The role of Hedgehogs (Hh) in murine skeletal development was studied by 
overexpressing human Sonic Hedgehog (SHH) in chondrocytes of transgenic mice 
using the collagen II promoter/enhancer. Overexpression caused a lethal 
craniorachischisis with major alterations in long bones because of defects in 
chondrocyte differentiation.
INTRODUCTION: Hedgehogs (Hhs) are a family of secreted polypeptides that play 
important roles in vertebrate development, controlling many critical steps of 
cell differentiation and patterning. Skeletal development is affected in many 
different ways by Hhs. Genetic defects and anomalies of Hhs signaling pathways 
cause severe abnormalities in the appendicular, axial, and cranial skeleton in 
man and other vertebrates.
MATERIALS AND METHODS: Genetic manipulation of mouse embryos was used to study 
in vivo the function of SHH in skeletal development. By DNA microinjection into 
pronuclei of fertilized oocytes, we have generated transgenic mice that express 
SHH specifically in chondrocytes using the cartilage-specific collagen II 
promoter/enhancer. Transgenic skeletal development was studied at different 
embryonic stages by histology. The expression pattern of specific chondrocyte 
molecules was studied by immunohistochemistry and in situ hybridization.
RESULTS: Transgenic mice died at birth with severe craniorachischisis and other 
skeletal defects in ribs, sternum, and long bones. Detailed analysis of long 
bones showed that chondrocyte differentiation was blocked at prehypertrophic 
stages, hindering endochondral ossification and trabecular bone formation, with 
specific defects in different limb segments. The growth plate was highly 
disorganized in the tibia and was completely absent in the femur and humerus, 
leading to skeletal elements entirely made of cartilage surrounded by a thin 
layer of bone. In this cartilage, chondrocytes maintained a columnar 
organization that was perpendicular to the bone longitudinal axis and directed 
toward its outer surface. The expression of SHH receptor, Patched-1 (Ptc1), was 
greatly increased in all cartilage, as well as the expression of parathyroid 
hormone-related protein (PTHrP) at the articular surface; while the expression 
of Indian Hedgehog (Ihh), another member of Hh family that controls the rate of 
chondrocyte maturation, was greatly reduced and restricted to the displaced 
chondrocyte columns. Transgenic mice also revealed the ability of SHH to 
upregulate the expression of Sox9, a major transcription factor implicated in 
chondrocyte-specific gene expression, in vivo and in vitro, acting through the 
proximal 6.8-kb-long Sox9 promoter.
CONCLUSION: Transgenic mice show that continuous expression of SHH in 
chondrocytes interferes with cell differentiation and growth plate organization 
and induces high levels and diffuse expression of Sox9 in cartilaginous bones.