Project description:A comparative single cell transcriptional analysis of murine CXCL12-abundant reticular stromal cells isolated from ablated and control bone marrow
Project description:In order to assess the descendants of hypertrophic chondrocytes, we utilized Collagen10-Cre;Rosa26-tdTomato mouse bone marrow harvested at 2 months of age by centrifugation and light Collagenase II digestion. After sequencing and downstream analysis using Seurat, we observed clusters of cells with gene profiles matching classically defined skeletal stem and progenitor cells as well as CXCL12 abundant reticular (CAR) cells. These cells appear to be upstream of both osteoblasts and adipocytes. We conclude that hypertrophic chondrocytes dedifferentiate to this progentior stage before further differentiation.
Project description:The chemokines CXCL13 and CXCL12 are reported to be important for the germinal center reaction. Since CXCL12-deficient mice are embryonically lethal, here we took advantage of the Cxcl13-Cre/TdTomato mouse models to genetically ablate CXCL12 from B cell-interacting reticular cells and examine the molecular consequence on germinal center B cells. Spatial segregation of follicular dendritic cells, germinal center B cells and follicular helper T cells is impaired in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice. Single cell transcriptomic analysis revealed that all germinal center B cell subsets (corresponding to distinct stages of the germinal center response) are present in draining lymph nodes of immunized CXCL12-conditionally deficient mice. While most transcriptional regulators of the germinal center response are unperturbed by the genetic perturbation of CXCL12, Bach2 levels were elevated in germinal center B cells from lymph nodes of Cxcl12fl/fl mice. Moreover, single cell B cell receptor sequencing revealed that germinal center B cells in Cxcl13-Cre/TdTomato Cxcl12fl/fl mice harbour a lower mutational burden when compared to germinal center B cells isolated from immunized control mice. Gene expression profiles were validated by flow cytometry and suggest that the provision of CXCL12 by reticular cells governs efficient germinal center responses.