Limits...
Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development.

Joeng KS, Long F - Bone Res (2014)

Bottom Line: Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development.Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh(-/-); Gli3(-/-) embryo.Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine , St Louis, MO, USA ; Division of Biology and Biomedical Sciences, Washington University in St. Louis , St Louis, MO, USA.

ABSTRACT
Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh(-/-) mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh(-/-) mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh(-/-); Gli3(-/-) embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

No MeSH data available.


Related in: MedlinePlus

Analyses of hypertrophic cartilage vascularization in the humerus of E18.5 littermate embryos. (a1–c1) H&E staining of humerus sections from Ihh+/− (a1), Ihh−/−; R26Wnt7b/+ (b1) and Ihh−/−; Col2-Cre3; R26Wnt7b/+ (c1) embryos. (a2–c2) High-magnification images of boxed areas in a1–c1. Red arrows point to red blood cells. (d1–d3, e1–e3, f1–f3) In situ hybridization on humerus sections. Hybridization signal shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4472126&req=5

fig4: Analyses of hypertrophic cartilage vascularization in the humerus of E18.5 littermate embryos. (a1–c1) H&E staining of humerus sections from Ihh+/− (a1), Ihh−/−; R26Wnt7b/+ (b1) and Ihh−/−; Col2-Cre3; R26Wnt7b/+ (c1) embryos. (a2–c2) High-magnification images of boxed areas in a1–c1. Red arrows point to red blood cells. (d1–d3, e1–e3, f1–f3) In situ hybridization on humerus sections. Hybridization signal shown in red.

Mentions: To test whether Wnt7b mediates Ihh function during endochondral bone development, we produced Col2-Cre3; Ihh−/−; R26Wnt7b/+ (termed Wnt7b-rescue) embryos by crossing the Col2-Cre3; Ihh+/− and Ihh+/−; R26Wnt7b/Wnt7b mice. The Ihh−/−; R26Wnt7b/+ embryos, like the Ihh−/− embryos as previously reported, showed little vascularization of the hypertrophic cartilage at E18.5, even though the control littermate had developed a bone marrow cavity (Figure 4a1, 4a2, 4b1 and 4b2). The long bones of the Wnt7b-rescue embryos appeared to be similar in gross morphology to those of Ihh−/−; R26Wnt7b/+ embryos (Figure 4b1 and 4c1, and data not shown). However, they displayed clear vascularization in their hypertrophic cartilage, as evident by the presence of red blood cells, even though a marrow cavity was not formed (Figure 4c1 and 4c2). To gain further insight about the vascularization phenotype, we performed in situ hybridization with molecular markers for hypertrophy and vascularization. Normally, Col10a1 is most robustly expressed by the early and intermediate hypertrophic chondrocytes, whereas Mmp13 demarcates the terminal hypertrophic cells as well as osteoblast-lineage cells, and Mmp9 marks the leading edge of vascular invasion in the bone marrow cavity (Figure 4d1–4d3). In the Ihh−/−; R26Wnt7b/+ embryo, the core of the cartilage element contained a hypertrophic domain expressing Col10a1 peripherally and Mmp13 centrally in a concentric manner, but no Mmp9 expression was detected, confirming no vascularization of the hypertrophic cartilage (Figure 4e1–4e3). In contrast, expression of Col10a1 and Mmp13 in the hypertrophic cartilage of Wnt7b-rescue mice assumed a linear instead of concentric arrangement more similar to the normal pattern (Figure 4f1 and 4f2). More importantly, the Wnt7b-rescue cartilage expressed Mmp9 within the hypertrophic region, indicative of vascular invasion (Figure 4f3). Likely as a result of the vascularization, the Mmp13-positive domain was no longer contiguous in the central hypertrophic region in the Wnt7b-rescue embryo, in contrast to the Ihh−/−; R26Wnt7b/+ mutant (Figure 4e2 and 4f2). Overall, forced expression of Wnt7b was sufficient to induce hypertrophic cartilage angiogenesis in the absence of Ihh.


Wnt7b can replace Ihh to induce hypertrophic cartilage vascularization but not osteoblast differentiation during endochondral bone development.

Joeng KS, Long F - Bone Res (2014)

Analyses of hypertrophic cartilage vascularization in the humerus of E18.5 littermate embryos. (a1–c1) H&E staining of humerus sections from Ihh+/− (a1), Ihh−/−; R26Wnt7b/+ (b1) and Ihh−/−; Col2-Cre3; R26Wnt7b/+ (c1) embryos. (a2–c2) High-magnification images of boxed areas in a1–c1. Red arrows point to red blood cells. (d1–d3, e1–e3, f1–f3) In situ hybridization on humerus sections. Hybridization signal shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4472126&req=5

fig4: Analyses of hypertrophic cartilage vascularization in the humerus of E18.5 littermate embryos. (a1–c1) H&E staining of humerus sections from Ihh+/− (a1), Ihh−/−; R26Wnt7b/+ (b1) and Ihh−/−; Col2-Cre3; R26Wnt7b/+ (c1) embryos. (a2–c2) High-magnification images of boxed areas in a1–c1. Red arrows point to red blood cells. (d1–d3, e1–e3, f1–f3) In situ hybridization on humerus sections. Hybridization signal shown in red.
Mentions: To test whether Wnt7b mediates Ihh function during endochondral bone development, we produced Col2-Cre3; Ihh−/−; R26Wnt7b/+ (termed Wnt7b-rescue) embryos by crossing the Col2-Cre3; Ihh+/− and Ihh+/−; R26Wnt7b/Wnt7b mice. The Ihh−/−; R26Wnt7b/+ embryos, like the Ihh−/− embryos as previously reported, showed little vascularization of the hypertrophic cartilage at E18.5, even though the control littermate had developed a bone marrow cavity (Figure 4a1, 4a2, 4b1 and 4b2). The long bones of the Wnt7b-rescue embryos appeared to be similar in gross morphology to those of Ihh−/−; R26Wnt7b/+ embryos (Figure 4b1 and 4c1, and data not shown). However, they displayed clear vascularization in their hypertrophic cartilage, as evident by the presence of red blood cells, even though a marrow cavity was not formed (Figure 4c1 and 4c2). To gain further insight about the vascularization phenotype, we performed in situ hybridization with molecular markers for hypertrophy and vascularization. Normally, Col10a1 is most robustly expressed by the early and intermediate hypertrophic chondrocytes, whereas Mmp13 demarcates the terminal hypertrophic cells as well as osteoblast-lineage cells, and Mmp9 marks the leading edge of vascular invasion in the bone marrow cavity (Figure 4d1–4d3). In the Ihh−/−; R26Wnt7b/+ embryo, the core of the cartilage element contained a hypertrophic domain expressing Col10a1 peripherally and Mmp13 centrally in a concentric manner, but no Mmp9 expression was detected, confirming no vascularization of the hypertrophic cartilage (Figure 4e1–4e3). In contrast, expression of Col10a1 and Mmp13 in the hypertrophic cartilage of Wnt7b-rescue mice assumed a linear instead of concentric arrangement more similar to the normal pattern (Figure 4f1 and 4f2). More importantly, the Wnt7b-rescue cartilage expressed Mmp9 within the hypertrophic region, indicative of vascular invasion (Figure 4f3). Likely as a result of the vascularization, the Mmp13-positive domain was no longer contiguous in the central hypertrophic region in the Wnt7b-rescue embryo, in contrast to the Ihh−/−; R26Wnt7b/+ mutant (Figure 4e2 and 4f2). Overall, forced expression of Wnt7b was sufficient to induce hypertrophic cartilage angiogenesis in the absence of Ihh.

Bottom Line: Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development.Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh(-/-); Gli3(-/-) embryo.Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine , St Louis, MO, USA ; Division of Biology and Biomedical Sciences, Washington University in St. Louis , St Louis, MO, USA.

ABSTRACT
Indian hedgehog (Ihh) is an essential signal that regulates endochondral bone development. We have previously shown that Wnt7b promotes osteoblast differentiation during mouse embryogenesis, and that its expression in the perichondrium is dependent on Ihh signaling. To test the hypothesis that Wnt7b may mediate some aspects of Ihh function during endochondral bone development, we activated Wnt7b expression from the R26-Wnt7b allele with Col2-Cre in the Ihh(-/-) mouse. Artificial expression of Wnt7b rescued vascularization of the hypertrophic cartilage in the Ihh(-/-) mouse, but failed to restore orthotopic osteoblast differentiation in the perichondrium. Similarly, Wnt7b did not recover Ihh-dependent perichondral bone formation in the Ihh(-/-); Gli3(-/-) embryo. Interestingly, Wnt7b induced bone formation at the diaphyseal region of long bones in the absence of Ihh, possibly due to increased vascularization in the area. Thus, Ihh-dependent expression of Wnt7b in the perichondrium may contribute to vascularization of the hypertrophic cartilage during endochondral bone development.

No MeSH data available.


Related in: MedlinePlus