Fig. 8
Defective angiogenesis, cilia, Hh signaling and ECM in Adamts9Gt/Gt yolk sac. a E12.5 Adamts9Gt/Gt embryos have poorly formed yolk sac vasculature. White arrows point to vitelline vessel branch points. b En-face images of isolectin-B4-stained (green) whole-mount yolk sacs show abnormal vasculature in Adamts9Gt/Gt yolk sacs. c, d RNA in-situ hybridization shows robust Adamts9 expression (red) in E11.5 mesoderm (ME) but not yolk sac endoderm (En). Adamts20 (d) is not expressed in the yolk sac. e Primary cilia and centrosomes visualized by acetylated α-tubulin (green) and γ-tubulin (red) respectively, or SEM (f, arrowheads) show short bulbous-tipped primary cilia in Adamts9Gt/Gt yolk sac mesothelium. g Quantitation of primary cilium length by SEM in wild-type and Adamts9Gt/Gt yolk sac mesothelium (N = 3 yolk sacs from each genotype, ***p < 0.001, Student’s t test). h In-situ hybridization for Gli1, Gli2, and Ptch1 mRNA in E11.5 yolk sacs (red) shows absent hedgehog signaling in Adamts9Gt/Gt yolk sacs (N = 3 yolk sacs from each genotype). i E9.5 and E11.5 Adamts9Gt/Gt yolk sacs were stained for versican, cleaved versican (DPEAAE), hyaluronan (HAbp), fibronectin, and collagen-IV, and counterstained with Alexa-568 phalloidin (red) to outline cell layers. Versican, hyaluronan and fibronectin accumulated in yolk sac mesoderm whereas staining for versican neo-epitope DPEAAE was lost in the Adamts9Gt/Gt yolk sac. Collagen IV staining was unaffected (N = 3 yolk sacs from each genotype). j Toluidine blue staining of 1 μm plastic-embedded sections of E11.5 yolk sacs show a thicker mesoderm in the mutant resulting from expanded ECM. k TEM images of the yolk sac show increased ECM between mesoderm cells (red arrows) in the Adamts9Gt/Gt yolk sac, primarily noncollagenous in appearance. l qRT-PCR analysis for Vcan, Has2 and Fn1 shows comparable levels in mutant and wild-type yolk sac (N = 3 yolk sacs from each genotype, Student’s t test). Scale bars = 2 mm in a, 100 μm in b, 50 μm in c, d and h, i, 2 μm in e, f, 10 μm in j, k, and 1 μm in the lower panels of k. Dotplot shows mean and SD. In box and whisker plot, whiskers indicate the range and boxes mark upper and lower quartiles. The centerline indicates the mean
