Shoshkes Carmel lab has an opening for a passionate postdoctoral fellow in the field of Telocytes as niche cells. We are seeking for a candidate with a strong mouse genetics background. We apply “-omics”, genetics and live-imaging approaches to uncover key aspects in the cell biology of Telocytes, large stromal cells recently emerged to constitute the intestinal stem cell niche, and their role in intestinal homeostasis.
For more details please see: Shoshkes-Carmel M et al., Subepithelial telocytes are an important source of Wnts that support intestinal crypts. Nature 2018 May 2.
Please apply via email including a cover letter with a short statement of research interests and Curriculum Vitae to:
Dr. Michal Shoshkes-Carmel
Dept. of Developmental Biology and Cancer Research
The Harvey Laboratory is looking to employ motivated and talented postdoctoral fellows to study the role of the Hippo pathway in organ size control and cancer. Our research is situated at both the Peter MacCallum Cancer Centre and Monash University in Melbourne, Australia.
We employ a range of techniques including Drosophila genetics, advanced microscopy, transcriptomics, bioinformatics, molecular biology and cancer cell biology. You will work in a supportive team, be a good communicator and also have the ability to work independently. You will possess expertise in a range of molecular and cell biology, biochemical and genetic techniques. Experience in Drosophila genetics is advantageous but not essential.
Peter MacCallum Cancer Centre is Australia’s largest specialist cancer centre with 600 research staff and students. We are located in Australia’s largest and most vibrant biomedical precinct with more than 10,000 researchers across multiple Universities, Research Institutes and Hospitals. Melbourne is a multi-cultural city with great food, weather, culture and sport and is often voted the world’s most liveable city.
For more information visit this link: https://www.petermac.org/research/labs/kieran-harvey
Poon et al., (2018). A Hippo-like signaling pathway controls tracheal morphogenesis in Drosophila melanogaster.Developmental Cell.47: 564-575.
Manning et al., (2018). Dynamic fluctuations in subcellular localization of the Hippo pathway effector Yorkie in vivo.Current Biology.28: 1651-1660.
Degoutin et al., (2013).Riquiqui and Minibrain, regulators of the Hippo pathway downstream of Dachsous.Nature Cell Biology.15: 1176-1185.
Harvey et al., (2013). The Hippo pathway and human cancer. Nature Reviews Cancer.13: 246-257.
Poon, et al.,(2011). The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Salvador-Warts-Hippo pathway. Developmental Cell.21: 896-906.
Birds are a dominant group of land Vertebrates (probably the largest in numbers with +10000 species described), highly successful and diverse. Birds originated from members of the Theropoda: the meat-eating dinosaurs that included famous forms like T. rex or Velociraptor, well-known from the movies. The fact that birds are a kind of dinosaur has been a matter of debate not except from controversy, but largely accepted today. The two most important lines of evidence that allowed us to understand the true identity of birds have been the (incredibly detailed) fossil record of the non-avian to avian dinosaur transition, and birds’ embryological development. Embryology reveals the dinosaur within birds and in cases development can parallel in short time what we see to have happened in millions of years of fossil record. Alexander Vargas’s lab at the University of Chile focuses on this evolutionary transition, employing both fossils and embryos to understand the workings of evolution.
Researchers of comparative anatomy have documented how embryos show the dinosaur-bird link since the times of Darwin, mostly focusing on the postcranial skeleton. With this idea in mind, we set to study the development and evolution of the dinosaur skull. The head of birds is very unique, and two key differences when comparing birds to other reptiles are their skull and the huge brains and eyes birds possess. The bones composing the skull of birds are thin, light and mostly fused together in the adult. In addition, two bones (the postorbital and the prefrontal, located behind and in front of the eye respectively), were lost at different moments during the evolution of the dinosaurs leading to modern birds. Both in the fossil record and during embryonic time, how and when these bones disappeared, and if there were remnants of their presence as suggested by previous reports, were some of the questions we wanted to resolve in our recent paper in Nature Ecology and Evolution.
Fig.1: Traditional view of the evolution of the theropod skull and the loss of prefrontal and postorbital bones in the line leading to modern birds
By looking at the fossil record of Theropods, we found two distinct “moments” when these bones were lost, at the origin of two recognizable clades: the postorbital was lost at the Euornithes node, close to modern birds; while the prefrontal was lost at the Pennaraptora node, right at the base of the clade containing dinosaurs like Oviraptor, Velociraptor, Archaeopteryx and modern birds. This bone has an interesting story, as in most early members of this clade, the prefrontal was not present, while the lacrimal bone acquired a T-shape (in contrast to the inverted L-shape of other dinosaurs that did possess a prefrontal). This T-shape, in which the T’s posterior tip occupies the position otherwise used by the prefrontal, suggested these bones were the result of fusion between the prefrontal and the lacrimal. The surprising part was that some dinosaurs seemed to be reverting into having a separate prefrontal bone. Some specimens of different species of these dinosaurs were showing a separate prefrontal bone, while at the same time losing that “tip” of the lacrimal that could have been the fused prefrontal. Even members of the same species would show variability in the presence or absence of the adult bone, as Deinonychus or Archaeopteryx specimens could have a prefrontal separated. To understand the basis of these changes in the cranium, we went to embryos.
Fig. 2: In the specimen of Deinonychus MOR747, the lacrimal has an inverted-L shape and is accompanied by a separated prefrontal bone (yellow), just like in earlier theropod dinosaurs but unlike closely related species like Velociraptor.
Chicken embryos have been a preferred animal model for those willing to study embryonic development for more than 2000 years, and have provided the vast majority of the information we have today about skull development for birds. However, they are just one species among thousands, representing one of hundreds of distinct lineages. In an effort to include more of the disparity and diversity of birds, we collected embryonic series of six families of birds, including (of course) chicken, but also ducks, and species not normally used for developmental studies like lapwings, coots, budgerigar and tinamous (a member of the palaeognathae related to ostriches and emus), and also included embryonic stages of Alligator mississippiensis to broaden our sample to members of the two living archosaur groups, birds and crocodylians. By looking at the avian embryos and their developing ossifications alone, we confirmed the presence of more ossification centers than adult bones in the embryos of birds, but only by comparing them with alligator embryos and the fossil record were we able to interpret them in light of their evolutionary history.
Fig. 3: Authors in the hunt of red-gartered coot nests, with permission of the Agriculture Service of Chile.
In embryos of all the birds observed, two ossification centers develop and make up the adult lacrimal bone. These two ossifications were observed and identified as the lacrimal and prefrontal bones of chicken by Erdmann in 1940, but we also found two separate ossifications giving rise to the lacrimal of alligator embryos, which do form a separate prefrontal from another ossification center, meaning Erdmann’s identification of a prefrontal bone in the chick was mistaken. However, we did find an embryonic bone in one species, the Chilean tinamou, where in addition to the two ossification centers of the lacrimal, a third, well-developed ossification develops in a position similar to that of the embryonic Alligator and adult dinosaur prefrontal. This bone, however fused to the nasal bone, and becomes indistinguishable from its other pieces. In a way, the bone is doing what we think it did in dinosaurs like Velociraptor: developing as a separate center and later fusing to a neighboring bone. This separate embryonic origin can explain why it re-appeared in different specimens as a separate adult bone, as it likely failed to fuse. However, in the tinamou, it is doing something it did not do in Velociraptor as instead of the lacrimal, it is fusing to a different neighbor.
Fig. 4: The prefrontal ossification of archosaur embryos (tinamou and alligator) and the adult bone in dinosaurs. Taken from Figure 1 of the original paper.
We also found an “extra” ossification not corresponding to any adult avian bone and just behind the eye that looks like the embryonic postorbital ossification of Alligator and the adult postorbital bone of dinosaurs, but instead of remaining separate as in these animals, fuses to the back of the frontal bone.
Fig. 5: The postorbital ossification of archosaur embryos (duck and alligator) and the adult bone in dinosaurs. Taken from Figure 2 of the original paper.
In a way, we were expecting to find this ossification, since the frontal of birds has been described as being formed from two separate portions, derived of two distinct embryonic germ layers; the mesoderm and the neural crest. Bones in the skull of vertebrates come in two flavors, as the most-rostral ones derive from neural crest cells and the ones in the back of the skull come from the mesoderm. We have known of this for years, thanks to careful chick-quail chimera experiments, and the double origin of the frontal has in fact created a lot of debate on the frontal identity itself. In other vertebrates, bones usually derive from one of these embryonic sources, and the frontal in particular is made up cells of neural crest origin. While other researchers also proposed the mesodermal portion could correspond to a separate bone that ended up fusing to the frontal, the identification of this portion as the parietal bone did not agree with many morphological criteria, anatomical correspondences nor with the evidence presented by the fossils. Our suggestion that the back portion of the avian frontal comes from the ossification that gives rise to the postorbital in other reptiles is consistent with the compared embryology of birds and crocodylians, as well as with the fossil record. This proposed homology implies the postorbital of Alligator should derive from mesoderm cells, something not yet corroborated as fate mapping of crocodilian embryos has not yet been done.
Fig. 6: The postorbital and frontal ossifications of chicken make up the adult frontal bone. Taken from Supplementary figure 4 of the original paper
The history of transformations and fusions of the prefrontal and postorbital provides us with some interesting lessons on how evolution takes place, but also leaves us with a number of intriguing questions of how skull development is regulated. Upon fusing with a neighboring bone, both the prefrontal and postorbital apparently lost their own identities, becoming a non-independent part of the larger bone, not showing any morphological similarity to the separated bone of other species. Moreover, the cells that make up the independent ossification centers end up forming a structure in which there’s no trace or clue of their origin, even when coming from two very distinct embryonic sources as are the neural crest and mesoderm. Contrary to most of the bones in the body, which form a mold or cast of cartilage that is later replaced by bony tissue, many bones in the skull (particularly those of the face, skull roof, palate and jaws) develop directly into bone from mesenchymal condensations. We know a (staggering) lot more about how these cells invade the head and face or find their location than what we know about how the mesenchymal condensations are established and how they turn into individual bones. Between the migration of neural crest cells into the head, for example, and the onset of bone formation, there’s a gap in our understanding that has still to be bridged. Questions like what signals regulate the condensation mesenchyme, the beginning of bone formation or the spatial distribution of the ossification centers that form the bones are still poorly answered. Our knowledge on this kind of bone development derives from (and has been limited by) histological sections, which do not allow us to have a whole-picture of what’s going on in the whole head, and the study a few molecular markers that mostly label post-mesenchymatic condensation and pre-osteogenic stages of maturation. It is, in truth, by these limitations that our study relied only on bone staining to observe and compare the ossification patterns of different species. More understanding of the whole picture was not going to come from digging into deeper molecular mechanisms in the chicken, but from studying and comparing the development of more species, in a maybe simpler way, like Alizarin staining.
One interesting thing to consider is that one possible driver behind the huge modification of the skull in the evolution of birds might be the evolution of a big brain. Birds have enlarged brains compared to other reptiles and non-avian dinosaurs, and the enlargement of the brain would necessitate a re-structuring of the skull covering it. Other cases of evolution of big brains also result in evolution of the skull as a whole, and even loss of bones, maybe in a similar way than what we see in birds. In mammals, bones of mixed origin proved to be composites resulting from fusion of elements, including those supposedly lost long ago in the earliest mammalian lineage. Although these bones are located in the back of the skull, mammals also lost bones like the prefrontal or postorbital. The brain, being a huge structure lying directly underneath the developing skull-roof, can possibly influence mesenchymal condensations, bone deposition, ossification rate and overall timing and spatial development of the skull. How these structures interact and how they evolve in concert is yet to be studied and understood.
One last lesson we can derive from our results is the evolutionary meaning of the embryonic persistence of a structure. The ossification centers we found add to a longer list of stories in which the embryo retains at least a rudiment of a long-lost structure, which enables the evolution of new morphologies in later lineages. By retaining the prefrontal ossification, for example, some dinosaurs were (and still are) able to “experiment” a variety of morphological outcomes that include fusion with the lacrimal or nasal bones. It also worth noting that, at least in the case of the skull of birds, the adult bones end up fused together. So in a sense, the order or pattern in which ossification centers are beginning to fuse to each other could be less of adaptive significance than it is just a phenomenon of developmental drifting, in which a pattern is established and conserved. Itself, the high degree of bone fusion in the avian adult skull could be an extension of a trajectory started by those dinosaurs in which first two bones, then two others began fusing.
Fig. 7: Ontogeny and phylogeny. Possible developmental outcomes can be possible adult phenotypes of different lineages.
How does evolutionary change take place as shown by the fossil record and during embryonic time can be a question not just of “how dinosaurs looked like” but also an important source of understanding of developmental mechanisms at play every day.
Artwork by Luis Pérez López [CC BY 4.0]
Erdmann, K. (1940). Zur Entwicklungsgeschichte der Knochen im Schädel des Huhnes bis zum Zeitpunkt des Ausschlüpfens aus dem Ei. Zeitschrift für Morphologie und Ökologie der Tiere, 36(3), 315-400.
Le Lièvre, C. S. (1978). Participation of neural crest-derived cells in the genesis of the skull in birds. Development, 47(1), 17-37.
Noden, D. M. (1983). The role of the neural crest in patterning of avian cranial skeletal, connective, and muscle tissues. Developmental biology, 96(1), 144-165.
Evans, D. J., & Noden, D. M. (2006). Spatial relations between avian craniofacial neural crest and paraxial mesoderm cells. Developmental dynamics: an official publication of the American Association of Anatomists, 235(5), 1310-1325.
Piekarski, N., Gross, J. B., & Hanken, J. (2014). Evolutionary innovation and conservation in the embryonic derivation of the vertebrate skull. Nature communications, 5, 5661.
Maddin, H. C., Piekarski, N., Sefton, E. M., & Hanken, J. (2016). Homology of the cranial vault in birds: new insights based on embryonic fate-mapping and character analysis. Royal Society open science, 3(8), 160356.
Abzhanov, A., Rodda, S. J., McMahon, A. P., & Tabin, C. J. (2007). Regulation of skeletogenic differentiation in cranial dermal bone. Development, 134(17), 3133-3144.
Stockholm University, Sweden, invites applications for one postdoctoral position in the laboratory of Professor Mattias Mannervik at the Department of Molecular Biosciences, The Wenner-Gren Institute (http://www.su.se/mbw). The position is scheduled to start as soon as possible.
Transcriptional coregulators are proteins that facilitate communication between transcription factors and the basal transcription apparatus, in part by affecting chromatin through post-translational modification of histones. As such, they contribute to generation of cell-type specific gene regulatory networks and epigenetic control of animal development (see Mannervik et al. Science, 284, 606-609, Boija et al. Mol Cell, 68, 491-503). This laboratory is using genomic, genetic, and transgenic approaches in Drosophila melanogaster to elucidate the molecular mechanisms of transcriptional and chromatin regulator function during development. In this project two approaches are used to investigate the in vivo role of histone modifications in gene expression. A histone replacement system is used to examine the effects of amino acid substitutions in the histones on organismal development, and a modified CRISPR/Cas9 system is employed to target chromatin modifying enzymes to endogenous loci.
The position is available immediately and requires a recent Ph.D. as well as extensive experience in molecular biology techniques. The successful applicant should have a high-quality publication record, and motivation to study underlying mechanisms of gene regulation in development. The position will be funded with a fellowship, and includes health insurance.
Stockholm University is one of the largest and most prominent universities in Sweden, located in the nation’s capital city, beautifully surrounded by the first national city park in the world. For further information, see http://www.su.se/english/ and http://www.academicstockholm.se/
Application: Applications marked with reference number SU 465-0156-18 should be submitted electronically as a single PDF file to mattias.mannervik@su.se and to birgitta.olsson@su.se
The application deadline is February 1, 2019.
Applications should comprise the following:
1) a personal statement describing your interest in this project (1-2 paragraphs), research experience (1–2 paragraphs) and career goals (1-2 paragraphs)
2) curriculum vitae
3) bibliography
4) names, e-mail adresses, and phone numbers of three references
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 final report from the 2018 group of student awardees comes from Rachel Wong (student at Queen’s University Belfast) who undertook her research with Karen Liu(King’s College, London).
Analysis of Rapgerf5 and canonical Wnt signalling in embryonic mouse development
During the summer of 2018, I worked with Dr John Griffin in Dr Karen Liu’s lab at King’s College London. My focus was on the gene RAPGEF5, which was previously identified as a candidate gene for heterotaxy, a congenital disease affecting heart development and the spatial arrangement of organs. It is estimated that 1 in 10,000 people are diagnosed with heterotaxy, and is the cause of 3% of all congenital heart cases [1]. However, the genetics of heterotaxy are still unclear. Thus research is necessary to understand the disease mechanism in more detail.
Not much is known about RAPGEF5 protein, but we know it is involved in the canonical Wnt pathway, in the transportation of beta-catenin into the nucleus [2]. When Wnt is active, a cascade of chemical reactions prevent the degradation of beta-catenin in the cytoplasm, allowing it to bind to a transporter protein to enter the nucleus. Our current model suggests that in response to Rap-GDP conversion to Rap-GTP by RAPGEF5, beta-catenin can dissociate from the transporter protein. This frees beta-catenin, allowing it to interact with DNA-binding proteins to alter gene expression. Therefore, my project aimed to answer three key questions:
Where is canonical Wnt signalling active during embryonic development?
Where is RAPGEF5 expressed during embryonic development?
Does loss of RAPGEF5 lead to any developmental abnormalities such as heterotaxy?
To answer my first question, I used genetically modified mouse embryos carrying a TCF/Lef-dependent reporter to visualize the areas with active Wnt signalling. When Wnt is active, the transcription factors TCF/Lef are active and bind to specific binding sites on DNA. This activates a promoter that causes the GFP reporter gene to be expressed and produce proteins that fluoresce under a specific wavelength.
I dissected embryos from their sacs at weeks E11.5, E12.5, E13.5 and E14.5. They were then photographed using a fluorescence microscope. In general, fluorescence can be seen in the ears, edges of limbs, spine, branchial arches, whiskers and brain. The heart was only visible in the E11.5, as the skin over the heart was too thick at later stages (Figure 1.a). At week E14.5, fluorescence was only faintly visible at the ear, due to the thicker skin.
In response to question 2, I fixed and dehydrated the E11.5 embryos for whole mount mRNA in situ hybridization. A Rapgef5-specific probe was used to stain the embryo, and the result was shown in figure 1.b. RAPGEF5 mRNA was expressed in the heart, brain, spine and the tip of the hind limb.
Figure 1 – Both photographs are of a TCF/Lef E11.5 mouse embryo. The left shows GFP signal reporting active Wnt., and the right shows the whole mount in situ hybridization with Rapgef5 probe Structures highlighted are the heart (yellow arrows), mid brain (pink), ear (red), tip of hind limb (blue).
Figure 1 presents two photographs of an E11.5 embryo, one showing the distribution of RAPGEF5 specifically, and the other for active Wnt signalling. When compared, the distribution appears similar, however there are discrepancies such as the ear, and tip of the hind limb.
Finally, for my third question, we bred RAPGEF5 mutant mice and inspected them at stages E9.5, E10.5, E14.5 and 6 weeks after birth. Tail clippings were taken from the embryos and ear clippings from the pups for DNA extraction and PCR to confirm their genotype, as they could either be wild-type, heterozygous or homozygous. Unfortunately the genotyping was still in the stages of trial-and-error, as the bands in the gel electrophoresis did not match the reference DNA ladder, and further tweaking with the PCR temperature and primers is necessary.
However, there were some phenotypic changes found. Out of the total of 9 E9.5 amniotic sacs, 4 were had healthy embryos, 4 were empty and 1 was malformed and underdeveloped. It is possible that this embryo was in the process of being reabsorbed to match the 4 other empty sacs. For the E10.5, there were 8 embryos in total, and 2 had underdeveloped heads and lacked proper surface morphology. All 8 of the E14.5 embryos were phenotypically normal.
Figure 2
Interestingly, as seen in figure 2, when compared with the wild-type, the 6 week old pup had bald patches in a ‘Christmas tree’ pattern and a possible front limb deformity. Both were similar in size and behaviour.
I thoroughly enjoyed my summer studentship at King’s, and learnt many new techniques such as wax sectioning and mounting, in situ hybridisation and using fluorescence microscopy. I would like to thank Mr John Griffin, Dr Karen Liu and the Liu lab for taking time out of their schedule for their help and guidance.
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 seventh report from the 2018 group of student awardees comes from Lucienne Pullen (student at Oxford) who undertook her research with Duncan Sparrow (also at Oxford).
Do environmental teratogens influence craniofacial development? Exploring embryopathies and birth defects in the context of maternal diabetes
My name is Lucienne Pullen and I am a third year undergraduate studying Medicine at Merton College, at the University of Oxford. This summer, I had the immense privilege of working for a Gurdon/Company of Biologists Summer Studentship, working in the Sherrington building in the Sparrow lab. My supervisors, Dr Duncan Sparrow, BHF Senior Basic Science Research Fellow, and Dr Nikita Ved, Novo Nordisk Post-Doctoral Research Fellow, undertake research in the Department of Physiology, Anatomy, and Genetics, and are focussed on embryonic cardiac development and its perturbation by genetic and environmental factors. Dr Ved in particular specialises in how pre-existing maternal diabetes induces embryonic heart defects.
I have been interested in the pathophysiology of Diabetes Mellitus since our first year lectures on metabolism and the problems that arise when it is dysregulated, and further study in the second year allowed me to explore the autoimmune and mal-resolving inflammatory aspects of the disease in more detail. Entering the Final Honours School of my course, I was highly motivated to continue this exploration in a different area: the effects of maternal diabetes on the developing embryo. The BSDB Gurdon/The Company of Biologists Studentship project was designed to complement Dr Ved’s research and my FHS project into the effects of diabetes on the placenta.
It may not be widely appreciated, but maternal diabetes carries a highly increased risk of having a child with birth defects (the incidence of birth defects among women with Type 1 and Type 2 diabetes is around 3-5 times higher than among non-diabetic mothers); yet exactly how and when these defects arise during embryonic development has been relatively sparsely studied. The aforementioned statistic is particularly alarming given that routine diabetes testing does not occur until around the 24-28 week stage of pregnancy, despite organogenesis usually occurring within the first three to eight weeks of gestation. Therefore, integral to the project was adding to the existing body of evidence that diabetes is associated with embryopathy, and limb and craniofacial defects more specifically. Any support for a causative link between diabetes and embryopathies can help build a body of evidence to show that early intervention and potentially pre-emptive treatment (or at the minimum earlier screening) would be beneficial to maternal and foetal health, minimising the risks to the developing embryo.
I was personally motivated to undertake this project as I am hugely interested in a career in reproductive medicine, both clinically and in a research capacity. Entering a specialisation in this field in the future would allow me to engage with a research career into areas of developmental biology integral to improving women’s health. The ultimate goal of this project, Dr Ved’s research, and that of scientists in different groups around the world is to produce data that can change guidelines, therapies, or procedures for the benefit of women everywhere.
My project involved using an inducible mouse model system of diabetic embryopathy (the ßV59M mouse model), in which a variety of developmental defects can be seen, including heart, craniofacial, and skeletal defects. The accuracy of this model’s replication of clinical occurrences is supported by the incidence of these defects in human patients. Clinical evidence also indicates that other anomalies occur, including caudal regression syndrome, hypoplastic femur, clubfoot, and improper formation of the cranial bones.
I explored the relatively under-researched area of craniofacial defects; the embryos were investigated using MRI imaging, which provided detailed images which were measured and analysed using FIJI/ImageJ. 82 diabetic and 21 non-diabetic control embryos were analysed with four parameters: snout length, snout angle, tongue length, and lower jaw length of the diabetic mice.
Once the embryos have been collected by around E14.5, they are arranged in cylinders to be scanned using magnetic resonance imaging (MRI). The MRI scans can then be analysed, with four embryos per MRI ‘slice’ measured at a time. At first I found it a little unsettling and difficult to orientate myself with the mouse embryo image, however it was highly interesting and satisfying to learn to recognise key features, and begin to note down when and where I spotted additional abnormalities, such as hydrocephaly, anencephaly, and widespread oedema, which were present in several of the embryos. Furthermore, the analysis process helped me to develop key scientific research skills, such as having specific reference points and scrupulous attention to detail when making measurements; the integrity of the data was dependent on having comparable and standardised measurement techniques.
Difficulties sometimes arose when the craniofacial areas were so heavily deformed it was difficult to find these standardised reference points, however through speaking to my supervisors it was always possible to find a suitable compromise or alternative way of measuring the embryo. On some occasions it was not possible to make measurements due to extreme deformities of particular embryos, which was important in teaching me that research can sometimes be frustrating, and that experimental hurdles can arise that require patients and creativity to overcome.
Preliminary data obtained suggests that in this particular set of embryos, there are no significant differences in the four craniofacial measurements obtained, however further analysis is possible, including looking at alternative parameters and undertaking alternative measurements, including area analysis and transformation to 3D imaging to provide further angles to explore. It would also be interesting to look at this data in conjunction with a companion research project into the effects of placental insufficiency on embryo development in diabetic pregnancies.
I would like to wholeheartedly thank Dr Sparrow, Dr Ved, and the BDSB for this invaluable experience, which has helped me hone my research skills and broaden my horizons in terms of possible future clinical research careers. I would encourage those thinking of applying to do so without hesitation!
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.
Figure 1: Single-cell tracking of cells in the tailbud during the end of somitogenesis: (A) HCR of a zebrafish tailbud showing a mesodermal marker (Tbx16), a neural marker (Sox2), and DAPI in grey. (B) Individual tracks of cells in the tailbud with fates assigned: Red-Mesoderm, Blue-Neural, Yellow-unassigned, overlying a still image of the light-sheet tailbud movie (C) Cell fates (red-mesodermal, blue-neural) shown overlaying a still image of a light-sheet tailbud movie (D) Schematic representation of tailbud progenitor fates, red-mesoderm, blue-neural.
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.
Figure 2. Zebrafish embryo tailbuds with photo-labelled cells of the posterior presomtitic mesoderm (PSM). Embryos were injected with a photoconvertible protein mRNA, Kikume, at one cell stage. Using a confocal microscope, the protein was converted from green to red in the selected area of the PSM and successive photos were taken (A) Image taken immediately after photo-labeling at the 32 somite stage (ss) (B) Image taken 7 hours after initial photolabelling (C) Image taken 24 hours after initial photolabelling. Note, the cells at 32ss plus 24 hours are undergoing division presumably as the fin begins to develop after these stages.
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.
Figure 1. Wild type mouse (A) in comparison to a Pax6+/- mouse(B) displaying microphthalmia (4).
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:
Figure 2. Florescent Immunohistochemistry Images of E14.5 mouse embryo lens. Blue corresponds with DAPI. Green corresponds with EdU positive cells. Red corresponds with IdU positive cells. A-C from a Pax6+/+ developing mouse lens. D-F from a Pax6+/- developing mouse lens.
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.
Figure 3. Histogram showing the length of S phase in hours, with error bars representing the standard error of the mean (SEM)s. There was a trend for S phase to last longer in Pax6+/- compared to Pax6+/+.
Figure 4. Histogram showing the length of Cell Cycle Time in hours, with error bars representing SEM. Cell cycle time lasted longer in Pax6+/- compared to Pax6+/+
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.
A tiring but amazing 10-hour hike to Lochnager with the girls from my lab!
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.
Figure 1: Two E5 chick embryos 24hrs after injection and electroporation of a GFP-containing plasmid injected into the midbrain. Arrows indicate successful injections into the area of interest.
Figure 2: Midbrain floorplate section dissected from a chick in Fig. 1, viewed under confocal microscope. Neural progenitor cells are visible, labelled with GFP.
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.
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