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PhD opportunities

University funded PhD positions are advertised below. Please use the links on each project to apply.

*EASTBIO* Understanding and controlling cell fate decisions

Applications will be accepted until 5pm Monday 4th December 2017

1st Supervisor: Dr Sally Lowell
2nd Supervisor: Dr Kim Dale

Description

The aim of this project is to understand how pluripotent cells of the early embryo reliability select the correct differentiated fate at the appropriate time and place at gastrulation. We will use this knowledge to improve our ability to control differentiation of pluripotent cells (ES cells and iPS cells) in culture. The global signals that control cell fate at gastrulation are now well understood, yet these signals do not reliably direct differentiation in culture for reasons we do not understand [1].  We will explore the idea that local communication between individual cells also influences differentiation decisions.
The Lowell lab has developed quantitative image analysis tools for following differentiation at single cell resolution in embryos, organoid-type cultures, or conventional monolayer cultures.  These tools will be used to uncover transient local patterning in early cell fate decisions.  These patterns will give clues as to the particular modes of local cell-cell interactions that are operating in these tissues. This will be explored using the expertise in mathematical modelling from the Dale lab.
The project will focus on Notch signalling as a candidate regulator of these local interactions [2,3]. We already know that Notch becomes active in certain regions of the gastrulating embryo and that it is capable of influencing differentiation of pluripotent cells by adjusting responsiveness to instructive signals.  The Lowell and Dale labs have complementary expertise in studying the Notch pathway in a number of different contexts. This project will use a set of tools for monitoring and manipulating Notch that have been developed in both labs in order to uncover the mechanisms by which Notch modulates cell fate decisions.  These findings will help us to understand how local cell-cell interactions confer robustness on developmental patterning in vivo, and provide new approaches for generating useful cell times from ES and iPS cells in culture.

Training outcomes

  • Wet lab skills: differentiation of pluripotent cells in monolayer and organoid cultures, embryology, genome engineering (routine techniques in Lowell and Dale labs).
  • Imaging skills: confocal and light-sheet microscopy, use of high-level quantitative image analysis software (already custom written and in use within Lowell lab)
  • Quantitative and mathematical modelling skills (Dale lab).

References

  1. Malaguti M, Nistor PA, Blin G, Pegg A, Zhou X, Lowell S. Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin. Elife. 2013 Dec 17;2:e01197. doi: 10.7554/eLife.01197.
  2. Lowell S, Benchoua A, Heavey B, Smith AG. Notch promotes neural lineage entry  by pluripotent embryonic stem cells. PLoS Biol. 2006 May;4(5)
  3. Carrieri FA, Dale JK. Turn It Down a Notch. Front Cell Dev Biol. 2017 Jan 18;4:151.

Application information

Project and application details can be found on the website below. You must follow the instructions on the EASTBIO website for your application to be considered.
Deadline for applications: 4 December 2017. This opportunity is only open to UK nationals (or EU students who have been resident in the UK for 3+ years immediately prior to the programme start date) due to restrictions imposed by the funding body. http://www.eastscotbiodtp.ac.uk/how-apply-0

*EASTBIO* Challenging key concepts in neural differentiation: the role of neuromesodermal progenitors in patterning the head-to-tail axis in vertebrates

Applications will be accepted until 5pm Monday 4th December 2017

1st Supervisor: Prof Val Wilson
2nd Supervisor: Prof Kate Story

Description

Generating physiologically-relevant cells of interest in vitro is a major challenge faced by researchers making cells for therapy. Accordingly, precise knowledge of the fate choices that cells make during embryo development, and the environmental signals governing these choices, is paramount in designing efficient in vitro differentiation protocols.

The ‘textbook’ description of central nervous system development statest that neural tissue is first specified, then subsequently patterned into brain and spinal cord (the ‘activation-transformation’ hypothesis, generated first in amphibians). The activation-transformation hypothesis has formed the rationale for in vitro neural differentiation protocols from pluripotent cells. However we have shown that during embryo development, the spinal cord, backbone, and its associated musculature are built not by committed neural precursors, but by bipotent neural/mesodermal progenitors (NMPs) located at the tail end of the embryo, while the brain is likely to be formed by an independent cell population at the head end. In vitro culture conditions that allow NMP differentiation have now resulted in the production of lower spinal cord neural types, which had not previously been generated using differentiation protocols that follow the ‘activation-transformation idea’ of first specifying indeterminate neural tissue, then ‘transforming’ it to a posterior identity. However the in vivo (and in vitro), sequence of cell fate restrictions leading to brain and spinal cord production is still not clear: do pluripotent cells first commit to a neural identity, then separate into brain and spinal cord- generating NMPs, or are NMPs and brain separately specified from pluripotent cells?

In this project, this question will be investigated both in vitro in mouse pluripotent cells, and in two in vivo systems, mouse and chick embryos.  
NMPs are characterised by the coexpression of two transcription factors, T(brachyury) and Sox2. Meanwhile Sox1 expression characterises the site of future brain formation. Since we have fluorescent markers of these populations, this is a tractable in vitro system to follow the sequence of cell fate decisions needed to make brain and spinal cord at the single cell level. Analysis of single cell transcriptomes in parallel with analysis of their commitment will further characterise these cell fate decisions. Furthermore, in vitro culture of manipulated early mouse and embryos where future brain and spinal cord tissue are challenged by transplantation to spinal cord or brain environments respectively will test at what stage these cells are committed to brain or spinal cord fates: before or after their commitment to a neural identity. Our preliminary data suggests a rather similar sequence of events in each organism, but subtle differences in the generation of these anterior and posterior cell types may underpin some of the species differences; this will be analysed by comparison of chick and mouse transcriptomes.

This project therefore challenges a fundamental concept in developmental biology, but also is likely to lead to improvements in the in vitro differentiation of pluripotent cells into cell types of clinical interest for degenerative disease and/or injury.

Application information

Project and application details can be found on the website below. You must follow the instructions on the EASTBIO website for your application to be considered.
Deadline for applications: 4 December 2017. This opportunity is only open to UK nationals (or EU students who have been resident in the UK for 3+ years immediately prior to the programme start date) due to restrictions imposed by the funding body. http://www.eastscotbiodtp.ac.uk/how-apply-0

*EASTBIO* Understanding haematopoietic stem cell development through global single-cell gene expression analysis

Applications will be accepted until 5pm Monday 4th December 2017

1st Supervisor: Prof Alexander Medvinsky
2nd Supervisor: Dr Tamir Chandra

Description

Although the first blood cells in mammals appear in the yolk sac, haematopoietic stem cells (HSCs) which can self-renew and give rise to the adult haematopoietic system appear separately inside the embryo body within the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, Cell, 1996). HSCs emerge through a multi-step process, which involves sequential maturation of intermediate cell types. However, our understanding of genetic mechanisms underlying HSC specification and progression through developmental stages is limited partly due to in utero development. To overcome this problem, we have already established an analytical in vitro model system which allows us to replicate the process of HSC maturation in the AGM region (Taoudi et al., Cell Stem Cell, 2008; Rybtsov et al., Stem Cell Reports, 2014). Although we have characterized several consecutive embryonic HSC precursors by cell surface markers, these markers are shared with other blood progenitors. This hampers precise identification of cells of the developing HSC lineage and specific genes underlying step-wise HSC maturation.

Recent advancement in single-cell analysis technology have dramatically transformed our understanding of cell types and lineages. It permits exploration of heterogeneity of cell populations and importantly, enables identification of novel cell type markers and otherwise unrecognisable cell sub-types. Using highly parallel barcoding of individual cells and computational tools specifically developed for single-cell data, it is now possible to build developmental/ differentiation trees into which intermediates of cell types and cellular states can be placed (Macosko et al., Cell, 2015; Mohammed et al., Cell Reports, 2017).

In this interdisciplinary project, we aim to explore molecular mechanisms underlying embryonic HSC development in mammals. The student will explore the transcriptional heterogeneity of the developing haematopoietic system in mouse using system-wide single-cell global transcriptome analysis. Single-cell gene expression analysis will be performed using cell barcoding in a microfluidics device. Using computational biology methods the student will reconstruct the developmental tree of the haematopoietic system and will identify molecular signatures specific to clades of the developing HSC lineage. Candidate genes identified in the molecular signature that could potentially be involved in HSC development will be validated using functional in vitro and in vivo assays and their expression in the embryo will be characterised using confocal microscopy. By contrast to model organisms, analysis of human embryonic HSC development has lagged behind due to the limited availability of material, lack of an in vitro modelling system and poor characterisation of cell type markers. Our data-driven approach using cutting-edge single cell methodology in mouse is expected to be highly informative for identifying homologous cellular differentiation pathways in human and will have substantial predictive power for analysing mechanisms underlying human HSC development. This project will fill a crucial gap in our mechanistic understanding of HSC development, which could in future inform improved strategies for manipulating HSCs ex vivo.

This is a collaborative interdisciplinary project addressing important biological questions at the system-wide level, between the groups of Prof. Alexander Medvinsky, an expert in embryonic development of mouse and human HSCs and experts in single-cell biology (including computational analysis) Dr. Tamir Chandra and Dr. Kristina Kirchner. To enhance the outcome of the project we have established a collaboration with Prof. Bertie Gottgens in Cambridge, a leading expert in the analysis of blood transcriptional networks.

The student will work in a highly collaborative environment and will become proficient in advanced methods of early analysis of embryonic HSC development (embryo manipulations, fluorescence activated cell sorting and analysis, HSC culture, qRT-PCR, confocal microscopy) as well as single-cell analysis and computational biology methods. Importantly, UoE has been awarded a £650K MRC Discovery Award to implement Drop-Seq-like approaches to single cell transcriptomics. This creates an excellent opportunity for the student to become an expert in this rapidly evolving cutting-edge technology. The project will allow the student to obtain important insights into the analysis of complex biological systems and to acquire important interdisciplinary skills in systems biology approaches, which should have a highly positive impact on their future career in science.

Application information

Project and application details can be found on the website below. You must follow the instructions on the EASTBIO website for your application to be considered.
Deadline for applications: 4 December 2017. This opportunity is only open to UK nationals (or EU students who have been resident in the UK for 3+ years immediately prior to the programme start date) due to restrictions imposed by the funding body. http://www.eastscotbiodtp.ac.uk/how-apply-0

Funded PhD Studentships within Donal O’Carroll laboratory

Applications will be accepted until 5pm Friday 15th December 2017

1st Supervisor: Prof Donal O'Carroll
2nd Supervisor: tbc

We are looking to recruit two outstanding postgraduate candidates to join Donal O’Carroll laboratory, with entry as soon as possible. The Stem Cell Research PhD programme provides cutting edge, cross-disciplinary PhD training at the University of Edinburgh's College of Science and Engineering. Our programme aims to train the next generation of scientific leaders in Stem Cell Research. As a PhD student, you’ll study in a unique environment, thanks to our location within the MRC-funded Scottish Centre for Regenerative Medicine (SCRM).

Training programme

The Stem Cell Research PhD Programme is a 4-year training programme. During applications stage the topic of the proposed PhD research project will be discussed in detail. This unique cross-disciplinary initiative is aimed at studying the contribution of RNA-based regulatory mechanisms to stem and germ cell biology. Applicants who are interested in combining molecular biology and computational approaches to stem cell research are strongly encouraged to apply. To ensure comprehensive training you will attend lab meetings and seminars by internal and invited speakers throughout the programme.

Select publications from the O’Carroll laboratory

  1. Ivanova I, Much C, Di Giacomo M, Azzi C, Morgan M, Moreira PN, Monahan J, Carrieri C, Enright AJ, O'Carroll D. The RNA m6A Reader YTHDF2 Is Essential for the Post-transcriptional Regulation of the Maternal Transcriptome and Oocyte Competence. Mol Cell. 2017 Sep 21;67(6):1059-1067.e4.
  2. Morgan M, Much C, DiGiacomo M, Azzi C, Ivanova I, Vitsios DM, Pistolic J, Collier P, Moreira PN, Benes V, Enright AJ, O'Carroll D. mRNA 3' uridylation and poly(A) tail length sculpt the mammalian maternal transcriptome. Nature. 2017 Aug 17;548(7667):347-351.
  3. Vasiliauskaitė L, Vitsios D, Berrens RV, Carrieri C, Reik W, Enright AJ, O'Carroll D. A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming. Nat Struct Mol Biol. 2017 Jul;24(7):604-606. doi:10.1038/nsmb.3413.
  4. Carrieri C, Comazzetto S, Grover A, Morgan M, Buness A, Nerlov C, O'Carroll D. A transit-amplifying population underpins the efficient regenerative capacity of the testis. J Exp Med. 2017 Jun 5;214(6):1631-1641. doi: 10.1084/jem.20161371.

Studentship offers support for stipend, fees and research costs

Up to two PhD studentships are available for promising postgraduate candidates with a strong interest in RNA and stem/germ cell biology. Successful applicants are awarded full UK/EU scholarship as per UK research council rates which includes a stipend, tuition fees (at the UK/EU rate).
https://www.mrc.ac.uk/skills-careers/studentships/studentship-guidance/minimum-stipend-and-allowances/

Application information

Studentships are awarded competitively. Applicants should hold at least an upper second class degree or equivalent in relevant scientific disciplines. Applicants should submit the following documents to our e-mail address crm-training@ed.ac.uk
•    Personal statement about your research interests and reasons for applying
•    CV
All documents should be submitted as soon as possible but no later than Friday 15th December 2017. Short-listed candidates will be notified in writing. Informal enquiries can also be directed to us via e-mail to crm-training@ed.ac.uk

*DTP Precision Medicine* Integrated analyses of metabolomic-epigenomic axes for stratified medicine in blood cancers

Application Deadline: 5pm Wednesday 10th January 2018

1st Supervisor: Prof Kamil Kranc
2nd Supervisor: Dr David Vetrie, Dr Vignir Halgason and Dr Olive Maddocks

Description

In acute and chronic myeloid leukaemias (AML and CML, respectively) the disease is initiated and driven by leukaemic stem cell (LSCs). Given that current therapies fail to eradicate LSCs and do not cure leukaemia, novel therapies must be developed to target LSCs. Emerging evidence indicates that epigenetic and transcriptional processes which control leukaemic transformation are dependent on the cell’s metabolic state. One key area with immense therapeutic potential is the relationship between metabolism and methylation/demethylation of DNA, RNA and histones: intermediates of cellular metabolism are direct substrates and co-factors for the enzymes which modify the epigenome and epitranscriptome1. Recently the Kranc laboratory has revealed that deletion of the TCA cycle enzyme fumarate hydratase results in elevated levels of fumarate which dysregulates the histone methylation status and suppresses the development of AML LSCs2. In CML, the Helgason/Vetrie laboratories have recently demonstrated that LSCs have dependencies on oxidative mitochondrial metabolism4 and the histone mark H3K27me33,4, respectively. How these and other metabolic and epigenetic dependencies are linked together in AML/CML and how they impact on clinical outcomes in AML/CML remains unknown.

Hypothesis

The metabolic control of DNA, RNA and histone methylation is critical for LSC survival in AML and CML.

Aims

In this multidisciplinary project we intend to combine broad expertise at the intersection of pathology of leukaemia, genomics, metabolomics, computational biology/bioinformatics to address the following aims:

    1. To employ a candidate gene approach to define the global metabolic-epigenetic relationships in AML/CML. We have recently uncovered key novel metabolic regulators that impact on the epigenetic status of LSCs. We will employ CRISPR-Cas9-mediated gene knockouts or small molecule inhibitors in AML/CML cell lines to specifically target these metabolic regulators. We will combine global epigenetic/genomics and metabolic assays to determine how these perturbations affect leukaemic cells at the molecular level. We will next validate the 2-3 most promising drug-able targets for further perturbations in primary patient samples available to us from the Glasgow biobank. We will measure the functional, metabolic and epigenetic consequences of these perturbations and computationally integrate them. 
    2. To reveal the metabolic-epigenetic dependencies in vivo in AML/CML models. We will employ murine models of either AML or CML (depending on outcomes of aim 1), to provide phenotypic and molecular evidence on the metabolic-epigenetic axes in vivo. Briefly, we will use CRISPR-Cas9 gene editing approaches in primary murine LSCs transplanted into recipient mice, or directly treat leukaemic mice with relevant drugs/inhibitors, and study the clinical outcome of the disease paralleled with monitoring metabolic and epigenetic changes. We will investigate the effects that these interventions have on LSC survival, leukaemic burden, and on disease propagation in secondary transplants. 
    3. To employ computational biology approaches to integrate our datasets with publically available resource of human ‘omics’ datasets obtained from AML/CML patients. This approach will uncover whether differences in expression of the tested metabolic regulators (or mutations) impact on transcriptional and epigenetic status of AML/CML cells and determines the disease severity, drug resistance and prognosis at different stages of AML/CML. 

      By discovering novel metabolic-epigenetic dependencies and computationally comparing them with publically-available AML/CML ‘omics’ datasets we will reveal completely novel mechanisms of leukaemic transformation. We will harness this knowledge to identify and explore new therapeutic targets for LSC eradication.

    Training outcomes

    The student will be based in state-of-the-art world-class laboratories specialising in multidisciplinary basic and translational science and will be trained in:

    • Computational approaches/quantitative skills (statistics, computation, bioinformatics, ‘omics’ analyses, network construction); 
    • Leukaemia biology (murine models, patient-derived cell culture, flow-cytometry, CRISPR-Cas9-mediated knockout)
    • Epigenetics (RNA-seq, ChIP-seq, methyl-RNA-seq, bisulphite sequencing);
    • Metabolomics (state-of-the-art metabolic assays, liquid chromatography-mass spectrometry).

    References

      1. Maddocks, O.D., Labuschagne, C.F., Adams, P.D. & Vousden, K.H. Serine Metabolism Supports the Methionine Cycle and DNA/RNA Methylation through De Novo ATP Synthesis in Cancer Cells. Mol Cell 61, 210-21 (2016).
      2. Guitart, A.V., Panagopoulou, T.I., Villacreces, A., Vukovic, M., Sepulveda, C., Allen, L., Carter, R.N., van de Lagemaat, L.N., Morgan, M., Giles, P., Sas, Z., Gonzalez, M.V., Lawson, H., Paris, J., Edwards-Hicks, J., Schaak, K., Subramani, C., Gezer, D., Armesilla-Diaz, A., Wills, J., Easterbrook, A., Coman, D., So, C.W., O'Carroll, D., Vernimmen, D., Rodrigues, N.P., Pollard, P.J., Morton, N.M., Finch, A. & Kranc, K.R. Fumarate hydratase is a critical metabolic regulator of hematopoietic stem cell functions. J Exp Med 214, 719-735 (2017).
      3. Kuntz, E.M., Baquero, P., Michie, A.M., Dunn, K., Tardito, S., Holyoake, T.L., Helgason, G.V.*& Gottlieb, E.* Targeting mitochondrial oxidative phosphorylation eradicates therapy-resistant chronic myeloid leukemia stem cells. Nat Med (2017).   * joint senior authors
      4. Scott, M.T., Korfi, K., Saffrey, P., Hopcroft, L.E., Kinstrie, R., Pellicano, F., Guenther, C., Gallipoli, P., Cruz, M., Dunn, K., Jorgensen, H.G., Cassels, J.E., Hamilton, A., Crossan, A., Sinclair, A., Holyoake, T.L. & Vetrie, D. Epigenetic Reprogramming Sensitizes CML Stem Cells to Combined EZH2 and Tyrosine Kinase Inhibition. Cancer Discov 6, 1248-1257 (2016).

            Application process

            Application details can be found on the programme website. This will be updated with application information for 2018 entry.

            *DTP Precision Medicine* Differentiation of pluripotent cells: a quantitative multiparametric approach

            Application Deadline: 5pm Wednesday 10th January 2018

            1st Supervisor: Dr Sally Lowell
            2nd Supervisor: Prof David Robertson

            Description

            We already know the extrinsic signals that direct pluripotent cells into particular cell fates, but cells do not respond reliably to these signals for reasons we do not understand. We have accumulated evidence that a number of properties of cells can influence the way they respond to differentiation signals. These properties include the way cells are organised in 3D space (Blin et al., 2017) the way they stick to each other (Malaguti et al., 2013), and the way they interact with their immediate neighbours (Lowell et al., 2006; Zhou et al., 2013). Other likely influences include the shape or position of the cell, the activity of particular signalling pathways, and the expression of particular transcription factors. However do not understand which of these factors are most important, nor how they might act together to influence differentiation.

            The Lowell lab (School of Biological Sciences) have developed quantitative image analysis tools for following differentiation at single cell resolution in embryos, organoid-type cultures, or conventional monolayer cultures (Wellcome Trust-funded project, unpublished).  We can measure a large number of parameters in each individual cell within these tissues/cultures over time, including activity of signalling pathways, expression of transcription factors and the morphological and cell biological events that we believe are influencing differentiation. This type of analysis generates large multi-parametric datasets containing multiple measurements for thousands of cells, including information about cell-cell interactions over time.  The challenge now is to find ways to explore these data and extract combinations of parameters that correlate with or predict particular differentiated cell fates.

            The Robertson lab (School of Informatics) have expertise quantitative models of dynamic interactions. They have an ongoing collaboration with the Lowell lab to study the social and dynamic behaviors of stem cells based on quantitative imaging data from a simple 2D differentiation system (currently led by a second-year PhD student). The proposed PhD project extends this approach in order to explore how cell-cell interactions modulate differentiation within 3D tissues or organoid-type cultures.

            This analysis will generate testable hypotheses about the combinations of influences that may help us to control differentiation more reliably.  We will use our standard lab toolkit (genome engineering, biophysical manipulations, organoid- type differentiation of pluripotent cells, quantitative analysis of cell fates) in order to test these hypotheses.

            Aims

            • Gather multi-parametric single-cell-resolution data from pluripotent mouse cells undergoing differentiation within monolayer cultures, organoid cultures, or embryos.
            • Organise these data into a graph database that can be manually interrogated in order to explore specific hypotheses on combinatorial control of cellular behavoir.
            • Use statistical analysis and modelling approaches to identify particular combinations of parameters or interactions that correlate with or predict particular cell behaviours, and use these data to generate new hypotheses on combinatorial control of cellular behavoir.
            • Test these hypotheses using genome engineering, biophysical manipulations, and other standard wet-lab approaches to manipulate the appropriate cellular propertie

            Training outcomes

            • Wet lab skills: differentiation of pluripotent cells in monolayer and organoid cultures, embryology, genome engineering (routine techniques in Lowell lab).
            • Imaging skills: confocal and light-sheet microscopy, use of high-level quantitative image analysis software (already custom written and in use within Lowell lab)
            • Quantitative and computational tools: computational and statistical analysis of single-cell multi-parametric datasets, modelling.

            References

            1. Blin, G., Picart, C., Théry, M., Puceat, M., 2017. Geometrical confinement guides Brachyury self-patterning in embryonic stem cells. bioRxiv 138354. doi:10.1101/138354
            2. Lowell, S., Benchoua, A., Heavey, B., Smith, A.G., 2006. Notch Promotes Neural Lineage Entry by Pluripotent Embryonic Stem Cells. PLoS biology 4, e121.
            3. Malaguti, M., Nistor, P.A., Blin, G., Pegg, A., Zhou, X., Lowell, S., 2013. Bone morphogenic protein signalling suppresses differentiation of pluripotent cells by maintaining expression of E-Cadherin. Elife 2, e01197. doi:10.7554/eLife.01197
            4. Zhou, X., Smith, A.J.H., Waterhouse, A., Blin, G., Malaguti, M., Lin, C.-Y., Osorno, R., Chambers, I., Lowell, S., 2013. Hes1 desynchronizes differentiation of pluripotent cells by modulating STAT3 activity. Stem Cells 31, 1511–1522. doi:10.1002/stem.1426

            Application process

            Application details can be found on the programme website. This will be updated with application information for 2018 entry.

            *DTP Precision Medicine* Understanding haematopoietic stem cell development through global single-cell gene expression analysis

            Application Deadline: 5pm Wednesday 10th January 2018

            1st Supervisor: Prof Alexander Medvinsky

            Description

            Haematopoietic stem cells (HSCs) can self-renew and give rise to the adult haematopoietic system. These potent cells appear first inside the embryo body within the aorta-gonad-mesonephros (AGM) region (Medvinsky and Dzierzak, Cell, 1996). HSCs emerge through a multi-step process however, our understanding of genetic mechanisms underlying HSC development is limited partly due to in utero development. To overcome this problem, we have already established an analytical in vitro model system which allows us to replicate the process of HSC maturation in the AGM region (Taoudi et al., Cell Stem Cell, 2008; Rybtsov et al., Stem Cell Reports, 2014). Although we have characterized several consecutive embryonic HSC precursors by cell surface markers, these markers are shared with other blood progenitors. This hampers precise identification of cells of the developing HSC lineage and specific genes underlying step-wise HSC maturation.

            Recent advancement in single-cell analysis technology have dramatically transformed our understanding of cell types and lineages. It permits exploration of heterogeneity of cell populations and importantly, enables identification of novel cell type markers and otherwise unrecognisable cell sub-types. Using highly parallel barcoding of individual cells and computational tools specifically developed for single-cell data, it is now possible to build developmental/ differentiation trees into which intermediates of cell types and cellular states can be placed (Macosko et al., Cell, 2015; Mohammed et al., Cell Reports, 2017).

            Aims

            In this interdisciplinary project, we aim to explore molecular mechanisms underlying embryonic HSC development in mammals. The student will explore the transcriptional heterogeneity of the developing haematopoietic system in mouse using system-wide single-cell global transcriptome analysis. Single-cell gene expression analysis will be performed using cell barcoding in a microfluidics device. Using computational biology methods the student will reconstruct the developmental tree of the haematopoietic system and will identify molecular signatures specific to clades of the developing HSC lineage. Candidate genes identified in the molecular signature that could potentially be involved in HSC development will be validated using functional in vitro and in vivo assays and their expression in the embryo will be characterised using confocal microscopy. By contrast to model organisms, analysis of human embryonic HSC development has lagged behind due to the limited availability of material, lack of an in vitro modelling system and poor characterisation of cell type markers. Our data-driven approach using cutting-edge single cell methodology in mouse is expected to be highly informative for identifying homologous cellular differentiation pathways in human and will have substantial predictive power for analysing mechanisms underlying human HSC development. This project will fill a crucial gap in our mechanistic understanding of HSC development, which could in future inform improved strategies for manipulating HSCs ex vivo.

            Training Outcomes

            This is a collaborative interdisciplinary project addressing important biological questions at the system-wide level, between the groups of Prof. A. Medvinsky, an expert in embryonic development of mouse and human HSCs and groups of Dr. Chandra and Dr. Kirchner, experts in single cell analysis including computational analysis of single-cell data. To enhance the outcome of the project we have established a collaboration with Prof. Bertie Gottgens in Cambridge, a leading expert in the analysis of blood transcriptional networks.

            The student will work in a highly collaborative environment and will become proficient in advanced methods of early analysis of embryonic HSC development (embryo manipulations, fluorescence activated cell sorting and analysis, HSC culture, qRT-PCR, confocal microscopy) as well as single-cell analysis and computational biology methods. Importantly, UoE has been awarded a £650K MRC Discovery Award to implement Drop-Seq-like approaches to single cell transcriptomics. This creates an excellent opportunity for the student to become an expert in this rapidly evolving cutting-edge technology. The project will allow the student to obtain important insights into the analysis of complex biological systems and to acquire important interdisciplinary skills in systems biology approaches, which should have a highly positive impact on their future career in science.

            Application process

            Application details can be found on the programme website. This will be updated with application information for 2018 entry.

            *DTP Precision Medicine* Structural and functional analyses of Nanog and Sox2 in ESCs 

            Application Deadline: 5pm Wednesday 10th January 2018

            1st Supervisor: Prof Ian Chambers
            1st Supervisor: Dr Laura Spagnolo

            Description

            Pluripotent embryonic stem cells (ESCs) possess the defining attributes of self-renewal and multilineage differentiation. ESC self-renewal is regulated by transcription factors (TFs) including Nanog and Sox2 [1]. However, the mechanisms by which these TFs work remains unclear. 

            Biochemical characterization of Nanog shows that Nanog forms both homo-oligomers and hetero-oligomers with Sox2 [2, 3]. Nanog and Sox2 interact directly via identified amino acids with the Nanog-Sox2 complex being important for ESC function [3]. 

            Recently we developed a method of producing >10mg of Nanog. Preliminary electron microscopy analysis in the Spagnolo lab shows that these preparations are of sufficient quality for high resolution cryo-electron microscopy. An initial negative stain 3D structure has been determined and is consistent with a ~300 kDa macromolecular assembly. The angular distribution of particles covers the Euler sphere homogeneously, demonstrating that there is no preferential orientation of individual copies of the complex, often a rate-limiting step in 3D-EM projects.

            This project will couple electron microscopy and single cell sequencing to illuminate the function of Nanog and Sox2.

            Aims

            Using mutant forms of Nanog and Sox2 that do not co-associate [3] the Nanog-Sox2 interaction will be investigated at the single cell level in aims 1 and 2.

            1. To determine transcription and self-renewal changes in ESCs in which Nanog lacks a Sox2 interacting domain

            We have constructed Nanog-null cells carrying a tamoxifen inducible wild-type Nanog or a Nanog mutant that cannot interact with Sox2 (Nanog-W10A) [3]. These ESCs will be released from naïve culture in 2i/LIF by replating in FCS/LIF or in differentiation medium (N2B27). In LIF/FCS culture, cells reduce their self-renewal efficiency over 3 days, while in N2B27, self-renewal is lost within 2 days. Populations of single cells will be prepared at appropriate times after release from 2i/LIF for single cell RNA preparation and clonal self-renewal assays. These experiments will identify target genes sensitive to the absence of a Sox2 interaction domain on Nanog. 

            2. To determine transcription and self-renewal changes in ESCs in which Sox2 lacks a Nanog interacting domain

            As the Sox2 interacting domain on Nanog also interacts with other proteins, we will use CRISPR/Cas9 to make derivatives of the above cell lines in which wild-type Sox2 is replaced by a Sox2 point mutant lacking the Nanog interacting domain. These ESCs will be treated identically to those in aim 1 for single cell RNA preparation and clonal self-renewal assays. By combining analyses in ESCs in which (i) the Sox2 interaction domain is missing from Nanog, (ii) the Nanog interaction domain is missing from Sox2 and (iii) the Nanog-Sox2 interaction domains are missing from both proteins these orthogonal experiments will rigorously identify target genes sensitive to the absence of a functional Nanog-Sox2 interaction. Moreover, the contribution of the Nanog-Sox2 interaction to the rate of flux of ESC populations out of the naïve state will be revealed. 

            3. Determination of high resolution structures of Nanog and Nanog-DNA complexes

            Cryo-electron microscopy will be used to determine high resolution structures of oligomeric Nanog complexes in the presence and absence of DNA. Changes in Nanog structure induced by DNA binding will be determined, paying particular attention to structural changes outwith the canonical DNA binding domain. The cryo-EM work will occur in Glasgow, where a recently MRC-funded world-leading structural EM facility (SMIC, Scottish Macromolecular Imaging Consortium) will be fully functional by autumn 2018.

            Perspective
            We have also purified the Nanog-Sox2 complex in high yield. The studies proposed in aim 3 will lay the groundwork for future analysis of the Nanog-Sox2 complex in the presence and absence of DNA, providing further interpretative value to the outcomes of aims 1 and 2.

            Training Outcomes

            The student will receive state-of-the-art training in several generally applicable wet lab and computational methods. Several data-rich outputs will be generated, including cryo-electron microscopy and high throughput sequencing data from single cell RNA-seq. Importantly, available expertise in the techniques is available either in Edinburgh (Chambers, Centre for Regenerative Medicine) or in Glasgow (Spagnolo, Institute of Molecular, Cell and Systems biology). This broad experience will provide a unique combination of highly desirable skills. 

            References

            1. Chambers, I. and S.R. Tomlinson, The transcriptional foundation of pluripotency. Development, 2009. 136(14): p. 2311-22.
            2. Mullin, N.P., et al., The pluripotency rheostat Nanog functions as a dimer. Biochem J, 2008. 411(2): p. 227-31.
            3. Gagliardi, A., et al., A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal. EMBO J, 2013. 32(16): p. 2231-2247.

            Application process

            Application details can be found on the programme website. This will be updated with application information for 2018 entry.

            Tissue Repair PhD Programme

            Applications are open for this programme (deadline Wednesday 6th December 2017)

            Funded studentships available for Wellcome Trust PhD Programme inTissue Repair.

            The MRC Centre for Regenerative Medicine is one of five research centres at the Edinburgh Medical School involved in the four-year PhD Programme in Tissue Repair. This innovative, multi-disciplinary training programme seeks to train the next generation of scientific leaders in tissue repair by providing interdisciplinary training in basic and translational biomedical research. The programme is run by the University of Edinburgh and funded by the Wellcome Trust. For programme details please visit the Tissue Repair website.

            Tissue Repair website

            We encourage enquiries and applications from self-funded basic and clinical scientists and from candidates who intend to apply for external funding all year round.

            Instructions on how to apply as a self funded student can be found here.

            Please contact the relevant PI’s informally to discuss potential projects and visit our funding opportunities page.

            Centre Funded Studentships include:

            • Stipend for 3 or 4 years
            • Tuition Fees
            • Research Training Costs
            • Conference Travel Allowance

            Further information about MRC Studentships.

            Contact us for more information.