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. 2012 Jan;23(1):61-6.
doi: 10.1097/SCS.0b013e318240c8c4.

Calvarial Cleidocraniodysplasia-Like Defects With ENU-induced Nell-1 Deficiency

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Free PMC article

Calvarial Cleidocraniodysplasia-Like Defects With ENU-induced Nell-1 Deficiency

Xinli Zhang et al. J Craniofac Surg. .
Free PMC article

Abstract

Nell-1, first identified by its overexpression in synostotic cranial sutures, is a novel osteoinductive growth and differentiation factor. To further define Nell-1's role in craniofacial patterning, we characterized defects of the ENU-induced Nell-1-deficient (END) mice, focusing on both intramembranous and endochondral cranial bones. Results showed that calvarial bones of neonatal END mice were reduced in thickness and density, with a phenotype resembling calvarial cleidocraniodysplasia. In addition, a global reduction in osteoblast markers was observed, including reductions in Runx2, alkaline phosphatase, and osteocalcin. Remarkably, detailed analysis of endochondral bones showed dysplasia as well. The chondrocranium in the END mouse showed enrichment for early, proliferating Sox9⁺ chondrocytes, whereas in contrast markers of chondrocytes maturation were reduced. These data suggest that Nell-1 is an important growth factor for regulation of osteochondral differentiation, by regulating both Runx2 and Sox9 expression within the calvarium. In summary, Nell-1 is required for normal craniofacial membranous and endochondral skeletal development.

Figures

Figure 1
Figure 1
Craniofacial skeletal features of END mice and wildtype mice at newborn stage. (A) The skeletal staining of whole mount neonatal END mouse and wild type littermate showing overall changes of skeletal system (top panel), and the confirmation of lack of mRNA by real time PCR (middle panel) and Nell-1 protein by Western Blot (bottom panel) from the calvaria of END newborns. (B) Lateral view of craniofacial skeleton revealed wider coronal sutures (arrows in black), dysplasia of maxillary bones (arrows in yellow) and of auricular bones (arrows in red) in END mouse in comparison to wild type littermate. Scale bar: 1mm. (C) Lateral and Top view of microCT images of neonatal END and wild type skulls demonstrating the difference in cranial bone formation and sutural patency. The red arrows point to auricular bony capsule and the location of the anterior fontanel of calvaria is marked with red asterisk. END images demonstrate widely patent midline sutures and fontanels. Defective mineralization and bone formation is also noted in the END mice. F: frontal bone; M: mandible; P: parietal bone; iP: interparietal bone; sO: supraoccipital bone. Scale bar: 1mm. (D) The red line drawn through the most prominent points of both parietal bones (top image) indicating a cut-plane with labels d1-d4 (bottom image). The d1 represents sagittal suture width, while d2 through d4 are equal 0.5mm interval measuring points of parietal bone thickness starting from d2. Quantitative analysis of the average width and thickness are depicted graphically with significant differences (*P<0.05 and **P<0.01). (E) Depiction of the Head Measurement angulation measurement (left panel). Head angle (°) measured using the angle between two constructed planes, CVT, line representing the long axis of the cervical vertebrae parallel and MPL, mandibular plane line, line representing the mandibular plane. Quantitatative analysis of the head angulation at newborn stage is significantly greater (*P<0.05) in wild type than that of END mice (right graph).
Figure 2
Figure 2
Histological analysis of defective calvarial bones in END mice at newborn stage. (A and B) H&E coronal sections of wild type and (A’ and B’) END mouse heads at newborn stage. The highlighted area in red box magnifies the parietal bone (arrows) showing a much thinner bone plate in END (B’) than in wild type sample (B). (C and C’) Wild type and (D and D’) END calvarial bones (single arrow) and the osteogenic front of sutures (arrows) revealed significant difference in ALP activity with much weaker staining intensity in END samples. (E) Immunohistochemistry of Ocn in the osteogenic front of sutures and calvarial bones (arrows) in wild type sample demonstrated stronger intensity than (E’) in END calvaria. (F and F’) Nuclear staining of Runx2 in the osteogenic front of sutures (arrows) clearly indicated greater numbers of positive cells in wild type than in END calvaria. Results were quantitated by average pixel numbers positive staining per 400× field. (G) Ocn and (H) Runx2 mRNA level by real time PCR with calvarial bone tissue (**P<0.01). Scale bar: 500um for A and A’; 100um for B and B’; 50um for C,C’,D,D’,F and F’.
Figure 3
Figure 3
Chondrogenic characterization of END mice at newborn stage. (A and B) The close up views of middle ear bone (in blue box) and cartilage in craniobase (in yellow box) in Figure 2A showing increased hypertrophic chondrocytes (arrows) in wild type samples than (A’ and B’) in END mouse in Figure 2A’. (C and C’) The temporal cartilaginous tissue and (D and D’) Meckel’s cartilage were stained much stronger in wild type than in END mouse with Col 10 antibody (arrows). (E and E’) Immunohistochemistry of Sox9 in newborn wildtype cartilage was barely detectable while small number of Sox9 positive chondrocytes (arrows in insert) and perichondral cells was observed with a greater staining intensity in END cranial base. (F and F’) Immunohistochemistry of Runx2 in newborn wildtype cartilage was significantly stronger and have more positive cells (arrows in insert) while only few positive perichondral cells and chondrocytes were observed in END cranial base. Results of Sox9, Runx2, and PCNA immunohistochemistry were quantitated by average pixel numbers positive staining per 400× field. Scale bar: 100um for A, A’,B, and B’; 50um for C,C’,D,D’,E,E’,F and F’.
Figure 4
Figure 4
Runx2 and Sox9 expression in craniofacial bone and cartilage during embryonic development. (A and A’) Wild type embryos at E14.5 have more Runx2 positive cells along calvarial bone plates than in END tissue (arrows). (B and B’) The cartilages in craniobase at E14.5 and (C and C’) E18.5 exhibited more Runx2 positive cells in wild type than in END, particularly some prehypertrophic and hypertrophic chondrocytes at E18.5 in wild type mouse (arrows) were strongly stained with Runx2 as compared to END tissue (arrow). In contrast to Runx2, (D and D’) the temporal cartilaginous tissue and (E and E’) cranial base at E14.5 revealed large number of Sox9 positive cells in both wild type and END tissues (arrows). However, (F and F’) the temporal cartilaginous tissue and (G and G’) cartilage in cranial base at E18.5 in END mouse contained more Sox9 positive chondrocytes than wild type late embryos (arrows), but the staining intensity was significantly less than that at E14.5. Results of Runx2 and Sox9 immunohistochemistry were quantitated by average pixel numbers positive staining per 400× field. Scale bar: 25um for A-G’.
Figure 5
Figure 5
Gene expression profile in END mice compared to wildtype fetuses (E14.5, E16.5) and newborn in calvaria. The level of Nell-1 during the mouse calvarial development was quantitatively detected and further confirmed the knockout nature of Nell-1 in END mice or embryos. The osteoblastic differentiation marker genes including Opn, Bsp, Runx2 and the chondrogenic molecules including Col 2, Aggrecan, Col 10 and Sox9 were also detected at embryonic and neonatal stages. The data represents the mean ± SD from tests performed in triplicates. (*P < 0.05; **P < 0.01, UD refers to undetectable levels).
Figure 6
Figure 6
Schematic diagram of the effects by Nell-1 deficiency in context with alterations of Runx2 and Sox9. The horizontal solid arrows or “T” bar implicate activating or inhibitory effects by Nell-1 deficiency. The vertical solid arrows indicate the up and down gene expression or functional effects. The vertical dotted arrows point to the sequential differentiation. IMB: intramembranous bone formation; ECB: endochondral bone formation.

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