Figure 1.
Photomicrograph of a longitudinal section of 10-day-old rat proximal tibia stained with eosin for manual microdissection.
Superficial (SZ) and intermediate/deep (IDZ) zones of articular cartilage and resting (RZ), proliferative (PZ), and hypertrophic (HZ) zones of growth plate cartilage were isolated with a razor blade. To minimize cross-contamination, a segment of cartilage between zones was discarded. Higher magnification is shown in the middle panel with respective regions delineated by dashed lines. For microarray analysis, only SZ, IDZ, and RZ were used, whereas all zones were used for real-time PCR. In situ hybridization of the articular cartilage SZ marker, Prg4, and the hypertrophic chondrocyte marker, Col10a1 (ColX), was used as a visual guide for manual microdissection. Hybridization was detected using NBT/BCIP substrates (purple) and tissues were counterstained with methyl green.
Figure 2.
Bioinformatic comparison of articular and growth plate cartilage zones.
Bioinformatics was performed on microarray gene expression of SZ and IDZ of articular cartilage and RZ of growth plate cartilage. (a) Principal components analysis scatter plot in three dimensions retaining 68.3% of original sample variation. (b) Dendrogram following unsupervised hierarchical cluster analysis organizing samples by similarity and heat map visualization of gene expression using only genes differentially expressed between SZ and IDZ. Red corresponds to higher gene expression levels represented by z-score. SZ, superficial zone; IDZ, intermediate/deep zone; RZ, resting zone.
Table 1.
Top differentially expressed genes between superficial and intermediate/deep zones of articular cartilage.
Figure 3.
Number of spatially upregulated genes that overlap between articular and growth plate cartilage zones.
Pearson’s chi-square test was used to determine whether an overlap is statistically significant. Significance indicates that an overlap is more or less than expected by chance, whereas non-significance indicates that an overlap is what would be expected by chance. Based on overlaps, SZ is more similar to HZ than other growth plate cartilage zones (b, d–f) and more similar to PZ than RZ (a, c), whereas IDZ is more similar to RZ than other growth plate cartilage zones (g–i, k) and has similarities to both PZ and HZ (j, l). SZ, superficial zone; IDZ, intermediate/deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone; N.S., not significant.
Figure 4.
Spatially upregulated genes that overlap between articular and growth plate cartilage zones.
SZ, superficial zone; IDZ, intermediate/deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone. Left and right panels are photomicrographs of 10-day-old rat articular and growth plate cartilage, respectively, stained with Masson’s Trichrome. Colors: black, nuclei; blue, collagen; red, cytoplasm.
Table 2.
Top signaling pathways implicated by spatially upregulated genes that overlap between superficial and hypertrophic zones as well as intermediate/deep and resting zones.
Table 3.
Expression of growth plate cartilage zonal markers in superficial and intermediate/deep zones of articular cartilage.
Figure 5.
Real-time PCR of spatially upregulated genes that overlap between articular and growth plate cartilage zones.
Relative expression of selected genes that overlap between RZ and IDZ, including Bmp3, Grem1, and Sfrp5 (D–F), PZ and SZ, including Gdf10, Prelp, and Olfml3 (G–I), as well as HZ and SZ, including Adamts1, Mmp13, and Mmp9 (J–L), was determined in proximal tibial epiphyses of 10-day-old rats (n = 4) and values were normalized to 18S rRNA. Accuracy of the microdissection technique was validated by inspecting relative expression of Prg4, Col10a1, and Alpl (A–C). SZ, superficial zone; IDZ, intermediate/deep zone; RZ, resting zone; PZ, proliferative zone; HZ, hypertrophic zone; N.S., not significant; *P<0.05; **P<0.01; ***P<0.001.
Table 4.
Top differentially expressed genes between intermediate/deep zone of articular cartilage and resting zone of growth plate cartilage.
Table 5.
Top signaling pathways implicated by differentially expressed genes between intermediate/deep zone of articular cartilage and resting zone of growth plate cartilage.