Principal Supervisor: Dr. Andrew Nelson, School of Life Sciences

Co-supervisor: Dr. Sascha Ott, Department of Computer Science

PhD project title: Study of Hhex-mediated mechanisms underlying vertebrate endoderm organogenesis

University of Registration: University of Warwick

Project outline:

All tissues and organs of the body consist of many diverse specialized cell types. Both the function of individual cell types and their emergence during development are controlled by elaborate gene expression programmes, in part dictated by sequences-specific transcription factors which may either promote or repress gene expression in a context-specific manner. Understanding how individual transcription factors form protein complexes at specific DNA elements to correctly influence target gene expression is pivotal to elucidating mechanisms controlling cell fate decisions during development.This project will explore the mechanisms underlying requirement for the transcription factor Hhex during vertebrate endoderm formation. Endoderm is one of three germ layers of the embryo, and makes major contributions to the respiratory and gastrointestinal tracts and all associated organs. Hhex has highly conserved roles in the development of numerous organ systems. For example, it is required for formation of both the liver and exocrine pancreas in zebrafish and mouse [1-6], and also required in adults for correct pancreatic function [7] consistent with its association with type 2 diabetes [e.g. 8]. Hhex is also implicated in many cancers, where a small number of putative target genes and interacting factors have been identified [9]. Very little is understood, however, about the molecular mechanisms underlying Hhex-mediated control of endoderm formation.

The central aim of this project is to understand the functional basis for endodermal defects in hhex mutants using a highly interdisciplinary range of approaches including high-throughput proteomics and transcriptomics, as well as detailed computational and statistical analyses of the resulting data. This will involve in vitro use of mouse P19Cl6 cells and in vivo approaches using zebrafish. Zebrafish is an excellent model organism for studying control of vertebrate development, offering major advantages facilitating quantitative biology approaches. For example, it is highly genetically tractable allowing generation of knock-in fluorescent reporter lines to allow isolation of specific cell types from large clutches of embryos developing ex utero for functional genomic and transcriptomic profiling. Genome editing can also be applied to study gene function during development.

Objective 1 – Identification of Hhex interacting partners controlling endoderm formation

This will involve generation of Hhex-expressing hormone-inducible P19Cl6 embryonal cells for Hhex complex purification followed by mass spectrometry analysis. Expression patterns of the interacting proteins will be studied in vivo during zebrafish embryogenesis to establish the contexts in which they may interact during endoderm formation.

Objective 2 – Identification of Hhex target genes underlying organogenesis defects

This will involve use of CRISPR to generate spectrally distinct fluorescent knock-in null alleles for fluorescence activated cell sorting (FACS) of hetero- and homozygous mutant Hhex endodermal progenitors for single-cell RNA-seq transcriptome analyses. Affinity-tagged Hhex alleles will also be produced for pull-down and ChIP-seq identification of direct Hhex target genes.

Objective 3 – Genetic analysis of Hhex target genes in the developing endoderm

This will involve using CRISPR to perform a knock-down screen of putative Hhex interacting proteins and target genes, followed by detailed embryological analysis to study their function in zebrafish endoderm formation.

References:

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

Contact: Dr Andrew Nelson, School of Life Sciences