Characterizing the role CSF1R-expressing cells play in craniofacial development

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Colony-stimulating factor-1 receptor (CSF1R) is expressed by numerous phagocytic cells, including microglia, macrophages, and osteoclasts, and is required for their proliferation, differentiation, and survival. In mice, Csf1r knockout causes increased bone density, a toothless phenotype, growth retardation, CNS disruptions, and perinatal mortality. These germline knockouts suggest CSF1R-expressing cells have embryonic functions, but other means of transiently disrupting these cells is required to address their gestational roles. Accordingly, we established a pharmacological depletion model whereby the CSF1R inhibitor PLX5622 is fed to pregnant mice starting at E3.5 and continued until birth. We show that PLX5622-exposure across gestation results in craniofacial and dental abnormalities. These robust phenotypes are novel and have not yet been examined. The long-term objective of my research program is to understand the mechanisms through which phagocytic immune cells contribute to the normal development of the embryo. In the short-term, the proposed work will focus on characterizing craniofacial macrophage and osteoclast heterogeneity during embryogenesis and determining how depleting these cells amid gestation impacts craniofacial development. We hypothesize that CSF1R-expressing cells reside in a spatially restricted pattern across craniofacial tissues, and it is through interactions with surrounding cells that the phenotypic disruptions observed are produced. Using our pharmacological depletion mouse model, we will determine how the CSF1R inhibitor PLX5622 impacts macrophages and osteoclasts across embryogenesis and define the craniofacial phenotypes that result from depleting these CSF1R+ cells during gestation in Aim 1. To start, we will use IHC to study how PLX5622-exposure impacts macrophage and osteoclast proliferation and survival across craniofacial tissues. We will then examine how depleting these cells impacts the developing nerves and muscles in the head of these animals using IHC, and use micro-CT to visualize craniofacial shape changes. In Aim 2, we will characterize macrophage and osteoclast heterogeneity during embryogenesis and define the spatiotemporal expression of discrete populations by isolating CSF1R-expressing cells for single-cell RNA sequencing. Bioinformatic analysis of the resulting datasets will allow us to characterize macrophage and osteoclast heterogeneity across craniofacial tissues, which will be validated using FISH and IHC. In Aim 3, we will study the signalling effects of CSF1R-expressing cells during craniofacial development by analyzing changes in secreted factors between control and PLX5622-exposed craniofacial tissues, and apply these same factors to neural crest-derived spheres in cultures to assess their impact on proliferation and/or differentiation. These studies are important because they will provide novel insights into how CSF1R-expressing phagocytes contribute to normal craniofacial development.