Characterizing the role CSF1R-expressing cells play in craniofacial development

表征 CSF1R 表达细胞在颅面发育中的作用

• 批准号: RGPIN-2022-03718
• 负责人: Rosin, Jessica
• 金额: $ 2.04万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=761888
• 关键词: Characterizing; role; CSF1R; expressing; cells

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.

集落刺激因子-1受体（CSF 1 R）由许多吞噬细胞表达，包括小胶质细胞、巨噬细胞和破骨细胞，并且是其增殖、分化和存活所必需的。在小鼠中，Csf 1 r基因敲除导致骨密度增加、无牙表型、生长迟缓、CNS破坏和围产期死亡。这些生殖系基因敲除表明表达CSF 1 R的细胞具有胚胎功能，但需要其他瞬时破坏这些细胞的方法来解决其妊娠作用。因此，我们建立了一个药理学消耗模型，其中CSF 1 R抑制剂PLX 5622从E3.5开始喂养妊娠小鼠，并持续到出生。我们表明，PLX 5622在整个妊娠期的暴露导致颅面和牙齿异常。这些强大的表型是新的，尚未进行检查。我的研究计划的长期目标是了解吞噬免疫细胞促进胚胎正常发育的机制。在短期内，拟议的工作将集中在胚胎发生期间颅面巨噬细胞和破骨细胞异质性的特征，并确定在妊娠期间耗尽这些细胞如何影响颅面发育。我们假设CSF 1 R表达细胞在颅面组织中以空间受限的模式存在，并且通过与周围细胞的相互作用产生观察到的表型破坏。使用我们的药理学耗竭小鼠模型，我们将确定CSF 1 R抑制剂PLX 5622如何影响胚胎发生过程中的巨噬细胞和破骨细胞，并定义Aim 1中妊娠期间耗竭这些CSF 1 R+细胞导致的颅面表型。首先，我们将使用IHC来研究PLX 5622暴露如何影响巨噬细胞和破骨细胞的增殖和颅面组织的存活。然后，我们将使用IHC研究耗尽这些细胞如何影响这些动物头部发育中的神经和肌肉，并使用micro-CT可视化颅面形状变化。在目标2中，我们将表征巨噬细胞和破骨细胞在胚胎发生过程中的异质性，并通过分离CSF 1 R表达细胞进行单细胞RNA测序来定义离散群体的时空表达。对所得数据集的生物信息学分析将使我们能够表征颅面组织中巨噬细胞和破骨细胞的异质性，这将使用FISH和IHC进行验证。在目标3中，我们将通过分析对照和PLX 5622暴露的颅面组织之间分泌因子的变化来研究颅面发育过程中CSF 1 R表达细胞的信号传导作用，并将这些相同的因子应用于培养物中的神经嵴衍生球体，以评估其对增殖和/或分化的影响。这些研究很重要，因为它们将为表达CSF 1 R的吞噬细胞如何促进正常颅面发育提供新的见解。