Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures

Abstract

Notch signaling is an evolutionarily conserved mechanism that plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis during embryogenesis. The limb bud high-density culture is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) showed that Notch and its related genes were expressed in such cultures on day 1 and reached a peak between day 3 and day 5, when cell condensation and nodule formation were initiated. Immunohistochemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), to analyze the function of Notch signaling in the culture system. The blocking of Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. DAPT treatment also induced chondrogenic markers and bone morphogenetic protein (BMP)-related molecules, including type II collagen, Sox9, GDF5, and Id1. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.

RBPjκ-dependent Notch signaling regulates mesenchymal progenitor cell proliferation and differentiation during skeletal development

“…DAPT treatment significantly enhanced cartilage nodule formation ( Fig. 2A), suggesting that Notch inhibition accelerates commitment of MPCs to the chondrocyte lineage, a finding that is consistent with a prior study (Fujimaki et al, 2006). We also assessed the effect of DAPT on the expression of the chondrogenic markers Sox9, Col2a1 and Agc1 via real-time RT-PCR.…”

Dermal condensation formation in the chick embryo: Requirement for integrin engagement and subsequent stabilization by a possible Notch/integrin interaction

“…Fusion of primordia might reflect a dynamic requirement of Notch signalling for the maintenance of the dermal condensation. The most common hypothesis of the role of the Notch system in boundary formation and in segregation of one cell type from another has been previously described in other models such as axial skeletal formation (Bulman et al, 2000) and chondrogenesis (Fujimaki et al, 2006). Here we show that Notch signalling appears to be implicated not in the initial choice between the two fates of embryonic skin, but in a later step to allow stabilization of the newly formed pattern.…”

Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development

“…There are, however, several cell culture studies showing that Notch signaling functions as an inhibitor of the early stage of chondrocyte differentiation (13,27,28). In vivo studies on genetic gain-and loss of function of the signaling using paired-related homeobox gene 1 (Prx1) promoter-driven Cre-transgenic (Prx1-Cre) mice also showed that activation of Notch receptors and RBPjκ in mesenchymal progenitor cells inhibited their differentiation into chondrocytes and osteoblasts, and thereby suppressed cartilage and bone formation, respectively (29,30).…”