ABSTRACT

First Person is a series of interviews with the first authors of a selection of papers published in Journal of Cell Science, helping early-career researchers promote themselves alongside their papers. Nina Short and Max Butler are co-first authors on ‘Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure’, published in JCS. Nina is a Molecular and Cellular Bioscience MRes student at Imperial College London, UK, where she studies developmental biology and immunology. Max is a medical student at The University of Cambridge, UK, where his primary research interests are developmental biology and medical imaging.

Max Butler

Nina Short

How would you explain the main findings of your paper in lay terms?

N.S. and M.B.: In the early mammalian embryo, a flat area of cells stretching from the head to tail folds to form a closed tube called the neural tube. Failure of closure causes defects such as spina bifida. This study focused on the role of the enzyme Rho-associated protein kinase (ROCK) in closing the posterior neuropore (PNP), a region of the neural tube that develops into the spinal cord. Previous studies had shown that ROCK inhibition disrupted regulation of the actin cytoskeleton in future spinal cord cells, called the neuroepithelium. Specialised F-actin structures are known to facilitate PNP closure, including F-actin cables that extend along the neural folds. However, we did not know whether ROCK facilitates PNP closure by driving formation of these F-actin structures. This project evolved into three components. We initially used mouse embryo culture to assess the effects of ROCK inhibition on PNP morphology and F-actin (led by Nina). Finding that the F-actin cables require ROCK activity led us to interrogate force-generating mechanisms (i.e. biomechanics) during PNP closure, and we concluded that ROCK promotes neuroepithelial apical constriction (led by Max). Unexpectedly, we observed that apical constriction did not happen evenly in all cells, but preferentially in cells exiting from the M-phase of the cell cycle.

Were there any specific challenges associated with this project? If so, how did you overcome them?

N.S.: I started this project as a second-year undergraduate student, and as such it was my first experience of working in a biomedical research environment. During the project, I had to learn a number of diverse research techniques in a short period of time (embryo microdissection, confocal microscopy, image analysis and molecular biology techniques). The most challenging of these tasks was embryo microdissection for whole-embryo culture. This is quite a delicate procedure and required a lot of practice before I was happy with the results.

M.B.: Just like Nina, I began the project at the end of my second undergraduate year, and there was a steep learning curve to get the hang of different experimental techniques. In addition, as part of the project I intended to use architectural design programs to model groups of individual cells. Difficulties with immunostaining prevented this at first, but refining the staining techniques and image processing advice from Dr Galea meant I could eventually create the models.

When doing the research, did you have a particular result or ‘eureka’ moment that has stuck with you?

N.S.: To me, every result felt like a ‘eureka’ moment, because it was the first time that my experiments were answering questions that no one knew the answer to.

M.B.: Nina's work demonstrated that briefly culturing embryos in a ROCK inhibitor resulted in widening of the posterior neuropore. Further experiments led us to believe that the effects were cell cycle dependent. We decided to test this by co-culturing embryos in hydroxyurea, thinking that arresting the cell cycle could mitigate the effects of ROCK inhibition. I was anxious this would be too toxic for any cells to survive, but was delighted to find that this resulted in PNPs with a normal width!

Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure. (A) Illustrative examples of mouse PNPs (green circle on the embryo image) in embryos cultured for 8 hours in vehicle or with the Rock inhibitor Y-27632. The asterisk indicates the most recently closed portion of the neuropore (the ‘zippering point’). (B) Schematic summary of key data showing that the apical dimensions of PNP neuroepithelial cells decrease between early (pHH3+) and late M-phase (pS780-pRB+). This apical constriction event does not happen when Rock is inhibited, resulting in accumulation of cells with wide apical surfaces and widening of the PNP.

Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure. (A) Illustrative examples of mouse PNPs (green circle on the embryo image) in embryos cultured for 8 hours in vehicle or with the Rock inhibitor Y-27632. The asterisk indicates the most recently closed portion of the neuropore (the ‘zippering point’). (B) Schematic summary of key data showing that the apical dimensions of PNP neuroepithelial cells decrease between early (pHH3+) and late M-phase (pS780-pRB+). This apical constriction event does not happen when Rock is inhibited, resulting in accumulation of cells with wide apical surfaces and widening of the PNP.

Why did you choose Journal of Cell Science for your paper?

N.S. and M.B.: We chose JCS collectively. It is a journal we all knew publishes highly regarded papers and, being part of The Company of Biologists, supports the academic community (e.g. through travel grants). We felt that our manuscript offered a number of findings that would be of broad interest to cell and epithelial biologists across disciplines so wanted to make sure it was visible to a broad audience rather than a developmental biology-specific readership. JCS is particularly highly regarded for publishing microscopy-heavy papers and the studies we described have only recently become possible thanks to advances in imaging technology.

Have you had any significant mentors who have helped you beyond supervision in the lab? How was their guidance special?

N.S.: I received excellent support from my supervisor, Dr Gabriel Galea, as well as from other members of the team. Of particular note is Dr Lucy Culshaw, a PhD student at the time who was always happy to help me get to grips with different research techniques. Lucy was particularly skilled at microdissection, which was a major component of her PhD and happened to be the technique I found hardest to master.

M.B.: Rosie Marshall, a PhD student, gave me invaluable feedback regarding presenting my work, and useful advice about research careers. In addition, Dr Gabriel Galea has been very supportive; aside from patient lab supervision, he taught me a great deal about imaging techniques, and how to judge which farfetched ideas are worth pursuing!

What motivated you to pursue a career in science, and what have been the most interesting moments on the path that led you to where you are now?

N.S.: Watching nature documentaries as a child led me to develop a strong interest in science across scales. At the ecosystem level, I have worked at a nature reserve in the Seychelles monitoring and tagging turtle and sea bird populations. Back in the UK, I then worked on science policy and learned about the inner fabric of science funding. My lab-based research has allowed me to work on the molecular and cellular aspects of embryonic development, and motivated me to continue my research as a postgraduate student.

M.B.: My journey has been a little unusual; I did a degree in architecture before deciding to pursue a career as a clinician-researcher. My final-year thesis project involved the use of novel bio-materials to minimise construction waste, and it was through reading about tissue morphogenesis that I became fascinated by research and developmental biology. Later, while taking part in an observership programme at Great Ormond Street Hospital, I was inspired by the close relationship between the hospital and the research at the Institute of Child Health next door; I knew I had to get involved.

Who are your role models in science? Why?

N.S.: It was David Attenborough's fascination with and seemingly endless knowledge about the natural world that first drew me towards science. As well as being an inspirational natural historian, he is a fantastic communicator who has reached and influenced a huge number of individuals.

M.B.: I admire scientists that have made discoveries by stepping back from established dogma and returning to first principles. An example of this is William Harvey's work on the cardiovascular system. Using only a few simple calculations, he showed that blood must be recirculated in the body, disproving long held theories proposed by the Greek physician Galen.

What's next for you?

N.S.: I am continuing my postgraduate training.

M.B.: After medical school, I hope to do an academic foundation placement, and an academic clinical fellowship (both feature protected research periods alongside clinical training), followed by a PhD.

Tell us something interesting about yourself that wouldn't be on your CV

N.S.: After science, my second passion is languages. I currently speak four languages and am in my fourth year of learning Mandarin. Given the important role of international research collaboration in solving global problems, I aim to combine these two passions as much as possible.

M.B.: I enjoy seeing unusual architectural sites, particularly pieces of infrastructure that normally are overlooked. Highlights include visiting the Victorian sewer network under London, and the rotary ‘bascule’ chamber inside Tower Bridge! I also like to make art that has been inspired by science; for example, I recently made a series of drawings based on patterns seen in microbial culture plates.

Is it possible for undergraduate students to generate data publishable in the Journal of Cell Science?

N.S. and M.B.: Yes! With the right project, work ethic and supervision…

Nina Short's contact details: Imperial College London, South Kensington, London SW7 2AZ.

Max Butler's contact details: The University of Cambridge, The Old Schools, Trinity Lane, Cambridge CB2 1TN.

E-mails: nina.short15@imperial.ac.uk; mbb35@cam.ac.uk

Reference

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Rho kinase-dependent apical constriction counteracts M-phase apical expansion to enable mouse neural tube closure
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J. Cell Sci.
132
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230300
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