A Mir338-CDK8-mitochondrial axis governs lineage-specific osteogenesis and defines Runx2 pleiotropy in cranial sutures.

INTRODUCTION

Developmental pattern formation critically depends on gene regulatory networks that enable context-dependent interpretation of molecular cues, yet how cells from distinct embryonic lineages in shared microenvironments produce divergent outcomes remains a central unresolved question. The murine skull provides a key model: the neural crest-derived posterior frontal (PF) suture fuses, while the mesoderm-derived sagittal (SAG) suture remains patent. A recently identified paradox exists: Runx2 haploinsufficiency impacts both sutures, but Mir338 cluster ablation selectively rescues the SAG suture, suggesting lineage-specific regulatory constraints.

OBJECTIVES

This study aims to resolve this paradox by investigating how embryonic origin dictates osteoprogenitor responses to Runx2-Mir338 signaling.

METHODS

We employed multi-omics (miRNA-seq, RNA-seq, scRNA-seq) and lineage tracing to define transcriptional heterogeneity, dissect Runx2 and Mir338-mediated lineage-specific regulation of osteoblastic subclusters, and identify differentially expressed mitochondrial-related genes and Cyclin-dependent kinase 8 (Cdk8). These findings were functionally validated through in vivo and in vitro assays.

RESULTS

We uncover that embryonic origin dictates osteoprogenitor responses to Runx2-Mir338 signaling through lineage-specific mitochondrial dynamics. In mesoderm-derived SAG sutures, Runx2 haploinsufficiency disrupts mitochondrial fragmentation, a process critical for ossification, via dysregulation of NADH: ubiquinone oxidoreductase subunit S5 (NDUFS5). We identify CDK8 as a novel direct target of Mir338 and a Runx2-activated gene that promotes mitochondrial fragmentation. This dual regulation of CDK8 establishes a dosage-sensitive "toggle switch" unique to SAG sutures, where restoring mitochondrial function via direct mitochondrial transplantation rescues ossification defects. In contrast, neural crest-derived PF sutures exhibit intrinsically low CDK8 expression, partially modulated by DNA methylation, rendering them unresponsive to Runx2-Mir338 modulation and mitochondrial rescue.

CONCLUSION

Our findings resolve the paradox of Runx2 pleiotropy by bridging positional identity, mitochondrial metabolism, and miRNA-mediated regulation, offering a redefined conceptual framework for understanding developmental processes and informing precision therapeutic strategies for congenital skeletal disorders.