Fig 1: Expression of syndecans and HS biosynthesis enzymes in human incisor tooth germ during the late bud (7/8 w) (A,E,I,M,Q,U), cap (11/12 w) (B,F,J,N,R,V), and early bell stage (14 w) cervical loop (C,G,K,O,S,W) and inner enamel epithelium area (D,H,L,P,T,X) (immunofluorescence). Note that Sdc1, EXT1, and NDST2 expression domains display almost a mirror image compared with expression domain of NDST1 during the late bud stage (A,M,Q,U). Arrows point to color scatterplots for co-localization of factor (GREEN channel) with 4'6'-diamidino-2-phenylindole (DAPI) nuclear stain (RED channel) for characterization of expression patterns (nuclear, non-nuclear/cytoplasmic/intercellular). Framed areas on color scatterplots for EXT1 (O,P), NDST1 (S), and NDST2 (W,X) point to co-localization (YELLOW, nuclear expression pattern) and thus nuclear expression pattern of these factors in cervical loops and inner enamel epithelium. Examination on maximum magnification showed that this is due to high density of cells in the tissues, and that EXT1, NDST1, and NDST2 in fact display perinuclear expression pattern. Magnification: (A–X) × 40, scale bar: 60 µm. Designations: oe, oral epithelium; dl, dental lamina; tb, tooth bud; m, jaw mesenchyme; oee, outer enamel epithelium; iee, inner enamel epithelium; cl, cervical loop; sr, stellate reticulum; si, stratum intermedium; dp, dental papilla; df, dental follicle.
Fig 2: 3D surface plots and intensity plot profiles for all investigated factors in human incisor tooth germs during the late bud (7/8 w), cap (11/12 w) and early bell stage (14 w) cervical loop and inner enamel epithelium area. Far Left: colorized full-range intensity scale with pixel values and corresponding values in OD units. Arrows point to merged intensity plot profiles of scanned photo-micrographs (image size calibrated in cm) for each investigated factor. Distance/cm (x-axis) represents image height (scanned vertically), intensity in OD (y-axis). Increase in OD value corresponds to decrease in intensity (opposite from pixel value). Intensity plot profiles are informative of staining patterns. Well-limited expression patterns (nuclear, perinuclear, non-diffuse cytoplasmic) display curves with closely aligned spikes (such as EXT1, NDST1, NDST2, and HPSE1 known to be located in ER, Golgi or lysosomes), whereas flat staining (diffuse cytoplasmic, membranar and inter-nuclear expression patterns) produces stretched curves (see Sdc1, Sdc2, and Sdc4 plot profiles).
Fig 3: 2D plot profiling for quantification and correlation of spatial gradients of IF signals from primary antibodies against HS GAG, HS GAG biosynthesis enzymes and common leukocyte antigen (CD45) in histological sections of gingiva from patient with advanced generalized periodontitis (sample: IP-DJD-2CHP). (Magnification: × 10; scale bar: 1000 µm). Spatial gradients of IF signals are plotted simultaneously on a single T-D plot (A) to devise the regression model. The purpose of the model is to disclose to how the presence of inflammatory cell infiltrate correlates with the expression of HS GAG and HS GAG biosynthesis enzymes. The spatial gradient for CD45 IF signal is set as dependent variable (y), whereas the spatial gradients for IF signals from HS 3G10, HS 10E4, EXT1, EXT2, NDST1 and NDST1 are designated as independent variables (x). Based on the regression function, a predicted CD45 spatial gradient is calculated (MODEL CD45) and plotted with the actual values of CD45 spatial gradient on several different plots (B–D) to check for the goodness of fit. The model is statistically significant (a = 1 × 10–8, P < 1 × 10–8) and reveals that the presence of inflammatory infiltrate can be well correlated with the expression of HS GAG and HS GAG biosynthesis enzymes (linear model: R = 0.95576; R2 = 0.91348; polynomial curve fitting: R = 0.96883; R2 = 0.93844). As visualized on the bar-chart of correlation coefficients (E), the deviation of the model (C,D) can be attributed to the low individual correlation of particular independent variables such as HS 3G10 (R = 0.32808; R2 = 0.10763), HS 10E4 (R = 0.02445; R2 = 0.00059), EXT2 (R = 0.02191; R2 = 0.00048) and NDST1 (R = 0.07752; R2 = 0.00601). IF signals of HS 3G10, HS 10E4, EXT2 and NSDT1 have larger expression domains and display different patterns than those of EXT1 and NDST2 (more confined to stromal compartment with CD45) (top row). Thus, the spatial gradients of EXT1, NDST2 display stronger correlation (EXT1–R = 0.52064; R2 = 0.27107) (NDST2–R = 0.55022; R2 = 0.30275) with CD45. (Image created in Adobe Photoshop CC 2014, ver. 6.3; https://www.adobe.com/products/photoshop.html).
Fig 4: Intensity distribution hotmaps for syndecans and HS biosynthesis enzymes in human incisor tooth germ during the late bud (7/8 w) (A,E,I,M,Q,U), cap (11/12 w) (B,F,J,N,R,V), and early bell stage (14 w) cervical loop (C,G,K,O,S,W) and inner enamel epithelium area (D,H,L,P,T,X). Far Left: colorized pixel intensity scale paired with seven corresponding mean values from Kodak No. 3 step calibration tablet. Arrows point to magnified framed areas on hotmaps for Sdc1 (A), Sdc2 (F), Sdc4 (J), EXT1 (P), NDST1 (S), and NDST2 (W) at different stages of tooth development. Magnification: (A–X) × 40, scale bar: 60 µm. Intensity range (pixel value): 15–30 (dark blue), 30–45 (bright blue), 45–55 (dark green), 55–70 (bright green), 70–85 (dark red), 85–95 (bright red), and 95–255 (yellow).
Supplier Page from Abcam for Anti-NDST2 antibody