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.

Our sixth report from the 2018 group of student awardees comes from Marketa Novotna (student at Dundee) who undertook her research with Pauline Schaap (also at Dundee). 

Like many other lucky students, I had the chance to participate in real cutting-edge research this summer thanks to the Gurdon Studentship award. Until then, I had spent my time learning the essential theory and mastering various lab techniques. What I was missing, however, was doing actual research that leads to brand new findings, rather than predictable results I’d get in a practical, which had been tried many times before. To me, the summer project represents a transition from only learning a theory and lab techniques to joining a team of scientists in a real-life lab and producing new data, that can advance the field.  That is really important to me because contributing to the general pool of knowledge has always been my greatest motivation to study science.

I was hosted by the lab of Prof Pauline Schaap in the University of Dundee. The lab concentrates on several species of slime moulds that are members of the Dictyostelia clade, in particular the model organism Dictyostelium discoideum. These social amoebas are unicellular under normal conditions but environmental stress – especially lack of nutrients or draught – can trigger formation of multicellular fruiting bodies that consist of many hundreds differentiated cells that are derived from the individual amoebas. Some individuals within the structure encapsulate and survive the harsh conditions in form of spores that germinate when environmental conditions improve. The formation of fruiting body is a complex process, which involves intricate cell signalling that ensures a coordinated movement and differentiation of cells.
The long-term mission of the Schaap lab is to understand how this and similar processes evolved from ancestral pathway controlling encystation in more primitive, solitary amoebas and thus partially uncover how multicellularity evolved.

Fruiting body formation in Dictyostelium discoideum

D.discoideum is a species of social amoeba that is unicellular under normal conditions. However, low concentration of food source in a combination with a high density of amoebas in the surrounding environment leads to an exit from the unicellular life cycle. The signals they produce activate PKA (cAMP-dependent kinase), which results in cAMP production. As cAMP diffuses to the environment it acts as a chemoattractant. Individual cells not only respond to this attractant, migrating closer to the source, but they also produce more cAMP, causing pulses of this chemical, which drive more amoebas towards the source. This results in aggregation of individuals that form a mound, which elongates and eventually topples over to create a migrating slug-like structure. Populations of cells start to differentiate into different cell types like pre-spore and pre-stalk cells. (1)

The slug follows environmental cues such as light or warmth to move towards the soil surface. Once it reaches the destination, the cells differentiate further into terminal cell types as the fruiting body develops. Some cells differentiate to form the stalk that serves as a scaffold to hold a mass of differentiated spore cells. A basal disc structure is formed at the base to support the stalk and cells also form upper and lower cups to support the spore head attachment to the stalk. (1)

My project

I worked under a day-to-day supervision of an amazing, patient PhD student Gillian. She’s been studying potential marker genes for distinct parts of the fruiting body (stalk, basal disc, lower cup, upper cup and spores) and the signalling pathways linked to formation of these structures.

Previous work done in the lab identified a number of genes that could play an important role in formation of one of the fruiting body structures due to their enrichment in a specific cell type. Out of these, I studied two genes that looked most promising and went on to establish whether they are expressed in the same parts of fruiting body as hypothesised. For simplicity, I will call them gene A and B.
I used PCR to multiply the promoter sequence of each studied gene then inserted it into a plasmid, which I then used for transformation of E. coli. As the bacteria proliferated, I was able to obtain enough DNA to sequence it and confirm I had the correct sequence. The confirmed promoter sequence could then be inserted into a plasmid I used for transforming D.discoideum. For this purpose, I used a plasmid containing the LacZ reporter gene directly after the promoter-insertion site. This LacZ gene encodes the enzyme β-galactosidase; therefore, since the expression of LacZ was controlled by promoter of the studied gene, the B-gal production mirrored expression of the studied gene.

Next, the gene expression could be visualised using the β-galactosidase substrate, X-gal. After addition of X-gal, a blue precipitate forms at the areas of the fruiting body where the promoter was activated. Hence, allowing us to locate where our gene of interest is expressed and determine whether they are cell-type specific. (2)

The genes that I worked on – A and B – were hypothesised to be expressed in the stalk and cup, respectively. After I’d spent a great deal of time on optimisation of PCR conditions and several attempts to transform D.discoideum, I acquired transformed amoebas on which I could perform the X-gal staining.

You can see the result of this experiment in Figure 1. In the case of gene A, the stalk was clearly stained, while fruiting bodies of amoebas transformed with gene B promoter showed staining of cup cells (both upper and lower cup). Therefore, the hypothesis was confirmed for both genes.

If I had more time, it would be interesting to find out if these genes are essential for formation of the respective structures by knockout experiments. Furthermore, it could be tested what signalling molecules trigger expression of these genes to further investigate their role in fruiting body formation.

I would like to thank the lab of Prof Pauline Schaap for hosting me and offering a great amount support within a friendly environment. I am also very grateful to my day-to-day supervisor Gillian, who has taught me so much during my placement.

Finally I would like to thank and appreciate the British Society for Developmental Biology for making this experience possible by selecting me for the Gurdon Studentship award. The summer project made me realise that I would really like to pursue a PhD and I would strongly recommend this scheme for any student who is considering a career in science.

References

• Schaap, P. (2011). Evolutionary crossroads in Developmental Biology: Dictyostelium discoideum. Development 138, 387-396.
• Dingermann T, Reindl N, Werner H, Hildebrandt M, Nellen W, Harwood A, Williams J, Nerke K. Optimization and in situ detection of Escherichia coli beta-galactosidase gene expression in Dictyostelium discoideum. Gene. 1989 Dec 28;85(2):353-62.

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.

Our fifth report from the 2018 group of student awardees comes from Courtney Lancaster (student at Durham) who undertook her research with Ben Steventon (University of Cambridge). 

Deciphering the dynamics of neuromesodermal progenitors at the end of axis elongation in the zebrafish embryo: A tail of a summer research project

I was introduced to developmental biology in the second year of my undergraduate degree at Durham University. From the first lecture, I was amazed and I left the lectures with more questions than answers on many occasions. This curiosity stemmed my quest to find a lab where I could begin to answer some of my questions. Of particular interest to me is the elongation of the vertebrate body axis, with a key question: How does the body axis stop elongating?

I was lucky enough to come across the Steventon lab, who focus their research on a population of bipotent stem cells called neuromesodermal progenitors (NMps). NMps co-express Sox2 and Brachyury (neural and mesoderm markers respectively) and they provide progenitor cells for the spinal cord and mesoderm during vertebrate axis elongation (Steventon and Martinez-Arias., 2017). Furthermore, NMps are a conserved source of spinal cord amongst vertebrate species making comparative studies to be of great interest (Steventon et al., 2016).

In mouse and chick embryos, NMps are a population of bipotent, self-renewing progenitor cells whose derivatives undergo a great deal of cell proliferation that is one of the main drivers of axis elongation. On the other hand, zebrafish body axis extension is more a product of cell movements rather than of volumetric growth (Steventon et al., 2016). Therefore, a key question is: To what degree do NMps self-renew in the zebrafish embryo to give rise to both neural and mesodermal cell fates? To begin answering this question, I carried out lineage tracing at single-cell resolution from a light-sheet movie of the zebrafish tail. This analysis meant that I got to grips with embryo mounting and I worked closely with computer scientists and engineers from the Cambridge Advanced Imaging Centre (CAIC). I was lucky to have access to a light-sheet microscope in which the stage position continually corrected to follow the tail. This allowed the tail to stay in view whilst the embryo was undergoing development, which is essential for long-term imaging of an elongating structure.

From the light-sheet dataset, all nuclei were segmented and automatically tracked using TGMM tracking software (Amat et al., 2014). To select my area of interest, the tailbud cells, computer scientists at CAIC wrote a Matlab script allowing me to select the area I wanted to track. I was then able to manually correct the tracks of interest using a Fiji plug in called MaMuT (Figure 1B) and assign fates according to gene expression data (figure 1A). The cells were tracked from 21 somite stage to the completion of somitogenesis, the stages where NMps contribute to the final stages of the body axis (Steventon and Martinez-Arias., 2017). From this data we concluded that little cell division occurs in the NMp population and that NMps are mono-fated progenitor cells, either giving rise to the neural or mesodermal lineages, but not both (Figure 1B, C, D). Therefore, in zebrafish, NMps do not undergo vast amounts of cell division to continually contribute to the elongating body axis.

Apoptosis takes place at the end of axis elongation in chick embryos. This occurs following a rise in retinoic acid signaling and the loss of FGF dependent mesoderm identity (Olivera-Martinez., 2012). Considering the different cell behaviors and contribution of NMps to the body axis in zebrafish, I next asked the question: Does apoptosis have a role in terminating the body axis in zebrafish embryos? To gain insight into this, cells at the end of the presomitic mesoderm (PSM) were photo-labelled by injecting embryos, at one cell stage, with a photoconvertible fluorescent protein mRNA called Kikume. A confocal microscope was then used to shine UV light onto the posterior PSM to convert the cells from green to red (figure 2A) and these cells could then be followed over time (figure 2B, C). It was found that the labelled cells did not noticeably undergo apoptosis and they did not segment (figure 2). It will be essential to confirm this finding with antibody immuno-labelling of apoptosis associated proteins such as caspase 3 or with a TUNEL assay. Nonetheless, morphological analysis suggests that apoptosis does not precede the termination of axis elongation in zebrafish embryos.

As well as the two main projects above, I also began to analyse the light-sheet data to understand which cell movements are responsible for elongating the body axis. This was carried out on Imaris software, which allowed me to view the tailbud in 3D and to select different tissues (e.g. PSM) for analysis. Further to this, I used photolabelling (method previously described) to look at the contribution of different tailbud progenitor populations to the PSM and somites.

This experience has been invaluable to me and I have thoroughly enjoyed mixing lab experiments with computational analysis, which are both important in science.

My interaction with academics from CAIC made me realize the importance of interdisciplinary science in order to make scientific research more productive. It allows biologists to gain better insights as well as improving computational techniques for the field as a whole. I have also attended lab meetings and had the opportunity to present my work. Further to this, I attended a fantastic conference “Engineering Multicellular Self-Organisation III”. When I thought that my summer could not get any better, my light-sheet data and analysis presented here has been accepted as part of a research article in the Development journal. Together, the skills gained here have taught me to be more critical and have begun to equip me for an exciting career in research.

I would like to thank Ben Steventon for making this incredible experience possible and for the great advice and discussions throughout the project. Thank you to the Steventon group: Lewis, Tim, Kane, Susie, Berta, Carolina, and John for creating a welcoming and enthusiastic working environment, not forgetting the tea breaks and pub nights! Last but not least, thank you to the BSDB Gurdon Studentship for financial support.

References

Amat F, Lemon W, Mossing, D. P, McDole K., Wan Y, Branson K, Myers E. W, Keller, P. J. (2014). Fast, accurate reconstruction of cell lineages from large scale fluorescence microscopy data. Nat. Methods 11: 951–958.

Olivera-Martinez I, Harada H, Halley PA, Storey KG (2012) Loss of FGF-Dependent Mesoderm Identity and Rise of Endogenous Retinoid Signalling Determine Cessation of Body Axis Elongation. PLoS Biol 10(10): e1001415.

Steventon, B., Duarte, F., Lagadec, R., Mazan, S., Nicolas, J.-F. and Hirsinger, E. (2016). Species tailoured contribution of volumetric growth and tissue convergence to posterior body elongation in vertebrates. Development 143(10): 1732–41.

Steventon, B. and Martinez Arias, A. (2017). Evo-engineering and the cellular and molecular origins of the vertebrate spinal cord. Dev. Biol. 432(1): 3-13.

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.

Our fourth report from the 2018 group of student awardees comes from Melissa Gomez (student at The University of Aberdeen) who undertook her research with Martin Collinson  (also at Aberdeen). 

The Development of microphthalmia in Pax6 mutant mice

Every 4 ½ minutes, a neonate in the USA is born with a birth defect, this totals up to 120,000 babies per year from the USA alone (1). As an embryology student, the underling mechanisms which cause birth defects are of prime interest to me, in the hope that understanding the cause of the defect can potentially lead to prevention. This summer I was able to conduct my own research on a birth defect called microphthalmia.

Microphthalmia is a disorder in which one or both eyes are abnormally small. This birth defect is closely associated with a gene called Pax6, the so-called ‘master regulator gene’ of the eye. Pax6-/- in both mouse and human causes anophthalmia (absence of the eye) due to failure of lens placode induction (2). Pax6+/- causes microphthalmia (Figure 1), as well as aniridia (absence of the iris), cataract and corneal opacity (2). In mice, all Pax6+/- have microphthalmia, but in humans, Pax6+/- individuals usually have aniridia, cataract and corneal problems but tend to have normal size eyes. However, it has been shown that point mutations in Pax6 are strong risk factors in the development of microphthalmia (6)(7). The mechanism for this is not clear, but it can be studied in mice because all Pax6+/- develop microphthalmia. Microphthalmia seems to arise due to the lens being sensitive to Pax6 dosage, for instance it has been found Pax6+/- mice have a 50% reduction in the number of cells in the lens during early embryogenesis (2). The lens is crucial in eye development as it secretes growth factors, thus a reduction in lens cells leads to a decrement in growth factors (3). In addition to this, Pax6+/- lens cells tend to undergo one less round of division when compared to Pax6+/+ mice (2). Why this occurs in Pax6+/- lens remains a mystery, however it can be hypothesised that cell cycle time is increased leading to fewer cell divisions. Thanks to the BSDB Gurdon’s Studentship I was able to further investigate this hypothesis alongside Professor Martin Collinson at the University of Aberdeen.

My projected started with timed matings of Pax6+/- x Pax6+/- mice. At E14.5, the pregnant mice received an injection of iododeoxyuridine (IdU), a thymidine analogue that is incorporated into the replicating DNA of cells in S-phase of the cell cycle. 60 minutes later a second injection of ethynyl-deoxyuridine (EdU), another thymidine analogue, was given. 30 minutes after, mice were killed, and embryos were harvested and fixed in paraformaldehye. The genotypes were confirmed by PCR and electrophoresis using a small piece of tissue from each mouse embryo, Pax6-/- embryos were not used in this experiment because they do not have eyes. After the genotype of each embryo was confirmed, I dehydrated the embryos in ascending concentrations of ethanol (75%, 80%, 95%, 100%) and placed them in xylene. Next, my favourite part of the experiment: paraffin wax embedding. Once the embryos were embedded, a cryotome was used to cut sections which were placed onto poly-L-lysine slides.

Immunohistochemistry was conducted on these slides, Anti- EdU conjugated with a green flurophore (Alexa 488) and Anti-IdU conjugated with a red fluorophore (Alexa 594) were used to detect EdU and IdU labelled cells respectively (Figure 2). DAPI staining was used to visualise all nuclei present in the mouse embryo (Figure 2). The images produced were magnificent, as shown below:

By counting the proportion of single labelled IdU or EdU cells in the lens epithelium, and the proportion of double labelled cells, the cell cycle time and length of S phase could be calculated using the following equations:

Length of S phase (Ts):
Ts= Ti / (L cells / S cells

Cell Cycle time (Tc):
Tc = Ts / (S cells / P cells)

(Ti = Length of IdU exposure, L cells = IdU only positive cells, S cells = EdU only positive cells, P cells = total number of cells) (5)

On average Ts and Tc lasted longer in Pax6+/+ than Pax6+/- lenses (Figure 3 and 4), however, when a t-test was conducted there was no significant difference shown between Pax6+/+ and Pax6+/- (t- test values; Ts = 0.11947 and Tc = 0.19446). The Pax6+/- lenses were very variable, which perhaps reflects the clinical variability of Pax6 mutation symptoms. If I had extra time, I would repeat the experiment with more embryos to allow for an increased statistical power for Ts and Tc between Pax6+/+ and Pax6+/- mouse embryos.

Looking back at my time in the lab, I can’t believe how much I have learned. I could have never imagined myself conducting research independently as an undergrad, however, from week 2 I felt confident enough to proceed with protocols on my own accord. My 8-week project has sadly come to an end, but I feel more excited than ever for a future in developmental research. I would like to thank Professor Martin Collinson and the Gurdon Summer Studentship for giving me this opportunity and making my summer in Aberdeen a lot less grey.

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.

Our third report from the 2018 group of student awardees comes from Natalie Dugdale (student at The University of Liverpool) who undertook her research with Thomas Butts (also at Liverpool). 

The cerebellum provides a good model for the study of the cell cycle and differentiation of neurons in the brain as a whole due to the great number of neural progenitors it produces. Tom’s lab focuses on the bone morphogenetic protein (BMP) signalling pathway and how the manipulation of proteins within this pathway affects the timing of a neural progenitor cell’s exit from the cell cycle, leading to terminal differentiation. When BMPs bind with their corresponding receptor on the cell surface, SMAD proteins are activated and these can initiate transcriptional changes through downstream signalling within the cell. By introducing an altered SMAD protein via vector into chick embryos through microinjection, the team was able to follow the effect of permanently “turned on” SMAD proteins in the embryo’s cerebellar development. To begin with in my project, we injected plasmids containing GFP or Tomato to perfect our aim and competence with electroporation.

Chicks are great model organisms for developmental research due to the ease of access to their embryos and their significant genome homology with humans. The cerebellum, the focus of the lab’s research, is an area of the brain where birds and mammals share a similar physiology. Chicks and humans share a highly proliferative external granule layer; the origin of a great number of cerebellar neural progenitor cells, which form our densely packed cerebellum, buckling into the highly folded structure seen in both mammals and birds.

As an undergraduate student at The University of Liverpool I was lucky enough to be awarded the Gurdon/BSDB Summer Studentship this year. This allowed me to spend two months in Dr Tom Butts’ lab within the Department of Cellular and Molecular Physiology at the University of Liverpool, researching neuronal development within the cerebellum. In my first few days in the lab, I was mostly shadowing Graham and Hal, both Masters students nearing the end of their projects and helping Wen, another undergraduate on her summer placement. Whilst watching Graham injecting E4 embryos I was a little stunned when he finished an embryo and asked me if I’d like to try a few. Suddenly feeling very much in the deep end, I took him up on the offer and spent an exceptionally long time tentatively breaking through membranes (in constant fear I would damage the embryo) and shakily making my first injection and electroporation of the rhombic lip within the hindbrain, from which the cerebellum develops. Although a delicate procedure, I was quite surprised I was able to accomplish it, then even more surprised to see the following day that one of my embryos had taken up some DNA in the brain (surely beginner’s luck!). With the continual support and enthusiasm of everyone in the lab, the “deep end” I had felt I was in quickly became much shallower.

As time went on and I became more practiced (and more importantly, more confident) working on the chicks, I was finishing electroporation of half a dozen eggs in less than half the time it took me to do two in the first week. I spend a lot of time early on practising dissection of the chick embryos to
establish the skill required to create histological brain sections for imaging under confocal microscope. This was a far trickier procedure than the injections primarily due to the size of the embryo (weighing a tiny ~0.05g at E4) and it required a knowledge of chick physiology as to isolate the correct section of the brain. After the first month we also began targeting our injections to the floor plate of the mid-brain, of which some of my results can be seen below. With every good result I found myself more eager to get in each morning and analyse the previous day’s electroporations. I also had my first attempt at presenting a journal club, more good experience for someone who wants to pursue a career in research.

Wen, Lydia (another undergraduate on summer studentship) and I also worked on mapping a vector. We did not have the exact sequence and wanted to confirm the presence and order of the inserts such as the promoter, GFP label and the MCS. This was a project I felt more confident starting on with my background in genetics and we were mostly free to design the primers and run our PCRs and gels ourselves, with a little guidance from Tom. Throughout the plasmid analysis, I had to produce a comprehensive report of the primers used and results gained to allow future students to continue our work. This gave us a chance to work as an independent group, rather than following a rigid step-by-step guide with a demonstrator watching over us, as is easy to become accustomed to in lab sessions at university.

Having spent many hours peering down a dissecting microscope performing injections, electroporations and dissections, I have had a real confidence boost in my capabilities in the lab which I’m sure will go a long way as I move into my final year of university and begin my dissertation project. I have finished my summer studentship with a newfound eagerness to continue from university into a career in research in developmental biology. I am greatly appreciative to Dr Butts and everyone I worked in the lab for giving me a fascinating and fully immersive working lab experience, and very thankful to have been awarded the Gurdon/BSDB Summer Studentship, allowing me to take up this position over summer.

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.

Our second report from the 2018 group of student awardees comes from Oliver Beaven (student at Durham), who undertook his project with Colin Jahoda, also at Durham. 

Do embryonic mouse tails regenerate?

This summer, with the incredible help of the Gurdon/The Company of Biologists Summer Studentship, I was lucky enough to work under the supervision of Prof. Colin Jahoda in his lab at Durham University. The project I worked on aimed to determine whether epimorphic regeneration occurs in the tips of embryonic mouse tails.

Epimorphic regeneration has scarcely many examples within mammalian systems. The most famous cases among animals are found in the limbs of salamanders, which can regrow fully functional limbs at any stage of their life cycle (Godwin & Rosenthal, 2014). Mammals are far more restricted, with cases found in nail buds, and ear pinnae (Seifert & Muneoka, 2018), but none in hugely complex developmental organs, such as limbs.

The process of epimorphic regeneration begins as the wound site re-epithelializes, and the surrounding tissue dedifferentiates to form a proliferative regeneration blastema. This is then followed by the re-initiation of developmental growth and patterning (morphogenesis). Late last year, the Jahoda lap reported both rapid wound sealing and growth in E13.5 embryonic mouse tails (being studied for other purposes). These highly novel observations therefore correspond with the definition of epimorphic regeneration. Curious, we decided to take a closer look.

The main bulk of my project worked towards elucidating whether the growth which had been observed previously could be classified as actually proliferative, regenerative growth, or simply growth by cellular expansion (not regeneration). I examined this using EdU proliferation markers – a thymidine analogue incorporated into DNA molecules during replication – along with immunohistochemical methods.

The experiments took place with mouse tails removed from E13.5 embryos. Most of the culturing took place on collagen filters, with an EdU pulse three hours before they were frozen down for sectioning. An issue we faced with the collagen filters is the tendency for the wounded end of the ablated tails to adhere to their surface. This would mean of each litter, most tails fail to close fully, blocking us from observing any regeneration in these tails. We tried overcoming this design limitation through the use of 3D hanging drops. While this generally got good results for shorter cultures, long term tail cultures appeared unhealthy. By the end, we tried merging the two methods of short and long-term culture, which seemed to work (unfortunately my time ended before we could generate a refined organ culture method).

Most tails which survived from culture to sectioning contained an EdU profile and an immunohistochemical stain of either fibronectin, or collagen IV, allowing us to identify the position of the EdU stain relative to the basement membrane. We were looking for staining behind the basement membrane, with pronounced upregulation at the tip, which would correspond to the forward growth just beneath the wound site (i.e. within the regeneration blastema). There were several good examples of this taking place when the sections were cut in the middle of the tail (fig. 1 – note that this tail did not have an additional stain). Something notice in this figure is the horizontal orientation of cells right beneath the tip – this is synonymous with patterns seen within regeneration blastema’s, giving support to our hypothesis that epimorphic regeneration was taking place at the tip.

Unfortunately, my finite time in the lab, combined with the harrowing challenge of producing perfect sections at the very centre of the tail, meant our dataset was limited. Consequently, we were unable to confidently state whether the proliferation was congruous with regeneration. This became something of a theme throughout my project; but something I have since come to respect about the nature of research.

We briefly attempted to find whether dedifferentiation occurred on any large scale towards the tip. Our methods again applied immunohistochemistry to observe this epigenetic phenomenon. We used three separate, global histone methylation markers, alongside our EdU analysis. Unfortunately, our markers were too general to notice any significant patterns of dedifferentiation within our samples. It seems the restricted extent to which tissues de-differentiate was matched in their extent of epigenetic repatterning.

Although we are still a way away from positively characterising these phenomena as regenerative processes, it has opened many exciting questions to be explored and debated before a firm statement can be made. Personally, this experience has been exceptionally beneficial for my understanding of research, and has taught me to appreciate new ways to carefully interpret results from a critical perspective. It was also a lot of fun! I would like to send the warmest thanks to Colin and Adam for being endlessly helpful, patient and welcoming, and look forward to hearing about what more results come through in the future.

References

Seifert AW, Muneoka K (2018) The blastema and epimorphic regeneration in mammals. Developmental Biology 433(2):190-199
Godwin JW, Rosenthal N (2014) Scar-free wound healing and regeneration in amphibians: Immunological influences on regenerative success. Differentiation 87(1-2):66-75

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.

Our first report from the 2018 group of student awardees comes from Annabel Adams (student at King’s College London), who undertook her project with Andrea Streit, also at King’s. 

As a second-year neuroscience undergraduate, I wanted specialist involvement in a working lab to progress my knowledge beyond the practical classes offered on my course.  The Gurdon Studentship provided the support which allowed me to translate my interest in research into tangible experience. I was therefore very grateful to be able to join Andrea Streit’s lab at King’s College London for eight weeks. During my studies, my embryology module caught most of my attention. In particular, part of my coursework focused on a paper that used the chick model to observe the development of craniofacial features. When reading around the subject I encountered various techniques used in chick and wanted to see their implementation first hand. Therefore, the Streit lab was ideal because not only do they use some of these methods, but they were also able to take me on as an intern.

One of the objectives of the Streit lab is to investigate the mechanisms behind how progenitor cells become committed to a certain lineage, specifically focusing on sense organs like the ear. They have previously characterised a circuit of eight transcription factors at the top of the gene network that governs the way in which cells progressively commit to ear identity. (Chen et al., 2017)

To build upon this discovery, the question my project aimed to address is whether or not this transcription factor module is active elsewhere in the chick during development, and if so how its architecture changes. In humans mutations in some of these factors not only result in deafness, but also in kidney and limb abnormalities. This suggests these regions could share common features with the ear module. To begin to establish if this is the case, I first performed in situ hybridisation (ISH) on a range of chick stages in order to analyse the expression patterns of these factors. I used antisense probes complimentary to the mRNA of each of six transcription factors present in the ear module: Lmx1a, Prdm1, Sox8, Sox13, Pax2 and Zbtb16. The probes were labelled with DIG (digoxigenin) allowing the use of anti-DIG antibodies followed by a colorimetric reaction to detect where each gene is expressed.

The procedure itself is relatively straight forward. However, the process is quite slow since several steps require a long time to complete. Therefore, planning and managing time pressure is one of the main challenges I came across. This was apparent when juggling several experiments at once. At first, it was a difficult to balance the fragility of the samples with furiously pipetting washes to get them in the incubator before the day’s end. The ability to multitask was a skill I had not previously considered to be important as a scientist. However, my experience has helped me see how I need to improve my organisation in order to get the most out of lab hours. Learning this lesson early stands me in good stead for my research project in third year. Eventually, I became more efficient at managing my time which lead to me feeling able to attempt a more complicated procedure. Having established expression patterns, the next challenge was to assess whether some of the regulatory relationships in the ear circuit are maintained in other organs. For example, the Pax 2 enhancer that is active in the ear, but does it also show activity in the kidney or limb? This was accomplished through the electroporation of a reporter plasmid, where the enhancer drives GFP, followed by fluorescence imaging. This involved successful embryo culture, plasmid injection and transfer of the plasmid using a current – many steps involving numerous opportunities for things to go wrong. The samples were a challenge to handle owing to their small size. Therefore, it took several attempts to build up enough dexterity and confidence to execute each step properly. Initially, it was frustrating when most of the cultures didn’t survive or the plasmid injections missed their target. Yet, this was outweighed by the satisfaction of when it all came together and I saw the fluorescence through microscope signifying my first successful electroporation.

Overall, an internship was a big time commitment but one that was invaluable in helping me validate my desire to pursue a career in research myself.  My time in a supportive working lab has taught me not only how to obtain results but also how to interpret them. Each day I was becoming more accustomed to techniques and equipment simply through practice. However, through engaging with the lab and their work as a whole, both in formal meetings and in conversation, I became more familiar with what a scientist looks for in their experiments, and crucially, assessing how what they found could apply to a wider context. I have learnt the importance of being aware of the work outside one’s own. As a student, going beyond a surface level understanding of what you are doing begins with immersing yourself in the field. An internship in a lab working on a project you have interest in is a great way to start.

 References

1. Chen, J., Tambalo, M., Barembaum, M., Ranganathan, R., Simões-Costa, M., Bronner, M. and Streit, A. (2017). A systems-level approach reveals new gene regulatory modules in the developing ear. Development, 144(8), pp.1531-1543.

Closing Date: 15 March 2021