Inactivation of <i>Hes1</i> in Skeletal Undifferentiated Cells Increases Bone Volume

Leptin receptor positive (LepR+) cells are multipotent stromal cells and a source of osteogenic and adipogenic cells. Inactivation of Notch signaling in LepR+ cells increases bone mass in mature mice, but the target gene responsible was not identified. Because in LepR+ cells the expression of the Notch target gene Hes1 prevails over that of other genes, we explored the role of the Hes1 deletion in LepR+ cells. To this end, LepR-Cre;Hes1Δ/Δ mice were compared to Hes1loxP/loxP littermates. Male and female 5-month-old LepR-Cre;Hes1Δ/Δ mice exhibited an increase in femoral bone volume/total volume due to an increase in trabecular number; vertebral (L3) and cortical bone was not affected. Bone histomorphometry demonstrated decreased osteoclast number and eroded surface, decreased osteoblast number only in male mice, and no changes in bone formation. Neither osteogenesis nor adipogenesis was modified by the Hes1 deletion in bone marrow stromal cell cultures, although Tnfsf11 (encoding RANKL) was suppressed in osteogenic cultures of Hes1Δ/Δ cells. Single-cell RNA sequencing of femurs from 5-month-old LepR-Cre;Hes1Δ/Δ and control mice revealed the presence of 23 cell clusters including clusters composed of hematological cells (myeloid, B cells, and neutrophils), endothelial cells, and osteoblasts. There were no substantial differences in gene expression, cluster distribution, or trajectory finding between control and Hes1 inactivated cells. In conclusion, Hes1 inactivation in LepR+ cells results in an increase in bone mass secondary to a decrease in RANKL, osteoclast number, and bone resorption, but HES1 has little influence on osteogenesis or adipogenesis in bone.