c-kit Signaling Inhibits Bone Development by Regulating Skeletal Stem Cell Precursors in Fetal Bone Marrow. c-kit Signaling Inhibits Bone Development by Regulating Skeletal Stem Cell Precursors in Fetal Bone Marrow

c-kit signaling plays pivotal roles in regulating the self-renewal and/or differentiation of many adult stem cells, such as hematopoietic stem cells. However, it remains controversial whether c-kit is expressed by and contribute to skeletal stem cells (SSCs). To test this, we lineage-traced c-kit+ cells and investigated the physiological importance of c-kit+ cells and c-kit signaling in bone development. We found that c-kit was not expressed by postnatal SSCs, but by fetal SSC precursors at the growth cartilage. Lineage-tracing of fetal c-kit+ cells marked approximately 20% of Lepr+ bone marrow stromal cells, generating nearly half of all osteoblasts in adult bone marrow. Disruption of mTOR signaling in c-kit+ cells impaired bone formation. Conditional deletion of Kitl (c-kit ligand, also known as Scf) from fetal, but not adult bone marrow stromal cells increased bone formation. Together, our work identified c-kit+ SSC precursors as an important source of bones formed during development. The osteogenic differentiation of these cells is temporally inhibited by Kitl from the bone marrow microenvironment. Overall design: We performed single-cell RNA sequencing (scRNA-seq) of CD45-Ter119-CD31-PDGFRα+ bone marrow stromal cells from 2-month-old KitMerCreMer; R26tdTomato mice that had been treated with tamoxifen at E12.5/14.5

c-kit信号通路（c-kit signaling）在调控多种成体干细胞（如造血干细胞）的自我更新与/或分化过程中发挥关键作用。然而，c-kit是否由骨骼干细胞（skeletal stem cells, SSCs）表达并参与其功能调控，目前仍存在争议。为解答这一争议，我们对c-kit阳性（c-kit+）细胞进行了谱系示踪，并探究了c-kit+细胞及c-kit信号通路在骨骼发育中的生理意义。研究发现，出生后的骨骼干细胞并不表达c-kit，而生长软骨中的胎儿骨骼干细胞前体细胞可表达该蛋白。对胎儿c-kit+细胞进行谱系示踪后可见，其可标记约20%的瘦素受体阳性（Lepr+）骨髓基质细胞，并可分化为成年骨髓中近半数的成骨细胞。在c-kit+细胞中干扰mTOR信号通路，会损害骨形成功能。从胎儿而非成年骨髓基质细胞中条件性敲除Kitl（c-kit配体，亦称干细胞因子Scf），可增强骨形成能力。综上，本研究确认c-kit阳性的骨骼干细胞前体细胞是发育过程中骨形成的重要来源。骨髓微环境分泌的Kitl会在时间维度上抑制这些细胞的成骨分化。实验整体设计：我们对在胚胎第12.5/14.5天经他莫昔芬处理的2月龄KitMerCreMer; R26tdTomato小鼠的CD45-Ter119-CD31-PDGFRα阳性骨髓基质细胞进行了单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。