marcin Rylski, Jacek Jaworski, Lars Holmgren, Tomasz J. Proszynski
Morphogen-Lineage Selector Interactions During Surface Epithelial Commitment
Sandra P Melo, Jillian M Pattison, Samantha N Piekos, Jessica L Torkelson, Elizaveta Bashkirova, Maxwell R Mumbach, Charlotte Rajasingh, Hanson Hui Zhen, Lingjie Li, Eric Liaw, Daniel Alber, Adam J Rubin, Gautam Shankar, Howard Y Chang, Paul A Khavari, Anthony E Oro
Francesconi, et al.’s transdifferentiation/reprogramming schema
Active fluctuations modulate gene expression in mouse oocytes
Maria Almonacid, Stephany El-Hayek, Alice Othmani, Isabelle Queguiner, Fanny Coulpier, Sophie Lemoine, Leïla Bastianelli, Christophe Klein, Tristan Piolot, Philippe Mailly, Raphaël Voituriez, Auguste Genovesio, Marie-Hélène Verlhac
Genome-scale oscillations in DNA methylation during exit from pluripotency
Steffen Rulands, Heather J Lee, Stephen J Clark, Christof Angermueller, Sebastien A Smallwood, Felix Krueger, Hisham Mohammed, Wendy Dean, Jennifer Nichols, Peter Rugg-Gunn, Gavin Kelsey, Oliver Stegle, Benjamin D Simons, Wolf Reik
How ladybirds get their spots, from Gautier, et al.’s preprint
The genomic basis of colour pattern polymorphism in the harlequin ladybird
Mathieu Gautier, Junichi Yamaguchi, Julien Foucaud, Anne Loiseau, Aurelien Ausset, Benoit Facon, Bernhard Gschloessl, Jacques Lagnel, Etienne Loire, Hugues Parrinello, Danny Severac, Celine Lopez-Roques, Cecile Donnadieu, Maxime Manno, Helene Berges, Karim Gharbi, Lori Lawson-Handley, Lian-Sheng Zang, Heiko Vogel, Arnaud Estoup, Benjamin Prud’homme
Transcriptomic atlas of mushroom development highlights an independent origin of complex multicellularity
Krisztina Krizsan, Eva Almasi, Zsolt Merenyi, Neha Sahu, Mate Viragh, Tamas Koszo, Stephen Mondo, Brigitta Kiss, Balazs Balint, Ursula Kues, Kerrie Barry, Judit Cseklye, Botond Hegedus, Bernard Henrissat, Jenifer Johnson, Anna Lipzen, Robin A. Ohm, Istvan Nagy, Jasmyn Pangilinan, Juying Yan, Yi Xiong, Igor V. Grigoriev, David S. Hibbett, Laszlo G. Nagy
Systematic Characterization of RhoGEF/RhoGAP Regulatory Proteins Reveals Organization Principles of Rho GTPase Signaling
Paul Markus Mueller, Juliane Rademacher, Richard D Bagshaw, Keziban Merve Alp, Girolamo Giudice, Loise E Heinrich, Carolin Barth, Rebecca L Eccles, Marta Sanchez-Castro, Lennart Brandenburg, Geraldine Mbamalu, Monika Tucholska, Lisa Spatt, Celina Wortmann, Maciej T Czajkowski, Robert William Welke, Sunqu Zhang, Vivian Nguyen, Trendelina Rrustemi, Philipp Trnka, Kiara Freitag, Brett Larsen, Oliver Popp, Philipp Mertins, Chris Bakal, Anne-Claude Gingras, Olivier Pertz, Frederick P Roth, Karen Colwill, Tony Pawson, Evangelia Petsalaki, Oliver Rocks
Kilohertz frame-rate two-photon tomography
Abbas Kazemipour, Ondrej Novak, Daniel Flickinger, Jonathan S Marvin, Jonathan King, Philip Borden, Shaul Druckmann, Karel Svoboda, Loren L Looger, Kaspar Podgorski
Precise tuning of gene expression output levels in mammalian cells
Yale S. Michaels, Mike B Barnkob, Hector Barbosa, Toni A Baeumler, Mary K Thompson, Violaine Andre, Huw Colin-York, Marco Fritzsche, Uzi Gileadi, Hilary M Sheppard, David JHF Knapp, Thomas A Milne, Vincenzo Cerundolo, Tudor A Fulga
Reproducible big data science: A case study in continuous FAIRness
Ravi K Madduri, Kyle Chard, Mike D’Arcy, Segun C Jung, Alexis Rodriguez, Dinanath Sulakhe, Eric W Deutsch, Cory Funk, Ben Heavner, Matthew Richards, Paul Shannon, Gustavo Glusman, Nathan Price, Carl Kesselman, Ian Foster
Applications are invited for a four-year funded PhD studentship to conduct research in developmental neurobiology under the supervision of Dr Alexander Fletcher and Prof. Marysia Placzek at the University of Sheffield.
This project is on the development of the hypothalamus, a brain structure with very similar anatomy across vertebrate species. We know that the hypothalamus is very important for mediating physiological homeostasis, yet its development remains poorly understood. This project will address this through a combination of gain-and loss-of function studies in vivo and ex vivo 3D culture with computational modelling.
This project is a great opportunity for a student interested in developmental neurobiology, who is keen to tackle new techniques and work in a truly interdisciplinary environment, acquiring programming and modelling skills and gaining expertise in tissue culture and imaging.
A four-year fully-funded EPSRC studentship is available to home students (British or EU nationality based in the UK) starting in October 2018. Please get in touch with Alexander Fletcher (a.g.fletcher@sheffield.ac.uk) for more details.
(No Ratings Yet)
Loading...
(4 votes)
(13 votes)
