Constructing a Spatially Resolved Single-Cell Reference Atlas of the Murine Gastrointestinal Tract with MERFISH

Abstract

This dissertation investigates the advancement and application of imaging-based spatial transcriptomic techniques, specifically MERFISH, to high-RNase, cell-dense tissues such as the gastrointestinal (GI) tract. The study addressed critical experimental and computational challenges in RNA integrity, data quality control, as well as cell segmentation, and produced an all-cell-type, multi-region, spatially resolved single-cell reference atlas for both the specific- pathogen-free (SPF) and germ-free (GF) gut for the purpose of understanding gut cell type and spatial organization, small molecule sensation, and microbial interaction.

Chapter One: Introduction

The introductory chapter outlines the interdisciplinary field of imaging-based spatial transcriptomics, data processing, and spatial analysis methods within the context of gut biology and the microbiome. It identifies key technical challenges in RNA integrity, data quality control, and cell segmentation, as well as open biological inquiries regarding gut cell type organization, spatial niches and gradients, and the remodeling that happens in the absence of the microbiome. Among the discussion of technical challenges, a specific focus is given to comparing recent computational algorithms for cell segmentation in imaging-based spatial transcriptomic datasets.

Chapter Two: Technological Advancements to MERFISH

This chapter focuses on the technological hurdles encountered when applying MERFISH to the GI tract, a tissue characterized by high RNase activity and cellular density. Adjustments to existing brain MERFISH protocols were necessary to preserve RNA integrity in fresh frozen gut tissues. A novel data quality filtering approach was developed and benchmarked against traditional threshold-based methods. The limitations of existing cell segmentation techniques were addressed by enhancing the Baysor algorithm, incorporating Cellpose priors, and refining downstream processing steps such as low confidence RNA removal and doublet score screening. The qualitative, semi-quantitative, and quantitative metrics for assessing segmentation quality are discussed in this chapter, together with recommendations for tuning Baysor parameters for optimal segmentation performance.

Chapter Three: A Spatially Resolved, Single-Cell Atlas of The Gastrointestinal Tract

The MERFISH gut atlas revealed significant insights into the spatial organization and sensory capabilities of the mouse lower digestive tract. By constructing a spatially resolved single-cell atlas, the study identified both expected and novel cell types, their spatial distributions across four lower GI regions, and the receptor expression gradients that drive spatial heterogeneity. The absence of the microbiome was shown to selectively remodel these spatial features, highlighting the dynamic interplay between gut cells and microbial populations. These findings offer a comprehensive view of the molecular and cellular organization of gut sensation as well as microbial interaction, with potential pharmacological implications.

Chapter Four: Conclusions

The concluding chapter summarizes the research achievements of this Ph.D. work, emphasizing the technological advancements in MERFISH application to cell-dense, high-RNase tissues and the biological discoveries that enhance our understanding of gut organization and function.