Until recently, the role of Notch signaling during skeletogenesis has mainly been limited to its role in patterning and somitogenesis. The involvement of the Notch signaling pathway in somitogenesis was first revealed by the finding that Notch1 and its ligand Dll1 were highly expressed in the presomitic mesoderm (PSM) of mouse embryos. Subsequently, additional cycling genes, such as Lunatic fringe
, and Hes1
were identified in different species to be components of this pathway [9
]. All of these genes are linked to the Notch signaling pathway suggesting that Notch-signaling activity in the PSM is itself oscillating and is either controlled by the segmentation clock or is a central component of it. In support of this, Notch1
null mouse embryos exhibited significantly delayed and disorganized somitogenesis[11
]. The Rbp-Jκ
null embryos displayed slightly more severe somitogenesis due to complete loss of the Notch sigaling [12
]. Knock-out models and spontaneous mutations of Notch signaling components such as Delta-like 1
, Lunatic fringe
also led to somitic phenotypes (). Together, these studies clearly implicate Notch signaling in the direct regulation of segmentation. Consistent with this, two human disorders, Spondylocostal dysostosis (SCD) and Alagille syndrome (AGS), caused by mutations in Notch pathway genes, exhibit vertebral column defects indicating that correct, cyclic function of the Notch pathway within the vertebrate segmentation clock is essential for proper somitogenesis in both mice and humans [13
Summary of mouse mutant phenotypes of Notch signaling components and related human diseases.
Apart from its role in somitogenesis, several in vitro
studies with conflicting results implicated the Notch pathway in the regulation of osteoblast differentiation. In these studies, the expression of Notch1 in MC3T3-E1 osteoblastic cells at early differentiation stages was detected. When Notch1 ICD was delivered by an adenovirus vector to osteoblastic MC3T3-E1 cells, a significant increase in calcified nodule formation was observed. Similarly, when the C3H10T1/2 multi-potent mesenchymal cell line was infected with an adenovirus expressing EGFP-Notch1ICD, stimulation of osteoblastic differentiation and inhibition of adipogenesis was observed, suggesting that osteoblastic differentiation was influenced by Notch [16
]. In support of this, the Notch target gene Hes-1 was shown to interact with Runx2 and increase its transactivation effect on an osteoblast specific enhancer [17
]. On the other hand, while these studies support a role for Notch in the stimulation of osteoblastogenesis, other groups have reported the opposite results also in vitro
]. In these studies, over expression of Notch1 in stromal and mesenchymal cells impaired osteoblastic differentiation.
The possibility of Notch regulation of osteoclastogenesis was also investigated in an in vitro
study, in which osteoclastogenesis was shown to be inhibited by an immobilized Notch ligand, Delta-1 [21
]. Constitutively active Notch1-transfected stromal cells showed increased expression of RANKL and OPG genes, and strong inhibition of M-CSF expression, resulting in negative regulation of osteoclastogenesis thus providing the first clue of cross talk between osteoblasts and osteoclasts that might be mediated by Notch signaling.
The distribution of Notch receptors and their ligands during articular cartilage development has also been reported [22
]. In vitro
studies suggested that Notch negatively regulates the initiation of pre-chondrogenic condensation and nodule formation, and later differentiation and proliferation of early chondrogenic cells are suppressed by Notch [23
]. Notch signaling has been shown to be required for the chondrogenic specification of mouse mesencephalic neural crest cells. The expression of Sox9, a transcription activator of collagen type II, was up-regulated by Notch activation and this activation of Notch signaling thereby promoted differentiation of proliferative and prehypertrophic chondrocytes [26
]. On the other hand, transduction of human mesenchymal stem cells hMSCs with an adenovirus expressing Jagged-1 activated Hey-1 expression, which resulted in up-regulation of type II collagen expression. Jagged-1-transduced hMSCs, which exposed to continuous elevated expression of Jagged-1, showed a complete inhibition of chondrogenesis [27
]. Thus, Jagged-1-mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed. In vivo
, in chick, Notch ligand Delta-1 negatively regulates the transition from pre-hypertrophic to hypertrophic chondrocytes during cartilage formation [28
]. The exact role of Notch and its temporal effects in chondrogenesis and osteoblastogenesis remains to be further demonstrated. We will discuss recently created animal models which shed light on Notch function in the two primary types of bone cells, namely osteoblasts and osteoclasts.