Role of Indian Hedgehog Signaling in Palatal Osteogenesis

Abstract

Introduction Cleft lip and palate are common birth defects and represent a large biomedical burden. Through the use of animal models, the molecular underpinnings of cleft palate are becoming increasingly clear. Indian Hedgheg (Ihh) has been shown to be associated craniofacial development and active in the palatine bone. We hypothesize that Ihh activity plays a role in osteogenesis within the secondary palate and that defects in this pathway may inhibit the osteogenesis of the secondary palate. Methods Palates were isolated from wild type mice during the period of palate development (e9.5-e17.5). qRT-PCR was used for detecting gene expression during osteogenic differentiation and cellular differentiation (Shh, Ihh, Ptc1, Gli1, Gli2, Gli3, Runx2, Alp, Col1a1). Next, palates were analyzed by H&E, Aniline Blue, Pentachrome, and In situ Hybridization to assess osteogenesis of the palatal shelf and expression of hedgehog pathway genes. Finally, the palate of Indian Hedgehog null mice was analyzed to determine the effect of genetic deficiency on palatal development osteogenesis. Results Increased Indian Hedgehog and osteogenic signaling coincided with ossification and fusion of the palate in wild-type mice. This included a 5-150 fold peak in expression of Hedgehog elements, including Ihh, at e15.5 as compared to e9.5. Contrarily, loss of Indian Hedgehog by genetic knockout (Ihh −/−) resulted in decreased secondary palate ossification. Conclusion Our results suggest a role for Hedgehog signaling during palatal ossification. The hedgehog pathway is activated during palatal fusion and deletion of Indian hedgehog leads to diminished ossification of the secondary hard palate.

Dexamethasone induces osteogenesis via regulation of hedgehog signalling molecules in rat mesenchymal stem cells

“…It has been proven that differentiation induced by 10 −8 mol/L DEX induces osteogenesis in MSCs [15]. Our study confirmed that DEX promoted osteoblast differentiation in rat mesenchymal stem cells at the concentration of 10 −8 mol/L from seven days to 21 days, demonstrated by up-regulation ALP activity and expression of osteoblast-associated markers type I collagen.…”

“…After pre-culture for 24 hours, the MSCs were allowed to culture in osteogenesis culture medium (including 10 −8 mol/L Dexamethasone, 10 mmol/L β-glycerophosphate, 50 μg/ml ascorbic acid and 50 μg/ml vitamin C) according to the experimental requirements for up to 21 days [15]. All MSCs were incubated in 5 % CO2 at 37°C, and the medium was replaced every three days before harvest.…”

Palatogenesis

“…In addition, effects of various protein ligands on palatal gene expression can be studied. 48,49 The disadvantage, of course, is that in vitro conditions do not completely mimic in vivo correlates, and thus palate cultures are better for analyzing pathways than morphogenesis.…”

Targeted Mutations of Genes Reveal Important Roles in Palatal Development in Mice

“…90 For example, at E16.5 and E17.0, an appreciable decrease in size of palatine bones was observed in the Ihhj/j mice as compared with Ihh+/+ littermates, which caused a cleft in the midline of the palatine bone. 91 Shh is the most broadly expressed member of the hedgehog signaling, and is expressed in the palatal mesenchyme and epithelium. 92 Previous studies have shown that Shh inhibition results in a cleft palate, 92 and abrogation of Shh function in the palatal epithelium generates cleft palate.…”