Spatial Transcriptomics of Early Tooth Morphogenesis in Formalin-fixed Paraffin-embedded Mouse Embryonic Tissue

Summary

The current protocol describes the use of formalin-fixed, paraffin-embedded sections from E13.5 and E15.5 craniofacial regions of mouse embryos to analyze the differential gene expression profiles using spatial transcriptomics.

Abstract

The developing tooth comprises diverse and highly specialized cell populations that work together to maintain proper form and function. Elucidating the interactions among these cells and their surrounding microenvironment is critical for understanding the regulatory mechanisms underlying normal tooth development. Perturbations in these processes can result in congenital disorders such as tooth agenesis, dentinogenesis imperfecta, and amelogenesis imperfecta. Despite the substantial progress enabled by single-cell RNA sequencing (scRNA-seq) in revealing cellular heterogeneity, it does not preserve the spatial context of cells within tissues, limiting the ability to relate gene expression to tissue architecture. Spatial transcriptomic technologies address this limitation by integrating high-resolution gene expression profiling with the preservation of native tissue architecture, enabling the in situ localization of molecular signatures. Here, we describe a step-by-step protocol for the collection, fixation, and paraffin embedding of mouse embryonic craniofacial tissue suitable for downstream spatial transcriptomic applications. The workflow details optimized sectioning and handling of formalin-fixed, paraffin-embedded tissue to preserve RNA integrity and tissue morphology for high-resolution spatial analysis. This method is compatible with sequencing and image-based spatial transcriptomics platforms, enabling reproducible spatial transcriptomic profiling of early tooth morphogenesis in mouse embryos. This approach offers powerful insights into the spatial organization and functional dynamics of craniofacial structures in both developmental and pathological states, providing a critical framework for linking molecular mechanisms to tissue morphology.

Introduction

Tooth development relies on a highly coordinated sequence of morphogenetic processes during early embryonic growth^1,2,3,4. Although numerous key genes and signaling pathways have been identified through genetic and developmental studies, our understanding of how these factors interact to shape individual craniofacial structures remains limited. Notably, even with substantial progress in linking specific genetic variants to both syndromic and non-syndromic tooth disorders, the detailed molecular mechanisms underlying structure-specific morpho....

Protocol

All animal procedures were approved by the National Institutes of Health, National Institute of Child Health and Human Development Animal Care and Use Committee (ACUC), under Animal Study Protocol #21-031.

1. Preparation of experimental animal and collection of tissue

1. Pair healthy, fertile male and female Mus musculus for timed matings. Identify pregnant female mice by the presence of a vaginal plug, designated as embryonic day (E) 0.5.
2. Euthanize pregnant female mice via CO₂ inhalation followed by cervical dislocation. Position the animal's supine on a sterile absorbent surgical surf....

Results

This method outlines the processing of freshly dissected mouse embryonic heads to generate FFPE samples of craniofacial tissues, including the developing tooth, that can be readily sectioned by microtome while maintaining RNA integrity (Figure 1). This protocol was successfully applied to E13.5 (embryonic day 13.5), E15.5, and E16.5 murine embryo heads for high-resolution image-based (Figure 2) and sequencing-based spatial transcriptomics (F.......

Discussion

In this work, we present a detailed protocol for preparing FFPE blocks of mouse embryonic heads optimized for use with high-resolution spatial RNA imaging platforms, including sequencing- and imaging-based spatial transcriptomics. A key objective of this protocol is to preserve both tissue morphology and nucleic acid integrity across whole-head sections, with particular focus on the developing craniofacial region. Ensuring this level of preservation is crucial for accurately resolving spatial gene expression patterns wit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Thermo Fischer	10010023	Use  to perform washes during the workflow
50 mL conical tubes (Ambion) RNAse free	Thermo Fischer	AM12502	Use to store samples in different solutions
Advanced orbital shaker	VWR	6683-470	Use to shake tissues in fixation solution during incubation
Alcohol, 70%, Fisherbrand, HistoPrep	Fisher Scientific	HC-1000-1GL	Use to clean and disinfect all the work space
Automated vacuum tissue processor	Leica Biosystems	ASP300S	Use to clear, dehydration, rehydration and wax infiltration of samples
Cover Glass Thickness 1.5, 25 mm x 25 mm	Corning	2850-25	Use for mounting of slide in Visium HD workflow
Dako Bluing Buffer, Ready-to-use	Agilant Technologies	CS70230-2	Use for H&E staining
Eosin-Y with phloxine	Fisher Scientific	22050198	Use for H&E staining
Hematoxylin, Mayer's, Ready-to-use aqueous solution	Agilant Technologies	S330930-2	Use for H&E staining
HistoCore Water Bath	Leica Biosystems	HIS2326	Use to float the sections at 40-43 °C to remove wrinkles from FFPE sections
Loupe browser  9.0.0	10X Genomics, Inc.		Use to analyze Visium HD data
Low-Profile Disposable Blades DB80LX	Leica Biosystems	14035843496	Use to section FFPE blocks
Neutral Buffered Formalin 10%	Azer Scientific	NBF-4-G	Use to fix the tissues
RNaseZap RNase Decontamination Solution	Thermo Fischer	AM9782	Use to clean and remove RNase
Semi-Automated Rotary Microtome	Leica Biosystems	RM2245	Use to section FFPE blocks as reported in the guidelines.
Slide Warmer with Cover	Premiere	XH2004	Use for incubation of slides at different temperatures
Superfrost Plus Slides	Fisher Scientific	12-550-15	Use to attach sections for Vsium HD
Surgical blade No. 11	Integra Miltex	4-311	Use for scoring of FFPE tissues
Surgipath Paraplast	Leica Biosystems	39601006	Use to carry out tissue infiltration and embedding of tissues
TISsue culture DISH 100X20MM 500/CS	Fisher Scientific	877222	Use for collecting and dissecting samples in 1x PBS
UltraPure Glycerol	Thermo Fischer	15514011	Use for Visium HD slide mounting of coverglass before CytAssist
Visium CytAssist	10X Genomics, Inc.	PN-1000442	Use for Visium HD workflow experiments
Visium HD Spatial RNA-sequencing	10X Genomics, Inc.	1000676	Use to perform spatial transcriptomic experiments
Xenium 5K In Situ RNA Localization	10X Genomics, Inc.	PN-1000724	Use to perform spatial transcriptomic experiments
Xenium Analyzer	10X Genomics, Inc.	PN-1000481	Use perform Xenium and Xenium 5K RNA imaging
Xenium Explorer 4	10X Genomics, Inc.		Use to analyze Xenium data
Xenium In Situ RNA Localization	10X Genomics, Inc.	1000672	Use to perform spatial transcriptomic experiments