Mechanical regulation of cell division in developing tissues: Speed Vs Strength

During embryogenesis, dynamic mechanical forces act on developing tissues, inducing cellular mechano-responses. These changes in cellular behaviours such as cell division, adhesion, and motility are a vital aspect of tissue morphogenesis and homeostasis.

This summer, I was given the opportunity to work under Dr. Woolner at the University of Manchester’s Division of Cell Matrix Biology & Regenerative Medicine. Using a multidisciplinary approach, Dr. Woolner’s lab examines the cellular response of developing tissues to an applied mechanical force and seeks to identify the underlying molecular basis. This placement was an incredible and unique opportunity for me, as I was able to receive training and experience in a variety of new techniques used in biomechanics, mathematics, biomodelling and developmental biology.

Previous work by the Woolner lab demonstrated that the rate of cell division increases in epithelial cells following the application of a low-magnitude, uniaxial tensile force¹. Work in other systems has shown that similar mechanically-induced increases in proliferation occur due to upregulation of the ERK1/2 pathway downstream of the stretch-activated calcium channel Piezo1, culminating in an upregulation of cyclin B². Additionally, it is known that the orientation of cell division aligns with the axis of stretch¹.

However, all current studies investigating the cellular response to tensile force involve rapid, instantaneous tissue stretching. Under physiological conditions, changes to mechanical tension in the developing embryo occur over a period of minutes to hours rather than seconds. In tumourigenesis, mechanical changes may take place over years. It is not currently known how cell division rate differs between fast and slow stretch regimes. Preliminary work suggests that slow-stretch regimes may not elicit the same division responses that are seen with instantaneous stretching.

My project aimed to help shed light on whether the speed or strength of an applied mechanical force is the major factor in altering cell division rate.

Using a tissue stretching apparatus, we applied an instantaneous, uniaxial stretch with reduced strength to tissues. For these experiments, Xenopus laevis embryonic tissue was used. Xenopus laevis embryos are a robust model organism for use in biomechanical research as they are large, develop externally and are easily visualised. I was very grateful for the opportunity to shadow members of the lab working with the Xenopus colony throughout the project. They are a unique model animal (I also have a few as pets!) and it was great to see how they are cared for and used responsibly in a research setting.

In order to visualise the cell edge and nucleus, Xenopus embryos were injected at 2-cell stage with GFP-tubulin and Cherry-histone RNA. Straight away I was given the chance to jump in and get involved with the experiments, as I helped Gina (the Woolner Lab’s Research Assistant) with DNA miniprep and mRNA preparation. We proceeded with microinjection, which involved inserting a microscopic needle tip into each cell under an optical microscope. This was a very tricky procedure at first but by practicing alongside Gina, I was eventually able to go from struggling to inject 10 embryos in an hour to injecting over 50 in half the time!

Following overnight incubation, embryos were staged at early gastrula and the animal caps were dissected. Isolated animal cap explants are a versatile tissue able to survive and develop ex-vivo, making them ideal for live imaging. Dissecting the animal cap was done through an optical microscope using two sets of forceps. This was the most technically challenging aspect of my lab work, as it required a steady hand and patience but couldn’t be done too slowly or the embryos would become too developed. It was very rewarding to eventually get a perfect set of animal cap explants.

Following incubation on a fibronectin-coated silicone membrane, the animal caps were stretched and imaged. Shown in Figure 2 is a single frame from one of our live movies captured using confocal fluorescence microscopy. This was great experience, as imaging science was always of great interest to me but I had never previously had the chance to put my theoretical knowledge into practice. I also used image analysis software to calculate the mitotic index, as well as try cell population tracing. The Woolner lab uses tracing alongside vertex modelling^3,4 to measure cell shape and infer mechanical stress across the tissue. The data collected during my project will be used to determine whether an increased cell division rate acts to relieve tensile stress across the tissue.

Alongside my core project work, I also successfully titrated the CDK-1 inhibitor RO-3306 to find the optimal concentration for cell division inhibition in Xenopus embryos. It is currently known that mechanical tension may increase cell division in fast-stretch regimes by promoting G1 to S phase transition⁵, which the Woolner lab will be investigating in slow-stretch regimes using a Fucci probe coupled with RO-3306 inhbition. Towards the end of my studentship, I was really grateful to have the opportunity to attend the 18^th International Xenopus Conference. This was a great chance to discover the wide array of biomedical research using Xenopus currently being conducted worldwide and make valuable connections.

I would like to thank everyone for all their support, guidance, patience and coffee & cake sessions throughout the internship. I am very grateful that I was able to receive the Gurdon/BSDB Summer Studentship and would recommend any student interested in developmental biology research to apply. Gaining first-hand lab experience in this field has given me invaluable skills and insight and has opened many doors for my future career.

References

1. Nestor-Bergmann A., Stooke-Vaughan G.A., Goddard G.K., Starborg T., Jensen O.E. and Woolner S. (2019) Decoupling the roles of cell shape and mechanical stress in orienting and cueing epithelial mitosis. Cell Reports 26: 2088-2100
2. Gudipaty S.A., Lindblom J., Loftus P.D., Redd M.J., Edes K., Davey C.F., Krishnegowda V., Rosenblatt J. (2017) Mechanical stretch triggers rapid epithelial cell division through Piezo1. Nature 543, 118-121.
3. Nestor-Bergmann A., Goddard G., Woolner S. and Jensen O.E. (2017) Relating cell shape and mechanical stress in a spatially disordered epithelium using a vertex-based model. Mathematical Medicine and Biology 35 (Supplement 1): 1-27
4. Jensen O.E., Johns E. and Woolner S. (2020) Force networks, torque balance and Airy stress in the planar vertex model of a confluent epithelium. Proceedings of the Royal Society A 476: 2237
5. Benham-Pyle B.W., Pruitt B.I and Nelson W.J. (2015) Mechanical strain induces E-cadherin-dependent Yap1 and β-catenin activation to drive cell cycle entry.  Science 348(6238): 1024–1027.

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Numerous efforts have been made to establish bona fide iPSCs from companion animals such dogs and cats. Generation of iPSCs from companion animals would provide useful unrestricted cell resources with a vast scientific potential. To name a few applications, they can be exploited as new models for regenerative medicine and as therapeutic veterinary tools to replace tissues; in veterinary pharmacology for drug development assays, and to elucidate function(s) of genetic variants that are associated with disease.

While protocols for producing human and mouse iPSCs are established, protocols for derivation of iPSCs from domestic animals are slowly developing due to difficulties encountered presumably in their reprogramming process. Only a few studies have indeed focused on the possibility of producing iPSCs from these companion species, and despite some describing their production, the burden of proof is largely lacking.

As an undergraduate student at the University of Edinburgh, this summer despite the current pandemic situation making it harder to find a lab-based studentship, I was lucky enough to have the opportunity to work in Drs. Schoenebeck’s and Burdon’s labs. The Gurdon/BSDB award allowed me to spend two months at Roslin Institute, a pioneering center for genetics and stem cell studies, collaborating with research groups with extensive experience in stem cell research (Dr. Tom Burdon’s) and canine genetics and genomics (Dr. Jeffrey Schoenebeck’s). During my time at Roslin I contributed to the research of an efficient protocol to derive canine iPSCs, supervised by the joint effort of these two excellent lab groups.

Based on the findings that iPSC generation is enhanced by P53 suppression and replacement of L-MYC with C-MYC(Okita et al., 2011) in the set of conventional reprogramming factors (OCT4, SOX2, KLF4, and C-MYC, collectively termed “OSKM” factors); recently, Yoshimatsu et al. (2021) have presented a study which provides insights on the possibility to facilitate canine cell reprogramming. They provided evidence of reprogramming somatic fibroblasts from a canine using an integration-free method. Their 8 episomal (Figure 1) vectors contain the OSKM factors including L-MYC, other pluripotency genes (LIN28 and NANOG), genes that have been shown to facilitate reprogramming (GLIS1and KDM4), and a dominant-negative form of the mouse TRP53 (mP53DD), which was shown to suppress endogenous P53 expression in human cells, and presumably should operate the same in canines.  

The episomal vectors contain OriP/EBNA1 sequences derived from Epstein-Barr virus (EBV), which ensure the stable extrachromosomal replication of the vectors, hence high expression of the reprogramming factors carried along, which facilitate the production of iPSC. However, the full applicability of this EBV-based system is still unclear as only two dogs were used to prove its actual functionality. 

The aim of my project was to assess the ability of the aforementioned system to reprogram canine fibroblasts, testing the capability of facilitating reprogramming by the inclusion of dominant-negative P53. After being introduced to the fundamental cell culturing techniques and practiced such skills on mouse feeder cells, I expanded canine fibroblast from testis in feeder medium prior to transfection of the EBV-based vectors. I then electroporated such cells with 2 different mixtures of vectors, one consisting of the 8 plasmids including the dominant-negative P53 (+mP53DD), and the other without it, consisting of 7 plasmids (-mP3DD). Right after transfection the medium used to feed the cells was changed to M10. Since one of the transfected vectors carried EGFP, I took GFP imaging to directly assess if the transfection was successful, comparing the transfected fibroblast with the non-transfected control (NTC). Images (Figure 2) show a high extent of cell death following electroporation of the cells, while GFP expression in a high proportion of the survived cells indicate uptake of the vectors. Cell recovered and showed prolonged GFP expression until day 14.

Following pre-expansion for 8 days after transfection, fibroblasts were transferred onto STO feeder cells and changed medium with NSM for induction of iPCS colonies. During reprogramming I sampled cells periodically to harvest their RNA (days 4, 8, and 14). From such RNA samples I obtained cDNAs that I used to perform subsequent RT-qPCR analyses. Using canine specific primers – some of which I personally designed and formerly validated – for endogenous expression of pluripotency markers (POU5F1, NANOG, and SOX2) and other genes of interest (CDH1, CD44, CDNK1A), I was able to assess the reprogramming status of the cells during the process.

Changes in expression of two markers of reprogramming (CDH1 and CD44) was consistent with what shown in another study (O’Malley et al., 2013). CDH1 (E-cadherin) is upregulated (Figure 3A) indicating mesenchymal-to-epithelial transition which is a typical behavior of the cells entering reprogramming. Furthermore, consistently with O’Malley et al. (2013) CD44 was found to be upregulated at day 4 and progressively downregulated passing the time (Figure 3B), the final population of iPSC are expected to be indeed CD44^–.

Unfortunately, I was not able to identify any iPSC colony by day 14, as showed in the Yoshimatsu et al. (2021) study, or later in time under either condition (+/- mP53DD). Upregulation of the core pluripotency markers POU5F1, NANOG, and SOX2 during the experiment, demonstrate the ability of the used EBV-based vector system to induce endogenous expression of pluripotency genes in canine cells; however, such expression dissipates throughout time (Figure 4).  The reason I could not obtain any iPSC colony might be that this vector system was shown to not maintain sustained enough endogenous expression of the pluripotent genes to overcome the full barrier of reprogramming. 

The two vector mixtures +/- mP53DD did not show distinguishable effects, since CDNK1A, direct target of P53showed no difference in expression levels between the two conditions (Figure 5). This suggest that either mP53DDwas expressed at not effective levels or not at all, or that this dominant-negative form of P53 do not interact with the canine form of P53.

Ultimately, this project surely helped to broaden my knowledge in stem cells and reprograming methods, as to learn numerous lab techniques fundamental to pursue hopefully my research career in the future.

References:

• O’Malley, J. et al. (2013) High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.
• Okita, K. et al. (2011) A more efficient method to generate integration-free human iPS cells. Nature methods. [Online] 8 (5), 409–412.
• Yoshimatsu, S. et al. (2021) Non-viral Induction of Transgene-free iPSCs from Somatic Fibroblasts of Multiple Mammalian Species. Stem cell reports. [Online] 16 (4), 754–770.

(1 votes)

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Maria Diaz de la Loza is an Andalusian researcher and scientific illustrator who has worked in genomic instability and development in both Spain and the UK. Her passion for science has been a huge artistic inspiration and has led to her illustrations and videos being used in scientific publications and events. She is currently working in London as a freelance illustrator and fly technician at King’s College London. Her work can be seen at http://be.net/MariaDiaz_delaLoza and you can follow her on Twitter at @Maria_Diaz_Loza

Where are you originally from and what do you work on now?

I am originally from Chipiona, a little town by the seaside in the South of Spain. Thanks to the support of my family, I was able to move to the capital of my region to complete my degree in Biology followed by a PhD in Genetics, studying genomic instability in yeast, at the University of Seville. I was ready for a change for my first postdoctoral position, and I moved to a new field to work on development in the fruit fly, in the Andalusian Centre for Developmental Biology. That was a life changing decision. The fly community is remarkably creative, and it truly inspired me to further apply my artistic side to science. For my second and last postdoc, I moved to London, to the new Francis Crick Institute, where I continued working in development. These years as a scientist put me in contact with many different (and amazing) people, mentors and experiences, and they inspired me to pursue a career that combined my two sides: art and science. Now, I work as a freelance illustrator, collaborating with other scientists around the world to illustrate their work. I find it extremely satisfying. I did not want to completely leave academia, and I am lucky to maintain a direct contact with the research community by working as a fly technician at King’s College London, where my supervisor is delighted with my illustrator side and gives me the flexibility that I need to balance my two jobs.

Were you always going to be a scientist?

I always wanted to be a biologist (although, at nine years old, I also fancied the idea of being a vet or a hairdresser). Being in close contact with the sea all my life, I thought I would specialise in marine biology. However, after taking my first genetics class I was totally fascinated, and I started to take all the genetics subjects I could. It was after getting in closer contact with that community that I considered the possibility of starting a PhD, and I was lucky they gave me that opportunity in the Department of Genetics of the University of Seville. Even though I started my PhD without a clear idea of how an academic career works, I found that I liked the environment a lot and I have enjoyed many aspects of it since. Being a scientist is not only about science, it allows you to meet all kind of people, to see the world and to grow as a person, and of course you gain an incredible number of new skills, so I will say it is a path worth taking.

And what about art – have you always enjoyed it?

Absolutely! I have been creating and drawing since I was a little. My mother was an avid reader who liked to copy illustrations from her favourite books, and she always encouraged me to do both. I continued drawing through school as a way to learn and embellish my homework, and I started to learn what I could about visual arts by myself. I even had a moment of doubt, just before starting University, when I played with the idea of enrolling into an Art Bachelor. In the end, biology won – I wanted to do that my entire life – but I never stopped painting and drawing. During my PhD, it was a pleasant surprise to discover how both science and art complement each other. Science is more easily understood with the help of an image, and illustrations are not only useful for communicating your work to others, but also an excellent tool to improve our insight into the process you are studying. I can say that through science, I continued learning and developing my artistic skills.

“During my PhD, it was a pleasant surprise to discover how both science and art complement each other.”

What or who are your most important artistic influences?

Absolutely everything! I get obsessed with anything that catches my eye and learn from it. Sometimes it is about visual arts, mostly classic and modern painters, or some visually attractive movies. Another major influence is nature itself and certain urban landscapes; I am completely fascinated with the amazingly eclectic London architecture since I moved here. I usually take photographs that I can use for future projects, and I often navigate through them to come up with ideas. Same goes for science, as a developmental biologist I have always worked with microscopy images, and I learn a lot from other people’s work. But inspiration can be found everywhere, sometimes I spend hours watching crafts, makeup, or tattoo videos, and very often I have used everyday objects to come up with a design. I think you can pick up ideas from enjoying any kind of artistic manifestation, which later, can offer you exactly what you need to make a design.

How do you make your art?

As a freelance illustrator, what really fulfil me is the process of illustrating someone else’s work and making it beautiful and precise.  The process usually starts by having a chat with the authors to understand their work and the general idea of what they want (which is made easier by my scientific background). Then I start to think about alternative options. At some point, there it is the perfect one. The drawing starts then. First, I prepare some pen and paper sketches, to show my concept to the client, and once we have decided the major points, I move to digital platforms. One of the great aspects of working with other scientists is that they are used to being very clear and concise in what they want. This means my preliminary ideas are usually well received, and the first feedback from the authors helps improved them substantially. The process continues with some back and forth with the authors, to be sure that I am showing exactly what they want. The final product usually consists of digital diagrams and illustrations for publications, journal covers or events, but I also like to combine them with different media. One of my favourite techniques is to take real photographs of everyday objects and combine them to illustrate a biological process as an analogy. Recently I have had some fun working with several labs to create video abstracts, and I am eager to explore more in that direction.

Does your art influence your science at all, or are they separate worlds?

I think that what inspires me the most to create art, is to apply it to something practical. When I started in academia, art and science started to evolve together and now my artistic side is almost completely focussed on science. I chose to study development because it is strongly based on imaging (I was jealous of their microscopy images!), and I used my artistic side to improve the way I acquire and show my results. Also, I think designing diagrams was a deal-breaker for me. I started to create my own diagrams early on during my PhD, primarily to try to understand what I was doing, but also to help to explain my work to others, and with time I started to do the same for my colleagues. Now I barely attend any kind of talk without drawing their ideas on my notebook. Working with scientific images also helped me to better understand photography, specially of very tiny things, and my connection with other laboratories is very helpful to get specific material for my illustrations. The last step was to officially combine them both, and I am extremely grateful that I have been able to do this during the last two years.

What are you thinking of working on next?

I always wonder how it would be to work in illustration full-time; for a journal or a company. Being a freelancer allows you to interact with people around the world and learn about many different projects, which is exciting and it is something that I will miss for sure. However, I have learnt a lot from other illustrators during my freelance stage, and I think working in a department dedicated to science illustration could improve my training further. For now, I am thrilled of working in different projects as a freelancer, but who knows what the future has in store for me!

Thanks to Maria and all the other SciArtists we have featured so far. We’re looking for new people to feature in this series – whatever kind of art you do, from sculpture to embroidery to music to drawing, if you want to share it with the community just email thenode@biologists.com (nominations are also welcome!)

(9 votes)

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Can you hack your hormones to be happier or boost your height? Could a hormone supplement be the key to beating ageing? Are humans heading for a fertility cliff? And is the ‘manopause’ real?

Hormones: The Inside Story, the podcast from the Society for Endocrinology uncovering the facts about hormones and health with an expert-led, myth-busting and entertaining format, is back for a second series.

Hormones affect growth, sleep, body fat, fertility and almost every aspect of our daily lives and health. Sadly, the mainstream media is brimming with misinformation and potentially dangerous advice from a host of non-experts and dubious commercial enterprises.

Building on the huge success of last year’s debut series, the Society for Endocrinology and First Create The Media have just released series two, which continues examining the stories and the science behind hormones, cutting through the myths and misinformation, providing real facts and enabling you to make better decisions about your health.

With the help of presenter Georgia Mills, this series uncovers the truth about how hormones affect our growth, weight, mood, how we age and our declining fertility. Speaking with leading experts, she’ll be finding out about the controversies around the male menopause, fasting and weight loss, whether there really is a fertility crisis and if you can beat the aging process or boost your happiness by hacking your hormones.

Tune in to learn how monkey testicles could link to the fountain of youth, why Irish giants are not just the stuff of legend, the secrets of lengthy prairie vole ‘romance’ and how brushing your teeth could save your life.

Listen and subscribe now on Podbean, Spotify, Apple Podcasts or just search for ‘Hormones: The Inside Story’ wherever you get your podcasts.

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How to apply: https://www.bmh.manchester.ac.uk/study/research/bbsrc-dtp/apply

• Application deadline: 21 January 2022
• Interviews: week commencing 7 March 2022

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Faculty of Biology, Medicine and Health, The University of Manchester

PhD Research Project Competition Funded Students Worldwide

Andreas Prokop  

Viki Allan

Matt Ronshaugen

Details

Axons are slender extensions of neurons which can be meter-long and form the biological cables that run through our nerves and brains to hardwire the nervous system. In humans, axons must survive for up to a century; we lose ~40% of axons towards high age and many more in neurodegeneration, but the causes are poorly understood.

To understand axon maintenance and pathologies, we are focussing on the bundles of microtubules (MTs) that run all along axons – be it in a tiny fly or in a human [Ref1]. These bundles determine axon structure and form the highways for life-sustaining axonal transport. Accordingly, MT bundle decay causes axon degeneration. But the mechanisms that maintain these bundles (and might fail in pathology!) are little understood [Refs2,3].

Here we will test the long-standing, but poorly proven hypothesis that axonal bundles are cross-linked by MT-binding proteins. For this, we will study potential architectural functions of MAP1B/Futsch proteins, known to be enriched in axons across the animal kingdom [1]. They show an intriguing evolutionary profile: N- and C-terminal regions are potentially MT-associating and evolutionary well conserved, whereas middle regions show enormous sequence and length differences [FigS1 in Ref1]. MAP1B/Futsch proteins may therefore act as flexible spacers, with their length differences explaining the variations in MT spacing observed in different species [1].  

To study MAP1B/Futsch, you will use inter-disciplinary experimental approaches that equip you with a wide range of skills relevant for the biomedical sciences and evolution biology. (a) To determine the precise sub-cellular localisation of MAP1B/Futsch you will use CRISPR/Cas9-mediated protein tagging and apply expansion/electron microscopy; molecular mechanisms will be determined via biochemical and in vitro assays. (b) To study MAP1B/Futsch family evolution you will use computational bioinformatics retrieving and analysing Futsch sequences from multiple species. (c) To determine the functional consequences of evolutionary variability you will generate hybrid proteins and assess their impacts on axon architecture.

The project will be supervised by experts in the field. Andreas Prokop has studied the Drosophila nervous system for 30 years, has long-standing experience with electron microscopy, and is also an expert in science communication (https://poppi62.wordpress.com/publications). Viki Allan studies MT-based transport with expertise in biochemical and in vitro assays to dissect functions of MT-associating proteins [Ref4]. Matthew Ronshaugen specialises on evolutionary biology and his lab is equipped to perform the computational analyses and generate CRISR/Cas9 variants [Ref5].

Entry Requirements

Applicants must have obtained or be about to obtain a First or Upper Second class UK honours degree, or the equivalent qualifications gained outside the UK, in an appropriate area of science, engineering or technology.

Applicants interested in this project should make direct contact with the Primary Supervisor to arrange to discuss the project further as soon as possible.

Equality, Diversity and Inclusion

Equality, diversity and inclusion is fundamental to the success of The University of Manchester and is at the heart of all of our activities. The full Equality, diversity and inclusion statement can be found on the website.

Funding Notes

Funding will cover UK tuition fee and stipend only. The University of Manchester aims to support the most outstanding applicants from outside the UK. We are able to offer a limited number of scholarships that will enable full studentships to be awarded to international applicants. These full studentships will only be awarded to exceptional quality candidates, due to the competitive nature of this scheme.

References 1. Prokop, 2020, J Cell Biol 219, e201912081ff. — https://doi.org/10.1083/jcb.201912081
2. Prokop, 2021, Cytoskeleton 78, 52ff. — https://doi.org/10.1002/cm.21657
3. Hahn et al., 2019, Neural Dev 14, 10.1186/s13064ff. — https://doi.org/10.1186/s13064-019-0134-0
4. Korabel et al., 2018, PLoS One 13 e0207436 — https://doi.org/10.1371/journal.pone.0207436
5. Gallicchio et al., 2020, Genes|Genomes|Genetics 11 — https://doi.org/10.1093/g3journal/jkaa010

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How to apply: https://www.bmh.manchester.ac.uk/study/research/mrc-dtp/apply/

• Application deadline: 12 November 2021
• Interviews: week commencing 10 January 2022 

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Faculty of Biology, Medicine and Health, The University of Manchester

PhD Research Project Competition Funded Students Worldwide

Martin Humphries

Andreas Prokop

Sid Banka

Tom Millard

Details

Background: We discovered that integrins and Rac1, form a pathway essential for axon growth during normal nervous system development. Axon growth establishes connections between neurons across the nervous system, essential for normal cognitive function.  Accordingly, integrin mutations cause intellectual disability in mouse and humans [1,2], and we discovered that germline RAC1 mutations cause a human genetic disorder likewise characterised by intellectual disability [3].

Aims: In this project the student will identify further components of the integrin-Rac1 pathway involved in axonal growth, establish their potential disease links and test therapeutic strategies.

Methods:  

(a)  The student will identify further axon growth-relevant components of the integrin-Rac1 pathway using mass-spectrometry experiments and their bioinformatic analysis.

(b)  Candidate proteins will be experimentally assessed in neuronal cell culture and in vivo to assess whether and how they function within the integrin-Rac1 pathway; this will involve extracellular matrix extraction, primary neuron cultures, brain dissections, Drosophila genetics and advanced imaging.

(c)  The student will identify disease-relevant mutations in integrin-Rac1 pathway components through interrogating large human genomic datasets via computational bioinformatics analysis and clinical correlation.

(d)  Through in vivo studies, the student will establish whether/how candidate proteins impact on axon growth leading to miswiring as a cause for intellectual disability.

(e)  The student will have the opportunity to test approved drugs to assess potential ameliorating effects on aberrant axon growth caused by these mutations.

Training: The experimental approaches used are highly inter-disciplinary and will equip the student with a wide range of skills relevant for the biomedical sciences. To guarantee high quality training and optimal progress, the project will be supervised by experts in the field. Martin Humphries is an expert on integrins and the mass-spectrometry analysis of integrin complexes [4]. Andreas Prokop has 30 years experience of applying genetics, cell biology and imaging approaches to study the Drosophila nervous system, integrins and the cytoskeleton [5], and is also an expert in science communication (https://poppi62.wordpress.com/publications); he has established the cellular model used here. Siddharth Banka is an expert in human genetics, analysis of the human genome and has discovered more than 20 novel human disorders [3]. Tom Millard is highly experienced with Drosophila genetics, molecular cloning and imaging and has established a set of disease-relevant Rac1 mutations available for this project [3].  

Entry Requirements

Applicants must have obtained or be about to obtain a First or Upper Second class UK honours degree, or the equivalent qualifications gained outside the UK, in an appropriate area of science, engineering or technology.

Equality, Diversity and Inclusion

Equality, diversity and inclusion is fundamental to the success of The University of Manchester, and is at the heart of all of our activities. The full Equality, diversity and inclusion statement can be found on the website https://www.bmh.manchester.ac.uk/study/research/apply/equality-diversity-inclusion

Funding Notes

Funding will cover UK tuition fee and stipend only. The University of Manchester aims to support the most outstanding applicants from outside the UK. We are able to offer a limited number of scholarships that will enable full studentships to be awarded to international applicants. These full studentships will only be awarded to exceptional quality candidates, due to the competitive nature of this scheme.

References

[1] Schuch et al., 2014, Gene 553, 24ff. — https://doi.org/10.1016/j.gene.2014.09.058
[2] Ellegood et al., 2012, Front Psychiatry 3, 10.3389/fpsyt.2012.00037ff. — https://doi.org/10.3389/fpsyt.2012.00037
[3] Reijnders et al., 2017, Am J Hum Genet 101, 466ff. — https://doi.org/10.1016/j.ajhg.2017.08.007
[4] Chastney et al., 2020, J Cell Biol 219, ff. — https://doi.org/10.1083/jcb.202003038
[5] Qu et al., 2019, eLife 8, e50319ff. — https://doi.org/10.7554/eLife.50319

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Welcome to our light-hearted look at the goings on in the world of developmental biology in the last two weeks (or so).

COP26 related news

This news is not all from the last two weeks, but relates to the COP26 meeting in Glasgow

• New study shows only 4% of biotech and pharma companies are on track to meet Paris 2030 climate goals

https://www.mygreenlab.org/blog-beaker/my-green-lab-measures-carbon-impact-of-biotech-and-pharma

• Nobel laureates call for action

https://www.nationalacademies.org/news/2021/04/nobel-prize-laureates-and-other-experts-issue-urgent-call-for-action-after-our-planet-our-future-summit

• The Sustainable Conferencing Initiative from The Company of Biologists

https://sustainability.biologists.com/blog/why-you-should-choose-a-train-journey-instead-of-a-flight/

Find out more about the initiative when Viktoria talks to the Caring Scientist podcast

• $10 million grant for research into lab-grown meat

https://www.eurekalert.org/news-releases/933866

Talking points on Twitter

• The DEVIL is in the detail

• When your gels are not smiling on you #GelsofTwitter

• What to look for in your next lab (or best practice when hiring)

prelight in #devbio

Shuttling centrioles down the nose – local maturation for multiple sensory cilia formation

CHD8 haploinsufficiency could be responsible for both autism and leaky gut in mice

Thanks to the #DevBio community for sharing their thoughts, especially on twitter. If you have some news that you think we should share on our blog, please get in touch at thenode@biologists.com. If you are interested in getting involved with writing preLights you can find out more here.

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Doing great science depends on teamwork, whether this is within the lab or in collaboration with other labs. However, sometimes the resources that support our work can be overlooked. In our new series, we aim to shine a light on these unsung heroes of the science world. The first article in the series is by Lindsay Henderson (NARF Academic Liaison and Post-Doctoral Scientist at Roslin Institute) who describes the work of the National Avian Research Facility, UK.

What is the NARF and where is it based?

The National Avian Research Facility (NARF) was founded in 2013 and is based at The Roslin Institute, on the University of Edinburgh’s Easter Bush campus, UK. The NARF produces and maintains poultry lines for use in scientific research and consists of two units, a conventional facility and a facility that has specified pathogen free (SPF) status.

What services does the NARF offer for developmental biologists?

The NARF supplies fertile eggs from our range of chicken lines that have a broad utility in developmental biology. These include, commercial layer and broiler chicken lines, and Japanese quail.

Researchers within the Roslin Institute Chicken Embryology (RICE) group have generated a number of genetically-altered (GA) fluorescent reporter chicken lines that are valuable tools for developmental biology². These lines carry fluorescent reporter transgenes that are expressed ubiquitously or are inducible in cells of the developing embryo and are used for fate mapping, cell lineage tracing and tissue grafting. At this time the NARF has three ubiquitous reporter chicken lines; the Roslin Green (eGFP)³, Flamingo (TdTomato)⁴ and Membrane GFP⁵, and an inducible reporter chicken line, the Chameleon (Cytbow)².

The NARF also maintains fluorescent reporter chicken lines that are gene specific. This includes a number of immune cell reporter lines, such as the CSF1R-reporter lines⁶, that can be used to visualise immune cells, including macrophages and dendritic cells.

The NARF also holds a spontaneous mutation line, the talpid³ line that is a classical recessive embryonic lethal chicken mutant with abnormal limb patterning and malformations^7–9. The talpid³ line has provided direct insights into clinical genetics and specifically the genes responsible for limb abnormalities and ciliopathies in humans^9,10.

The NARF maintains one of the only SPF inbred lines, Line 0, that is free of Avian Sarcoma-Leukosis Virus (ASLV) ev loci. Embryos from this line may be particularly suitable for research using RCAS retrovirus vectors¹¹.

Who are the developmental biologists based at the Roslin Institute that use the NARF?

RICE is a collective of research groups based at the Roslin Institute that use the chicken embryo to study vertebrate embryonic growth and development. RICE includes; the Balic Group that has produced the first transgenic chicken lines which allow specific sub-population of chicken immune cells to be visualised. The Davey Group examines the causative alterations of gene expression which lead to variations in phenotype using comparative anatomy, genomics and embryonic manipulation of avian species, with a particular interest in the molecular anatomy of the developing limb bud. The Headon Group focuses on the development, maintenance and repair of the skin and its appendages. The Rainger Group’s research is focused on the fusion of tissues in the developing retina using the chicken embryo as an experimental model system. The McGrew Group has made major advances in and continues to explore gene editing of avian germ cells and biobanking. The Clinton Group investigates the molecular control of sex determination and gonadal development, and the mechanisms underlying sexual dimorphisms in birds. The Sang Group has a broad focus on the development and application of technologies for genome engineering of the chicken.

What are the strengths of the chick model system in today’s developmental biology?

Recent advances in genome modification in the chicken led by Mike McGrew using the NARF facilities, have made possible the rapid and cost-effective production of GA chicken lines. In addition to new methods to create precise and targeted gene modifications, like CRISPR/Cas9 technologies, this greatly advances the potential of gene editing in chickens for developmental biologists. These advances in gene editing and transgenesis in conjunction with the experimental accessibility of chick embryos, and the ease of live culture and imaging, make the chick a powerful model for future developmental biology research.

Gene editing in chickens can be used to create targeted mutations, to knock-out genes of interest and enable inducible ablation of cell types that contain specific genes¹². Recently, the NARF’s gene editing technologies have made possible research investigating avian sex determination using targeted mutations in the DMRT1 gene¹³. Gene editing in the chicken will soon be used to explore the genes responsible for defects of the optic fissure closure in the eye that cause ocular coloboma¹⁴.

How can researchers get involved with the NARF?

The NARF can supply fertile eggs for research and can supply a quote for eggs for inclusion in grant applications. Please find the full list of the lines we provide here. To order eggs from any of the poultry lines or to obtain costings for grant applications, contact narf@roslin.ed.ac.uk.

We continue to develop new GA chicken lines to aid in research under both conventional and SPF conditions. We welcome new collaborations with developmental biologists based at other research institutes. If you have any questions or are interested in a collaboration please contact Lindsay Henderson the NARF Academic Liaison.

The NARF is supported by the University of Edinburgh and the UKRI-Biotechnology and Biological Sciences Research Council.

Publications

1. Macdonald, J. et al. Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc. Natl. Acad. Sci. U. S. A. 109, 8803 (2012).
2. Davey, M. G., Balic, A., Rainger, J., Sang, H. M. & McGrew, M. J. Illuminating the chicken model through genetic modification. Int. J. Dev. Biol. 62, 257–264 (2018).
3. McGrew, M. J. et al. Localised axial progenitor cell populations in the avian tail bud are not committed to a posterior Hox identity. Development 135, 2289–2299 (2008).
4. Ho, W. K. W. et al. Feather arrays are patterned by interacting signalling and cell density waves. PLoS Biol. 17, (2019).
5. Rozbicki, E. et al. Myosin-II-mediated cell shape changes and cell intercalation contribute to primitive streak formation. Nat. Cell Biol. 17, 397–408 (2015).
6. Balic, A. et al. Visualisation of chicken macrophages using transgenic reporter genes: Insights into the development of the avian macrophage lineage. Development 141, 3255–3265 (2014).
7. Ede, D. A. & Kelly, W. A. Developmental abnormalities in the head region of the talpid3 mutant of the fowl. J. Embryol. Exp. Morphol. 12, 161–182 (1964).
8. Ede, D. A. & Kelly, W. A. Developmental abnormalities in the trunk and limbs of the talpid3 mutant of the fowl. J. Embryol. Exp. Morphol. 12, 339–356 (1964).
9. Fraser, A. M. & Davey, M. G. TALPID3 in Joubert syndrome and related ciliopathy disorders. Current Opinion in Genetics and Development 56, 41–48 (2019).
10. Davey, M. G., Towers, M., Vargesson, N. & Tickle, C. The chick limb: Embryology, genetics and teratology. Int. J. Dev. Biol. 62, 253–263 (2018).
11. McNally, M. M., Wahlin, K. J. & Canto-Soler, M. V. Endogenous expression of ASLV viral proteins in specific pathogen free chicken embryos: Relevance for the developmental biology research field. BMC Dev. Biol. 10, 106 (2010).
12. Ballantyne, M. et al. Direct allele introgression into pure chicken breeds using Sire Dam Surrogate (SDS) mating. Nat. Commun. 12, 659 (2021).
13. Ioannidis, J. et al. Primary sex determination in birds depends on DMRT1 dosage, but gonadal sex does not determine adult secondary sex characteristics. Proc. Natl. Acad. Sci. 118, e2020909118 (2021).
14. Rainger, J. Novel approaches to define tissue fusion mechanisms in embryonic development. Funder: UK Research and Innovation Available at: https://gtr.ukri.org/projects?ref=MR%2FS033165%2F1.

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A press release from Universität Zürich on Lienkamp lab paper, published in Development

Using cutting-edge genetic engineering, UZH researchers have developed a model to study hereditary kidney disease with the help of tropical frogs. The method allows them to collect large amounts of data on anomalies, which can then be analyzed using artificial intelligence. The research opens up new opportunities in the search for new treatment approaches for the hitherto incurable disease.

Frogs’ anatomy and organ function are strikingly similar to those of humans. An international team led by Soeren Lienkamp, professor at the Institute of Anatomy at UZH, has now exploited this similarity by using a tiny tropical frog called Xenopus tropicalis to model human genetic diseases. The researchers focused on polycystic kidney disease, a congenital and currently incurable form of progressive kidney deterioration, and replicated it in frogs.

Observing disease processes in real time

Using CRISPR/Cas9, a methodology for turning off gene function, the scientists targeted genes known to play a role in cystic kidney disease. “Our novel frog models develop cysts in the kidneys within only a few days, allowing us to observe these disease processes in real time for the first time,” says lead author Thomas Naert. While most genetic studies are performed on mice, frogs have features that make them well-suited for larger scale studies. “One frog couple can produce hundreds or even thousands of eggs,” says Naert. “That’s why you see such large numbers of tadpoles in the Swiss lakes in springtime.” Similarly, in the lab large numbers of Xenopus tropicalis tadpoles can be manipulated to develop cystic kidney diseases.

AI analyzes data from light-sheet microscopy

To analyze the data from such a large number of animals, the team employed a technique called light-sheet microscopy, which produced a 3D reconstruction of the entire tadpole and all its organs. Much like magnetic resonance imaging, light-sheet techniques make it possible to see through tissues in tadpoles to find disease-affected organs. The collected data was then processed using artificial intelligence to allow rapid, automated assessment of disease. “While it would normally take my team several days or even weeks to analyze data from hundreds of tadpoles, artificial intelligence can now do this task in a matter of hours,” says Lienkamp.

The findings from frog models analyzed in this way provide new insights into the early processes of polycystic kidney disease. These insights will form the basis for developing new treatment approaches for affected patients.

Literature

Thomas Naert et al. Deep learning is widely applicable to phenotyping embryonic development and disease. Development (2021) 148 (21): dev199664.

Funding:

The study was funded by the Swiss National Science Foundation (SNSF), NCCR Kidney.ch, and ERC Horizon2020 (Starting Grant and Marie Skłodowska-Curie Program).

Contact

Prof. Soeren Lienkamp,

University of Zurich

Institute of Anatomy

Phone: +41 (0)44 635 53 48

E-mail: soeren.lienkamp@uzh.ch

Media Relations

University of Zurich

Phone: +41 44 634 44 67

E-mail: mediarelations@kommunikation.uzh.ch

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Many internal organs fill and empty periodically while carrying out their normal physiological function. Associated peripheral neurons act as specialised mechanoreceptors to detect changes in organ volume, and relay this information to the brain, where it is processed, and used to evoke appropriate physiological and behavioural response(s). For example, during a meal the stomach expands to accommodate ingested food, triggering neural circuits to inhibit feeding behaviour, promote digestion, and evoke feelings of satiety, fullness, nausea or pain, depending on the size of the meal. Despite being central to normal physiological function many basic questions remain about the mechanisms of organ volume sensing. 

In this project you will explore how mechanisms of gut volume sensing control physiology and life cycle in a classic insect model—the blood-sucking bug Rhodnius prolixus. You will use state-of-the-art techniques in genomics, imaging, genetic manipulation, and gene/protein expression analysis to identify the molecular and cellular mechanisms of mechanotransduction in the Rhodnius gut and define the relevant neural circuits that act to control profound changes in this creature’s physiology and development in response to feeding.  

This project is a curiosity-driven exploration into a fundamental question relating to how animals sense and respond to their internal world. The molecular mechanisms of organ volume sensing are not well understood for any animal, including humans, but are likely to be conserved.  Therefore, the project is likely to provide insight into physiological processes that are key for maintaining health in humans, illuminating areas relating to appetite, overeating and disorders connected to visceral pain. 

Specific details about the project: Rhodnius prolixus is a blood-sucking bug of immoderate feeding habits. It can take from its host a volume of blood sufficient to increase its own weight by about tenfold. The food-swollen gut distends the abdomen and sets in train a series of endocrinological processes that culminate in profound changes to the physiology and life cycle of the animal. These include (i) a rapid diuretic response, enabling the animal to jettison excess salts and water, returning the animal to a more comfortable state, and (ii) stimulation of body growth and maturation that precede the transition to the next life phase (an older nymph or metamorphosis to adult form). 

A major goal of the current project is to determine how abdominal distension is sensed and transduced to the brain to elicit such dramatic changes.

For more details please contact: Barry.Denholm@ed.ac.uk

Application Procedure 

Download application and reference forms from:

http://www.eastscotbiodtp.ac.uk/how-apply-0

Completed application form along with your supporting documents should be sent to our PGR student team at sbms-postgraduate@ed.ac.uk by 16 December 2021. References: Please send the reference request form to two referees. Completed references for this project should also be returned to sbms-postgraduate@ed.ac.uk by the closing date: 16 December 2021. 

It is your responsibility to ensure that references are provided by the specified deadline.

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