Specific bone cells produce DLL4 to generate thymus-seeding progenitors from bone marrow.
Bottom Line: Production of the cells that ultimately populate the thymus to generate α/β T cells has been controversial, and their molecular drivers remain undefined.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.
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
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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.
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.