Images were captured with a widefield fluorescence microscope (Nikon Eclipse E800) with prefigured triple-band filter settings for DAPI/FITC/TRITC, and merged with Spot Advanced Software (Spot Imaging) or an automated fluorescent microscope with a whole-slide scanning platform (TissueFAXS, TissueGnostics). wherein chondrocytes appear and proliferate. Chondrocytes in the growth plate continue to proliferate well into adulthood in mice1. In the center of the developing cartilage mold, chondrocytes stop proliferating and become hypertrophic chondrocytes. These cells signal to induce the migration of mesenchymal cells into the marrow space; these cells then differentiate into osteoblasts that then form bone on top of the cartilaginous matrix. Perichondrial precursors expressing osterix (Osx) invade into the cartilage template along with blood vessels and eventually become both osteoblasts and stromal cells in the marrow space2. However, mesenchymal cells constituting earlier cells of the osteoblast lineage than Osx+precursorsin vivohave not been fully characterized. The transcription factor Sox9 is expressed in mesenchymal condensations, and osteochondroprogenitors are derived from Sox9+cells, asSox9-cremarks all chondrocytes and osteoblasts3. Sox9 binds to genes encoding major cartilaginous matrix proteins such as aggrecan (Acan) and type II collagen-1(Col2a1)and regulates their expression. How these early osteochondroprogenitors and their descendants relate to mesenchymal precursors in adult bone is unknown. In adult endochondral bones, the source of osteoblasts and stromal cells has been proposed to be mesenchymal stem cells (MSCs) or bone marrow stromal/mesenchymal progenitor cells (BMSCs), which are traditionally defined as cells capable of forming coloniesin vitro(CFU-Fs: colony forming unit-fibroblasts) that can undergo multilineage differentiationin vitroand upon transplantation4. CFU-Fs are enriched among various adult marrow populations such asnestin (Nes)-GFP+cells5, platelet-derived growth factor receptor- (PDGFR)+Sca1+CD45Ter119(PS) cells6and leptin receptor (LepR)/LepR-cre+cells7in mice and CD146+pericytes in humans8. In this study, we sought to identify cells that robustly supply osteoblasts and stromal cells in the metaphyses of growing bones, and suggest the possibility that these cells might be the source of MSCs/BMSCs in adult bone marrow. == Results == == Col2-cre/Col2-creER are expressed in early cells of the osteoblast lineage in fetal mice == We chose to explore the hypothesis that cells defined by activities of theSox9,aggrecan (Acan), andtype II collagen (Col2)gene promoter/enhancers might encompass mesenchymal precursors of osteoblasts and stromal cells. Previous studies indicate that osteochondroprogenitors are marked bycrerecombinases driven by theCol2a1promoter912. First, we mapped cell fates using aCol2-cre;R26R-tdTomato reporter13,14in combination withCol1(2.3kb)-GFP15as a Indibulin readout of osteoblastic cells. In this system, cells expressingCol2-creand their descendants become red, and if they concurrently expressCol1-GFP, they become yellow. In addition, a thymidine analog, EdU, was administered shortly before analysis to evaluate cell proliferation. At embryonic day 12.5 (E12.5), red cells were observed in the growth cartilage (Figure 1a, asterisks) and perichondrium (Figure 1a, sharps), where many of these cells were proliferating (See alsoSupplementary Figure 1afor an earlier day). At E14.5, almost allCol1-GFP+cells appearing in the osteogenic perichondrium were yellow (Figure 1b, arrowheads); thus, these perichondrial preosteoblasts were derived fromCol2-cre+cells. Active cell proliferation was observed in the Indibulin perichondrium, but not in its adjacent hypertrophic chondrocytes (Figure 1b). At E15.5,Col2-cre-derived cells proliferated within the marrow space, as the nascent primary ossification center was occupied by red cells (Figure 1c, asterisks). == Figure 1. Fate mapping ofCol2-cre+andOsx-cre+cells during endochondral ossification. == ac. Fate mapping ofCol2-cre+cells was performed usingCol1-GFP;Col2-cre;R26RTomatomice, with EdU administration prior to analysis. Indibulin (a) Embryonic day 12.5 (E12.5); sharps: Tomato+perichondrial cells, asterisks: Tomato+chondrocytes. (b) E14.5; arrowheads: Col1+Tomato+perichondrial cells. (c) Indibulin E15.5; asterisks: Tomato+cells in primary ossification center. Green: EGFP, red: tdTomato, blue: Alexa647, gray: DAPI. Scale bars: 100m. df. Fate mapping ofOsx-cre+cells was performed usingOsx-creGFP;R26RTomatomice, with EdU administration prior to analysis (e,f). (d) E12.5; arrows: Osx+perichondrium. (e) E14.5; arrows: Osx+Col1perichondrium, arrowheads: Osx+prehypertrophic chondrocytes. (f) E15.5; asterisks: Osx+cells in primary ossification center. Green: EGFP, red: tdTomato, blue: Alexa647, gray: DAPI. Scale bars: 100m. g. Fate mapping ofCol2-cre+cells in Runx2-deficient Indibulin bone anlage at E14.5 was performed usingCol1-GFP;Col2-cre;R26RTomato; Runx2/mice. Middle and right panels: dotted area of left panel revealing perichondrium. Arrows: Tomato+perichondrial cells, arrowheads: Tomato+chondrocytes beneath perichondrium. Green: EGFP, red: tdTomato, gray: DAPI and DIC (differential interference contrast). Scale bars: 100m (left panel) and 10m (center and right panels). h,j. Comparative fate mapping in postnatal day 3 (P3) bones was performed usingCol1-GFP;Col2-cre;R26RTomato(h) andCol1-GFP;Osx-cre;R26RTomato(j) mice. Green: EGFP, red: tdTomato, blue: DAPI. Scale bars: 500m. i,k. Comparative fate mapping in P3 bones was performed usingCxcl12-GFP;Col2-cre;R26RTomato(i) andCxcl12-GFP;Osx-cre;R26RTomato(k) mice. Shown are Tshr diaphyseal endocortices and bone marrows, stained for CD31. Green: EGFP, red: tdTomato, blue: Alexa633, gray: DAPI. Scale bars: 50m. l,m. Flow cytometry analysis was performed using dissociated bone cells harvested at P3. CD45fraction was gated for GFP. (l) Representative dot plots.