Defining the p63 regulatory network driving periderm development in the mouse

How cells transition between different cellular states is a fundamental biological question. In this context, the simple ectoderm passes through a dynamic series of cell states to produce a self-replenishing, multi-layered epidermis that protects the organism from dehydration, mechanical trauma, and microbial invasion. The first stratification event results in the formation of a layer of flattened periderm cells. Periderm development commences on the tail and limbs before spreading over the torso and face so that the embryo is covered by embryonic day (E)14; all stages of periderm development are therefore present in individual E11.5 embryos.

Periderm functions as an embryonic barrier that prevents pathological adhesion between epithelia during development. Failure of periderm formation underlies several birth defects that are characterised by webbing of the skin across the major joints, cleft lip and/or palate, fusion of the fingers and/or toes, and genital malformations.

While we have demonstrated that the transcription factor DeltaNp63Alpha (DNp63A) is a key regulator of periderm development, the regulatory networks controlling this fundamentally important cell layer are poorly characterised; this is a barrier to understanding a major event in epidermal development and in dissecting disease pathogenesis.

We have assembled a team of investigators with expertise in developmental biology, gene regulation, and computational biology with the aim of delineating the regulatory interactions that drive cells to delaminate from the ectoderm to form periderm. Two objectives will be addressed:

1. Predicting the regulatory networks driving mouse periderm formation at single-cell (sc) resolution: We will define the transcriptome of the developing periderm by performing scRNA-seq analysis of skin dissected from E11.5 wild-type mice in which all stages of periderm development are present. Clustering algorithms will be used to identify sub-populations of cells and expression of their 'marker genes' will be analysed to reconstruct the populations into a tissue context. Developmental trajectories will be inferred computationally allowing us to study the relationships of the clusters during development.

To identify regulatory elements, we will perform scATAC-seq analysis and infer the interactions of transcription factors and their target genes. We will link open regulatory regions to their target genes and use the scATAC-seq data to infer potential upstream regulators through differential enrichment of transcription factor binding motifs. Evolution of the regulatory networks driving periderm development will be analysed computationally.

Deliverables: We will predict the regulatory networks driving periderm development at single-cell resolution allowing us to formulate hypotheses about the DNp63A regulatory cascade.

2. Dissecting the molecular cascade downstream of the transcription factor DNp63A: We will use the data generated in Objective 1 to extend the DNp63A regulatory network. We will perform scRNA-seq and scATAC-seq analyses of skin dissected from E11.5 Tp63-null mice and perform computational analyses to investigate how the predicted regulatory networks are perturbed.

The inferred regulatory interactions will be verified experimentally. Direct transcriptional targets of DNp63A will be confirmed by ChIP-qPCR analysis of E11.5 skin. Subsequently, we will extend the ChIP-qPCR analyses to the second layer of network regulation. To analyse whether the transcription factors act as activators or repressors, we will perform luciferase reporter assays and deadCas9-mediated CRISPR activation/inhibition studies.

Deliverables: We will validate the DNp63A regulatory network driving periderm development at single-cell resolution.

The data will provide mechanistic insights into cell state transitions during development and will have potential impact in determining the pathogenesis of a series of congenital disorders.

We aim to dissect the regulatory interactions that drive cells to delaminate from the ectoderm to form periderm during embryonic development. By integrating single-cell and computational approaches, we will delineate the regulatory interactions driving periderm formation and use this information to dissect the molecular cascade downstream of the transcription factor DeltaNp63Alpha (DNp63A).

Objective 1: To predict the regulatory networks driving mouse periderm formation at single-cell resolution. Single-cell (sc) isolates prepared from skin dissected from E11.5 wild-type mice, in which all stages of periderm development are present, will be analysed using scRNA-seq and scATAC-seq approaches. The resulting data will be evaluated computationally to identify sub-populations of cells from each type of analysis. scATAC-seq data will be analysed further by aggregating data and identifying potential transcription factor regulators. Computational analysis of the scRNA-seq and scATAC-seq will allow inference of the regulatory networks driving mouse periderm development.

Objective 2: To dissect the molecular cascade downstream of the transcription factor DNp63A. We will perform scRNA-seq and scATAC-seq analyses of skin dissected from E11.5 Tp63-null mice and repeat the computational analyses to investigate how the predicted regulatory networks are perturbed. The computational predictions will be verified using a combination of expression analyses, chromatin immunoprecipitation, luciferase reporter assays, and deadCa9-mediated genome-editing experiments.

The data will provide mechanistic insights into cell state transitions during development and will have potential impact in determining the pathogenesis of a series of congenital disorders.