The Notch pathway has recently been implicated in mesenchymal progenitor cell (MPC) differentiation from bone marrow-derived progenitors. However, whether Notch regulates MPC differentiation in an RBPjκ-dependent manner, specifies a particular MPC cell fate, regulates MPC proliferation and differentiation during early skeletal development or controls specific Notch target genes to regulate these processes remains unclear. To determine the exact role and mode of action for the Notch pathway in MPCs during skeletal development, we analyzed tissue-specific loss-of-function (Prx1Cre; Rbpjkf/f), gain-of-function (Prx1Cre; Rosa-NICDf/+) and RBPjκ-independent Notch gain-of-function (Prx1Cre; Rosa-NICDf/+; Rbpjkf/f) mice for defects in MPC proliferation and differentiation. These data demonstrate for the first time that the RBPjκ-dependent Notch signaling pathway is a crucial regulator of MPC proliferation and differentiation during skeletal development. Our study also implicates the Notch pathway as a general suppressor of MPC differentiation that does not bias lineage allocation. Finally, Hes1 was identified as an RBPjκ-dependent Notch target gene important for MPC maintenance and the suppression of in vitro chondrogenesis.

SUMMARYThe Notch signaling pathway has emerged as an important regulator of endochondral bone formation. Although recent studies have examined the role of Notch in mesenchymal and chondro-osteo progenitor cell populations, there has yet to be a true examination of Notch signaling specifically within developing and committed chondrocytes, or a determination of whether cartilage and bone formation are regulated via RBPj-dependent or -independent Notch signaling mechanisms. To develop a complete understanding of Notch signaling during cartilage and bone development we generated and compared general Notch gain-of-function (Rosa-NICD ). These data demonstrate, for the first time, that Notch regulation of chondrocyte maturation is solely mediated via the RBPj-dependent pathway, and that the perichodrium or osteogenic lineage probably influences chondrocyte terminal maturation and turnover of the cartilage matrix. Our study further identifies the cartilage-specific RBPj-independent pathway as crucial for the proper regulation of chondrocyte proliferation, survival and columnar chondrocyte organization. Unexpectedly, the RBPj-independent Notch pathway was also identified as an important long-range cell non-autonomous regulator of perichondral bone formation and an important cartilage-derived signal required for coordinating chondrocyte and osteoblast differentiation during endochondral bone development. Finally, cartilage-specific RBPj-independent Notch signaling likely regulates Ihh responsiveness during cartilage and bone development.

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