Temporal and spatial distribution of histone acetylation in mouse molar development

Mouse lines and procedures

C57BL/6 mice were provided from the Experiment Animal Center of Sichuan University, and used at various embryonic and early postnatal stages. All mice were housed in specific pathogen-free facilities at the Experimental Animal Core of West China Hospital Sichuan University, a temperature-controlled (25 °C) environment with a 12-h light/dark cycle, cotton batting, and free access to food and water as previously described (Yu et al., 2022). Mice were mated overnight to generate embryos for experiments. Vaginal plug discovery was taken as E0.5 at noon. Pregnant mice were euthanized by CO₂ followed by cervical dislocation at desired timepoints, and embryos were removed from the uterus. Both male and female embryos or postnatal pups from 3–4 different litters each timepoint were selected at random. Each experiment was performed at least three times with different embryos. The developmental stages of the tooth germs were judged from the tissue sections according to morphological criteria. All mice received humane treatment, and every possible measure was taken to reduce any potential distress. All experiments involving mice were approved by the Ethics Review Committee of the West China School of Stomatology, Sichuan University (WCHSIRB-D-2022-155).

Tissue preparation for sectioning

Tissue was prepared for paraffin sections by fixing embryonic heads or postnatal jaws in 4% paraformaldehyde (PFA) in PBS overnight at 4 °C and were then decalcifying in RNase-free EDTA for 3–5 days. Samples were embedded in paraffin and then sectioned at 6 µm.

Immunofluorescence and immunohistochemistry staining

Immunofluorescence staining is performed as previously described (Du et al., 2024). Briefly, paraffin sections were rehydrated through serial ethanol and water washes, and antigen retrieval was performed by incubation in pH 6.2 citric buffer containing 2 mM EDTA, 10 mM citric acid, 0.05% Tween 20 just below boiling temperature for 15 min followed by a 30-min cool-down to room temperature. Samples were blocked in 1X animal-free blocker (Vector Laboratories), supplemented with 2.5% heat inactivated goat serum, 0.02% SDS and 0.1% Triton-X for 1 h. Slides were then incubated with primary antibodies overnight at 4 °C. All the antibodies were diluted in the same blocking solution without serum. Primary antibodies and dilutions used are as follows: Histone H3 (acetyl K4+K9+K14+K18+K23+K27) (1:200; Abcam, ab300641), Histone H4 (acetyl K5 + K8 + K12 + K16) (1:200; Abcam, ab177790), Histone H3 (acetyl K27) (1:200; Abcam, ab4729), Histone H3 (acetyl K9) (1:200; Abcam, ab32129), Histone H4 (acetyl K5) (1:200; Abcam, ab51997), Histone H4 (acetyl K16) (1:200; Abcam, ab109463), CREBBP (1:100; Abcam, ab253202), HDAC1 (1:100; Abcam, ab109411), HDAC2 (1:100; Abcam, ab32117) and EP300 (1:100; Zenbio, 347220). Signals were detected using secondary antibodies conjugated with Alexa Fluor 555 (1:250; Thermo Scientific, Waltham, MA, USA) or horseradish peroxidase (HRP) followed by DAB staining kit (PK10006; Proteintech). Nuclear counterstaining was performed using DAPI (Thermo Scientific, Waltham, MA, USA) or hematoxylin, and mounted with antifade mountant (Solarbio) or neutral balsam. Images were taken using a confocal laser scanning microscope (FV3000; Olympus) or Slideview VS200 (Olympus).

scRNA-seq data analysis

scRNA-seq data was downloaded from the NCBI GEO database (GSE162413). Cell-level transcripts were processed using the Seurat package for normalization, quality control, dimensionality reduction, and clustering. The GSE162413 dataset comprises tooth germs collected at different developmental stages. Four stages were selected: bud stage (E12.5), cap stage (E14.5), bell stage (E16.5), and postnatal day 1 (PN1), including a total of 38,874 cells. The differentially expressed genes for each cluster, compared to other clusters at different time points, were identified by the FindAllMarkers function in Seurat, with a threshold of average log2FC > 0.25 and an adjusted p-value < 0.05 using two-sided Wilcoxon rank-sum test with a Bonferroni correction. Sub-clustering analyses were carried out using the Findneighbors function of the Seurat package with proper resolutions. Identified cell clusters and sub-clusters were visualized on UMAP plots, as previously described (Zeng et al., 2024). Clusters were annotated based on top expressed genes within each cluster and known canonical cell markers. After annotation, the expression of histone acetylation-related was further analyzed across epithelial, mesenchymal, and other cells types.

Image analysis

The average immunofluorescence pixel intensity of dental epithelium and mesenchyme or dental papilla (as defined in Fig. 1) were determined by measuring the total pixel intensity in the region of interest and divided by the total measured pixel area using the “Measure” function of ImageJ. The measured regional histone H3, histone H4, H3K9ac, H3K27ac, H4K5ac, H4K16ac, CREBBP, HDAC1, HDAC2 and EP300 levels in the epithelium were normalized across samples using averaged signals from the mesenchyme in the same section sample, respectively.

Statistical analysis

Data were collected as previously described (Du et al., 2024). Briefly, all experiments were repeated using three independent biological samples. Representative images were shown in figures (replicate staining images of independent biological samples are available in the author’s response letter in peer review reports from PeerJ website). Each data point in bar graphs represents a single biological sample without investigator blinding. No data were excluded. All statistical analyses were performed using the Prism 9 software and displayed as mean ± S.D (standard deviation) in graphs. All p values were calculated using unpaired two tailed Student’s t-test or one-way ANOVA followed by Dunnett’s test. Significance was taken as p < 0.05 with a confidence interval of 95%. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Spatial–temporal pattern of histone acetylation marks in mouse molar development

To investigate the distribution patterns of histone acetylation, we first performed immuno-fluorescence staining of histone 3 and 4 acetylation (H3Ac and H4Ac) in the mouse molar through the bud stage (E13.5), cap stage (E14.5), early bell stage (E16.5) and late bell stage (E18.5 and P3) (Figs. 1, 2, Figs. S1A, S2A). At E13.5, H3ac was weakly detected in both dental epithelium and mesenchyme, with few strong positive cells in the epithelium (Figs. 2A, 2F). In contrast, H4ac was ubiquitously expressed, pointing to a dominant role for H4ac in the early stage of molar development (Figs. 2K, 2P). At E14.5, both H3ac and H4ac were detected in the dental epithelium and the condense mesenchyme (Figs. 2B, 2G, 2L, 2Q). Instead of comparatively higher expression level in the condense mesenchyme at E14.5, H3ac localized in both dental epithelium and the dental papilla at similar expression level at E16.5 (Figs. 2C, 2H). While H4ac also localized in both dental epithelium and dental papilla at E16.5, it was weaker in the inner enamel epithelium (IEE) and the mesenchyme next to the IEE (Figs. 2M, 2R). However, the distribution of H3ac and H4ac were observed abundantly in the dental epithelium, but began to downregulate in the dental papilla at E18.5 (Figs. 2D, 2I, 2N, 2S). Of note, H3ac and H4ac distributions were apparently at a higher levels in the IEE, indicating the potential important role for participating amelogenesis. Similar distribution patterns of H3ac and H4ac were detected at later late bud stage, P3, with more apparent restricted in the IEE and ameloblasts. In the mesenchyme, H3ac and H4ac were mostly expressed in the odontoblasts (Figs. 2E, 2J, 2O, 2T).

We then set out to examine H3K9ac and H3K27ac as major histone 3 acetylation marks in mouse molar across different developmental stages (Fig. 3, Figs. S1B, S1C). At E13.5, weak signal of H3K9ac and H3K27ac was present in both dental epithelium and mesenchyme (Figs. 3A, 3F, 3K, 3P). At E14.5, H3K9ac was significantly upregulated in both the dental epithelium and mesenchyme (Figs. 3B, 3G). For H3K27ac, strong signal was restricted in the condense mesenchyme, with weak signal in the dental epithelium (Figs. 3L, 3Q). At E16.5, both H3K9ac and H3K27ac widely expressed in both the dental epithelium and mesenchyme. Specifically, H3K9ac in the IEE was much weaker than that in other cells, while H3K27ac was stronger in the IEE in the dental epithelium and dental papilla (Figs. 3C, 3H, 3M, 3R). At E18.5, H3K9ac was no longer highly expressed in the entire dental papilla, but rather in the entire dental epithelium including IEE and the mesenchymal cells underlying the IEE (Figs 3D, 3I). At this stage, H3K27ac was highly expressed in the IEE and the mesenchymal cells underlying the IEE, where weaker signal was also detected in other cells in the dental epithelium (Figs. 3N, 3S). At P3, H3K9ac was broadly expressed with higher expression in the IEE and the odontoblasts, and H3K27ac was ubiquitously expressed in both the dental epithelial and mesenchymal cells (Figs. 3E, 3J, 3O, 3T).

We next examined the distribution of histone 4 acetylation marks H4K5ac and H4K16ac in mouse molar development (Fig. 4, Figs. S2B, S2C). At E13.5, both H4K5ac and H4K16ac were barely detectable (Figs. 4A, 4F, 4K, 4P). Increased signal of both H4K5ac and H4K16ac were obtained at E14.5. At this stage, while H4K5ac was relatively stronger in the condense mesenchyme than that in the epithelium (Figs. 4B, 4G), H4K16ac was present extensively expressed in both the dental epithelium and mesenchyme. Of note, the presence of H4K16ac was stronger in the outer enamel epithelium (OEE) and lower in the stellate reticulum (Figs. 4L, 4Q). At E16.5, H4K5ac and H4K16ac remain broadly distributed. Interestingly, slightly weak H4K5ac was observed in the OEE and dental papilla, where significant high H4K16ac was detected (Figs. 4C, 4H, 4M, 4R). At E18.5, the distribution of H4K5ac and H4K16ac in the dental papilla weakened, whereas that in the dental epithelium continued to be highly expressed. Moreover, both H4K5ac and H4K16ac were highly expressed in the IEE (Figs. 4D, 4I, 4N, 4S). At P3, H4K5ac and H4K16ac were still widely expressed in the dental epithelium cells, concentratedly in the IEE and tall-columnar ameloblasts, with the highest expression near the cervical loop (Figs. 4E, 4J, 4O, 4T). In the mesenchyme, H4K5ac was mainly expressed in the odontoblasts and in the middle zone between the two lateral cervical loops (Figs. 4E, 4J), while H4K16ac was widely expressed (Figs. 4O, 4T).

Histone acetylation-related enzymes expressed in dental epithelium and mesenchyme during mouse molar development

Histone acetylation is accomplished by its modification enzymes, HATs and HDACs. To evaluate the specific alternations of HATs and/or HDACs, which medicate the acelylation of H3K9, H3K27, H4K5 and H4K16 (Fig. 5A) (Steunou, Rossetto & Côté, 2014; Fang et al., 2021), through the tooth development stages, we then utilized the published scRNA-seq data (Hu et al., 2022) for analysis. We found HATs and HDACs were widely present in both dental epithelium and mesenchyme during tooth development. Notably, at each timepoint, HATs and HDACs exhibited stronger signals in the mesenchyme, and Crebbp and Hdac2 were the prominent HATs and HDACs, respectively (Figs. 5B–5E). Rtt09, Hat1, Atac2, Sas2 and Esa1 were barely detectable in the scRNA-seq, suggesting that they are either expressed at very low levels or not at all. We next examined the distribution of CREBBP, EP300, HDAC1 and HDAC2, the enzymes with high expression levels in scRNA-seq, by immunohistochemistry (Fig. 6, Fig. S3). As expected, CREBBP, EP300, HDAC1 and HDAC2 were broadly distributed in both dental epithelium and mesenchyme.