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Supplemental Figure 7. DHFR inhibition resulted in disrupted orofacial development at Stage 40 (66 hpf). A. Schematic for experimental design for B-E. B. left: Schematic of facial dimensions measured (top), quantification of face height and intercanthal distance (bottom). right: Morphometric landmarks (top) and transformation grid (bottom) for control vs. MTX treated embryos at St. 40 (66 hpf) (n=37, *p<0.05)., C. Representative images of in situ hybridization for AP-2 in control (left) and MTX- treated (right) embryos. Top: frontal view, bottom: side view. cg= cement gland (n=20). D. Representative cell cycle profiles at st. 40 (66 hpf, top). The average percentages of cells in each fraction from 2 experiments (bottom, n=8 from 2 different experiments). E. top: representative images of immunohistochemistry in control (i) and MTX-treated (ii,iii) embryos for cleaved caspase-3 (red) (n=10, 1 experiment). bottom: immunohistochemistry in control and DHFR MO-injected embryos for cleaved caspase-3 (red). FITC-tagged MO shown in green. (n=10, 2 experiments). |
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Fig. 4. Inhibition of DHFR alters jaw muscle and cartilage, gene expression and total histone levels. (A) Schematic of the experimental design. (B–D) Representative ventral views of embryos labeled with phalloidin to mark muscle. Images are all at the same magnification (scale bar=500 μm), 2 experiments (n=14). (E–G) Representative dorsal views of embryos labeled with Alcian blue to show cartilage. Images are all at the same magnification (scale bar =500 μm), 2 experiments (n=20). (H) in-situ hybridization for neural crest marker, AP-2, frontal views (i, ii) and lateral views with anterior to the left (iii, iv). All images are at the same magnification (scale bar=175 μm). Asterisk designates the region measured and graphed in I. (I) Bar graph showing quantification of the AP-2 domain in the face. There is no statistical significance in 2 experiments (n=12, p value=0.64). (J) Bar graph showing quantification of AP-2 mRNA levels by qRT-PCR. Statistical significance designated by asterisk, 2 experiments. (K) mRNA levels of genes expressed in the face relative to GAPDH. Asterisks designate statistical significance (p values for significance are all≤0.005). (M–N) Immunohistochemistry of H3K4me3 in representative transverse sections of the face (2 experiments, n=10). Images are at the same magnification (scale bar=100 μm). (N) Representative western blot of H3K4me3, total H3 and actin (n=2 experiments). (P) The ratio of H3k4me3 to H3 from 2 experiments is not statistically different (p value=0.589). Abbreviations; gh=Geniohyoideus, ih=Interhyoideus, so=Subarcualis obliquus II, oh=Orbitohyoideus, qh=Quadrato-hyo-angularis, cb=Constrictores branchialium, Mk=Meckel's cartilage, ch=ceraohyal cartilage, bh=basihyal cartilage, pc=parachordal cartilage. |
