Established by the British Society for Developmental Biology in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding. You can read accounts from previous years here. If you’re interested in applying or hosting a student in 2020, applications need to be in by the end of March.

Our fifth report from the class of 2019 comes from Lauren Mulcahy (University of Sussex) who studied fly miRNAs in Sarah Newbury’s lab at the Brighton and Sussex Medical School.

Exploring the genetic control of microRNAs in Drosophila melanogaster

This summer, thanks to the BSDB Gurdon Studentship, I was able to work closely with a PhD student in the lab of Sarah Newbury at the Brighton and Sussex Medical School. Under their supervision I was able to get an applied, practical approach to lab work, much different from that experienced during my undergraduate course.

My project involved looking at the genetic control of microRNAs in Drosophila melanogaster, otherwise known as the common fruit fly. MicroRNAs are small-noncoding RNAs that participate in RNA silencing and regulation of gene expression. miRNAs are able to base-pair with their complementary target mRNA, and through this they are able to silence them, either by their subsequent degradation or prevention of their translation.

Previous studies have shown that exoribonucleases, known as Pacman (XRN1) and Dis3L2 degrade different microRNAs through the mRNA decay pathway, shown in Figure 1.

The project made use of wing imaginal discs, which are highly proliferative organs found in Drosophila that will eventually develop into the adult fly’s wings. Prior work in the lab had found that there were significant differences between the size of these discs in both pacman and dis3L2 mutants, with dis3L2 mutants having much larger discs (Towler et al. 2016), and pacman mutants having smaller ones (Waldron et al. 2015). Both exoribonucleases are conserved to humans and their defects have shown to be significant in human disease as well. For example, mutations of DIS3L2 in humans have been linked to Perlman syndrome, an overgrowth syndrome which presents as organomegaly and is associated to a high risk of developing Wilm’s tumours (Astuti et al. 2012). Together this suggested that there could be an involvement of these exoribonucleases in the control and regulation of cell proliferation and apoptosis within these discs, potentially through their degradation activity. Therefore, the aim of my project was to study the difference between levels of specific miRNAs (which were extracted from these WIDs) in both pacman and dis3L2 mutants, compared to their respective control wildtype.

My results, shown in Figure 2, were obtained through the use of qPCR (quantitative or real-time PCR). Also shown on Figure 2 are two other datasets from previous high-throughput RNA sequencing experiments, known as Cuffdiff and sRNAtoolbox.

Looking at my data, the pacman mutants showed that there was a negative fold change in the levels of two miRNAs, miR-A and miR-B when normalized to their wildtype’s levels. This indicated that the two miRNAs are downreguated in the pacman mutants. This confirmed the results obtained from the RNA sequencing, as they both showed that the miRNAs were downregulated as well.

Results from the dis3L2 mutants were less definite. For one of the miRNAs, miR-C, there was a negative fold change when normalized to the wildtype’s level. Again, this confirmed results obtained from the RNA-seq, but my data, and data from Cuffdiff, suggested a much stronger downregulation than that from sRNAtoolbox. For the other miRNA, miR-D, qPCR showed that there was no significant fold change in the mutants. This agrees with results from one of the RNA-seq, but not the other. Data from Cuffdiff showed a slight negative fold change of miR-D, suggesting it is downregulated in the mutants. However, my data, and that from sRNAtoolbox, suggested there is no change between the wildtypes and mutants.

Further areas of my project involved looking at the difference in volume of the abdomen and ovaries in flies with a defect in the gene of a TUTase known as Tailor, to see whether it was involved in controlling their size. TUTases, or Terminal Uridylyl Transferases, are enzymes that uridylate miRNAs, potentially serving as a signal for exoribonuclease-mediated degradation such as Dis3L2. This involved me freezing flies in liquid nitrogen, then photographing and dissecting out their ovaries. The limited number of ovaries measured suggested that there were no significant differences in the sizes between the wildtype and mutants.

In addition to this, I also looked at the differences in sizes of wings in Tailor mutant and double mutants of Tailor and Dis3L2 compared to wild types. This included dissecting wings and mounting them on a slide then taking measurements using computer software and a microscope. We hypothesised that the wings from the Tailor mutants would be the same size as the wildtype but the Tailor/Dis3L2 double mutants would have larger wings than the wildtype. My results here were too limited to draw a reliable conclusion but indicated there was no difference in wing sizes between the mutants and wildtype.

Overall, my time in the lab has proved highly valuable and interesting. Learning about the techniques in lectures and actually performing them yourself in a lab are completely different experiences and I am really grateful. I feel it was really important for me to experience the fly work alongside the molecular work as this has helped me to familiarize myself with numerous practises and therefore determine what is most interesting and suitable for me in the future. But not only did I learn how to perform these techniques, I was also introduced into the world of Drosophila, ranging from learning about their genome, recognising their phenotypes, and performing my own genetic crosses to learning their general upkeep and how to do simple things such as egglays or anesthetizing the flies. This experience for me has really increased my interest in developmental biology. Being able to produce my own data and learning to work independently in a lab have also strengthened my desire to do a PhD and I’m very appreciative that I got to experience something not many undergraduate students will. On top of all this, being inside a lab with such a friendly and welcoming group of people has made it all that more enjoyable.

A huge thank you to everyone in the lab for their support and assistance, and to the BSDB for making this opportunity possible through the Gurdon Studentship.

Sources

• Towler BP, Jones CI, Harper KL, Waldron JA, Newbury SF. 2016. A novel role for the 3′-5′ exoribonuclease Dis3L2 in controlling cell proliferation and tissue growth. RNA biology 13: 1286-1299.
• Waldron JA, Jones CI, Towler BP, Pashler AL, Grima DP, Hebbes S, Crossman SH, Zabolotskaya MV, Newbury SF. 2015. Xrn1/Pacman affects apoptosis and regulates expression of hid and reaper. Biology open 4: 649-660.
• Astuti D, Morris MR, Cooper WN, Staals RH, Wake NC, Fews GA, Gill H, Gentle D, Shuib S, Ricketts CJ et al. 2012. Germline mutations in DIS3L2 cause the Perlman syndrome of overgrowth and Wilms tumor susceptibility. Nature genetics 44: 277-284.

Closing Date: 15 March 2021

Established by the British Society for Developmental Biology in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding. You can read accounts from previous years here. If you’re interested in applying or hosting a student in 2020, applications need to be in by the end of March.

Our fourth report from the class of 2019 comes from Luca Argirò (University of Rome Sapienza) who studied plant symmetry in Laila Moubayidin’s lab at the John Innes Centre.

How to get radial: Unlocking the mechanisms for symmetry establishment across plant organs

The development of multicellular organisms depends on correct establishment of symmetry both at the whole-body scale and within individual tissues and organs. Setting up planes of symmetry must rely on communication between distant cells within the organism, presumably via mobile morphogenic signals. Although symmetry in nature has fascinated scientists for centuries, it is only now that molecular data to unravel mechanisms for symmetry establishment are beginning to emerge.

Over the summer, I had the opportunity of conducting a research project at the John Innes Centre (JIC) working under the supervision of Dr Laila Moubayidin, whilst participating in the amazing JIC/TSL/EI International Undergraduate Summer School.

Dr Moubayidin’s research looks at identifying a conserved “core machinery” necessary and sufficient to control symmetry establishment across plant organs. She is elucidating the underlying process of symmetry establishment at the cellullar, molecular and genetic level.

Unlocking the molecular mechanisms that underpin this regulation holds potential for understanding the processes that allow plant organs to reach their perfectly-optimized shape and function.

The project I worked on focused on gynoecium (the plant female reproductive structure) development, using Arabidopsis thaliana as a model system.

The gynoecium forms in the centre of the flower and it is derived from the fusion of two carpels. The gynoecium includes the ovary, which displays bilateral symmetry, and the style, at its apex, characterized by radial symmetry (Figure 1). The ovary is important for seeds production and the style is important for fertilization.

During its development, the distal end of the gynoecium becomes radially symmetric via a switch from bilateral to radial symmetry. Symmetry transitions are common during embryogenesis in all multicellular organisms. In most cases, the transition is from radial to bilateral symmetry, which is controlled by Hox and decapentaplegic genes in animals. The A.thaliana gynoecium, instead, is the only molecularly documented example of a developing structure that reprograms its development over time to achieve a bilateral-to-radial symmetry transition.

Growing evidence shows that coherent organ growth and symmetry establishment during organogenesis are influenced by post-translational modifications of specific nuclear and cytoplasmatic proteins. My project focused on the role of O-linked N-acetylglucosamine modification during gynoecium development. O-GlcNAcylation is a post-translational modification that consists of a single O-linked N-acetylglucosamine attached to either a serine or a threonine residue. It has been extensively studied in animals, where it regulates a wide range of developmental and metabolic processes.

O-GlcNAcylation is dynamically controlled by two enzymes: an O-GlcNAc transferase (OGT) and an O-GlcNAcase (OGA), which add and remove O-GlcNAc, respectively. O-GlcNAcylation occurs in the cytoplasm, nucleus and mithocondria, and it is implicated in cellular processes, including transcription, translation, signal transduction, nuclear pore function, epigenetic regulation and proteosomal degradation. Altered levels of protein O-GlcNAcylation in animals have been associated with neurodegeneration, diabetes, cardiovascular diseases and cancer.

Arabidopsis thaliana has two putative OGTs: SPINDLY (SPY) and SECRET AGENT (SEC) whose role is essential for plant development, since spy;sec double mutant is embryionically lethal, similar to the OGT knockout mutant in animals.

Exploring this interesting and at the same time quite complicated topic, gave me the chance of learning and mastering a wide range of molecular biology techniques, such as cloning and Yeast-two-Hybrid. It was amazing to experience practical methods and protocols I had studied during university courses in Rome and to understand how much work, attention to detail and care there is behind even repetitive actions (Figure 2).

Using the state-of-the-art JIC microscopy facilities, I had the opportunity to look at wild-type and mutant Arabidopsis gynoecia at different developmental stages using Scanning Electron Mycroscopy (SEM). Fixing samples for SEM analysis, dissecting flowers and using machines such as the Critical Point Dryer and the Gold Sputter Coater was a challenging experience, but the final result was worth the hard work. I was astonished whilst I was conducting SEM analysis as it allows observation of samples at incredible magnifications, providing details of a single cell surface (Figure 3).

At the end of this experience, I felt extremely grateful for several reasons.

Firstly, I greatly enhanced my knowledge of plant developmental biology. As a “Sapienza School for Advanced Studies” student, in the first year of university, I was keen to work on plants during my Research Project, so I applied to the International Undergraduate Summer School at the JIC. After being selected, I was urged by Dr Moubayidin to apply for a Gurdon/The Company of Biologists Summer Studentship offered by the British Society for Developmental Biology (BSDB). Moreover, working side-by-side an experienced, young principal investigator, such as Dr Moubayidin, has been a rare opportunity and a great inspiration for my future career. Dr Moubayidin has been recently appointed a prestigious Royal Society University Research Fellowship, therefore it has been an exciting time to be part of her lab, experiencing how a research group is built and the importance of teamwork.

Lastly, this summer placement has given me great insight into research career paths and has made me even more aware of how incredibly surprising and stimulating daily lab life can be. Each workshop organized by the JIC Summer School and every seminar held at the JIC has been a great chance for me to learn about new topics, uncovering the research world of an international centre and the beauty of working in a collaborative and stimulating atmosphere.

Altogether, this unexpected chain of events has lead me to immensely broaden my scientific and transferable skills, as well as my perspectives, not only as a scientist, but also as a “world citizen”. This Summer School experience, set in the incredibly positive environment of the JIC, has reaffirmed my desire of pursuing a career in science, despite all the hard work it actually requires. I wholeheartedly recommend the Gurdon/BSDB Summer Studentship and the possibility to carry on research abroad during university studies, visiting new countries and encountering new cultures. I believe every undergraduate student should be made aware of unique opportunities, like this, available to them.

References

• Moubayidin L., Østergaard L., Dynamic control of auxin distribution imposes a bilateral-to-radial symmetry switch during gynoecium development, Current Biology 24, 2743-2748, (2014).
• Moubayidin L., Østergaard L., Symmetry matters, New Phytologist (2015).
• Shou-Ling Xu, Robert J. Chalkley, Jason C. Maynard, Wenfei Wang, Weimin Ni, Xiaoyue Jiang, Kihye Shin, Ling Cheng, Dasha Savage, Andreas F. R. Hühmer, Alma L. Burlingame, and Zhi-Yong Wang, Proteomic analysis reveals O-GlcNAc modification on proteins with key regulatory functions in Arabidopsis, PNAS 114 (8) E1536-E1543, (2017)

Established by the British Society for Developmental Biology in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding. You can read accounts from previous years here. If you’re interested in applying or hosting a student in 2020, applications need to be in by the end of March.

Our third report from the class of 2019 comes from Isabel Swinburn (University of Birmingham) who studied zebrafish muscle development with Robert Knight’s lab at King’s College London.

Throughout my Biological Sciences degree, I came to realise that I want to pursue a career in medicine that combines clinical practice with research. After having thoroughly enjoyed my final year research project that focused on bacterial genetics, I decided to apply for a studentship in a biomedical research lab. This was with the hope that I’d be able to apply some of the experimental techniques I’d already learnt to understand complex processes in living organisms. Having a good grounding in organismal biology will be useful when applying a research perspective to human systems.

I was very fortunate to complete my Gurdon/The Company of Biologists Summer Studentship under the supervision of Dr. Robert Knight at King’s College London. His group uses zebrafish as a model to explore the molecular control of muscle repair and the regulation of muscle stem cells, otherwise known as satellite cells. These cells are characterised by their expression of the pax7b transcription factor (1). The zebrafish is a suitable model organism for their investigation, as it has a well-defined genome and is transparent in its larval stages. Therefore, genetic manipulations that affect cell and tissue dynamics can be readily visualised. Their work particularly appealed to me, as I am very interested in the use of reverse genetics approaches to investigate genetic diseases and how they arise due to the disruption of development.

Background to project:

The transmembrane Ret tyrosine kinase receptor is activated by the binding of Glial-derived neurotrophic factor family ligands (GFL) to Glial-derived neurotrophic factor receptors (Gfra), as this interaction stimulates Ret dimerisation and subsequently, its activation. Ret signalling has an important role in the development of the enteric nervous system and hepatic tissue, but its role in muscle development is poorly understood. Ret signalling has been implicated in facioscapulohumeral muscular dystrophy, which manifests as the weakening of the facial muscles in its initial stages (2). The binding of artemin2 (artn2), a GFL, to Grfa3 is an interaction required for the activation of Ret related to the development of the cranial muscles in zebrafish (3). My project set out to investigate the role and level of importance of Ret signalling in the development of cranial muscle satellite cells.

Experiments

1) Is an activating ligand for the Ret receptor able to alter cranial muscle development?

To test this hypothesis, I performed MF20 immunolabelling on zebrafish embryos exhibiting heat-shock inducible artn2 overexpression, as well as controls. Diaminobenzidine (DAB) staining was used to detect the signal from the antibodies, which made myofibres appear orange-brown (Figure 2a). The embryos were fixed and imaged using bright-field microscopy. The sizes of a subgroup of cranial muscles were measured and compared between the experimental and control samples (Figure 1). There did not appear to be a significant difference in muscle size between the two sample groups, suggesting that the activating ligand for the Ret receptor cannot alter cranial muscle development.

2) Is an activating ligand for the Ret receptor able to alter cranial muscle satellite cell formation?

To test this hypothesis, a transgenic line was used, in which pax7b-expressing cells were labelled with EGFP and artn2 was overexpressed. Embryos with this genotype, along with GFP+ embryos with the wild type artn2 genotype, were fixed and imaged using confocal microscopy. The number of GFP+ myofibres and myoblasts within the muscles of interest were quantified and compared between the experimental and control samples. I was unable to discriminate a significant difference, due to the control sample size being too small to be able to perform statistical tests. Therefore, more wild-type embryos need to be imaged and analysed to confirm whether artn2 can alter cranial muscle satellite cell formation.

3) How important are satellite cells for cranial muscle development?

To answer this question, I ablated pax7b-expressing cells during development. A GAL4;UAS system was used, in which the pax7b promoter induced expression of nitroreductase (NTR). Embryos which carried this system were treated with metronidazole, which reacts with NTR to produce a cytotoxic compound. The muscles of these embryos, as well as controls, underwent MF20 immunolabelling and these signals were detected using either DAB or tyramide FITC, to which a green fluorophore is conjugated. Depending on the detection method, the embryos were imaged using bright-field or confocal microscopy, respectively, and the myofibres in each image were quantified (Figure 2). Analysis of the bright-field images suggested a difference in the number of myofibres within the cranial muscles of the experimental and control sample groups. However, quantification analysis of the confocal images did not reveal a significant difference. Therefore, further experiments must be carried out to confirm the importance of satellite cells for cranial muscle development.

Outcomes:

At university, I had only used optical microscopy in practical sessions. However, following my time in the Knight lab, I now feel confident in using a much wider range of more advanced microscopy techniques to gather experimental data. I also appreciate much more the importance of analysing the data – quantifying cells and myofibres within images seemed like a simple task, but it took me weeks! Unlike at university, the data I collected whilst working in the lab was raw and unclean and strict parameters had to be set for its analysis. Although the task was daunting and sometimes difficult, it was very gratifying when the numbers and statistical test results allowed me to test the hypotheses generated and realise that I had made a scientific discovery.

I would like to say thank you to Robert for allowing me to work under his supervision, and to the British Society for Developmental Biology for providing the financial backing to enable me to do so. A special thank you also goes to the rest of the Knight group for all of their support and encouragement throughout. Despite my focus being primarily on a career in medicine, this experience has reinforced my desire to continue to contribute to the type of evidence that will drive medical practice. I would recommend anyone considering a career in biological research to apply for this programme.

References

• Pipalia, T. G. et al., 2016. Cellular dynamics of regeneration reveals role of two distinct Pax7b stem cell populations in larval zebrafish muscle repair. Diseases Models & Mechanisms, 9(6), pp. 671-684.
• Moyle, L. A. et al., 2016. Ret function in muscle stem cells points to tyrosine kinase inhibitor therapy for facioscapulohumeral muscular dystrophy. eLife, e11405.
• Knight, R. D. et al., 2011. Ret signalling integrates a craniofacial muscle module during development. Development, 138(10), pp. 2105-2024.
• Schilling, T. F. & Kimmel, C.B., 1997. Musculoskeletal patterning in the pharyngeal segments of the zebrafish embryo. Development, Volume 124, pp. 2945-2960.

Closing Date: 15 March 2021

Closing Date: 15 March 2021

Closing Date: 15 March 2021

Established by the British Society for Developmental Biology in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding. You can read accounts from previous years here. If you’re interested in applying or hosting a student in 2020, applications need to be in by the end of March.

Our second report from the class of 2019 comes from Grace Blakeley who studied spider segmentation in Alistair McGregor’s lab in Oxford Brookes University.

How to build a spider: investigating the role of Delta in posterior segmentation

Three animal phyla have segmented bodies – the vertebrate chordates, the annelid worms and the arthropods. To understand the evolution and development of these segmented bodies it is necessary to identify what mechanisms regulate segmentation and the similarities and differences of these mechanisms among phyla. Amongst arthropods there are two main different mechanisms for segmentation: long-germ arthropods, such as Drosophila melanogaster which develop their segments simultaneously; and short-germ arthropods,  constituting the majority of arthropods, which add their posterior segments sequentially from a segment addition zone (SAZ). Under supervision from Dr Anna Schonauer in Professor Alistair McGregor’s lab, I aimed to investigate the regulation of posterior segmentation in arthropods by further studying the Delta-Notch signalling pathway in the spider Parasteatoda tepidariorum.

Spiders are a useful model for answering questions about the regulation and evolution of segmentation as they develop their prosomal (anterior) segments simultaneous like Drosophila but their opisthosomal (posterior) segments sequentially in a manner analogous vertebrate segmentation. It is understood that the addition of posterior segments relies on the formation of the SAZ, which develops at around stage 6 in spider embryos through dynamic Wnt and Delta-Notch signalling. These genes are subsequently also required for segment addition. Specifically, Delta appears to differentially regulate the posterior and anterior regions of the SAZ to maintain Wnt8 in the posterior SAZ but lower its expression in the anterior SAZ to facilitate the formation of nascent segments. However, little is known about the genes downstream of Delta that are involved in segment addition.

The gene hairy is thought to be involved in segment addition and it has been shown to have a similar oscillatory expression pattern to Delta leading to the hypothesis that it may be regulated by Delta as part of a gene regulatory network (GRN) that results in segment addition. Previous studies of the role of Delta in segment addition used parental RNA interference to knockdown this gene. However, the consequential loss of the SAZ resulting in a truncated germ band impedes investigation into any specific, localised downstream effects of loss of Delta. To be able to investigate the relationship between Delta and hairy I therefore aimed to use embryonic RNAi to knockdown Delta in subsets of posterior cells. This would allow embryonic development to progress as normal without truncating the germband, but create Delta knockdown clones allowing me to see within an embryo if hairy expression is different in cells with and without a knockdown of Delta. Thus I would be able to test whether hairy does act downstream of Delta and if so, might be able to infer what potential role it may have in segment addition.

Microinjections of Delta double stranded RNA and biotin into single cells at embryonic stage 1F (when there are only 32 cells) were carried out with the aim of knocking down Delta and at the same time staining the clone of cells derived from the injected cell. The microinjection technique itself was technically demanding, requiring a lot of patience and practice and it took me two weeks before I was able to successfully inject a cell without it bursting. Once I had injected successfully into a single cell (Figure 1) I gained experience in the correct technique needed and I was then able to successfully inject about 15% of embryos in a cocoon (a cocoon containing approximately 200 embryos). The other main technique I used was in situ hybridisations (ISH), which used labelled RNA probes to bind to the hairy mRNA in all the cells in the embryo to show where hairy is being expressed. I carried out the ISH two days after the microinjections as this is when embryos develop their SAZ and begin to add their first segments. As a control, I stained embryos solely for Delta or hairy as this allowed me to see the wildtype expression of each (Figure 2). I also performed a double ISH for Delta and hairy which confirmed their overlapping expression in the posterior of the embryo.

No blastodermal cell fate map exists for P. tepidariorum and as such it was never guaranteed that I would get clones in the posterior opisthosomal segments. Unfortunately, all my Delta knockdown clones occurred in the anterior prosomal segments so I was unable to draw conclusions about any Delta-hairy interaction in the SAZ. My anteriorly located clones however, do suggest an interaction between Delta and hairy in the prosomal segments. Here, I was able to detect downregulation of hairy expression in the subset of Delta knockdown cells. Most of our clones occur in a single prosomal segment, however in a stage 8 embryo, the clone spanned four developing prosomal segments, and showed downregulation of hairy across all of them. This suggests that Delta directs hairy in patterning of the anterior segments (data not shown).

Over the course of this project I learned many different things, most importantly that in science, patience and perseverance are key. I was surprised with how involved each technique was and quickly learned to take the advised protocol amendments suggested by my peers. Being part of a team all of whom supported me, helped me and questioned my work ultimately encouraged me to become a better researcher. The experience has confirmed my ambition to pursue a PhD and focused my interest on understanding the fundamental and complex molecular interactions that ultimately regulate and drive development.

Closing Date: 15 March 2021