Conditional Deletion of Indian Hedgehog in Limb Mesenchyme Results in Complete Loss of Growth Plate Formation but Allows Mature Osteoblast Differentiation

Abstract

Indian hedgehog (Ihh) is widely recognized as an essential factor for proper skeletal development. Previous in vivo studies using mutant Ihh mouse models were limited by perinatal lethality or carried out after a growth plate formed. Thus the important role of Ihh in mesenchymal cell differentiation has not been investigated. In this study, we established Prx1-Cre;Ihh fl/fl mice to ablate Ihh specifically in limb mesenchyme to allow us to observe the phenotype continuously from prenatal development to 3 weeks of age. Mutant mice displayed severe limb abnormalities characterized by complete lack of secondary ossification center and growth plate, indicating an essential role for Ihh in the development of these structures. Interestingly, we discovered that osteoblast differentiation and bone formation could occur in conditions of deficient Ihh. This is a novel finding that has not been observed because of the early lethality of previous Ihh mutants. Mature osteoblasts expressing osteocalcin could be detected in the center of mutant bones at postnatal day 10 (P10). Osteoclasts and blood vessel formation were also present, suggesting active bone remodeling. Histomorphometric analyses show a significant increase in osteoclast number with no major changes in bone formation rate at 3 weeks of age. Mutant long bones in the limbs were deformed, with cortices comprised of irregular woven bone. Also, there was a marked decrease in gene expression of osteoblastic and osteocytic markers. Moreover, mutant long bones displayed bone dysplasia in which we observed increased osteoclast activity and partially reduced osteoblastic and osteocytic differentiation that lead ultimately to loss of bone structures at 3 weeks of age. In summary, our data show for the first time, the presence of mature osteoblasts in long bones of the limbs despite the complete loss of growth plate formation due to Ihh deficiency. These data indicate an important function for Ihh in regulating limb mesenchymal cell differentiation.

Limb derived cells as a paradigm for engineering self‐assembling skeletal tissues

“…The centre regions contained early hypertrophic cartilage cells that for the most part were roughly spherical and larger than cells in neighbouring regions Although the implants analysed resembled normal developing long bones morphologically and histologically, further verification of whether they displayed appropriate molecular signalling found during development was needed. To address this, IHH, a signalling molecule crucial for growth plate development and function (Amano, Densmore, & Lanske, 2015;Kronenberg, 2003) was first selected. Immunohistochemical staining for IHH revealed high expression in the proliferating and hypertrophic zones of the implants (Figure 2S), mirroring the signature expression pattern observed during normal development.…”

Signaling pathways regulating cartilage growth plate formation and activity

“…Ihh– deficient mouse embryos display severe skeletal growth retardation. They show reduced chondrocyte proliferation, and premature and abnormal hypertrophy [77] [78] [79]. Scube2 encodes a secreted and cell membrane-bound protein that mediates long-range Ihh signaling, probably through increasing Ihh solubility by binding to its cholesterol moiety.…”

Indian hedgehog in craniofacial neural crest cells links to skeletal malocclusion by regulating associated cartilage formation and gene expression

“…We used the second version of the Wnt1‐Cre driver since the original version of Wnt1‐Cre is reported to cause ectopic activation of Wnt signaling and subsequently affect the midbrain development 11 . The Ihh ‐floxed and R26‐tdTomato (as referred to Tomato ‐floxed in this paper) alleles were described previously 12,13 . We obtained the mouse model for this study by crossing male Wnt1‐Cre;Ihh fl/+ with female Ihh fl/fl ;Tomato fl/fl mice.…”

“…The skeletons were stained using 0.03% Alcian Blue solution (Wako, Osaka, JAPAN) and subsequently 0.01% Alizarin Red solution (Wako), as previously described 13 . Representative staining data from at least three individuals are shown in the figures.…”