During endochondral ossification, two secreted signals, Indian hedgehog (Ihh) 
and parathyroid hormone-related protein (PTHrP), have been shown to form a 
negative feedback loop regulating the onset of hypertrophic differentiation of 
chondrocytes. Bone morphogenetic proteins (BMPs), another family of secreted 
factors regulating bone formation, have been implicated as potential interactors 
of the Ihh/PTHrP feedback loop. To analyze the relationship between the two 
signaling pathways, we used an organ culture system for limb explants of mouse 
and chick embryos. We manipulated chondrocyte differentiation by supplementing 
these cultures either with BMP2, PTHrP and Sonic hedgehog as activators or with 
Noggin and cyclopamine as inhibitors of the BMP and Ihh/PTHrP signaling systems. 
Overexpression of Ihh in the cartilage elements of transgenic mice results in an 
upregulation of PTHrP expression and a delayed onset of hypertrophic 
differentiation. Noggin treatment of limbs from these mice did not antagonize 
the effects of Ihh overexpression. Conversely, the promotion of chondrocyte 
maturation induced by cyclopamine, which blocks Ihh signaling, could not be 
rescued with BMP2. Thus BMP signaling does not act as a secondary signal of Ihh 
to induce PTHrP expression or to delay the onset of hypertrophic 
differentiation. Similar results were obtained using cultures of chick limbs. We 
further investigated the role of BMP signaling in regulating proliferation and 
hypertrophic differentiation of chondrocytes and identified three functions of 
BMP signaling in this process. First we found that maintaining a normal 
proliferation rate requires BMP and Ihh signaling acting in parallel. We further 
identified a role for BMP signaling in modulating the expression of IHH: 
Finally, the application of Noggin to mouse limb explants resulted in advanced 
differentiation of terminally hypertrophic cells, implicating BMP signaling in 
delaying the process of hypertrophic differentiation itself. This role of BMP 
signaling is independent of the Ihh/PTHrP pathway.