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Abstract

Objective: Colony-stimulating factor-1 receptor (CSF1R) signalling facilitates commitment, proliferation, differentiation, and survival of microglia, macrophages, and osteoclasts. However, little is known about the role of CSF1R signalling in development of craniofacial structures. We hypothesized that disrupting CSF1R signalling during embryogenesis would impact the morphology of the skull, muscles of mastication, and tongue. Methods: Pregnant CD1 dams were administered the CSF1R inhibitor PLX5622 starting at embryonic day 3.5 and continuing until birth. For assessment of soft tissues, the heads of 6 PLX5622 diet exposed postnatal day 1 (P1) pups (3 males and 3 females) and 10 control diet exposed P1 pups (5 males and 5 females) were collected, fixed with 4% paraformaldehyde, and all stained with 1% phosphotungstic acid (PTA) in a 90% methanol solution for 2 weeks. For assessment of skull bones, the heads of 8 PLX5622 diet exposed P1 pups (4 males and 4 females) and 6 control diet exposed P1 pups (3 males and 3 females) were collected. The head samples were micro-CT scanned. The skull bones and 5 muscles of mastication: internal and external pterygoids, temporalis, masseter, zygomatico-mandibularis, and tongue were segmented, landmarked, and subjected to dense correspondence (DeCA) and geometric morphometric (GM) analysis with 3D Slicer and R statistics software packages. Results: GM analyses of PLX5622 diet P1 pup head samples compared to control diet P1 pup head samples suggested the variation in shape of the skull bones, tongue, and muscles of mastication, except for the external pterygoids, correlated significantly with the diet the pregnant dam was exposed to (p<0.05); although to varying extents. However, this shape variation was not consistently observed in both the right and left muscles. Canonical variate plots revealed segregation of PLX5622 diet and control diet specimens. DeCA analysis produced heatmaps suggestive of surface-level shape variation of the skull, tongue, and muscles of mastication in the PLX5622 diet animals compared to control diet animals. Conclusions: Disrupting embryonic CSF1R signalling impacts the morphogenesis of both the skull bones and craniofacial muscles, including the tongue and muscles of mastication, to varying extents in P1 mouse offspring.