Developing PUFFFIN: a novel neighbour-labelling system for studying cell-cell interactions in developmental biology

Abstract

Cell communication plays a central role in coordinating developmental processes, maintaining tissue homeostasis, and contributing to disease progression. Understanding how cells influence each other within their local environment is therefore crucial for interpreting biological systems. In this thesis, I introduce Positive Ultra-bright Fluorescent Fusion For Identifying Neighbours (PUFFFIN), a novel neighbour-labelling system that provides a simple, sensitive, and highly customisable approach for profiling non-cell-autonomous responses to changes in cell identity or behaviour—whether naturally occurring or experimentally induced.

The first chapter establishes mouse embryonic stem cells (mESCs) as a tractable in vitro model for early mammalian development and describes the exit from naïve pluripotency as a representative developmental transition. It provides an overview of cell-cell communication, illustrated through well-characterised signalling pathways. I then examine current strategies for identifying neighbouring cells, so-called neighbour-labelling systems. These approaches are often limited by technical complexity, restricted specificity, or poor scalability. In response to these challenges, I introduce two candidate proteins—supercharged +36GFP and the HaloTag—highlighting their potential for developing next-generation neighbour-labelling technologies.

Chapter 2 offers a comprehensive and instructive description of all experimental methods and reagents used in this study. This section is intended as a practical reference for other researchers aiming to apply or adapt neighbour-labelling techniques in diverse model systems.

The following three chapters present the core results of my PhD research on the development, validation, and application of PUFFFIN.

In Chapter 3, I assess the suitability of various protein components and regulatory elements for the construction of an efficient gene expression cassette, focusing on the s36GFP-mNG label as the key to PUFFFIN neighbour labelling. I describe the modular cloning strategy that underpins PUFFFIN’s flexible design, facilitating its rapid adaptation to different experimental needs. I also present data from pilot experiments that guided the optimisation of the final construct.

Chapter 4 demonstrates PUFFFIN’s strong performance in cellular assays. I show that the system enables robust and specific labelling of neighbouring cells in HEK293 cultures, and I characterise its labelling range and timing. I further explore how integration with the HaloTag technology platform enhances detection sensitivity and expands the functional versatility of the system.

Chapter 5 explores PUFFFIN’s utility in developmental biology. I apply the system to both in vivo and mESC-based model systems, demonstrating its ability to uncover local cell-cell interactions that drive or coordinate developmental transitions. I focus in particular on the local coordination of the exit from naïve pluripotency, providing a proof-of-principle for PUFFFIN’s use in studying emergent changes in cells and their influence on the cellular neighbourhood.

In the final chapter, I reflect on the broader implications of PUFFFIN for the study of cell-cell communication and community effects. I discuss the strengths and limitations of using supercharged GFP as a transferable label and consider potential avenues for further optimisation. Finally, I outline opportunities for applying PUFFFIN across a wide range of biological questions and suggest directions for future development of the toolkit.