Multiplex Fluorescent mRNA In Situ Hybridization of Developing Craniofacial Tissues in FFPE Mouse Samples.

Craniofacial development depends on the coordinated activity of diverse, specialized cell populations that interact within intricate tissue architectures to form and maintain normal tissue structure and function. Understanding how these cells communicate with each other and with their local microenvironment is essential for defining the molecular mechanisms that regulate their formation and homeostasis. Disruption of these processes can lead to congenital craniofacial conditions, including tooth agenesis, cleft palate, and cleft lip, either alone or as part of syndromic disorders. While quantitative techniques like RT-PCR offer high sensitivity and dynamic range, they require RNA extraction from homogenized tissue, resulting in a loss of spatial and cellular context, an especially critical limitation for heterogeneous craniofacial tissues, where the cellular origin of transcripts cannot be resolved. Multiplex fluorescent RNA in situ hybridization overcomes this limitation by enabling sensitive and specific detection of multiple mRNA transcripts while maintaining tissue architecture. This advanced approach allows precise localization of gene expression at the cellular level, providing valuable insight into the spatial regulation of developmental processes and disease mechanisms in craniofacial biology.