The Effects of Indian Hedgehog Deletion on Mesenchyme Cells: Inducing Intermediate Cartilage Scaffold Ossification to Cause Growth Plate and Phalange Joint Absence, Short Limb, and Dwarfish Phenotypes

Abstract

The endochondral ossification plays a critical role in vertebrate limb development and skeletal homeostasis, where limb mesenchyme cells form an intermediate cartilage scaffold that develops into growth plates and then replaced by bone. Although Indian hedgehog (Ihh) is known to control the hypertrophic differentiation process of chondrocytes, its role from the mesenchyme cells to the early stages of chondrogenesis is unclear. To define the function of Ihh in the mesenchymal cell's early stages of chondrogenesis, we specifically delete Ihh in Prx1-expressed mesenchyme cells at E9.5 using Prx1-Cre;Ihh;Rosa26 mice. We found that deleting Ihh in the mesenchyme cells results in an early and quick ossification of the intermediate cartilage scaffold, causing the growth plate and phalange joint absence, short limbs, and dwarfishness. The green fluorescent protein (GFP)-positive cells derived from deleted Ihh mesenchyme cells overlap with von Kossa- and osteocalcin-positive staining area. These deleted Ihh/GFP-positive cells isolated from Prx1-Cre;Ihh;Rosa26 newborn mice had osteogenic differentiation by showing a positive Alizarin red and von Kossa staining, as well as an enhanced Col1a1, osteocalcin, and Runx2 expression. Our findings demonstrate that deleting Ihh in mesenchyme cells during early limb development promotes intermediate cartilage scaffold ossification, which prevents growth plate formation that causes phalange joint absence, short limb, and dwarfish phenotype.

Hsa_circularRNA_0079201 suppresses chondrocyte proliferation and endochondral ossification by regulating the microRNA‑140‑3p/SMAD2 signaling pathway in idiopathic short stature

“…Wang et al ( 31 ) observed that SMAD2 could inhibit IHH mRNA and protein expression levels and significantly reduced chondrocyte proliferation and hypertrophy in the neonatal mouse growth plate. Multiple studies have confirmed that downregulated IHH mRNA and protein expression levels could inhibit chondrocyte proliferation and impede longitudinal bone growth ( 18 , 40 , 41 ); therefore, the present study investigated the role of SMAD2 in ISS. As expected, Starbase identified that SMAD2 and miR-140-3p have three binding sites with a high score, whilst luciferase assays verified that miR-140-3p binds to SMAD2.…”

“…Numerous studies have shown that non-coding (nc)RNAs regulate the growth plate ( 14 - 16 ) and thus, may affect longitudinal bone growth. For example, it has been reported that microRNAs (miRNA/miR) could regulate the endochondral ossification of the growth plate and longitudinal bone growth ( 17 , 18 ).…”

Quick and Abnormal Ossification Results in No Growth Plate Formation and Skeletal Dysplasia in Postnatal Mice Induced by Conditional Knockout of Indian Hedgehog

“…The results of our study demonstrate that the early intermediate cartilage scaffold ossi cation in Ihhde cient mice is responsible for the absence of growth plate and the second ossi cation center formation. Based mainly on in vivo studies with the use of genetically modi ed mice, several studies and our data have revealed that Ihh is indispensable for the process of growth plate organization [5,6,[9][10][11][12].…”

“…They believe an initial cartilaginous fusion in digits and the initial cartilage gradually resorbed and replaced by bone are speci c mechanisms that lead to the loss of epiphyseal growth plate [10,11]. Recently, we found that deleted Ihh on mesenchyme cells accelerated pathological matrix calci cation and results in the intermediate cartilage scaffold ossi cation, which prevents growth plate and phalange joint formation causing short limb and dwar sh phenotypes [6]. This further supports that the deleted Ihh results in a loss of growth plate due to abnormal ossi cation of the cartilage scaffold.…”

“…Earlier studies have suggested Ihh expression in the postnatal chondrocytes is essential for maintenance of the growth plate and the articular surface and for sustaining a primary spongiosa [5]. We also generated Prx1-Cre; Ihh / ; Rosa26 − ZsGreen1 mice to precisely delete Ihh in the mesenchyme cells and found Ihh deletion on mesenchyme cells results in the intermediate cartilage scaffold ossi cation, which prevents growth plate and phalange joint formation causing short limb and dwar sh phenotypes [6]. However, due to Prx1 are speci cally expressed at E9.5 in limb development [7], the role of Ihh in postnatal bone growth and remodeling could not be studied.…”