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. 2014 May 12;29(3):340-9.
doi: 10.1016/j.devcel.2014.03.013.

Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development

Toshihide Mizoguchi  1 , Sandra Pinho  2 , Jalal Ahmed  3 , Yuya Kunisaki  2 , Maher Hanoun  2 , Avital Mendelson  2 , Noriaki Ono  4 , Henry M Kronenberg  4 , Paul S Frenette  5
Affiliations

Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development

Toshihide Mizoguchi et al. Dev Cell. .

Abstract

Mesenchymal stem and progenitor cells (MSPCs) contribute to bone marrow (BM) homeostasis by generating multiple types of stromal cells. MSPCs can be labeled in the adult BM by Nestin-GFP, whereas committed osteoblast progenitors are marked by Osterix expression. However, the developmental origin and hierarchical relationship of stromal cells remain largely unknown. Here, by using a lineage-tracing system, we describe three distinct waves of contributions of Osterix(+) cells in the BM. First, Osterix(+) progenitors in the fetal BM contribute to nascent bone tissues and transient stromal cells that are replaced in the adult marrow. Second, Osterix-expressing cells perinatally contribute to osteolineages and long-lived BM stroma, which have characteristics of Nestin-GFP(+) MSPCs. Third, Osterix labeling in the adult marrow is osteolineage-restricted, devoid of stromal contribution. These results uncover a broad expression profile of Osterix and raise the intriguing possibility that distinct waves of stromal cells, primitive and definitive, may organize the developing BM.

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Figures

Figure 1
Figure 1. Lifelong contribution of Osx+ cells to the BM cells in developing bones
(A–I) Z-stack confocal images of thick bone sections of iOsx/Tomato mice administered with tamoxifen (Tam) at embryonic day 13.5 (E13.5) (A–C), postnatal day 5 (P5) (D–F), and 8 week-old (G–I) mice analyzed at the indicated periods. Bone sections were stained with VE-cadherin (VE-Cad), PECAM-1 antibodies (green) and Hoechst 33342 (blue). Right panels are magnified views of the boxed areas. Scale bars: 100 µm. Arrows: iOsx-derived Tomato+ (iOsx/Tomato+) stromal cells. Arrowheads: iOsx/Tomato+ osteolineage cells. See also Figures S1 and S2A-G.
Figure 2
Figure 2. Osx+ cells in the neonatal bone give rise to Nes-GFP+ MSPCs
Analysis of Nes-Gfp/iOsx/Tomato mice administered with tamoxifen (Tam) at P5. (A and B) Z-stack confocal images of thick bone sections at 1 day (A) and 15 weeks (B) after Tam injection. Bone sections were stained with VE-cadherin (VE-Cad) and PECAM-1 antibodies (white). Right panels show the magnified confocal images within the area defined by the rectangle. Arrows: Nes-GFP and iOsx-derived Tomato (iOsx/Tomato) double-positive cells. (C and D) Representative FACS plots showing the percentages of the Nes-GFP+ cells within the iOsx/Tomato+ (CD45−Ter119−CD31−) population in the bone (C) (n=3) and in the BM (D) (n=3) after 1 day and 15 weeks of chase, respectively. Blue and red lines represent the WT control and Nes-GFP, respectively. (E and F) CFU-F activity of sorted iOsx/Tomato+ and iOsx/Tomato− populations in the BM stroma harvested 4 weeks after Tam injection. Quantification (E) and representative image of CFU-F colony (F; Giemsa staining on the left panel and Tomato fluorescence on the right panel). n=3 independent experiments. *P<l0.05. (G–I) Differentiated phenotypes of clonal iOsx/Tomato+ BM stromal cells shown by Alizarin Red S: osteoblasts (G), lipid droplets and staining with FABP4 antibody: adipocytes (H), and Alcian Blue: chondrocytes (I). Nuclei were detected by DAPI (blue). (J) Confocal image of Nes-GFP and iOsx/Tomato double-positive BM stromal cells stained with Lepr antibody in 15 week-old mice (white). Arrows: Nes-GFP, iOsx/Tomato, and Lepr triple-positive cells. (K) Representative FACS plots showing the percentages of Nes-GFP and Lepr double-positive cells and the expression of PDGFRαand PDGFRβ in the iOsx/Tomato+ (CD45−Ter119−CD31−) BM stromal population in 15 week-old mice. Blue and red lines represent isotype controls and antibodies against the markers indicated, respectively. n=3. In (C, D, E, and K), data are represented as mean ± SEM. Scale bar: 5000 µm (G), 500 µm (F: left panel and I), 200 µm (A and B: left panels), 100 µm (F: right panel), 30 µm (A and B: right panels, H, and J). See also Figure S2H-J.
Figure 3
Figure 3. Lepr-cre -derived BM stromal cells give rise to bone-lineages in the adult stage
(A) Z-stack confocal images of thick bone sections of Nes-Gfp/Lepr-cre/Tomato mice in the indicated stage, stained with Osx antibody (white). *: Primary ossification center. Arrows: Lepr-cre-derived Tomato+ (Lepr/Tomato+) cells in the epiphysis. Arrowheads: Lepr/Tomato+ cells in the periosteum. (B) Z-stack confocal images of thick bone sections of Lepr-cre/Tomato mice in the indicated stages. Arrows: Lepr/Tomato+ osteoblasts. Arrowheads: Lepr/Tomato+ osteocytes. (C and D) Confocal images of bone tissues from 15 week-old Lepr-cre/Tomato mice stained with osteocalcin (green) (C) and DMP1 antibodies (green) (D). (E) Z-stack confocal images of thick bone sections in 6 week-old Col1(2.3)-Gfp mice stained with Lepr antibody (red). Right panels show a magnified view the area of around the cortical bone. Arrows: Col1(2.3)-GFP-positive mature osteoblasts. (F) Z-stack confocal images of thick bone sections in 15 week-old Leprcre/Tomato mice stained with Lepr antibody (white). Arrows: Lepr/Tomato+ osteocytes. Arrowheads: Lepr/Tomato+ osteoblasts. Nuclei were detected by Hoechst 33342 (blue). Scale bar: 500 µm (E: left panel), 200 µm (B), 100 µm (A), 30 µm (E: right panels and F), 10 µm (C and D). See also Figure S3.
Figure 4
Figure 4. P5-iOsx-derived BM stromal cells contribute to tissue remodeling after tissue injury
(A–F) Z-stack confocal (A and D) and confocal (B, C, E, and F) images of thick bone sections at day 6 post-irradiation stained with BODIPY (493/503) (green) (A, B, D, and E) or Perilipin antibody (green) (C and F) from 8 weeks Lepr-cre/Tomato mice (A–C) or P5 labeled iOsx/Tomato mice after 15 weeks chase (D–F). Arrows: Tomato+ adipocytes. (G–L) Images of bone sections at day 8 post-bone fracture from P5 labeled iOsx/Tomato mice after 32 weeks chase (G–I) or 15 week Lepr-cre/Tomato mice (J–L). Serial sections stained with Toluidine blue (G and J). Z-stack confocal (H and K) and confocal (I and L) images of thick bone sections stained with Sox9 antibody (white). The numbered squares indicate the area of respective zoomed right panels (H and K). Arrows: Tomato and Sox9 double-positive chondrocytes. #: Mark of the needle used for bone stabilization during bone fracture. *: Fracture callus. Magnified confocal images in H1 and K1’ panels (I and L). Nuclei were detected by Hoechst 33342 (blue). Scale bar: 500 µm (G, H, J, and K), 200 µm (A and D), 50 µm (I and L), 10 µm (B, C, E, and F). See also Figure S4.
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