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Fig. 4. Analysis of BMP signaling and Chordin protein localization in embryos depleted of Tolloid (Tld MO) or Sizzled (Szl MO). All embryos were siblings allowed to develop for the same period; all images were processed identically. (A–F) pSmad1/5/8 (Upper) and Chordin (Lower) immunostainings of sagittal optical sections. The gradient of BMP activity was complementary to that of Chordin localization in WT embryos (Left, n = 6 and n = 4, respectively). When translation of the Chordin-degrading enzyme Tolloid was inhibited in Tld MO-injected embryos (Center, n = 5 and n = 3, respectively), BMP activity was decreased on the dorsal side and increased accumulation of Chordin was observed in Brachet’s cleft (arrowheads). Conversely, when Sizzled was depleted (Szl MO), BMP activity (nuclear pSmad1/5/8) was greatly increased in the embryo, and Chordin failed to accumulate in the Brachet’s cleft (Right, n = 3). Diffuse accumulation of Chordin in dorsal ectoderm is indicated by the arrow. (G–L) pSmad1/5/8 (Upper) and Chordin (Lower) immunostainings of embryos sectioned transversely through the animal–vegetal axis at late gastrula (stage 12). pSmad1/5/8 staining was decreased in Tld MO embryos (H, n = 6) and increased in Szl MO embryos (I, n = 4) compared with WT (G, n = 6). Tld MO increased (K, n = 7) and Szl MO decreased (L, n = 5) Chordin staining in Brachet’s cleft compared with WT (J, n = 7). Image published in: Plouhinec JL et al. (2013) Copyright © 2013. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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