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Deciphering the principles underlying specification of endoderm versus mesoderm in mouse embryonic stem cells

  • Full or part time
    Dr C Hill
  • Application Deadline
    Tuesday, November 12, 2019
  • Funded PhD Project (Students Worldwide)
    Funded PhD Project (Students Worldwide)

Project Description

This 4-year PhD studentship is offered in Dr Caroline Hill’s Group based at the Francis Crick Institute (the Crick).

Our lab focuses on the transforming growth factor beta (TGF-b) superfamily of ligands which comprises the TGF-bs, Activins, Nodal, BMPs and GDFs. These ligands control many aspects of embryonic development and adult tissue homeostasis, and deregulated signalling is associated with cancer and fibrosis [1, 2]. Ligands bind to a type II receptor, which complexes with and transphosphorylates a type I receptor that subsequently phosphorylates the receptor-regulated Smads (R-Smads), the effectors of the pathway. The activated Smads then accumulate in the nucleus where they regulate gene transcription in conjunction with other DNA-binding transcription factors [3]. Projects in the lab are focused on understanding how these pathways function normally during vertebrate development to induce and pattern different tissues, and how deregulated signalling contributes to human disease.

One aspect of our work is focused on how sustained Nodal signalling, in conjunction with other signalling pathways, determines the balance between mesoderm and endoderm specification in early vertebrate embryos. This role of Nodal signalling is conserved in vertebrates and we are exploring it in both a zebrafish model and in mouse embryonic stem cells (ESCs). The project will use a mouse ESC line engineered to contain fluorescent reporters which provide a live readout of mesoderm and endoderm differentiation. These cells can be differentiated along the mesoderm and endoderm lineages using cocktails containing Activin to mimic sustained in vivo Nodal signalling [4]. We want to understand the transcriptional network downstream of Nodal/Activin signalling that leads to these cell fate decisions, as well as understanding the interplay between Nodal/Activin signalling and other signalling pathways.

We have recently shown that SMAD2 and SMAD3, which are highly homologous to each other [3], have distinct roles in mesoderm and endoderm differentiation. CRISPR-induced knockout of SMAD2 and SMAD3 in mouse ESCs has indicated that SMAD2 is absolutely required for endoderm differentiation, whilst SMAD3-null cells differentiate to mesoderm, but are deficient in endoderm differentiation. The student will use RNA-seq in the different knockout cell lines to obtain a global picture of which genes depend on which SMADs, and ChIP-seq for SMAD2 and SMAD3 to identify specific enhancers bound by these SMADs at different timepoints. Footprinting of ATAC-seq data on these enhancers will aid identification of other collaborating transcription factors for SMAD2 and SMAD3, and allow us to identify the transcriptional networks downstream of sustained Activin/NODAL signalling.

The mESC system also provides the ability to perform unbiased screens. Therefore, as a means of discovering novel regulators of endodermal and mesodermal fate downstream of sustained Activin/NODAL signalling, we will perform a pooled lentiviral-based CRISPR/Cas9 selection screen coupled to next-generation sequencing in Activin/Nodal-induced differentiating mESCs, using the integrated fluorescent reporters as readouts. Hits from the screen will be functionally validated in the mESCs, and also in the zebrafish system, which is the developmental model used in the lab [5].

Candidate background
This project will suit a student with a degree in biological sciences, with a particular interest in signal transduction, developmental biology and regenerative medicine.

Talented and motivated students passionate about doing research are invited to apply for this PhD position. The successful applicant will join the Crick PhD Programme in September 2020 and will register for their PhD at one of the Crick partner universities (Imperial College London, King’s College London or UCL).

Applicants should hold or expect to gain a first/upper second-class honours degree or equivalent in a relevant subject and have appropriate research experience as part of, or outside of, a university degree course and/or a Masters degree in a relevant subject.

APPLICATIONS MUST BE MADE ONLINE VIA OUR WEBSITE (ACCESSIBLE VIA THE ‘APPLY NOW’ LINK ABOVE) BY 12:00 (NOON) 13 NOVEMBER 2019. APPLICATIONS WILL NOT BE ACCEPTED IN ANY OTHER FORMAT.

Funding Notes

Successful applicants will be awarded a non-taxable annual stipend of £22,000 plus payment of university tuition fees. Students of all nationalities are eligible to apply.

References

1. Wakefield, L. M. and Hill, C. S. (2013)

Beyond TGFβ: roles of other TGFβ superfamily members in cancer.

Nature Reviews Cancer 13: 328-341. PubMed abstract

2. Wu, M. Y. and Hill, C. S. (2009)

TGF-β superfamily signaling in embryonic development and homeostasis.

Developmental Cell 16: 329-343. PubMed abstract

3. Schmierer, B. and Hill, C. S. (2007)

TGFβ-SMAD signal transduction: molecular specificity and functional flexibility.

Nature Reviews Molecular Cell Biology 8: 970-982. PubMed abstract

4. Rothová, M., Hólzenspies, J. J., Livigni, A., Villegas, S. N. and Brickman, J. M. (2016)

Differentiation of mouse embryonic stem cells into ventral foregut precursors.

Current Protocols in Stem Cell Biology 36: 1G.3.1-1G.3.12. PubMed abstract

5. van Boxtel, A. L., Economou, A. D., Heliot, C. and Hill, C. S. (2018)

Long-range signaling activation and local inhibition separate the mesoderm and endoderm lineages.

Developmental Cell 44: 179-191 e175. PubMed abstract

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