Defects in the cellular organization of the notochord and PNBM structure of U0126-treated embryos. In 24 hpf. U0126-treated embryos notochordal cells adhere to the sheath at preferential focal points which results in the bending of the NC (red arrow in panel B). Phalloidin stainings reveal smaller amount of fibrillar actin present between NC cells but not in the overlying neural tube. Notice the large vacuolar cells and the periferial positioning of nuclei in U0126-treated embryos (panels C, D). By 48 hpf. the NC of U0126-treated embryos with milder phenotypes contains smaller rounded cells which become apoptotic (yellow arrows in panel F and see also in Fig. 3/X). Embryos with stronger phenotypes have large bulges protruding where the flat sheath cells surrounding the large vacuolated cells are missing (see blue arrows in panel E for normal sheath cells and in panel H marking the last sheath cells surrounding the bulge; red arrows mark the region devoid of sheath cells). Laminin expression and ultrastructure of the inner layer of the PNBM (marked with blue arrowheads) composed mainly of laminins are normal in NC treated embryos as demonstrated by whole-mount immunhistochemistry (I, J) and TEM pictures (K, L). The middle (green bracket) and outer (yellow bracket) layers of the PNBM composed mainly of circumferential and longitudinal collagen fibers respectively show decreased length and/or disorganization in the orientation of fibers (K, L). Panels A, C, E, G, I, K: wild type embryos. Panels B, D, F, H, J, L: U0126-treated embryos.
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