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 systems, and supported through collaboration with Prof. Patricia Murray (University of Liverpool) who is an expert on hESC differentiation to neural cell populations.

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

Techniques

• Stem cell maintenance, differentiation and manipulation
• CRISPR/Cas9 genome editing in human stem cells and zebrafish
• Embryological phenotyping
• Confocal imaging and image analysis of both embryos, and 2D and 3D cell cultures
• Immunostaining
• Flow cytometry
• Genome bioinformatics