EHMT2 Controls Neural Crest-Derived Craniofacial Development but is Dispensable in Limb Development

Abstract

Post-translational modifications of histones, such as methylation of histone H3 at lysine 9 (H3K9), play critical roles in regulating chromatin structure and gene expression. EHMT2 (also called G9A), a histone methyltransferase, mediates H3K9 mono- and dimethylation and has been implicated in both transcriptional repression and context-specific gene activation. Although global knockout of the mouse Ehmt2 gene results in early embryonic lethality, tissue-specific knockouts have uncovered diverse roles in organ development. However, how EHMT2 contributes to skeletal development in a lineage-specific manner remains to be fully elucidated. Here, we investigated the role of EHMT2 in skeletal development by conditionally inactivating Ehmt2 in neural crest-derived and mesoderm-derived progenitors using Wnt1-Cre and Prx1-Cre mouse lines, respectively. Loss of Ehmt2 function in neural crest cells led to postnatal growth failure and craniofacial defects, including delayed intramembranous ossification and malformations of the jaw and cranial base. Transcriptomic analysis of neural crest cells revealed disrupted chromatin regulatory networks, reduced expression of proliferation-associated genes, and upregulation of inflammatory pathways. In contrast, inactivation of Ehmt2 in Prx1-expressing mesodermal progenitors had minimal impact on limb and cranial bone development, with no significant alterations in bone mass or osteoblast function. Together, these results reveal a lineage-specific requirement for EHMT2 in neural crest-derived skeletal tissues, suggesting that distinct progenitor populations exhibit differential dependency for bone development.

Figure 1.. Ehmt2 ablation in neural crest cells causes craniofacial hypoplasia and cranial base defects.

(A) Micro-computed tomography (μCT) analysis of E17.5 embryos revealed microcephaly and delayed ossification of the nasal, frontal, and parietal bones in Wnt1-Cre; Ehmt2^f/f embryos compared to controls (Ehmt2^ff). (B) Alcian Blue and Alizarin Red S staining of whole-mount skeletal preparations highlighted defects in cranial morphology. Wnt1-Cre; Ehmt2^f/f embryos exhibited impaired chondrogenic differentiation in the nasal region and severe mandibular malformations, including a shortened and thickened lower jaw. The paranasal cartilage (PNC), premaxillary (PX), and maxillary bones were also significantly affected. Furthermore, ossification of the basisphenoid (BS) and basioccipital (BO) bones was markedly reduced, and the spheno-occipital synchondrosis (SOS) appeared abnormally widened, indicating disrupted cranial base development.

Figure 2.. Ehmt2 ablation alters transcriptional programs in neural crest cells.

(A) Unsupervised hierarchical clustering of differentially expressed genes (DEGs) in Wnt1-Cre; Ehmt2^f/f (KO) vs. control (Ehmt2^f/f, WT) NCCs (E10.5; n=2 per group). Euclidean distance demonstrates distinct transcriptional profiles. (B) Principal component analysis (PCA) confirms separation of KO and WT NCCs. (C) Volcano plot of DEGs (|log2FC| > 1 and p-value < 0.05). (D-E) Gene set enrichment analysis (GSEA) of Hallmark and REACTOME pathways. Top enriched terms in KO NCCs include inflammatory and neuromodulatory pathways, while WT NCCs exhibit proliferative and cell cycle signatures. NES: normalized enrichment score; FDR: false discovery rate. (F) Heatmap of select deregulated transcription factors (TFs) and NCC functional genes. Meis1 and EMT/migration genes (Zeb1, Cdh2, Cdh11) are downregulated, while the innate immune gene Aim2 is upregulated in KO NCCs. (G) MA plot showing differentially expressed TE loci in Wnt1-Cre; Ehmt2^f/f (KO) versus Ehmt2^f/f (WT) neural crest cells. TE expression was quantified using SQuIRE. Only intergenic and intronic TEs were included in the analysis. Significance thresholds were set at p < 0.05 and |log₂ fold change| > 1.

Figure 3.. μCT analysis of cranial and femoral bones in Prx1-Cre; Ehmt2^f/f and Ehmt2^f/f control mice.

(A) μCT imaging of the cranial skeleton shows no differences in bone morphology or mineralization between Ehmt2^f/f (Con) and Prx1-Cre; Ehmt2^f/f (Ehmt2 cKO) mice at 1 month of age. (B) Representative μCT images of femoral trabecular bone (upper panels) and cortical bone (lower panels) from 2-month-old male Con and Ehmt2 cKO mice. (C) Quantification of trabecular bone parameters of both sexes, including bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.Sp), and trabecular thickness (Tb.Th). (D) Cortical bone parameters, cortical thickness (Cs.Th), bone area/total area ratio (B.Ar/T.Ar), total cross-sectional area (T.Ar), and bone area (B.Ar) were compared between genotypes (ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4.. BMSC ex vivo osteogenic differentiation.

(A) RT-qPCR analysis of relative Ehmt2 and Runx2 expression in 1-month-old Ehmt2^f/f (Con) and Prx1-Cre; Ehmt2^f/f (Ehmt2 cKO) hindlimb bone osteoblasts. Statistical significance was assessed using Student’s t-test on Ct values (n = 3; ns = not significant; *p < 0.05; **p < 0.01; ***p < 0.001). (B) Ex vivo osteogenic differentiation of BMSCs from 1-month-old male Ehmt2^f/f (Con) and Prx1-Cre; Ehmt2^f/f (Ehmt2 cKO), cultured in 24-well plates. (C) Calcein/alizarin double labeling of the cortical midshaft of femurs from 2-month-old Ehmt2^f/f (Con) and Prx1-Cre; Ehmt2^f/f (Ehmt2 cKO) mice was used to assess dynamic bone formation. No significant differences were observed in mineral apposition rate (MAR) or other formation parameters. (D) Calcein/alizarin labeling of trabecular bone in the distal femur also showed no significant differences in Mineral Apposition Rate (MAR) or bone formation rate between genotypes. (E) Publicly available ENCODE RNA-seq data from embryonic day 15 (E15) mouse tissues showed high expression of Ehmt2 and Ehmt1in neural crest-derived tissues, but relatively low expression in limb mesenchymal cells. This differential expression may help explain the distinct skeletal phenotypes observed with tissue-specific deletion of Ehmt2.