Figure 16.

Adtrp deficiency leads to increased expression of matrix metallopeptidase‐9 (Mmp9) mRNA, protein, and activity levels in zebrafish embryos and newborn mice. A, Whole‐mount in situ hybridization for mmp9 (blue) in 72 hours postfertilization (hpf) control and adtrp zebrafish morphants. Blue arrow: specific signal accumulation. Black arrow: caudal vascular plexus (CVP). n, biological replicates. Bars, 100 μm. B, Immunofluorescence staining with anti‐Mmp‐9 (Cy3, red) IgG on cross‐sectioned CVP (green) of 72‐hpf Tg(fli1:EGFP)y1 (EGFP, fluorescent vascular reporter) control and adtrp morphants±human ADTRP mRNA. Bars, 100 μm. C, Total lysates of 72‐hpf zebrafish embryos incubated ±100 μmol/L of pan‐MMP inhibitor GM6001 for 24 hours and analyzed by 10% gelatin gel electrophoresis. Left: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitometry and statistics (mean±SEM by t test). *P<0.05. Each marker represents a group of at least 10 embryos. D, In situ zymography with MMP fluorescence substrate QXL™ 570‐KPLA‐Nva‐Dap(5‐TAMRA)‐AR‐NH2 (red, dequenched fluorescence) on cross‐sectioned CVP (green) of 48‐hpf Tg(fli1:EGFP)y1 embryos injected with Control or adtrp morpholinos (MOs). Arrows: MMP activity. E, Fluorescence confocal microscopy of 72‐hpf Tg(fli1:EGFP)y1 embryos injected with control or adtrp MOs (left panels; EGFP, fluorescent vascular reporter)±GM6001 as for C. Bars, 100 μm. Quantification of vascular defects (right panels): scatter plots and statistics (1‐way ANOVA with Bonferroni's multicomparison test) of the percentage of defective intersegmental vessels (ISVs) and percentage area of CVP dilation normalized to tail area. Data are mean±SEM of biological replicates (n). ****P<0.0001 between all groups. F, Double immunofluorescence and whole‐mount confocal microscopy with anti‐MMP‐9 (Cy3, red) and either anti‐CD31 or anti‐GR‐1 (FITC, green) in cephalic skin; or with anti‐MMP‐9 (Cy3, red) and anti‐mast cell (MC) Tryptase (FITC, green) in lung of wild‐type (WT) and Adtrp ^−/− P₀ pups. MMP‐9 associates with both CD31 and GR‐1–positive cells in the skin (lower panels, white arrow and inset), and with MC (lung panels, yellow arrows). Bars, 100 μm. G, Zymography (as in C) of conditioned media of primary fibroblasts cultured from P₀ WT and Adtrp ^−/− cephalic skin explants. Upper panel: representative zymogram (grayscale inverted image). Histogram: semiquantitative densitometry and statistics using t test. Data are mean±SEM of biological replicates. ****P<0.0001. Adtrp indicates androgen‐dependent tissue factor pathway inhibitor regulating protein; EGFP, enhanced green fluorescent protein; IgG indicates immunoglobulin G.