Do common ancient gene regulatory programmes govern both neural and pancreatic fates throughout vertebrate evolution?

Secondary Supervisor(s): Professor Timothy Saunders

University of Registration: University of Warwick

BBSRC Research Themes: Understanding the Rules of Life (Neuroscience and Behaviour, Stem Cells, Systems Biology)

Project Outline

The vertebrate body is inherently complex, containing diverse tissues consisting of multiple distinct cell types. Assignment of specific cellular identities at the correct location and time during embryogenesis is essential to establishing a viable vertebrate body plan. Consequently, genes controlling cell identities must be stringently controlled in terms of timing, location, and levels of expression. Experimental and theoretical studies suggest that genes dictating cell fates are often controlled by regulatory sequences known as highly conserved non-coding elements (HCNEs), which show a high degree of DNA sequence conservation across hundreds of millions of years of vertebrate evolution.

We have performed functional genomics analyses comparing progenitors of developing zebrafish endoderm to human embryonic stem cells (hESCs) induced to form endoderm populations including pancreatic progenitors. Our analyses reveal HCNEs likely to act as enhancers linked to many genes encoding transcription factors (TFs) controlling pancreas development in fish and mammals. Remarkably, most of these TF genes also control key aspects of nervous system development. We have tested the ability of a subset of HCNEs to drive gene expression in zebrafish embryos. Despite having been discovered in pancreatic progenitors, we find they can drive expression in the zebrafish nervous system. This has led to the hypothesis that a common gene regulatory programme mediated by these HCNEs may govern both neural and pancreatic development throughout vertebrate evolution.

We will analyse the function of a select subset of HCNEs in both zebrafish and human developmental systems. To test developmental requirements for these HCNEs we will use CRISPR genome editing to delete HCNEs in hESCs, followed by directed differentiation to neural and pancreatic fates, combined with phenotypic analysis. We will analyse expression of TF genes presumed to be regulated by the HCNEs, and also compare HCNE deletion phenotypes to deletion of their presumptive target genes. We will perform equivalent experiments in zebrafish and study subsequent embryonic development. To test sufficiency of HCNEs to drive gene expression in the pancreas and nervous system we will use fluorescence-based reporter assays combined with imaging to determine when and where these HCNEs can drive gene expression during zebrafish development and in vitro hESC differentiation. Moreover, to more precisely understand how these HCNEs control gene expression, we will perform reporter assays using HCNEs with TF binding sites (TFBSs) mutated to determine which sites are of greatest importance. The student will benefit from the expertise of the Saunders lab in imaging and image analysis, to extract quantitative data that can be mapped onto the genetic results.

The outcome will be valuable new knowledge of how gene expression is controlled during both pancreas and nervous system development, and whether the same HCNEs and TFBSs control gene expression in each context. This will yield valuable insights into the evolution and development of these two highly distinct tissue types, and potentially reveal how mutations in such sequences lead to human developmental disorders.

The project will be supervised by Dr Andrew Nelson and Prof. Timothy Saunders who both work with zebrafish and human stem cell and organoid systems.

References

1. Nelson & Wardle (2013) Conserved non-coding elements and cis regulation: actions speak louder than words. Development 140: 1385-1395.

2. Figiel et al. (2021) Investigating the molecular guts of endoderm formation using zebrafish. Brief Funct Genomics 20: 394–406.

3. Martinez-Morales (2016) Toward understanding the evolution of vertebrate gene regulatory networks: comparative genomics and epigenomic approaches. Brief Funct Genomics 15:315–321.

Epigenetics of early human placenta and embryo development: From molecular mechanisms to regenerative medicine

Secondary Supervisor(s): Dr Wolfram Gruhn, Dr Yolanda Markaki

University of Registration: University of Warwick

BBSRC Research Themes:

• Understanding the Rules of Life (Stem Cells, Systems Biology)
• Integrated Understanding of Health (Regenerative Biology)

Project Outline

The defining genetic difference between human males and females are their distinct pair sex chromosomes – females are XX and males XY. It is essential that one of the female X chromosomes is switched off in a process known as X chromosome inactivation (XCI). That way females equalize gene dosage of X-linked genes with XY males. The process of XCI is an incredible event of whole chromosome silencing which occurs during embryonic development. Without this act of self-silencing of the X, female embryos won't survive, while X-inactivation can influence human health, from genetic diseases to cancer.

But how does this "switch off" button work? And what happens if it malfunctions? Interestingly, there is increasing evidence that XCI is different in the placenta compared to other tissues and organs, leading to differences in gene expression between male and female pregnancies and possibly sex-based differences in pregnancy disorders. However, the precise details and mechanisms governing XCI in the placenta are not completely understood. In this project, we will use advanced interdisciplinary techniques and human stem cells to dissect the mechanisms of XCI in placenta vs other tissues.

What You'll Explore

XIST-SMACs: The Markaki lab has recently discovered these tiny molecular machines, which are key players in the silencing of the X chromosome. You will investigate how XIST-SMACs form and control X-inactivation during human embryonic development when the process is established.

Frontline Tech: Dive deep into human development using human pluripotent stem cells and trophoblast stem cells combined with super-resolution microscopy to observe changes on the inactivating X chromosome.

Next-generation sequencing Revelations: Harness the power of omics technologies to unpick the transcriptional output of XIST-SMACs and how they regulate genes on the inactive X.

Genomic Resets: Experiment with cutting-edge genome editing tools to reset X-inactivation, paving the way for improved cell therapies.

Why This Matters

Many pregnancies terminate during the mysterious time of XCI, while human pluripotent stem cells exhibit defects in the maintenance of the silenced X when being cultured and are thus inappropriate for regenerative medicine applications. With your help, we can unravel why this happens and develop new therapeutic strategies for X-linked diseases, while also exploring how XCI differs between the placenta and the embryo.

Where You'll Thrive

You will be based in the Interdisciplinary Biomedical Research Building at University of Warwick, and have access to our excellent world class facilities, and developmental biology and stem cell community.You will collaborate with the Department of Molecular and Cell Biology at University of Leicester and the Leicester Institute of Structural and Chemical Biology (LISCB), an institute of excellence also offering access to world class facilities. Through the guidance of our expert teams in developmental epigenetics, imaging and transcriptomics you'll embark on a holistic learning journey, mastering stem cells, genome editing, super-resolution microscopy, and more!

References

Dror I, Chitiashvili T, Tan, SYX, Cano CT, Sahakyan A, Markaki Y, Chronis C, CollierAJ, Deng W, Liang G, Sun Y, Afasizheva A, Miller J, Xiao W, Black DL, Ding F, Plath K.XIST directly regulates X-linked and autosomal genes in naive human pluripotent cells. Cell. 2024.

Markaki Y*, Chong JG, Wang Y, Jacobson EC, Luong C, Tan SYX, Jachowicz JW, Strehle M, Maestrini D, Dror I, Mistry BA, Schöneberg J, Banerjee A, Guttman M, Chou T*, Plath K*. Xist nucleates local protein gradients to propagate silencing across the X chromosome. Cell. 2021.

Kraus F, Miron E, Demmerle J, Chitiashvili T, Budco A, Alle Q, Matsuda A, Leonhardt H, Schermelleh L, Markaki Y. Quantitative 3D structured illumination microscopy of nuclear structures. Nat Protoc. 2017.

Turco, M.Y. and Moffett, A. Development of the human placenta. Development 2019.

Techniques

• Stem cell maintenance, differentiation and manipulation.
• Gene editing and bioengineering techniques using CRISPR/Cas9 Embryological phenotyping.
• Cloning and other molecular biology methods.
• Gene editing and bioengineering techniques using CRISPR/Cas9.
• RNA/DNA Fluorescence In Situ Hybridization (FISH), immunofluorescence.
• Super-Resolution and Confocal Laser Scanning Microscopy.
• Biochemical protein-RNA/protein-protein interaction assays and affinity purification.
• Next-generation sequencing, transcriptomics.
• Data analysis and visualization in Fiji, R and Python.