CSF1R+ Macrophages and Osteoclasts Are Essential for Limb Bone Development During Embryogenesis

Colony-stimulating factor-1 receptor (CSF1R) provides essential signals for macrophage and osteoclast proliferation, differentiation, and survival, but the roles of CSF1R+ macrophages and osteoclasts during limb morphogenesis are understudied. Here, we utilized a pharmacological model by feeding the CSF1R inhibitor PLX5622 to pregnant mice across gestation to examine how CSF1R disruption impacts embryonic limb development. CSF1R-expressing cells were significantly depleted in response to PLX5622 exposure, including a complete loss of embryonic osteoclasts and osteoclastic activity in the developing limb bones. Although the gross morphology of limb nerves, muscles, cartilage, and bone appeared intact between embryonic day 11.5 (E11.5) and E15.5, prenatal PLX5622 exposure resulted in a completely penetrant truncated phenotype for all postnatal day 1 (P1) limb bones analyzed, suggesting that CSF1R+ cells play important roles in mediating limb bone formation during late embryogenesis. Interestingly, strain-specific defects were observed in the heel, where most of the CD1 mice presented with absent talus and underdeveloped calcaneus bones, while the C57BL/6 mice presented with milder developmental disruptions in both bones. Taken together, our data demonstrate that PLX5622 effectively depletes CSF1R-expressing macrophages and osteoclasts in embryonic limbs and suggest an essential role for embryonic CSF1R+ cells in driving limb bone morphogenesis.