Cell fate mediators Notch and Twist in mouse mandibular condylar cartilage

Abstract

Objective: The objectives of this study were to examine if Twist and Notch 1 are present in the mandibular condylar cartilage (MCC) and whether their gene expression can be altered by exogenous FGF-2 and TGF-β2.

Design: Half-heads from CD-1 mice pups harvested at embryonic day 17 (E17) were fixed, decalcified, and sectioned in the sagittal plane for immunohistochemical detection of Notch and Twist using confocal microscopy. Other mandibular condyles and adjacent ramus from E17 mice were cultured in serum-free DMEM containing 0, 3, or 30 ng/mL of FGF-2 (10-12 condyles per treatment group). This experimental design was repeated with medium containing 0, 3, or 30 ng/mL of TGF-β2. After 3 days of culture, the pooled RNA from each group was extracted for examination of Notch and Twist gene expression using quantitative real-time RT-PCR.

Results: Immunohistochemical examination revealed that Notch and Twist were localized to the prechondroblastic and upper chondroblastic layers of the cartilage. Exogenous FGF-2 up-regulated Notch 1, Twist 1 and Twist 2 gene expression in MCC explants from E17 mice, whilst TGF-β2 had the opposite effect.

Conclusions: The gene expression data demonstrate that MCC explants are sensitive to growth factors known to affect Notch and Twist in other tissues. The subset of cells in which Twist and Notch immunoreactivity was found is suggestive of a role for FGF-2 and TGF-β2 as regulators of cell differentiation of the bipotent MCC cell population, consistent with the role of Notch and Twist as downstream mediators of these growth factors in other tissues.

Figure 1

Fig. 1A Immunolocalization of Notch1 in mandibular condylar cartilage (MCC); articular surface is inferior. Cell nuclei are in red, Notch1 in green. Upper left: low power (10X) view showing strong localization of Notch1 to periosteum and perichondrium., upper right: Propidium iodide only; lower left: merged image. Fig. 1B: Higher power (40X) view of condylar cartilage showing primary localization of Notch 1 to prechondroblastic and upper chondroblastic layers of the MCC. Tissue at lower border of image is the articular disc, separated from the articular surface by the inferior joint space.

Figure 1

Fig. 1A Immunolocalization of Notch1 in mandibular condylar cartilage (MCC); articular surface is inferior. Cell nuclei are in red, Notch1 in green. Upper left: low power (10X) view showing strong localization of Notch1 to periosteum and perichondrium., upper right: Propidium iodide only; lower left: merged image. Fig. 1B: Higher power (40X) view of condylar cartilage showing primary localization of Notch 1 to prechondroblastic and upper chondroblastic layers of the MCC. Tissue at lower border of image is the articular disc, separated from the articular surface by the inferior joint space.

Figure 2

Fig. 2A Immunolocalization of Twist in mandibular condylar cartilage (articular surface is at right). Cell nuclei are in red, Twist in green. A low power view showing localization to prechondroblastic and upper chondroblastic layers. Fig. 2B: High power view showing predominantly cytoplasmic reactivity of Twist in MCC cells.

Figure 2

Fig. 2A Immunolocalization of Twist in mandibular condylar cartilage (articular surface is at right). Cell nuclei are in red, Twist in green. A low power view showing localization to prechondroblastic and upper chondroblastic layers. Fig. 2B: High power view showing predominantly cytoplasmic reactivity of Twist in MCC cells.

Figure 3

Fig. 3A: Notch1 expression with increasing concentration of FGF-2; Fig. 3B: Notch1 expression with increasing concentration of TGF-β2. Y-axis shows the relative expression of mRNA levels in experimental and control groups. All calculations were normalized to 18S, and then the control values normalized to 1.0. Each bar represents the mean ± SEM of the RT-PCR in triplicate.

Figure 3

Fig. 3A: Notch1 expression with increasing concentration of FGF-2; Fig. 3B: Notch1 expression with increasing concentration of TGF-β2. Y-axis shows the relative expression of mRNA levels in experimental and control groups. All calculations were normalized to 18S, and then the control values normalized to 1.0. Each bar represents the mean ± SEM of the RT-PCR in triplicate.

Figure 4

Fig. 4A: Twist1 expression with increasing concentration of FGF-2.; Fig. 4B: Twist1 expression with increasing concentration of TGF-β2.

Figure 4

Fig. 4A: Twist1 expression with increasing concentration of FGF-2.; Fig. 4B: Twist1 expression with increasing concentration of TGF-β2.

Figure 5

Fig. 5A: Twist2 expression with increasing concentration of FGF-2.; Fig. 5B: Twist2 expression with increasing concentration of TGF-β2.

Figure 5

Fig. 5A: Twist2 expression with increasing concentration of FGF-2.; Fig. 5B: Twist2 expression with increasing concentration of TGF-β2.