Date of Award
Doctor of Philosophy (PhD)
Indian hedgehog (Ihh) is an essential regulator for endochondral skeletal development. Although Gli2 and Gli3 are known as primary mediators for hedgehog signaling, the function of each factor in endochondral bone development is not clear. Our previous study showed that removal of Gli3 fully restored chondrocyte proliferation and maturation in Ihh-null embryos, but osteoblast differentiation and cartilage vascularization were not fully rescued. Interestingly, Gli2-null embryos revealed both reduced bone formation and delayed cartilage vascularization. Based on these results, I hypothesized that the Gli2 activator mediates Ihh signaling to regulate osteoblast differentiation and cartilage vascularization. To test this hypothesis, I generated a R26-δNGli2 mouse strain which can be used to express a constitutively activated form of Gli2 (termed δNGli2) in any tissue depending on a CRE recombination. Subsequently, I generated Col2-CRE; Ihh-/-; R26δNGli2/+ embryos (termed δNGli2-rescue embryo) to perform the rescue experiment. The δNGli2-rescue embryos showed a rescue in cartilage vascularization, but δNGli2 did not rescue Ihh-dependent osteoblast differentiation. The lack of Ihh-dependent osteoblast differentiation in the δNGli2-rescue embryo prompted me to hypothesize that Ihh-dependent osteoblast differentiation requires both de-repression of Gli3 and activation of Gli2. To test this hypothesis, I generated Col2-CRE; Ihh-/-; Gli3-/-; R26δNGli2/+ embryos (termed double-rescue embryo). As I expected, the double-rescue embryos exhibited fully-rescued Ihh-dependent osteoblast differentiation. Based on these results, I conclude that Ihh controls endochondral skeletal development primarily through de-repression of Gli3 and activation of Gli2. Whereas de-repression of Gli3 by Ihh ensures proper chondrocyte maturation and proliferation, activation of Gli2 by Ihh controls cartilage vascularization. Finally, both Gli3 de-repression and Gli2 activation are required for Ihh-dependent osteoblast differentiation.
Although I showed Gli2 and Gli3 mediate Ihh signaling to regulate all aspects of endochondral bone development, the direct target of each Gli protein was not identified. Because none of osteoblast, chondrocyte, and angiogenesis markers are known as direct targets of Gli transcription factors, I speculated that other signaling pathway mediate Ihh signaling to regulate endochondral bone development. Our expression screening revealed that the expression of Wnt7b is dependent on Ihh signaling in perichondral cells. Based on the data, I hypothesized that Wnt7b is a specific Wnt ligand mediating Ihh signaling to regulate endochondral bone development. To test this hypothesis, I generated a R26-Wnt7b mouse strain which can express Wnt7b in any tissue depending on the CRE recombination. First, I tested the function of Wnt7b in Wild-type by generating Col2-CRE; R26Wnt7b/+ embryos (termed C2Wnt7b). Morphological and molecular analysis showed that ectopic expression of Wnt7b increased bone formation. To perform the rescue experiment, I generated Col2-CRE; Ihh-/-; R26Wnt7b/+ embryos (termed Wnt7b-rescue embryo). The Wnt7b-rescue embryos showed enhanced cartilage, but the expression of Wnt7b did not rescue chondrocyte development and Ihh-dependent osteoblast differentiation. Because the phenotypes in Wnt7b-rescue embryos were similar to those in δNGli2-rescue embryos, I hypothesized that Ihh-dependent osteoblast differentiation requires both de-repression of Gli3 and expression of Wnt7b. To test this hypothesis, I generated Col2-CRE; Ihh-/-; Gli3-/-; R26Wnt7b/+ embryos (termed Wnt7b-double-rescue embryo). Although Ihh-dependent differentiation was not rescued in Wnt7b-double-rescue embryos, more increased bone formation was observed. Based on these results, I conclude that Wnt7b mediates Ihh signaling to regulate cartilage angiogenesis and later stage of osteoblast development.
Although a large number of genetic studies have shown that Indian hedgehog signaling is essential for endochondral bone development, the function of Hedgehog (Hh) signaling in postnatal bone formation is not clear. In a previous study, I generated the R26-δNGli2 mouse strain which can express a constitutively activated form of Gli2 (termed δNGli2) in a CRE-dependent manner. To study the function of Hh signaling in postnatal bones, I initially generated Col2-CRE; R26ΔNGli2/+ mice by crossing R26ΔNGli2/+ mice with Col2-CRE mice which express Cre in both osteoblasts and chondrocytes. The Col2-CRE; R26ΔNGli2/+ animals showed a disorganized growth plate and severe osteopenia at 2 and 4 months of age. However, the disorganized growth plate precluded the analysis of direct effects of Hh signaling on bone. To avoid the growth plate phenotype, I generated Osx-CRE; R26ΔNGli2/+ mice (termed OsxΔNGli2) by crossing R26ΔNGli2/+ mice with Osx-CRE mice which specifically express CRE in osteoblasts. The OsxΔNGli2 animals exhibited a dramatic decrease (6-fold by uCT) in bone mass without any obvious growth plate phenotype at 4 and 6 weeks of age. Further analysis showed that the reduced bone mass in OsxΔNGli2 animals was due to decreased osteoblast number and activity. To study the molecular basis for the osteoblast phenotype, I performed in situ hybridization with osteoblast markers in neonatal OsxΔNGli2 animals. Whereas early osteoblast markers, Runx2 and Osterix (Osx) , were expressed normally, the mature osteoblast marker, Osteocalcin (Oc) , was rarely detectable in OsxΔNGli2 animals. These data indicate that ectopic activation of Hh signaling by ΔNGli2 inhibits differentiation of Osx+ progenitors to Oc+ osteoblast. To examine the role of physiological Hh signaling in Osx+ progenitors, I generated Osx-CRE; Smo c/c mice in which Smoothened (Smo) , the indispensable mediator for hedgehog signaling, was specifically knocked out in osteoblasts. Remarkably, Osx-CRE; Smo c/c animals showed a notable increase in bone mass (7-fold by uCT). Consistent with the notion that loss of Hh signaling promotes differentiation of Osx+ progenitors, in situ hybridization in neonatal Osx-CRE; Smo c/c animals detected a decrease in Osx signal in the primary spongiosa. Overall, through both gain- and loss-of-function studies, I have uncovered a new function for Hh signaling in osteoblast development. I speculate that the newly identified role of Hh signaling may be important for maintaining an adequate pool of progenitors for osteoblast.
Chair and Committee
Jason C Mills
David C Beebe, Fanxin Long
Joeng, Kyu Sang, "The Function of Hedgehog Signaling in Embryonic and Postnatal Bone Development" (2010). Arts & Sciences Electronic Theses and Dissertations. 505.
Available for download on Friday, August 15, 2110