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

Closing Date: 15 March 2021

When I tell my friends that I spent my summer looking down a microscope at worms they often give a snigger. My enthusiasm on the subject quickly earned me the affectionate (I hope) nickname, ‘Wormboy’. Yet the Wormboys and girls of Richard Poole’s lab instilled in me a great thirst for scientific study over the 8 weeks I spent with them, which I will certainly carry forward in my career. The lab studies the nematode worm C. elegans, and my job was to help characterise a glia-to-neuron transition they discovered in the male tail that occurs during sexual maturation. During my time, I learned how to handle worms, keep them healthy, perform crosses, use a fluorescent microscope and present my data to an audience. All of this as part of my first lab experience was not only invaluable, but very enjoyable, and I came away wanting more.

Worm death was a regular part of lab life. Saving them from starvation and disease was a constant uphill battle, one that at first I found insurmountable. Along with those natural causes of death, they also had to contend me, often crushing them clumsily under my pick and purging potentially contaminative stragglers on said pick in the Bunsen. Happily for the worms, such fates became less and less frequent, and I was eventually able to sustain healthy populations from which I selected young males to dunk in the intensely toxic chemical sodium azide, all the better to view them under fluorescent microscopy. A necessary sacrifice.

This microscopy work was the first step in characterising the glia-to-neuron transition of phasmid socket 1 (PhSo1) into the neuron PHD, which occurs in the male tail during sexual maturation. The lab had previously identified a similar transition of the amphid socket to MCM neuron in the head of the male (Sammut et al., 2015), though the PHD transition differs in how the neuron is formed. Where the amphid socket divides asymmetrically to generate the MCM, the PHD derives from PHso1 by direct trans-differentiation, without a division, while its sister- PHso2- remains glial. This new neuron’s activity was found to be linked to initiation of a novel behaviour employed by males during mating thought to improve their chance of spicule insertion. To track the changing identity of PHso1, I compared its expression of glial and neuronal markers linked to GFP and RFP respectively with that of PHso2. The two glial markers I used were grl-2 (socket specific protein) and mir-228 (pan-glial microRNA), and the neuronal marker a nuclear synaptic fusion protein, rab-3.

The glia-to-neuron transition appears to start in males of larval instar 4 (L4), while the gonad is taking shape. Through picking many, many worms, I showed a clear gradient of change of marker expression in PhSo1 compared with PhSo2:

• In late L3/early L4 GFP brightness is equal in the two sockets
• By mid/late L4, GFP is dimmer in PHso1
• The PHso1 of day 1 adults is beginning to express neuronal marker, displaying coexpression of GFP and RFP
• By day 2, all GFP has dissipated from the newly formed PHD
• Both glial markers displayed the same trend (fi g.1 and 2)
• Some animals classified mid/late L4 displayed coexpression of markers, but these were all on the cusp of adulthood, undergoing moult at the at the time of imaging (fig. 3)

Of course, fluorescent proteins do not perfectly match levels of the markers they represent, rather they give a general overview of the transformation. At the very least, this result shows PhSo1 might partially dedifferentiate before it eventually acquires neuronal characteristics when the worm becomes an adult. Understanding trans-differentiation events such as this is key to understanding how nature itself reassigns cell fate. And if you’re trying to do something yourself in cell biology, it often pays to learn how nature beat you to it. To determine the mechanics of this trans-differentiation would require a more quantitative technique: single molecule fluorescent in situ hybridisation (smFISH).

SmFISH employs fluorescently tagged RNA oligomers, antisense to an mRNA (or miRNA) of interest. Combined, these oligos fluoresce strongly enough that individual RNAs can be resolved and counted within PHso1. Tracking change in RNA quantity rather than GFP fluorescence would tell you whether or not glial expression stops before neuronal expression begins. Does the cell become completely naive? Perhaps only partially? Or maybe there’s no dedifferentiation at all, and the cell passes through a totally novel identity- part glia, part neuron. Though I didn’t have time to perform these experiments, I did create the necessary strains. By crossing a markerless strain with my GFP/RFP animals, and then selecting against RFP over a couple of generations I rendered worms expressing only one of the two GFP markers so that the RFP reporter used in smFISH would be visible.

Though the work I was doing represented a project in its early stages and was at times very laborious, I quickly got an appetite for it. There was a thrill to it, knowing that no one had ever done what I was doing. I felt that buzz again when I arranged the prettiest pictures I could muster into figures to show to the lab, and then again when they ceremoniously scoured my findings with the same level of scrutiny I had seen them exact on each other. Alongside my data, I showed off a particularly pretty picture of both the worm’s glia-to-neuron transitions occurring at once in the same animal, which I think will remain my crowning achievement in science for a long time (Richard even kindly offered to steal it for his presentations). The BSDB’s grant has allowed me a taste of what a career in academia can offer. Straightforward experiments fraught with unforeseen difficulties. Working on weekends when, infuriatingly, age-specific experiments simply weren’t possible on Friday. Enough money to live off (just). But above all, the enormous reward in discovering something new. And getting to work with some really, really great people. Thanks guys.

References

Sammut, M., Cook, S., Nguyen, K., Felton, T., Hall, D., Emmons, S., Poole, R. and Barrios, A. (2015). Glia-derived neurons are required for sex-specific learning in C. elegans. Nature, 526(7573), pp.385-390.