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

Objective: To observe the dynamic effect of conditional knockout of Indian hedgehog (Ihh) on ossication of postnatal growth plate, skeletal abnormalities and the related signaling factors in mice. Methods: P0 Col2a1-Cre ER T2 ; Ihh / mice were randomly divided into tamoxifen (TM) injection to delete Ihh and oil injection as control. Real-time PCR (RT-PCR) was used to detect Ihh knockout rate. X-ray and micro-CT were used to evaluate gross and knee joint morphology of 8-week-old mice. The hind knee joints were harvested at P2-P14 after the animals were euthanized to observe the dynamic evolution of the growth plate. Safranin-O and Von Kossa staining were performed to assess growth plate development and the mineralization of bone respectively. The costal cartilage cells of mice were cultured in vitro on postnatal day 6. Changes in the expression of parathyroid hormone-related protein (PTHrP) and bone morphogenetic protein 6 (BMP-6) were detected by immunohistochemistry and RT-PCR. Results: RT-PCR results conrmed that the knockout rate of Ihh was 76.83%. X-ray and micro-CT scans showed that the short-limb deformity of the experimental group was associated with abnormal development of the epiphysis. Saffron O staining showed a disorder of cell arrange in the growth-plate area of Ihh d/d mice; Von Kossa staining showed an early and premature growth-plate ossication that prevents the growth plate formation in Ihh d/d mice starting at P8. Immunohistochemistry and RT-PCR showed signicantly decreased PTHrP expression (P<0.05) and signicantly increased BMP-6 expression (P<0.05). plate


Background
The growth plate is an epiphyseal cartilage region constituted by chondrocytes and cartilage matrix between the metaphysis and the metaphysis [1]. In mammals, the growth plate is composed of three types of highly organized and specialized cartilage: resting zone chondrocytes, proliferative zone chondrocytes, and hypertrophic zone chondrocytes. The growth plate provides a continuous supply of chondrocytes for endochondral ossi cation, whereby limb mesenchyme cells form an intermediate cartilage scaffold that develops into growth plate and is then replaced by bone, and any failure in this process causes a wide range of skeletal disorders [2]. The formation and function of the growth plate is a particularly important topic of research in the prevention of skeletal abnormalities.
The Hh homologous proteins in mammals are Sonic hedgehog, Indian hedgehog (Ihh), and Desert hedgehog, which all share the same signaling pathway [3]. Ihh, which is primarily released from the prehypertrophic chondrocytes of growth plate [4], plays an important regulatory role in the proliferation and differentiation of chondrocytes during endochondral bone formation. 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.
To avoid delete Ihh in the embryonic stage, we generated Col2a1-Cre ER T2 ; Ihh / mice, in which Ihh was time selectively ablated from chondrocytes. This unique and powerful genetic animal model provided the in vivo tool to investigate whether early intermediate cartilage scaffold ossi cation contributes to absence of growth plate and dwar sh phenotypes in the postnatal mice lacking Ihh expression, speci cally in chondrocytes. Thus, we are able to observe the growth plate continuously and dynamically at different time points and determine the role of Ihh in postnatal bone growth and remodeling in vivo.
Understanding the role of Ihh during growth plate development will be bene t to the diseases related to abnormal ossi cation and growth plate development.
Random numbers were generated using the standard = RAND ( ) function in Microsoft Excel. In the TM group, Col2a1-Cre ER T2 ; Ihh / mice were intraperitoneal injected with TM (5 μL of 20 mg/mL for 3 consecutive days) to delete Ihh. In the no TM group, mice were injected with 3 doses of solvent (corn oil) as a control. The experimenter could not be blinded to whether the animal was injected with TM or with corn oil due to severe phenotype. Animal were killed at different age and right hindlimbs were harvested immediately after the mice were killed. All study animals were bred at the Experimental Animal Center of Shanxi Medical University. Approval of the animal experiments was obtained from The Institutional Animal Care and Use Committee of Shanxi Medical University.

PCR Analysis for Genotyping
Total DNA were extracted from mouse tail and genotyping of mice was performed by conventional PCR. The quanti cation of DNA was performed by real-time PCR using the SYBR Premix Ex TaqTM kit (Takara, Dalian, China) with the IQ50 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Each reaction was performed in triplicate. The following primers were used: Cre allele; forward, 5′-ATCCGAAAAGAAAACGTTGA-3′, reverse, 5′-ATCCAGGGTACGGATATA GT-3′; Ihh allele; forward, 5′-AGCACCTTTTTTCTCGACTGCCTG-3′, reverse, 5′-TGTTAGGCCGAGAGGGATTTCGTG-3′. Gross Appearance and Radiographical Analysis 8-week-old mice in each group were anesthetized by intraperitoneal injections of ketamine hydrochloride (100 mg/kg body weight) and plain radiographs were taken using a Faxitron X-ray apparatus (Faxitron, Tucson, AZ). After the animals were euthanized, hind limbs were harvested and the length of the femur and tibia were measured.
Micro-CT Analyses 8-week-old mice in each group were sacri ced by cervical dislocation, the tibias of mice were collected. Three-dimensional (3D) reconstructions of the tibial bone from each group were generated from images acquired on vivaCT 80 (SCANCO MEDICAL, Bassersdorf, Switzerland), following recommended guidelines from Bouxsein et al. [23]. For trabecular bone analysis, a region of interest (0.5 mm below the growth plate) were selected for analysis. The following morphometric parameters of bone volume to total volume (BV/TV), trabecular number (Tb. N), trabecularnseparation (Tb. Sp) and trabecular thickness (Tb. Th) were were calculated using builtin software.

Histology
After the animals were euthanized, the hind knee joints from each group were harvested and immersed in 10% formalin for 24 hours at postnatal days 6, 8, 10, 12, 14. The para n sections were prepared through normal procedures. Safranin O staining was performed using 0.5% Safranin-O solution and counterstaining of 0.2% fast green to assess glycosaminoglycan production. Von Kossa staining was performed using 4% silver nitrate solution with counterstaining of 1% fast red solution to evaluate the mineralization of bone [10]. Immunohistochemistry was used to detected PTHrP and BMP-6 using 6 μm thick section.

In Vivo Ectopic Bone Formation in Nude Mice
BALB/c nude mice at 3 weeks of age were purchased from Charles River (China). They were acclimated to our animal facilities for 1 week and then used in experiments. Murine costal chondrocytes were isolated from the ventral parts of the rib cages of 6-day-old mice and cultured in F-12 media with 10% fetal bovine serum (Thermo Fisher Scientifc, Inc., Waltham, MA) as previously described [24,25]. A total of 1 × 10 6 Ihh / or Ihh d/d costal chondrocytes cells were seeded in 200 lL Matrigel (BD Biosciences, San Jose, CA, USA) and implanted subcutaneously into athymic nude mice at 4 weeks of age as previously described (n = 4 per group) [8]. The dynamic bone formation was monitored weekly by Faxitron X-ray (Faxitron, Tucson, AZ).

Quantitative Real-Time PCR Analysis
Total RNA was extracted from cartilage by using the RNeasy Mini Kit (Qiagen, Inc., Valencia, CA) and reversely transcribed into complementary DNA by using the PrimeScript RT-PCR Kit (Takara, Dalian, China). Real-time PCR was conducted utilizing SYBR Premix Ex TaqTM (Takara, Dalian, China) according to the manufacturer's instructions. Levels of gene expression were normalized to Glyceraldehyde 3phosphate dehydrogenase (GAPDH) expression. The speci c primers used for gene expression analysis are listed in Table 1. The cycle threshold value for target gene was measured and calculated by computer software IQ50 (Bio-Rad Laboratories, Hercules, CA, USA). Relative mRNA level was calculated as x = 2 −ΔΔCt , in which ΔΔCt = ΔCt E − ΔCt C, and ΔCt E = Ctexp − Ct18S, and ΔCt C = CtC − Ct18S [26]. Each sample was analyzed in triplicate. Table 1 Information of qPCR primers used in this study

Statistical Analysis
Data for each group were derived from at least three independent samples, and all sampling was repeated thrice in each study group. Data are presented as the mean ± standard deviation. Student's t-test was used to compare the difference between Ihh / and Ihh d/d . The signi cance level was set at α = 0.05.
The Graph Pad Prism 5 software was used for creating statistical graphs.
In our present study, RT-PCR was used for genotyping to con rm disruption of both Ihh alleles and the presence of Col2α1-Cre (Fig. 1a). There was a 392-bp band in wild type, 392-bp and 484-bp bands in the Ihh /heterozygote, and a 484-bp band in the Ihh / homozygote. As a reference, the Cre-recombinant enzyme has a 374-bp band. Our real-time PCR results con rmed a statistically signi cant deletion of Ihh (76.83%) in mutants (Fig. 1b). Col2a1-Cre ER T2 ; Ihh / mutant mice and Ihh / control mice were intraperitoneal injected with tamoxifen (5 μL of 20 mg/mL) for 3 consecutive days at P0. 8 weeks after birth, mutant mice displayed a severe phenotype that restricted their general body size, limbs, and tail, while phalange joint and digit number were not affected (Fig. 2a, b). As expected, radiographic analysis con rmed a complete loss of the growth plate and abnormal growth of the epiphysis in endochondral long bones in mutant after TM was injected (Fig. 2c, d). Mice in the TM group displayed substantial limb shortening, resulting in signi cantly shorter femoral and tibial lengths when compared to the control group (Table 2). Our observations are in accord with previous ndings where Cre recombinase-mediated deletions of Ihh gene expressed in chondrocytes [5].

Characterization of Ihh-De cient Bones
To study the potential effect of Ihh-de cient on bone remodelling and homeostasis, Micro-CT scanning and 3D reconstruction were performed to assess changes in the bone micro-architecture in vivo. Micro-CT 3D images revealed that deleted Ihh in Col2α1-Cre ER T2 ; Ihh / mice had more and disorganized trabecular architecture on epiphysis of tibia compared with the control mice, while a signi cantly decreased trabecular bone mass in mutant mice compared to control littermates (Fig. 2c, d and Fig. 3b, e). We select 0.5 mm below the growth plate as region of interest for trabecular bone analysis. Because of the lack of a growth plate on mutant mice, we had to carefully choose a section area below the distal ends of the mutant tibia for comparisons (Fig. 3b, e). Bone histomorphometric analyses indicated that Ihh d/d mice displayed a signi cantly lower bone volume/tissue volume (BV/TV) and trabecular number (Tb.N) in the distal femur when compared to control mice. The trabecular thickness (Tb.Th) in the distal femur was also lower in Ihh d/d mice than in control mice, but did not reach statistical signi cance. On the other hand, the trabecular spacing (Tb.Sp) was signi cantly higher in mutant mice than in control mice (Fig. 3g).

Accelerated Abnormal Ossi cation Results in No Growth Plate Formation
To investigate the mechanism of deleting Ihh in chondrocytes results in complete loss of growth plate, after injecting TM at P0 to delete Ihh, histological examination of the tibia growth plate was checked at postnatal days 6, 8, 10, 12, and 14. The Safranin O staining results showed proliferating columns of cartilaginous cells in the tibial growth plate of mutant mice was disordered. In the control group, the boundary between primary ossi cation center and secondary ossi cation center was apparent at P10, and the growth plate is composed of three types of highly organized and specialized cartilage. While the columnar structure of chondrocytes were not found in the tibia of mutant mice, the mutant growth plate was primarily composed of irregular hypertrophic chondrocytes. In addition, mutant mice did not form the second ossi cation center, while the control mice had a second ossi cation center at P10 (Fig. 4a)

Deleting Ihh in Chondrocytes can Differentiate into Osteoblasts
To determine whether isolated deleted Ihh chondrocytes can form bone in vivo, we isolated murine costal chondrocytes from the ventral parts of the rib cages of 6-day-old mice and implanted the deleted Ihh chondrocytes into immunocompromised nude mice using a well-characterized subcutaneous ectopic bone formation model (Fig. 5a) [8]. As shown in Fig. 5b, X-ray results con rmed costal chondrocytes alone did not form bone over an 4-week period. However, deleted Ihh chondrocytes induced robust bone formation in vivo. Immunohistochemistry was performed to determine the expression of PTHrP and BMP-6. In the in mutant mice, PTHrP expression in chondrocytes was less than in the control group. In contrast, BMP-6 content was higher in cartilage in the mutant mice than in the control mice (Fig. 6a). Consistent with the immunohistochemical data, real-time PCR revealed a signi cant decrease in PTHrP expression in Ihh-deleted mice, whereas the relative expression of BMP-6 mRNA increased (Fig. 6b).

Discussion
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].
Mice carrying null mutations of the Ihh gene show a severely disrupted growth plate with abnormal chondrocyte proliferation and maturation at embryonic stages [12]. The conditional ablation of Ihh in the chondrocytes by using Col2a1 promoter reveals severe skeletal deformities with loss of a normal growth plate [9]. However, global Ihh knockout mice and Ihh deletion from collagen type 2 alpha1-expressing transgenic mice were perinatal lethal, limiting observations of postnatal growth plate development. To overcome the perinatal lethal due to deletion of Ihh, Lanske have generated tamoxifen-inducible Col2a1-CreER transgenic mice and demonstrate ablation of the Ihh gene from postnatal chondrocytes cause premature closure of the growth plate: disrupted columnar structure of chondrocytes, and the appearance of abnormal maturation of hypertrophic chondrocytes near the articular surface [5]. However, the mechanism of deleting Ihh results in complete loss of growth plate is still unclear.
After injecting TM at P0 to delete Ihh using Col2a1-Cre ER T2 ; Ihh / models, we checked the growth plate of mutant mice at different speci c times continuously and dynamically. The von Kossa staining con rmed that functional knockout of the Ihh gene causes future growth plate cartilage scaffold to mineralize at P8, leading to lack of normal growth plate and secondary ossi cation center. Our in vivo ectopic bone formation model further demonstrated deleting Ihh in chondrocytes can result in an ectopic ossi cation. Recent evidence has emerged demonstrating that at least some of growth plate chondrocytes do not die by apoptosis, but instead transdifferentiate directly into the full osteogenic lineage in developing bone [13][14][15][16][17]. Thus, we hypothesize that Ihh knockout may be associated with premature hypertrophy of chondrocytes and transformed into osteoblasts.
In mammals, the growth plate originates from intermediate cartilage scaffold by the condensation of undifferentiated limb bud mesenchymal cells. Ihh is secreted by pre-hypertrophic chondrocytes in the growth plate, while PTHrP is expressed in periarticular resting cells and proliferating chondrocytes adjacent to the Ihh expressed pre-hypertrophic zone. Ihh and PTHrP signaling pathways form a negative feedback loop that both synchronizes and regulates growth plate activities and endochondral bone growth [18,19]. In endochondral ossi cation, hypertrophic chondrocytes secrete extracellular matrix, which eventually becomes mineralized and causes the elongation of the bone [16]. BMP6 is expressed in hypertrophic chondrocytes in developing endochondral bones [20]. After Hh-dependent lineage speci cation into osteoblasts, BMP signaling may be involved in the transition of the Runx2-positive osteoblast precursor into the Sp7-positive precursor and act downstream of Hh signaling in mammalian osteoblast development. [21]. Therefore, in this study, we established an animal model of Ihh gene knockout to investigate whether or not conditional skeletal dysplasia is caused by the regulation of BMP-6 signaling factor expression by the Ihh-PTHrP signaling axis. Our immunohistochemistry and RT-PCR results showed signi cantly decreased PTHrP expression and signi cantly increased BMP-6 expression. Taken together, these observations suggest that BMP-6 and Ihh-PTHrP signaling collaboratively regulate the growth plate ossi cation and BMP signaling is likely to accelerate hypertrophic chondrocytesosteoblasts differentiation in conditional deleted Ihh mutant mice.
We also used Prx1-Cre; Ihh / mice to investigate the mechanism of deleting Ihh results in complete loss of growth plate. An early closure of the growth plate has been reported by Amano et al. in the Prx1-Cre; Ihh / mice, and they demonstrate that Ihh and PTH1R signaling in limb mesenchyme is essential to regulate digit structure development. 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.  AuthorChecklistE10only.pdf