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Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.

Yu VW, Saez B, Cook C, Lotinun S, Pardo-Saganta A, Wang YH, Lymperi S, Ferraro F, Raaijmakers MH, Wu JY, Zhou L, Rajagopal J, Kronenberg HM, Baron R, Scadden DT - J. Exp. Med. (2015)

Bottom Line: Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells.Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function.These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02215 Harvard Stem Cell Institute, Cambridge, MA 02215 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02215.

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Reduced DLL4 in the bone marrow of Ocn+ cell depleted mice led to decreased Notch signaling in CLP. (A) Intracellular Notch signaling was assessed by flow cytometry in the indicated cell populations. Representative flow plots are shown, and data are summarized by dot plot (right). Three independent experiments; n = 16–17 mice/group. Data show mean ± SEM. (B) Expression of the indicated Notch target genes in Ly6D− CLP from OcnCre+/−;iDTR mutants and controls. Mutant and WT mice were treated with DT for 28 d before being subjected to flow cytometry for Ly6D− CLP isolation and quantitative PCR. Two independent experiments; n = 12 mice/group. Data show mean ± SEM. (C) Expression of the Notch ligands in the bones of OcnCre+/−;iDTR DT-treated and control animals was assessed by qPCR. Mice were examined after 28 d after DT treatment. Three independent experiments; n = 12–14 mice/group. Data show mean ± SEM. (D) DLL4 expression in bone sections from control or Ocn+ cell deleted animals was assessed by immunohistochemistry. Two independent experiments; n = 6 mice/group. (E) Osx-mCherry cells and Ocn-Topaz cells were isolated from the OsxCre-mCherry;OcnCre-Topaz double transgenic mice by flow cytometry and expression of the indicated Notch ligands was assessed by qPCR. Experiment performed twice; n = 3/experiment. Data show mean ± SEM. (F) Gene set enrichment analysis (GSEA) comparing Ly6D− CLP versus MPP microarray data. Experiment performed twice; n = 3/experiment. (G) GSEA comparing Ly6D− CLP microarray data obtained from OcnCre;iDTR control versus mutant mice. Experiment was performed twice independently; n = 3–4/experiment.
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fig5: Reduced DLL4 in the bone marrow of Ocn+ cell depleted mice led to decreased Notch signaling in CLP. (A) Intracellular Notch signaling was assessed by flow cytometry in the indicated cell populations. Representative flow plots are shown, and data are summarized by dot plot (right). Three independent experiments; n = 16–17 mice/group. Data show mean ± SEM. (B) Expression of the indicated Notch target genes in Ly6D− CLP from OcnCre+/−;iDTR mutants and controls. Mutant and WT mice were treated with DT for 28 d before being subjected to flow cytometry for Ly6D− CLP isolation and quantitative PCR. Two independent experiments; n = 12 mice/group. Data show mean ± SEM. (C) Expression of the Notch ligands in the bones of OcnCre+/−;iDTR DT-treated and control animals was assessed by qPCR. Mice were examined after 28 d after DT treatment. Three independent experiments; n = 12–14 mice/group. Data show mean ± SEM. (D) DLL4 expression in bone sections from control or Ocn+ cell deleted animals was assessed by immunohistochemistry. Two independent experiments; n = 6 mice/group. (E) Osx-mCherry cells and Ocn-Topaz cells were isolated from the OsxCre-mCherry;OcnCre-Topaz double transgenic mice by flow cytometry and expression of the indicated Notch ligands was assessed by qPCR. Experiment performed twice; n = 3/experiment. Data show mean ± SEM. (F) Gene set enrichment analysis (GSEA) comparing Ly6D− CLP versus MPP microarray data. Experiment performed twice; n = 3/experiment. (G) GSEA comparing Ly6D− CLP microarray data obtained from OcnCre;iDTR control versus mutant mice. Experiment was performed twice independently; n = 3–4/experiment.

Mentions: Since Notch is an essential determinant of T-lymphoid commitment and is required for progression through the DN stage (Sambandam et al., 2005; Tan et al., 2005; Visan et al., 2006; Rothenberg et al., 2010; Love and Bhandoola, 2011), we hypothesized that Notch may be participating in the phenotype observed. Notch participation in T-lymphopoiesis before thymic location was previously discounted (Krueger and von Boehmer, 2007), but the cells that were examined have since been shown to be precursors of extrathymic maturation and do not represent thymic progenitors (Luche et al., 2013). In contrast, some studies have reported Notch signaling in bone marrow T cell progenitors (Harman et al., 2005; Maeda et al., 2007). We assessed bone marrow CLP by flow cytometry for evidence of activated, or cleaved Notch and lower levels were observed in OcnCre+/−;iDTR mutants (Fig. 5 A). This was validated by a decrease in several Notch target genes by qPCR (Fig. 5 B). The five mammalian Notch ligands, Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4 were evaluated by qPCR and DLL4 was selectively decreased in bones from OcnCre+/−;iDTR mutant mice (Fig. 5 C). Using a OsxCre-mCherry;OcnCre-Topaz double transgenic mouse model, we selectively labeled Osx+ cells red, Ocn+ cells green, and Osx+Ocn+ cells yellow. This system allowed clear distinction and isolation of different osteolineage subsets within the same animal by flow cytometry. Immunohistochemistry (Fig. 5 D) and gene expression data (Fig. 5 E) showed that Ocn+ mature osteoblasts, but not other osteolineage cells, potently expressed DLL4, and both mRNA and protein were markedly reduced in OcnCre+/−;iDTR mice with protein notably decreased at the endosteal interface. This change in DLL4 was associated with corresponding alterations in Notch target genes in CLPs in the bone marrow by gene set enrichment analysis (GSEA; Fig. 5, F and G). These data suggest that reduced DLL4 expression from Ocn+ cells led to reduced intracellular Notch activation in the hematopoietic precursors within the bone marrow, thereby limiting T-lineage specification.


Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.

Yu VW, Saez B, Cook C, Lotinun S, Pardo-Saganta A, Wang YH, Lymperi S, Ferraro F, Raaijmakers MH, Wu JY, Zhou L, Rajagopal J, Kronenberg HM, Baron R, Scadden DT - J. Exp. Med. (2015)

Reduced DLL4 in the bone marrow of Ocn+ cell depleted mice led to decreased Notch signaling in CLP. (A) Intracellular Notch signaling was assessed by flow cytometry in the indicated cell populations. Representative flow plots are shown, and data are summarized by dot plot (right). Three independent experiments; n = 16–17 mice/group. Data show mean ± SEM. (B) Expression of the indicated Notch target genes in Ly6D− CLP from OcnCre+/−;iDTR mutants and controls. Mutant and WT mice were treated with DT for 28 d before being subjected to flow cytometry for Ly6D− CLP isolation and quantitative PCR. Two independent experiments; n = 12 mice/group. Data show mean ± SEM. (C) Expression of the Notch ligands in the bones of OcnCre+/−;iDTR DT-treated and control animals was assessed by qPCR. Mice were examined after 28 d after DT treatment. Three independent experiments; n = 12–14 mice/group. Data show mean ± SEM. (D) DLL4 expression in bone sections from control or Ocn+ cell deleted animals was assessed by immunohistochemistry. Two independent experiments; n = 6 mice/group. (E) Osx-mCherry cells and Ocn-Topaz cells were isolated from the OsxCre-mCherry;OcnCre-Topaz double transgenic mice by flow cytometry and expression of the indicated Notch ligands was assessed by qPCR. Experiment performed twice; n = 3/experiment. Data show mean ± SEM. (F) Gene set enrichment analysis (GSEA) comparing Ly6D− CLP versus MPP microarray data. Experiment performed twice; n = 3/experiment. (G) GSEA comparing Ly6D− CLP microarray data obtained from OcnCre;iDTR control versus mutant mice. Experiment was performed twice independently; n = 3–4/experiment.
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fig5: Reduced DLL4 in the bone marrow of Ocn+ cell depleted mice led to decreased Notch signaling in CLP. (A) Intracellular Notch signaling was assessed by flow cytometry in the indicated cell populations. Representative flow plots are shown, and data are summarized by dot plot (right). Three independent experiments; n = 16–17 mice/group. Data show mean ± SEM. (B) Expression of the indicated Notch target genes in Ly6D− CLP from OcnCre+/−;iDTR mutants and controls. Mutant and WT mice were treated with DT for 28 d before being subjected to flow cytometry for Ly6D− CLP isolation and quantitative PCR. Two independent experiments; n = 12 mice/group. Data show mean ± SEM. (C) Expression of the Notch ligands in the bones of OcnCre+/−;iDTR DT-treated and control animals was assessed by qPCR. Mice were examined after 28 d after DT treatment. Three independent experiments; n = 12–14 mice/group. Data show mean ± SEM. (D) DLL4 expression in bone sections from control or Ocn+ cell deleted animals was assessed by immunohistochemistry. Two independent experiments; n = 6 mice/group. (E) Osx-mCherry cells and Ocn-Topaz cells were isolated from the OsxCre-mCherry;OcnCre-Topaz double transgenic mice by flow cytometry and expression of the indicated Notch ligands was assessed by qPCR. Experiment performed twice; n = 3/experiment. Data show mean ± SEM. (F) Gene set enrichment analysis (GSEA) comparing Ly6D− CLP versus MPP microarray data. Experiment performed twice; n = 3/experiment. (G) GSEA comparing Ly6D− CLP microarray data obtained from OcnCre;iDTR control versus mutant mice. Experiment was performed twice independently; n = 3–4/experiment.
Mentions: Since Notch is an essential determinant of T-lymphoid commitment and is required for progression through the DN stage (Sambandam et al., 2005; Tan et al., 2005; Visan et al., 2006; Rothenberg et al., 2010; Love and Bhandoola, 2011), we hypothesized that Notch may be participating in the phenotype observed. Notch participation in T-lymphopoiesis before thymic location was previously discounted (Krueger and von Boehmer, 2007), but the cells that were examined have since been shown to be precursors of extrathymic maturation and do not represent thymic progenitors (Luche et al., 2013). In contrast, some studies have reported Notch signaling in bone marrow T cell progenitors (Harman et al., 2005; Maeda et al., 2007). We assessed bone marrow CLP by flow cytometry for evidence of activated, or cleaved Notch and lower levels were observed in OcnCre+/−;iDTR mutants (Fig. 5 A). This was validated by a decrease in several Notch target genes by qPCR (Fig. 5 B). The five mammalian Notch ligands, Jagged1, Jagged2, Delta-like 1 (DLL1), DLL3, and DLL4 were evaluated by qPCR and DLL4 was selectively decreased in bones from OcnCre+/−;iDTR mutant mice (Fig. 5 C). Using a OsxCre-mCherry;OcnCre-Topaz double transgenic mouse model, we selectively labeled Osx+ cells red, Ocn+ cells green, and Osx+Ocn+ cells yellow. This system allowed clear distinction and isolation of different osteolineage subsets within the same animal by flow cytometry. Immunohistochemistry (Fig. 5 D) and gene expression data (Fig. 5 E) showed that Ocn+ mature osteoblasts, but not other osteolineage cells, potently expressed DLL4, and both mRNA and protein were markedly reduced in OcnCre+/−;iDTR mice with protein notably decreased at the endosteal interface. This change in DLL4 was associated with corresponding alterations in Notch target genes in CLPs in the bone marrow by gene set enrichment analysis (GSEA; Fig. 5, F and G). These data suggest that reduced DLL4 expression from Ocn+ cells led to reduced intracellular Notch activation in the hematopoietic precursors within the bone marrow, thereby limiting T-lineage specification.

Bottom Line: Here, we report that specific deletion of bone-producing osteocalcin (Ocn)-expressing cells in vivo markedly reduces T-competent progenitors and thymus-homing receptor expression among bone marrow hematopoietic cells.Decreased intrathymic T cell precursors and decreased generation of mature T cells occurred despite normal thymic function.These data indicate that specific mesenchymal cells in bone marrow provide key molecular drivers enforcing thymus-seeding progenitor generation and thereby directly link skeletal biology to the production of T cell-based adaptive immunity.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02215 Harvard Stem Cell Institute, Cambridge, MA 02215 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02215.

Show MeSH
Related in: MedlinePlus