Gli1 labels progenitors during chondrogenesis in postnatal mice

Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identified Gli1+ chondrogenic progenitors (Gli1+ CPs), which were distinct from SSCs, were responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs led to cartilage defects and dwarfishness phenotype in mice. Furthermore, we found that the BMP signal played an important role in self-renewal and maintenance of Gli1+ CPs. The deletion of Bmpr1α caused the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis. Primary chondrocytes were stained with anti-CD45, anti-CD31 and anti-Ter119 primary antibody as described previously. Cell sorting was performed on a FACS Aria III cell sorter (BD Biosciences) to collect CD45-CD31-Ter119-tdTomato+ and CD45-CD31-Ter119-tdTomato- populations. The total RNA sample from each population was extracted separately using TRIzol and the RNeasy mini kit (Qiagen, Germany).

由软骨内成骨（endochondral ossification）驱动的骨骼生长，严格受软骨祖细胞的自我更新与分化调控。越来越多的研究证据表明，多种骨骼干细胞（skeletal stem cells, SSCs）参与软骨形成过程。然而迄今为止，尚无研究证实不同类型软骨中存在通用的长效软骨祖细胞。本研究鉴定出Gli1+软骨祖细胞（Gli1+ CPs），其与骨骼干细胞存在显著差异，可终身为生长板、椎骨、肋骨及其他软骨组织提供软骨细胞来源。小鼠体内Gli1+软骨祖细胞的缺失会引发软骨缺陷与侏儒表型。进一步研究发现，骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路在Gli1+软骨祖细胞的自我更新与维持过程中发挥关键调控作用。敲除Bmpr1α会导致Gli1+软骨祖细胞耗竭，进而破坏柱状软骨结构。综上，本研究数据证实，Gli1+软骨祖细胞是多种软骨组织中共用的长效软骨祖细胞，对于维持软骨稳态至关重要。按照此前报道的方法，使用抗CD45、抗CD31与抗Ter119一抗对原代软骨细胞进行染色。使用FACS Aria III流式细胞分选仪（BD Biosciences）进行细胞分选，收集CD45⁻CD31⁻Ter119⁻tdTomato⁺与CD45⁻CD31⁻Ter119⁻tdTomato⁻细胞群。使用TRIzol试剂与RNeasy微量试剂盒（Qiagen，德国）分别提取各细胞群的总RNA样本。