The Indian Hedgehog - PTHrP System in Bone Development
193
11.2.2 Expression of Pte and Gli
In a variety of organisms, hedgehog molecules have been shown to act
through a conserved signaling pathway including homologues of
Patched (Ptc) and Gli. Ptc has been identified as a subunit of the
hedgehog receptor complex (Goodrich et al. 1996; Marigo et al. 1996a;
Marigo et al. 1996c; Stone et al. 1996), and Gli (Marigo et al. 1996b;
Ruppert et al. 1988), is a member of a zinc finger transcription factor
family. Both genes have been shown to be upregulated in target tissues
responding to hedgehog signaling (reviewed in Tabin and McMahon
1997). During the early steps of endochondral ossification, both Ptc and
Gli are expressed in the condensing chondrocytes. Their expression
domains overlap with, but are wider than that of Ihh. (Vortkamp et al.
1998). With the refinement of the cartilage elements and the subsequent
onset of hypertrophic differentiation, Ptc and Gli become strongly expressed in the developing perichondrium flanking the Ihh expression
domain. However, weaker expression can still be found in the proliferating chondrocytes distal to the Ihh expression domain (Vortkamp et al.
1996; Vortkamp et al. 1998). These expression patterns suggest that Ihh
signals in two directions: to the flanking perichondrium and to the
proliferating and less differentiated chondrocytes.
11.3 Ihh and PTHrP Interact in a Negative Feedback Loop
Regulating Chondrocyte Differentiation
11.3.1 Ihh Misexpression
To analyze the role of Ihh during endochondral ossification, we used a
retroviral vector system to misexpress Ihh in the limbs of developing
chick embryos. The limbs were infected at the onset of limb outgrowth
at stage Hamburger-Hamilton 22 (HH 22) (Hamburger and Hamilton
1951), when the cartilage condensations start to form. The effect of Ihh
misexpression was analyzed at stages of hypertrophic differentiation, at
stage HH 34 and stage HH 36. These experiments revealed that misexpression of Ihh disrupts the process of endochondral ossification by
blocking hypertrophic differentiation of chondrocytes. Instead, the
chondrocytes remain in a proliferating state. More detailed analysis
193
11.2.2 Expression of Pte and Gli
In a variety of organisms, hedgehog molecules have been shown to act
through a conserved signaling pathway including homologues of
Patched (Ptc) and Gli. Ptc has been identified as a subunit of the
hedgehog receptor complex (Goodrich et al. 1996; Marigo et al. 1996a;
Marigo et al. 1996c; Stone et al. 1996), and Gli (Marigo et al. 1996b;
Ruppert et al. 1988), is a member of a zinc finger transcription factor
family. Both genes have been shown to be upregulated in target tissues
responding to hedgehog signaling (reviewed in Tabin and McMahon
1997). During the early steps of endochondral ossification, both Ptc and
Gli are expressed in the condensing chondrocytes. Their expression
domains overlap with, but are wider than that of Ihh. (Vortkamp et al.
1998). With the refinement of the cartilage elements and the subsequent
onset of hypertrophic differentiation, Ptc and Gli become strongly expressed in the developing perichondrium flanking the Ihh expression
domain. However, weaker expression can still be found in the proliferating chondrocytes distal to the Ihh expression domain (Vortkamp et al.
1996; Vortkamp et al. 1998). These expression patterns suggest that Ihh
signals in two directions: to the flanking perichondrium and to the
proliferating and less differentiated chondrocytes.
11.3 Ihh and PTHrP Interact in a Negative Feedback Loop
Regulating Chondrocyte Differentiation
11.3.1 Ihh Misexpression
To analyze the role of Ihh during endochondral ossification, we used a
retroviral vector system to misexpress Ihh in the limbs of developing
chick embryos. The limbs were infected at the onset of limb outgrowth
at stage Hamburger-Hamilton 22 (HH 22) (Hamburger and Hamilton
1951), when the cartilage condensations start to form. The effect of Ihh
misexpression was analyzed at stages of hypertrophic differentiation, at
stage HH 34 and stage HH 36. These experiments revealed that misexpression of Ihh disrupts the process of endochondral ossification by
blocking hypertrophic differentiation of chondrocytes. Instead, the
chondrocytes remain in a proliferating state. More detailed analysis