Ruiz i Altaba A , Cox C , Jessell TM , Klar A .

	FIG. 1. Comparison of the frog and rat F-spondin proteins. (Upper) Schematic representation of the domain structure of F-spondin protein. The black box represents the signal sequence. The gray boxes represent the six thrombospondin type I repeats (TSRs). (Lower) Amino acid alignment of the frog and rat F-spondin proteins. Underlined NH2-terminal sequences correspond to the putative signal sequences. Bars above the sequence delineate the TSRs, numbered 1-6 as in the schematic. The overall similarity of the frog and rat F-spondin proteins is 84%. The NH2-terminal domain of the proteins, which does not contain the TSRs, exhibits 86% identity, indicating that this do- main may have a role in the structure or function of the protein. Apart from the signal sequence, the only region of extensive sequence divergence is the highly charged segment between TSRs 5 and 6. Numbers at
	FIG. 2. Localization of F-spondin mRNA in frog embryos. (A) Whole-mount in situ hybridization of a X. laevis tadpole, stage 34-36, showing F-spondin RNA in the floor plate at the ventral midline of the neural tube and in cells of the hypochord. Note the higher F-spondin expression in hindbrain compared with spinal cord. (B) Whole-mount in situ hybridization of an exogastrulated embryo at a stage equivalent to that in A, showing that F-spondin RNA is not present in the ectoderm (ec). In contrast, F-spondin RNA is detected in cells of the hypochord found adjacent to the notochord (n). en, Endoderm; me, mesoderm. (C and D) High magnification of midbrain-hindbrain region of tadpole (stage 36) labeled in whole-mount, showing F-spondin RNA in the floor plate (fp) of the hindbrain and in the hypochord (h), located under the notochord (n). There is also F-spondin RNA in the lateral region of the midbrain. In contrast to the expression in rat embryos (17), in frogs F-spondin is not expressed by the most anterior floor plate cells located in the midbrain and there is a clear boundary of expression in the floor plate at the boundary of the midbrain (mb) and the hindbrain (hb) (arrowhead in D). F-spondin is expressed at low levels in the posterior part of the notochord and in branchial arches (data not shown). (E and F) Cross sections of the hindbrain of tadpoles (stage 36) after whole-mount in situ hybridization. F-spondin RNA is restricted to the floor plate (fp) at the ventral midline. In F, F-spondin RNA is detected in the hypochord (h), an embryonic endodermal structure located ventrally to the notochord (n). s, Somites. (G) Dorsal view of the anterior hindbrain of a tailbud-stage (stage 22) embryo after whole-mount in situ labeling, showing early expression of F-spondin by small groups of cells at the ventral midline of the neural tube. (H and I) Whole-mount in situ hybridization oflate blastula (stage 9) (H) and late gastula (stage 12.5) (I) embryos. (H) Vegetal view showing normal expression ofPintallavis in the organizer region, located dorsally, and the injected RNA (arrowhead). d, Dorsal; v, ventral. (I) Dorsal view of an embryo injected with Pintallavis RNA, showing normal expression ofPintallavis in the midline. a, Anterior; p, posterior. (J) Cross section of a tadpole (stage 36) injected with RLDx into one cell at the two-cell stage, showing unilateral restriction of the injected tracer. Note the labeling of decussated axons in the ventral funiculus of the unlabeled half. [Bar = 600 ,m (A and B), 150 ,um (C), 30 pm (D), 40 ,pm (E and J), 20 ,um (E and G), or 200 pm (H and I).]
	FIG. 3. Localization of F-spondin mRNA in Pintallavis mRNA-injected tadpoles. (A-G) Cross sections of the hindbrain of tadpole stage embryos injected with Pintallavis RNA at the two-cell stage, showing the localization of F-spondin RNA by whole-mount in situ hybridization. F-spondin RNA is expressed in floor plate (fp) cells at the ventral midline of the neural tube. Arrowheads depict the sites of ectopic F-spondin RNA expression. Embryos shown in A, B, D, and F show ectopic F-spondin RNA in cells adjacent to the dorsal midline, which form part of the roof plate. In B and D the dorsal cells were displaced slightly during the labeling procedure. The embryo shown in B was pigmented. Embryos shown in C, E, and G show an expanded area of F-spondin expression adjacent to the floor plate. C shows F-spondin RNA expression in hypochord cells under the notochord (n). As previously shown (8), injection of Pintallavis RNA leads to an increase in the amount of posterior neural tissue in affected embryos. C and E show enlarged neural tubes (arrows) and (in C) a secondary neurocoel. (H and I) Lateral view of tadpoles (stage 36) injected with Pinn tallavis RNA after whole-mount in situ localization of F-spondin RNA. Arrowheads point to the sites of ectopic F-spondin expression. The embryo in H faces right and that in I faces left. In all cases, dorsal side is up. [Bar = 10 ,um (A-G) or 200 Zm (H and I).]

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