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. 

Our fifth report from the 2017 group of student awardees comes from Jake Cornwall Scoones (student at University of Cambridge), who undertook his studentship with Anna Philpott at the Dept. of Oncology in Cambridge.

During development, cell-lineages sequentially accrue epigenetic modifications that consign them to their fate, transforming from a totipotent zygote to terminally differentiated cells. Waddington’s visual metaphor, envisaging this sequential differentiation as a ball rolling down a terrain of bifurcating valleys, helps to frame questions in developmental biology.⁴ Upon transplanting the nucleus of somatic Xenopus cells into enucleated eggs, Gurdon found that the resultant cell was pluripotent, capable of differentiating into all cell types.⁵ In so doing, Gurdon demonstrated that factors within an egg have the capacity to modify the epigenetic state of a nucleus, implying that the ball can roll back up the valley. In the case of pancreatic transdifferentiation, with sufficient knowledge of this terrain and with the correct molecular intervention, we should able to push the ball into our chosen valley.

Pancreatic development proceeds through two stages: the primary transition (embryonic day 9 (E9) to E12.5); and the secondary transition (E12 to birth).⁶ The primary transition sees endodermal thickening, followed by the pancreatic progenitor proliferation leading to pancreatic bud formation. Through a process of epithelial stratification and micro-lumen formation, the initial tubular morphology that will later characterise the pancreas begins to emerge. This initial expansion phase is followed by rounds of specification and patterning, forming a bipotent trunk and a multipotent tip. Adult pancreata are comprised of three different cell types, namely acinar, duct and endocrine cells, the former derived from tip progenitors and the latter two from trunk cells.

Endocrine development, part of the secondary transition, is triggered by the transient expression Neurogenin 3 (Ngn3) above a threshold level, and proceeds through delamination from the epithelium and aggregation into Islets of Langerhans. Ngn3⁺  cells are specified to one of five fates in a temporally regulated manner:⁷ α-cells produced upon earliest Ngn3-activation synthesising glucagon; followed by β-cells producing insulin; then δ-cells producing somatostatin; then PP-cells producing pancreatic polypeptide; and finally ε-cells producing ghrelin. Previous work from the lab by Azzarelli et al.⁸ demonstrated Ngn3 is phosphoregulated by proline-directed kinases (e.g. CDKs), regulating the balance between proliferation, (Ngn3 hyper-phosphorylation), and differentiation, (Ngn3 under-phosphorylation). β-cell maturation is promoted by the transcriptional regulator Pdx1, among others, whose expression remains confined to β-cells in the adult pancreas. Working in tandem with Pdx1, MafA transcriptionally regulates genes involved in insulin secretion and biosynthesis and it is thus considered a marker of mature β-cell identity.

Ngn3, crucial in assigning endocrine fate initially, and MafA and Pdx1, important in β-cell maturation, have been previously over-expressed in combination to induce transdifferentiation of acinar cells to β-fate in an in vivo model. These factors were used in our experiment to see if such transdifferentiation is possible in an in vitro model, specifically pancreatic organoids, a form of 3D cell culture grown in matrigel. Upon over-expression of these three factors, it is possible to test for changes in cell fate, by looking at symptomatic traits of β-cells, namely the production of insulin.

Over-expression can be achieved through the infection of organoids with lentiviral vectors carrying genetic loci that upon infection will be inserted into the cells’ nuclear genomes. The activation of these imported genes should be controllable so a transactivator is used which, when bound to the antibiotic doxycycline (Dox), becomes active, binding the promoter of our genes of interest, inducing their transcription. The three factors are necessarily expressed together through their transcription within a single ORF, containing MafA, Pdx, Ngn3 and GFP (a fluorescent marker) sequences, separated by loci encoding the cleavage peptide 2A, meaning upon translation, cleavage ensues resulting in four functional proteins. Hence a GFP signal is sufficient to determine the expression of all three factors, leaving other wavelengths free to test for pancreatic hormones through immunohistochemistry.

Two lentiviral vectors were used to infect organoids, one carrying the transactivator gene Tet3G, and the other carrying the locus of interest, Pdx-MafA-Ngn3-GFP (PMN), or GFP as our control. Viruses were produced by the transfection of HEK cells with several plasmids, each containing genes encoding different viral proteins plus the locus of interest, followed by incubation, viral purification and titration. Organoids were incubated with viruses for a week with Dox and then fixed with PFA. Immunohistochemistry was performed, staining organoids for insulin. Preliminary results are very encouraging and I look forward to seeing how my contribution fits into the larger project as a whole.

This studentship has been an invaluable experience, allowing me to gain an understanding of real-world lab science, a practice venturing into the unknown, far from the rushed experiments for which answers were already known performed as a part of my course. I have developed lab-skills, most notably having the rare opportunity to work with organoids, a relatively novel technology. I urge other students to consider applying for this fantastic opportunity in future years.

References

1.  Pagliuca, Felicia W., and Douglas A. Melton. “How to make a functional β-cell.” Development 140.12 (2013): 2472-2483.
2.  Zhou, Qiao, and Douglas A. Melton. “Extreme makeover: converting one cell into another.” Cell Stem Cell 3.4 (2008): 382-388.
3. Takahashi, Kazutoshi, and Yamanaka. “Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.” Cell 126.4 (2006): 663-676.
4. Waddington, C. H. (1957). The Strategy of the Genes. London : George Allen & Unwin
5. Gurdon, John B., Thomas R. Elsdale, and Michel Fischberg. “Sexually mature individuals of Xenopus laevis from the transplantation of single somatic nuclei.” Nature 182.4627 (1958): 64-65.
6. Benitez, Cecil M., William R. Goodyer, and Seung K. Kim. “Deconstructing pancreas developmental biology.” Cold Spring Harbor Perspectives in Biology 4.6 (2012): a012401.
7. Johansson, Kerstin A., et al. “Temporal control of neurogenin3 activity in pancreas progenitors reveals competence windows for the generation of different endocrine cell types.” Developmental Cell 12.3 (2007): 457-465.
8. Azzarelli, Roberta, et al. “Multi-site Neurogenin3 Phosphorylation Controls Pancreatic Endocrine Differentiation.” Developmental Cell 41.3 (2017): 274-286.

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. 

Our fourth report from the 2017 group of student awardees comes from Eleanor Sheekey (student at University of Cambridge), who undertook her studentship with Peter Rugg-Gunn at the Babraham Institute in Cambridge.

Named after the mythical “Land of the young”, Tír na nÓg, Nanog is a homeobox transcription factor expressed in embryonic stem cells (ESCs) aiding continual cellular proliferation alongside the other main pluripotency factors, Oct4 and Sox2. However, despite its contribution to pluripotency, much is still unknown of the mechanistic roles that Nanog plays within stem cells, since cells kept in culture deficient in Nanog retain the ability to self-renew ⁽¹⁾. Interestingly, it was recently found that Nanog provides a link between pluripotency and chromatin organisation suggesting important functions beyond transcriptional regulation ⁽³⁾. With the BSDB Gurdon Studentship, I was privileged to spend my 8 weeks at the Babraham Institute alongside Dr Clara Novo to continue to explore this link.

Centromeres and chromatin

A key hallmark of stem cells is their ability to divide. However, this division must be executed precisely to avoid aneuploidy, an abnormal chromosomal number in a cell, and prevent unregulated division and cancer formation. Centromeres play a crucial role in chromosome separation, composing centric heterochromatin (CH) for kinetochore formation and pericentromeric heterochromatin (PCH) to hold chromatids together ⁽²⁾. Although centromeres are thought to be defined epigenetically, the centro- and pericentromere contain minor and major satellite DNA repeats, respectively. Nanog binds the major satellite DNA repeats within the pericentromere, maintaining the PCH in a ‘more’ open state with increased transcription of major satellites and lower levels of H3K9me3⁽³⁾, bridging the gap between pluripotency and chromatin organisation. It also promotes the formation of chromocenters, which are clusters of major satellite repeats from several chromosomes. Deletion of Nanog leads to chromatin compaction including the PCH. Since PCH is essential for genetic stability and Nanog levels are known to fluctuate in stem cell cultures, the consequences of an altered PCH organisation will need to be understood before successfully developing more stably reprogrammed stem cells for future medical treatments ⁽⁴⁾.

Meet E14 and BT12

Throughout my project, I was working with mouse ESCs (mESCs). Much like in a vending machine, each of the different genetically modified mESCs are assigned a systematic name. E14 is like salted crisps, a male, wild type mESC cell line, originally isolated from the inner cell mass of a developing embryo. BT12, on the other hand is spiced up, no longer containing Nanog, but instead expressing a GFP transgene ⁽¹⁾. Maintaining these two cell lines in culture was essential for conducting my experiments to study the effect of Nanog^-/-.

FISH, IF and ChIPs

As Nanog was shown to affect heterochromatin at pericentromeres, we wondered if this effect extended to the centromere. To tease apart any differences in protein levels and localisation at the centromeres between E14 and BT12, we used a combination of techniques. Immunofluorescence (IF) provides a clear single cell insight and is beautiful to visualise under the microscope. Fluorophores had to be carefully chosen to avoid the wavelength clashing with that of GFP (already being expressed in BT12) and to differ from each other. Fluorescence in situ hybridisation (FISH) visualises specific locations of DNA using single-stranded probes and thus in combination with IF provides a good indication of whether proteins localise to a particular DNA region.

We were able to take some stunning images (Figure1) which could be used to quantify the levels of centromeric proteins assayed in wild-type and Nanog knockout cells. We then used the Imaris software ⁽⁵⁾ to quantify signals obtained from multiple 3D stacks, allowing for easy identification of fluorescent foci (Figure 2). Once the quantification of protein intensity was gathered I analysed it on R-studio to test for significant differences between both cell types (Figure 3).

To further this analysis, we used Chromatin immunoprecipitation (ChIP) that can answer many biological questions associated with DNA-protein interactions by fixing proteins to chromatin. In brief, by using antibodies specific to the protein of interest and magnetic beads that bind to these antibodies, a ‘pull-down’ of captured DNA can be isolated. The eluted DNA can then be analysed using ChIP-quantitative polymerase chain reaction (ChIP-qPCR) to quantify protein intensity at specific DNA regions. In our case, by using primers complementary to the major and minor satellites, we could assess if the binding of our proteins of interest differed between the cell lines.

Epilogue

Much like predicting the murderer in an Agatha Christie novel, one of my favourite aspects of the project was piecing together the experimental evidence to suggest hypotheses to explain our results. It is quite feasible for the absence of Nanog to have a diverse range of effects on the mESCs due to its interactions with DNA and other pluripotency factors, which could result in mislocalisation and/or altered post-translational modifications of its partners. Nevertheless, my preliminary results will require further validation and follow up before any conclusions can be made. However, in the manner of Shakespeare’s comedy, it has definitely been a fun experience, one from which I have gained much more lab confidence and exposure from, of which I am grateful both to the Rugg-Gunn lab at the Babraham Institute and the BSDB studentship that helped fund it.

References:

1. Chambers et. al. Nanog safeguards pluripotency and mediates germline development Nature 450, 1230-1234 (2007)
2. Probst, A. V. and Almouzni, G. (2008), Pericentric heterochromatin: dynamic organization during early development in mammals.Differentiation, 76: 15–23
3. Novo CL, Tang C, Ahmed K, et al. The pluripotency factor Nanog regulates pericentromeric heterochromatin organization in mouse embryonic stem cells.Genes & Development. 2016
4. Lopes Novo, C., & Rugg-Gunn, P. (2016). Crosstalk between pluripotency factors and higher-order chromatin organization.Nucleus, 7(5), 447–452.
5. http://www.bitplane.com/imaris/imaris

Closing Date: 15 March 2021

Closing Date: 15 March 2021

Established  by the British Society for Developmental Biology in 2014, The Gurdon/The Company of Biologists Summer Studentship scheme provides financial support to allow highly motivated undergraduate students an opportunity to engage in practical research during their summer vacation. Each year, ten successful applicants spend eight weeks in the research laboratories of their choices, and the feedback we receive is outstanding. 

Our third report from the 2017 group of student awardees comes from Rachael Adams (student at University of Cambridge), who undertook her studentship with Peter Lawrence at the Dept. of Zoology in Cambridge.

This summer, thanks to the Gurdon/The Company of Biologists Summer Studentship, I was fortunate to spend 8 weeks working in Dr Peter Lawrence’s lab in the Department of Zoology.

The group studies PCP, a pathway which coordinates cell polarity and helps to align epidermal patterns in the Drosophila abdomen. Drosophila larvae are covered with a cuticle decorated by denticles which help the larvae to grip substrate in order to move. These denticles form a specific pattern and changes in the pattern can be used to investigate the properties of the PCP pathway. PCP genes are highly conserved and have been identified as being involved in processes such as vertebrate gastrulation, demonstrating their fundamental importance in animal development and patterning.

My project aimed to investigate the importance of Rab GTPases to PCP. Much research has been carried out on the Rab family of proteins, as they act as master regulators of intracellular membrane trafficking. The accurate delivery of cargo between organelles is crucial for normal eukaryotic cell function. Rabs facilitate this by coordinating vesicle formation, transport and fusion.

In order to test the role of as many Drosophila Rabs as possible I carried out a UAS RNAi knockdown screen of 34 different Rab and Rab-related genes, see figure 1. This screen took 7 weeks to carry out as I had to cross a collection of 103 different UAS-RNAi lines with ptc.Gal4 virgin females. This involved maintaining a population of ptc.Gal4 flies in several bottles. I crossed the virgins with males obtained from each UAS-RNAi stock and incubated the flies at 29℃, the optimal temperature to see the effect of the RNAi. Once each cross was made it was approximately 5 days before third instar larvae could be collected. To identify any change in the phenotype, I mounted several third instar larval progeny of each Gal4-UAS- RNAi cross and examined the denticle pattern produced under the microscope. Most larvae exhibited a wild type pattern, this may be because most of the Rabs I examined don’t have a function in PCP or redundancy of Rab proteins may mask certain phenotypes. The low efficacy of some of the RNAi constructs may have also affected the proportion of successful knockdowns. However, I found that several of the Rab23 crosses produced an unusual phenotype where the tendon cell gaps appeared larger than normal, see figure 2. In order to test whether this result is repeatable, I have crossed the UAS-RNAi Rab23 stocks to different driver lines (sr.Gal4 and en.Gal4). As Gal4 expression is under the control of a different promoter in each driver line, this results in a different expression pattern of the RNAi, see figure 3. Whilst different drivers will not produce the same phenotype, if the denticle pattern is disrupted it may help in the investigation of the role of Rab23 in PCP.

The results from the ptc.Gal4- UAS-RNAi Rab 23 crosses raised the question of why the tendon cell gap appeared larger. Possibilities include: that the tendon cells were enlarged, divided more frequently or the surrounding cells were smaller. To investigate this phenotype, I set up a series of crosses and aim to ultimately generate larvae carrying the Rab23 knockdown and expressing spaghetti squash protein (sqh) labelled with the fluorescent marker mCherry. Sqh encodes the regulatory light chain of non-muscle myosin so is expressed throughout cells. The sqh.mCherry construct therefore allows individual cells to be visualised with a confocal microscope and could help identify why the tendon cell gap is expanded.

To further investigate the phenotype I set up crosses to produce marked clones in adult flies where Rab23 is knocked down, the resultant mosaic animal allows comparison with wild type cells. This is achieved by using the Flp-FRT system to bring about site-specific recombination, see figure 4.

I have really enjoyed my time in the lab and have learned a great deal. I am looking forward to continuing to investigate the Rab23 phenotype and seeing the results of my crosses as part of my Part II Zoology project this term. My special thanks to José Casal for supervising and to everyone in the lab for providing a great deal of help and advice.

Closing Date: 15 March 2021