Kcnj2 regulates electrical activity-induced gene networks in embryonic mouse palate shelves.

Cleft palate interferes with eating, drinking, breathing, and speech, causing significant human suffering. Fetal exposure to many medications that target ion channels increases the incidence of cleft palate. Cleft palate could be prevented by understanding how ion channels contribute to palatal development. Ion channels regulate the electrical properties of cells. We discovered that the mouse embryonic palate mesenchymal cells are electrically active, like neurons. In neurons, electrical activity regulates transcription cell-autonomously and regulates the secretion of chemical cues. We discovered that electrical activity regulates secretion of bone morphogenetic protein (BMP4) from mouse palate mesenchymal cells. The next important step is to determine how electrical activity affects transcription to control palate development. Loss of a potassium channel called Kcnj2 (Kcnj2KO/KO) alters electrical activity in palate mesenchyme cells and causes cleft palate in mice. We compared single cell RNA sequencing datasets from Kcnj2KO/KO and wildtype E13.5 mouse anterior palate shelves to define how electrical activity affects gene expression cell autonomously and in surrounding cells. Our data reveal that Kcnj2KO/KO alters a network of calcium-induced transcription factors and downstream effectors. These data also reveal that loss of Kcnj2 affects gene expression outside of the cells that express Kcnj2 consistent with disruption of BMP signaling. Overall design: Kcnj2KO/+ (FVB.129-Kcnj2tm1Swz/J Stock No. 005057/Kcnj2, Jackson Labs) dams were bred to Kcnj2KO/+ males. Kcnj2KO/+ mice are maintained as a colony on a C57BL/6J background in accordance with IACUC protocol #0139 at the University of Colorado Anschutz Medical Campus. Observation of the vaginal plug was considered embryonic day 0.5. On embryonic day 13.5, pregnant dams were sacrificed with isoflurane followed by cervical dislocation and embryos were harvested. Heads were kept in ice-cold PBS for dissection. Two sequenced WT embryos were female and one WT was male while the two Kcnj2KO/KO embryos were male. Anterior palatal shelves were dissected and dissociated. Embryos were genotyped using the established Jackson Labs protocol. Single cell suspensions were submitted to the University of Colorado Anschutz Medical Campus Genomics Shared Resource for droplet derivation and library preparation with the Chromium single cell 3' NextGen V3.1 chemistry (10X Genomics). Sequencing was performed on an Illumina NovaSeq6000 with V1.5 chemistry. 7976, 6994, and 7268 cells from 3 Wildtype embryos (22,238 cells in total across all embryos) and 7591 and 4067 cells from 2 Kcnj2KO/+ embryos (11,658 cells in total) were collected and sequenced at a depth of 100,000 reads per cell. Cells from different embryos were run separately and not pooled together during sequencing.