Delineating the IRF6 regulatory network driving periderm formation and function

During development, the ectoderm passes through a series of cell states to produce a self-replenishing, multi-layered epidermis. Initial stratification forms a single-cell layer of flattened periderm cells which act as a barrier to prevent pathological adhesions during embryogenesis. However, the molecular events that drive periderm formation are ill-defined.Recently, we have used a single cell, multiomic approach to analyse periderm development in wild-type and transcription factor P63 mutant embryos. Although we have shown that P63 primes periderm formation, the downstream regulatory interactions driven by its direct transcriptional target Interferon Regulatory Factor 6 (IRF6), remain incompletely characterised. We now aim to delineate the IRF6 regulatory network driving periderm formation using single-cell, spatial transcriptomic, computational, and functional approaches.

Our objectives are:

1. To analyse Irf6 mutant mice using multiomic approaches.

We will perform single-cell, multiome analysis of skin dissected from Irf6 mutant embryos. So that the data complement our wild-type multiome atlas, we will perform triplicate analyses of E10.5, E11.5, and E12.5 Irf6 mutant mice. The resulting data will be analysed computationally to: perform quality control; visualise cell populations; identify potential enhancer regions; predict the upstream candidate transcription factors; and link transcription factors, candidate enhancers and their target gene(s) to predict enhancer-driven regulatory networks. We will use the data to predict how the regulatory networks driving periderm formation are disrupted in Irf6 mutant mice.

2. To generate a spatially resolved cell atlas of periderm formation.

To generate a high-resolution atlas of gene expression during periderm development in wild-type mice and demonstrate how this is disrupted in Irf6 mutant embryos, we will perform imaging-based spatial transcriptomics analysis using the MERSCOPE platform. We will analyse ~500 genes in sections of E10.5-E12.5 lateral skin from wild-type and Irf6 mutant embryos. For each stage, we will generate three spatial imaging datasets enabling us to spatially resolve fine-grained cell types and changes in cellular architecture along the continuum of periderm development. We will use the MERSCOPE web visualiser to spatially explore the resulting data and employ Squidpy to perform downstream analyses. These analyses will explain how neighbouring cells interact and the role of spatial organisation of the cells in driving cell fate transition.

3. To perform functional analyses of the predicted IRF6 regulatory networks driving periderm formation.

A subset of the predicted IRF regulatory network will be verified experimentally. Initially, we will validate a proportion of the MERSCOPE data using immunofluorescence to analyse predicted IRF6 targets in E10.5, E11.5 and E12.5 wild-type mice and their Irf6R84C/R84C littermates. Predicted direct IRF6 transcriptional targets will be confirmed by ChIP-qPCR experiments. To analyse the regulatory function of IRF6, we will perform luciferase reporter assays and deadCas9-mediated CRISPR-based studies.

The data will provide insight into a fundamental developmental process and will have potential impact in dissecting the pathogenesis of a series of congenital disorders.

Training/techniques to be provided

Training will include: multiome analysis; computational analysis; spatial transcriptomics; immunocytochemical analysis and in situ hybridisation; ChIP-qPCR; luciferase assays.

Entry requirements

Candidates must hold (or be about to obtain) a minimum upper second class honours degree (or equivalent) in a related area/subject.

Before you Apply

Applicants must make direct contact with preferred supervisors before applying. It is your responsibility to make arrangements to meet with potential supervisors, prior to submitting a formal online application.

How To Apply

For information on how to apply for this project, please visit the Faculty of Biology, Medicine and Health Doctoral Academy website (https://www.bmh.manchester.ac.uk/study/research/apply/). Informal enquiries may be made directly to the primary supervisor. On the online application form select PhD Developmental Biology.

For international students, we also offer a unique 4 year PhD programme that gives you the opportunity to undertake an accredited Teaching Certificate whilst carrying out an independent research project across a range of biological, medical and health sciences. For more information please visit https://www.bmh.manchester.ac.uk/study/research/programmes/integrated-teaching/

Your application form must be accompanied by a number of supporting documents by the advertised deadlines. Without all the required documents submitted at the time of application, your application will not be processed and we cannot accept responsibility for late or missed deadlines. Incomplete applications will not be considered.

If you have any queries regarding making an application please contact our admissions team FBMH.doctoralacademy.admissions@manchester.ac.uk.

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