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

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 overfixed 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 (Figure 3) craniofacial regions. In sequencing-based spatial transcriptomics datasets, marker genes are identified by analyzing differential expression between spatial clusters, detecting transcripts enriched within specific clusters, and examining genes with spatially restricted expression patterns. This combined approach allows reliable assignment of genes to distinct cell types and spatial regions. Candidate marker validation enables confirming the genes identified from transcriptomic analyses are specific, robust, and biologically meaningful within intact tissue.

Figure 1: Schematic of the method used to obtain and prepare FFPE blocks of embryonic heads for spatial transcriptomic analysis of tooth development.

Abbreviation: FFPE = formalin-fixed paraffin-embedded. Please click here to view a larger version of this figure.

Figure 2: Spatial transcriptomic analysis of the E15.5 and E16.5 mouse whole head using image-based spatial transcriptomics platforms.

(A) In situ spatial transcriptomics analysis platform visualization of coronal sections from a wild-type E15.5 embryonic head processed using a custom panel (350 genes). Cell clusters and corresponding transcript counts are shown across craniofacial structures. Post-assay RNA quality is evaluated via high-confidence decoded transcripts, median transcripts per cell, number of cells, decoded transcripts per 100 μm², and spatial pattern consistency. (B) In situ spatial transcriptomics analysis platform visualization of sagittal sections from a wild-type E16.5 embryonic head processed using the image-based spatial transcriptomics panel (5,000 genes). Spatially resolved cell clusters and transcript counts across developing craniofacial tissues. Post-assay RNA quality is evaluated via high-confidence decoded transcripts, median transcripts per cell, number of cells, decoded transcripts per 100 μm², and spatial pattern consistency. This figure shows spatially resolved cell clusters and transcript counts across developing craniofacial tissues, illustrating that the protocol preserves tissue morphology and enables consistent, reliable detection of cell type–specific expression patterns. Please click here to view a larger version of this figure.

Figure 3: Spatial transcriptomic analysis of the E13.5 mouse craniofacial region, including developing palate and molar tooth buds, using sequencing-based spatial transcriptomics.

(A) High-resolution H&E-stained FFPE section prepared for sequencing-based spatial transcriptomics, illustrating the anatomical region of interest. (B) Loupe Browser visualization of sequencing-based spatial transcriptomics, graph-based spatial clusters, highlighting distinct cellular domains across the palate and tooth bud regions. (C) Differential gene expression heat map showing marker genes enriched within individual spatial clusters. This figure demonstrates that the protocol reliably preserves spatial transcriptomic patterns, allowing consistent identification of cluster-specific gene expression. Abbreviations: H&E = hematoxylin and eosin; FFPE = formalin-fixed paraffin-embedded. Please click here to view a larger version of this figure.

Sequencing-based and imaging-based spatial transcriptomics facilitate this process by detecting RNA molecules in situ at single-cell resolution while preserving spatial context. Following marker selection, their expression is evaluated to ensure it occurs in the expected anatomical locations, is restricted to the appropriate cell types, and is absent from unrelated cells. For example, during tooth development, genes such as Msx1 and Pax9 are specifically restricted to the molars at E15.5 (Figure 4). The prepared FFPE blocks are sectioned according to the protocol described above, and both histological quality and RNA integrity of the resulting sections were verified. Specifically, structural integrity and tissue morphology can be assessed by hematoxylin and eosin (H&E) staining, while RNA quality can be evaluated using RNAScope. For older or demineralized tissues, DV200 values above 50% are recommended, as this threshold is considered the minimum acceptable RNA quality for scRNA-seq or spatial transcriptomic analyses.

Figure 4: Visualization of tooth-specific transcripts in E15.5 mouse maxillary molar by image-based spatial transcriptomics platform.

DAPI stain and individual counts of Msx1 and Pax9 mRNA transcripts visualized in coronal sections of the maxillary molar by the image-based in situ hybridization at E15.5. Scale bar = 200 μm. Abbreviation: DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.

Visual success checkpoints include well-preserved tissue morphology with minimal tearing or folding, consistent section thickness, accurate placement of tissue within the capture area, and reliable attachment to the slide. Successful assays are further characterized by clearly defined, spatially resolved transcript signals that correspond to known anatomical features, low background signal, and reproducible spatial patterns across adjacent sections or biological replicates. Postassay RNA quality for image-based spatial transcriptomics is evaluated via high-confidence decoded transcripts, median transcripts per cell, spatial pattern consistency, and background signal. Postassay RNA quality for sequencing-based spatial transcriptomics is assessed using library and sequencing metrics, such as median genes per spot, percent mapped reads, sequencing saturation, and tissue alignment.

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 within complex, multi-tissue environments, such as the developing palate and tooth structures.

Recent studies highlight the importance of spatial transcriptomics for dissecting cellular heterogeneity and lineage specification during craniofacial development. For instance, the integration of single-cell RNA sequencing with spatial transcriptomics has enabled the construction of comprehensive transcriptional atlases of tooth development^14,15. These integrated datasets have defined distinct epithelial and mesenchymal cell populations in human embryonic teeth, uncovered molecular regulators of lineage commitment, and provided foundational reference maps for studying early odontogenesis. Similarly, analyses of the palatal mesenchyme using spatial RNA sequencing approaches have revealed dynamic transcriptional programs underlying osteoblast differentiation in the hard palate, including the spatially restricted activation of late-stage osteogenic markers¹⁶. These findings highlight that spatial transcriptomic technologies can provide important insights into craniofacial tissue development and differentiation. Prior studies have shown that integrating scRNA-seq reference atlases for cell-type deconvolution with image-based segmentation enhances the assignment of gene expression to individual cells and improves cell-level interpretation in sequencing-based spatial transcriptomics datasets^21,22. Such integration can help overcome limitations related to the relatively lower spatial resolution of sequencing-based platforms^21,22.

The workflow described here provides a practical and reproducible framework for generating FFPE sections suitable for such high-resolution spatial analyses. As spatial transcriptomic and spatial imaging platforms continue to advance, standardized sample preparation methods will become increasingly important. This protocol provides a reliable framework for FFPE-based investigations of tooth development, maintaining the integrity of delicate structures and allowing precise sectioning of small tissues. Its reproducibility and robustness make it particularly suitable for studies using mutant mouse models, including tooth agenesis and cleft palate, as well as broader craniofacial disease research, enabling high-resolution spatial and molecular analyses throughout developmental stages. In summary, this protocol has broad relevance for developmental studies and also for work in disease pathogenesis, tissue engineering, and regenerative medicine, all of which depend on maintaining precise anatomical and molecular context.

Practical troubleshooting for spatial transcriptomics requires careful attention at each step to ensure both tissue morphology and RNA integrity are preserved. During fixation, under-fixation can cause tissue collapse, while over-fixation can reduce RNA accessibility for probe hybridization or library preparation; thus, fixation times should be optimized based on tissue size and developmental stage. In paraffin embedding, incomplete infiltration can result in sectioning artifacts that compromise spatial resolution, making thorough dehydration and paraffin penetration essential. Sectioning quality directly affects spatial capture; blade angle, temperature, and section thickness must be consistent, and blades should be regularly replaced to avoid tissue tearing or folding. Minor adjustments in fixation duration, embedding, or section thickness may be made to accommodate tissue variability without compromising spatial transcriptomic performance. Using manufacturer-recommended slides and proper drying or baking at recommended temperatures is critical to maintain section integrity during staining, hybridization, or sequencing workflows and for ensuring compatibility with spatial transcriptomics instruments.

This workflow is optimized for small, delicate embryonic tissues and FFPE preservation. It may not perform well in large or highly calcified tissues, heavily decalcified samples, or tissues with extreme enzymatic activity. Compared to traditional fresh-frozen tissue preparation for spatial transcriptomics, this FFPE workflow improves preservation of tissue morphology and facilitates long-term storage, enabling reproducible sectioning of small craniofacial structures. While fresh-frozen protocols may provide higher RNA integrity for sequencing, they are more prone to tissue distortion, especially in developing heads. By contrast, our workflow balances morphological fidelity and RNA quality, making it robust for both imaging- and sequencing-based spatial assays. The reproducibility of this workflow is supported by several concrete features: standardized technique for tissue collection, fixation times, embedding conditions, sectioning and placement methods, and post-assay quality checks.

Critical Steps:

The success of this workflow depends on several critical steps, especially maintaining tissue morphology during fixation and embedding, and preserving RNA integrity throughout processing. Proper fixation, thorough paraffin infiltration, and accurate sectioning are essential to prevent tissue distortion, tearing, or RNA degradation. Careful placement of the tissue section within the recommended area on the slide is critical for spatial transcriptomics, as it directly influences assay performance, tissue coverage, and downstream data quality.