Indian hedgehog synchronizes skeletal angiogenesis and perichondrial maturation with cartilage development

Abstract

A null mutation in the morphogen Indian hedgehog (IHH) results in an embryonic lethal phenotype characterized by the conspicuous absence of bony tissue in the extremities. We show that this ossification defect is not attributable to a permanent arrest in cartilage differentiation, since Ihh-/- chondrocytes undergo hypertrophy and terminal differentiation, express angiogenic markers such as Vegf, and are invaded, albeit aberrantly, by blood vessels. Subsequent steps, including vessel expansion and persistence, are impaired, and the net result is degraded cartilage matrix that is devoid of blood vessels. The absence of blood vessels is not because the Ihh-/- skeleton is anti-angiogenic; in fact, in an ex vivo environment, both wild-type and Ihh mutant vessels invade the Ihh-/- cartilage, though only wild-type vessels expand to create the marrow cavity. In the ex vivo setting, Ihh-/- cells differentiate into osteoblasts and deposit a bony matrix, without benefit of exogenous hedgehog in the new environment. Even more surprising is our finding that the earliest IHH-dependent skeletal defect is obvious by the time the limb mesenchyme segregates into chondrogenic and perichondrogenic condensations. Although Ihh-/- cells organize into chondrogenic condensations similar in size and shape to wild-type condensations, perichondrial cells surrounding the mutant condensations are clearly faulty. They fail to aggregate, elongate and flatten into a definitive, endothelial cell-rich perichondrium like their wild-type counterparts. Normally, these cells surrounding the chondrogenic condensation are exposed to IHH, as evidenced by their expression of the hedgehog target genes, patched (Ptch) and Gli1. In the mutant environment,the milieu surrounding the cartilage - comprising osteoblast precursors and endothelial cells - as well as the cartilage itself, develop in the absence of this important morphogen. In conclusion, the skeletal phenotype of Ihh-/- embryos represents the sum of disturbances in three separate cell populations, the chondrocytes, the osteoblasts and the vasculature, each of which is a direct target of hedgehog signaling.

Indian hedgehog: Its roles and regulation in endochondral bone development

“…In Ihh À/À mice, endothelial cells had the ability to invade the partially mineralized cartilaginous matrix, but they were only briefly sustained [Colnot et al, 2005]. The same phenomenon was observed when bone rudiments isolated from these mice were transplanted under the renal capsule of wild type mice.…”

“…This study suggested that it was the lack of Ihh itself, rather than the cartilage environment that caused the disappearance endothelial cells in Ihh À/À animals. Ihh, Ptc1, and Gli1 are all expressed in the perichondrium during early chondrogenic development, and endothelial cells normally develop from cells adjacent to that area [Colnot et al, 2005]. It can be concluded that Ihh signaling during the early developmental stage affects subsequent endothelial cell development and retention in the cartilage matrix.…”

“…Initially it was observed that Ihh has a direct role in promoting osteoblast differentiation in the perichondrium, as there was no cortical bone development in Ihh À/À mice [St.-Jacques et al, 1999;Long et al, 2004]. It was subsequently found that Ihh À/À mice had a thinner perichondrium, suggesting that Ihh may regulate cell differentiation in the perichondrium, and therefore cortical bone defects may result from the lack of proper development of the perichondium [Colnot et al, 2005].…”

Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling

“…Despite the fact that Ihh is a potent mitogen in cartilage and other tissues (Long et al, 2001;Minina et al, 2001), no detectable effect on proliferation was observed in the Ihh null calvaria. In addition, Hedgehogs have a known pro-angiogenic effect, which is reduced with aberrant vascular invasion in the Ihh 2/2 growth plate (Colnot et al, 2005). However, PECAM-1 immunostaining in the calvaria showed no appreciable difference between wild-type and Ihh 2/ 2 littermates (data not shown).…”

Kinetics of tamoxifen‐regulated Cre activity in mice using a cartilage‐specific CreERT to assay temporal activity windows along the proximodistal limb skeleton

“…8 in Colnot et al, 2005). Col2CreER T expression is also shut-off in mature perichondrium, except in the articular surfaces where expression detected by Rosa26LacZ reporter activation persists even postnatally (Fig.…”