Single Cell Spatial Transcriptomics of the Murine Embryonic Palate Links Pax9 to Patterning and Organization of Extracellular Matrix Components

Despite advances in understanding the morphological disruptions that lead to defects in palate formation, the precise perturbations within the signaling microenvironment of palatal clefts remain poorly understood. To explore in greater depth the genomic basis of palatal clefts, we designed and implemented the first single cell spatial RNA-sequencing study in a cleft palate model, utilizing the Pax9-/- murine model at multiple developmental timepoints, which exhibits a consistent cleft palate defect. A novel platform for true single-cell resolution spatially resolved transcriptomics (Visium HD, 10x Genomics, Inc.) was employed using custom bins of 2x2 µm spatial gene expression data, then validated using Xenium In Situ mRNA spatial profiling and RNAscope Multiplex. Functional enrichment analysis revealed a palate cell-specific perturbation in Wnt signaling effector function in tandem with disrupted expression of extracellular matrix genes in developing mesenchyme. As a key step toward laying the framework for identifying key molecular targets these data can be used for translational studies aimed at developing effective therapies for human palatal clefts. FFPE tissue sections from the embryonic murine secondary palate, ages E12.5 (Pax9-/- vs. WT) and E13.5 (Pax9-/- vs. WT), were placed on Visium HD slide and prepared according to 10X Genomics protocols. After H&E staining, imaging and decrosslinking steps, tissue sections were incubated with mouse specific probes targeting 19,405 genes (10X genomics, Visium Mouse Transcriptome Probe Set v2.0 mm10-2020-A).