Unveiling critical signaling pathways in the murine salivary gland and the role of midkine

Salivary gland (SG) development is a complex process involving coordinated signaling between epithelial and stromal cell populations. While some growth drivers and branching mechanisms are known, many intercellular communication pathways that underpin embryonic SG morphogenesis remain unidentified. We leveraged single cell RNA-sequencing datasets, from murine submandibular salivary glands at three embryonic stages to identify critical signaling networks. Using CellChat, we mapped global ligand-receptor interactions among different cell populations revealing an evolving signaling landscape during gland maturation. Our analysis highlighted both well-established and understudied pathways, including midkine (MDK) signaling. Functional experiments using embryonic SG explants demonstrated that MDK regulates branching morphogenesis via Rho-associated coiled coil containing protein kinase 1 (Rock1) signaling. Additionally, the key lineage driving transcription factor p63 was shown to act as primary mediator of the MDK/Rock1 axis. These findings uncover a comprehensive signaling code that guides SG development and offers new targets for follow-up studies into SG regeneration.