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 morphogenesis are still not fully defined^1,2,3,4. This knowledge gap continues to limit the progress of targeted therapeutic strategies for tooth agenesis or other tooth aberrations.

Single-cell mRNA sequencing (scRNA-seq) has enabled the discovery of previously unrecognized cell types in both healthy and diseased tissues, as well as the identification of multiple cell subtypes based on distinct gene expression profiles. However, because scRNA-seq requires dissociation of tissues into single cells, information about each cell's original spatial position within the tissue is lost. As a result, these datasets do not capture how cells are arranged relative to the tissue architecture^5,6,7. In addition, predicted cell-to-cell interactions rely on the presence of complementary interactions of transcripts, but do not account for actual spatial information, which is important for determining whether such interactions occur in vivo^5,6,7.

Spatial transcriptomics technology enables high-throughput profiling of gene expression while retaining spatial information within tissue sections^8,9. Spatial transcriptomics technologies are either imaging-based^10,11,12 or sequencing-based^12,13. Imaging-driven spatial transcriptomics approaches employ FISH to detect and measure targeted gene expression with cellular or subcellular spatial precision. In contrast, sequencing-based technologies capture transcriptome-wide gene expression at a lower spatial resolution. Sequencing-based spatial transcriptomics employs a strategy to achieve near-single-cell resolution with whole-transcriptome coverage (~20,000 genes), supporting unbiased exploratory analyses, whereas image-based spatial transcriptomics targets gene panels (up to ~5,000 genes), enabling precise hypothesis-based analysis of specific pathways and cellular interactions¹⁴.

Technological advances in spatial transcriptomics now enable the analysis of gene expression within preserved tissue architecture. Formalin-fixed paraffin-embedded (FFPE) and cryosection-based workflows offer complementary approaches for spatial transcriptomics^15,16,17. FFPE provides superior preservation of tissue architecture, enabling accurate spatial mapping of gene expression in structurally complex tissues such as developing teeth and palatal shelves. In our study and others, FFPE fixation and archiving have yielded highly accurate and reproducible spatial transcriptomic data¹⁸. Cryosection workflows preserve RNA integrity but are more prone to tissue distortion¹⁸. Therefore, FFPE was selected for this study. While scRNA-seq success largely depends on retaining high RNA integrity, spatial transcriptomics also requires high-quality tissue morphology to accurately localize gene expression signals to specific histological features.

This protocol is best suited for FFPE mouse embryonic craniofacial tissues spanning early developmental stages, where preservation of fine tissue architecture is critical for spatial transcriptomic analysis. The workflow is likely to perform suboptimally in over-fixed samples, poorly oriented paraffin blocks, or tissues with compromised RNA integrity, which can result in reduced signal quality and loss of spatial resolution. Successful application of this method is indicated by intact tissue morphology, minimal section distortion, and robust, spatially resolved transcriptomic signals that align with histological features.

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 surface and disinfect the abdominal area with 70% ethanol along the intended incision site.
3. Make a midline abdominal incision using sterile surgical scissors, and carefully open the peritoneal wall with microsurgical scissors to expose the uterine horns. Gently exteriorize the uterine chain using blunt forceps. Isolate embryos by releasing the uterine tissue along the myometrium and severing at the oviduct and the median uterine horn attachment.
4. Immediately transfer whole embryos to ice-cold, sterile 1x PBS in a 10 cm Petri dish.
5. Carefully separate each embryo from the surrounding uterine, amniotic, and chorionic membranes, and transfer it into a fresh 10 cm dish containing ice-cold, sterile 1x PBS.
6. Dissect embryonic heads at the desired developmental stages using a sterile surgical blade, and rinse them once in ice-cold, 1x PBS before further processing.

2. Fixation and processing of the tissue

1. Dissect embryonic mouse heads at the desired developmental stages and fix in 10% neutral buffered formalin overnight at room temperature on a shaker.
2. Following fixation, remove the formalin, and rinse the samples with 1x PBS before being transferred to 70% ethanol for storage. Perform tissue dehydration, clearing, and paraffin infiltration using an automated tissue processor.
3. Embed embryo heads in paraffin with orientation in either the coronal or sagittal plane.
4. After solidification of wax, remove the paraffin blocks from the molds, place them in labeled airtight containers or zip-lock bags, and store at 4 °C until sectioning.
   NOTE: FFPE blocks can be stored for several years before sectioning without compromising tissue integrity.

3. Processing of FFPE tissue for spatial transcriptomics

1. Disinfect work surfaces and instruments with 70% ethanol, followed by treatment with RNase-decontaminating solution to minimize RNA degradation.
2. Prepare a water bath containing deionized water and maintain it at 40 °C, adjusting the temperature between 40 °C and 43 °C as needed based on embryonic stage or tissue characteristics.
3. Set up the microtome, install a new blade after gently wiping it with RNase-decontaminating solution, secure the blade, and set the clearance angle to 10°.
   NOTE: Ensure that the new blade is used for sectioning to achieve precise tissue slices. Changing the microtome blade angle can significantly impact section quality, affecting tissue integrity, thickness uniformity, and the preservation of morphological features.
4. Trim excess paraffin from around the embedded tissue, and mount the FFPE block onto the cold stage of the microtome.
5. Prepare and treat clean tweezers and brushes with RNase-decontaminating solution prior to use.
6. Align the paraffin block face with the microtome blade, and make trimming cuts of approximately 12 µm until the region of interest is approached (e.g., until the mesial part of molars becomes visible in coronal sections). Adjust block and blade positioning as necessary to ensure symmetrical, properly oriented whole-head coronal sections.
7. If RNA isolation of tissue sections is to be performed, directly collect 10 µm sections into 1 mL microcentrifuge tubes and process them according to the manufacturer's recommendations^19,20 for DV200 quality assessment.
8. Cut serial sections of 5 µm thickness, and continuously monitor proximity to the area of interest by examining test sections on a regular slide under a microscope.
9. Once near the region of interest, carefully score the tissue area using a No. 11 scalpel blade without fully cutting through, in two strategically spaced regions to prevent curling or adherence of sections. After sectioning, remove any attached paraffin using the bristles of a fine brush.
10. Float tissue sections onto the water bath at 41 °C to allow for gentle relaxation and flattening before picking them up on a slide.

4. Sequencing-based spatial transcriptomics

1. Collect tissue sections onto the recommended glass slides, ensuring that all regions of interest are contained within a 6.5 × 6.5 mm area. When multiple adjacent sections are placed on the same slide, briefly warm the slides on a slide warmer at 40 °C to promote adhesion and prevent detachment of previously placed sections. After all sections are positioned, incubate the slides in a covered slide warmer at 60 °C for 2 h to enhance tissue adherence.
   NOTE: Sections extending beyond the 6.5 × 6.5 mm area were not processed.
2. Following incubation, perform hematoxylin and eosin (H&E) staining using a standard H&E staining protocol, substituting deionized water for tap water to preserve RNA integrity.
3. Mount coverslips using 80% glycerol, and acquire high-resolution H&E images, providing a higher-quality reference image to replace the typical H&E image generated by the slide imaging and tissue transfer system.
4. After imaging, remove the coverslips by immersing the slides individually in 50 mL tubes containing deionized water, and prepare the slides for submission to the core facility for subsequent processing.

5. Image-based spatial transcriptomics

NOTE: Slides were supplied by the manufacturer²⁰, each containing a designated imageable area (outlined lines) measuring 12 mm × 24 mm, with an available sample placement region of approximately 235 mm² (10.45 mm × 22.45 mm).

1. Position the tissue sections entirely within this defined area, ensuring that sections, including residual paraffin, do not overlap. When placing multiple adjacent sections, briefly warm the slides on a slide warmer at 40 °C to promote adhesion and prevent detachment of previously mounted sections.
2. After all tissue sections are positioned, place the slides in a covered slide warmer and incubate overnight at 40 °C to ensure proper adherence.
3. After incubation, follow the manufacturer's¹⁹ protocol to process the slides and load them into the machine for RNA sequencing, with the protocol cross-checked against the most recent version, as procedural details are subject to periodic updates.
   NOTE: All hazardous materials generated during the protocol, such as formalin, ethanol, and RNase-decontaminating solutions, should be disposed of in accordance with institutional safety policies and local regulations, using proper chemical waste containers, biohazard disposal methods, and fume hood practices.

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