TGF-β Signaling in Mesenchymal Progenitor Cells during Development Plays an Essential Role in the Emergence of the Bone Marrow Niche

Abstract Hematopoietic stem cell (HSC) quiescence and function are regulated, in part, by signals generated by stromal cells in the bone marrow. Mesenchymal stromal cells implicated in HSC maintenance include CXCL12-abudndant reticular (CAR) cells, osteolineage cells, and adipocytes, all of which, are derived from mesenchymal stem cells. The factors that regulate the growth, differentiation, and function of mesenchymal stems in the bone marrow are largely unknown. Here, we focus on transforming growth factor beta (TGF-β), which regulates osteoblast differentiation and has been implicated in the development of myelofibrosis in myeloproliferative neoplasms. We previously reported that constitutive deletion of Tgfbr2 (which abrogates all canonical TGF-β signaling) in mesenchymal stromal cells targeted by Osx-Cre results in marked alterations in the bone marrow microenvironment. These alterations include the following: 1) a 85-fold increase in adipocytes; 2) a 5-fold increase in CXCL12-abundant reticular (CAR) cells; and 3) the near absence of osteocalcin+ osteoblasts. Surprisingly, hematopoietic stem cells were only modestly reduced and had normal long-term repopulating and self-renewal capacity. These data suggest that TGF-β signaling plays a crucial role in determining the lineage commitment of mesenchymal progenitors. To investigate mechanisms by which TGF-β signaling regulates mesenchymal stromal cells, we first generated mice in which Tgfbr2 deletion is limited to Dmp1-Cre targeted stromal cells, which is mostly restricted to osteolineage cells. No alteration in stromal cells or hematopoiesis was observed. We next used a conditional Osx-Cre transgene to post-natally delete Tgfbr2 starting on P1. Surprisingly, these mice also had no alteration in osteoblasts, CAR cells, or adipocytes. Together, these experiments suggested the hypothesis that TGF-β signaling in mesenchymal progenitors during development may determine their lineage specification. To test this hypothesis, we analyzed Osx-Cre Tgfbr2fl/fl Ai9 mice during development; in these mice, Osx-Cre targeted cells express TdTomato. On E14.5 the nascent long-bones of control and Tgfbr2 -deleted mice appeared similar. Specifically, the primary ossification center contained a similar number and distribution of aggrecan-positive chondrocytes. Likewise, the number of TdTomato+ cells in the surrounding perichondrial “bone collar” was similar. However, on E16.5, a striking accumulation of perilipin-positive adipocytes was observed in Tgfbr2 -deleted but not control mice. Consistent with this finding, RNA expression profiling of Osx-Cre targeted (TdTomato-positive) cells sorted from the E16.5 long bones of Tgfbr2 -deleted mice showed a significant increase in adipocyte marker genes compared to control mice. Expression of peroxisome proliferator-activated receptor-gamma PPARG), a key transcription factor regulating adipogenesis, was increased 2-fold in Tgfbr2 -deleted cells. This is consistent with prior studies suggesting that TGF-β signaling negatively regulates PPARG expression. Collectively, these data show that TGF-β plays a key and non-redundant role in specifying the lineage commitment of mesenchymal progenitors in the bone marrow during development. Specifically, TGF-β suppresses the adipogenic potential of mesenchymal progenitors, possibly by regulating PPARG expression. Whether TGF-β signaling regulates bone marrow adiposity in adults, especially in response to aging or inflammation, is an open and interesting question. Disclosures No relevant conflicts of interest to declare.