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Fig. 4. Hip as multifunctional antagonist of Shh, eFgf/Fgf-8 and Wnt-8 signaling pathways. (A) mHip1 inhibits Shh and eFgf pathways in animal cap explants. (a–e) Explants corresponding to NF stage 17. In situ hybridization with Ptc1 as a marker for active Shh signaling. (a, b, c) Control caps and caps either injected with Chordin (50 pg) or Shh (500 pg) mRNA do not express Ptc1. (d) Co-injection of Chordin and Shh mRNA (500 pg) induces Ptc1. (e) mHip1 mRNA (1 ng) suppresses Ptc1 induced by Chordin and Shh. (f–w) Explants stained for Xbra at NF stage 10.5 or stage 17, respectively. (f) Control caps and caps injected with mHip1 mRNA (k, p) do not express Xbra. (g, l) Xbra expression induced by BMP4 mRNA (1 ng) or by activin mRNA (5 pg; i, n) is not blocked by mHip1 in early explants (h, j) but in late explants (m, o). The induction of Xbra by either eFgf mRNA (10 pg; q, u) or Fgf-8 (150 pg; s) is inhibited by the co-injection of mHip1 (r, t, v), but Xbra expression is rescued by Elk mRNA (w, 300 pg). (B) (a–d′) Early gastrula stage embryos (NF stage 10), vegetal view. (a, b, b′) Xbra in situ hybridization and (c, d, d′) immunostaining of phosphorylated MAPK (MAPK-P). (a, c) Control embryos, Xbra and MAPK-P staining around the blastopore. Microinjection of mHip1 mRNA (750 pg) into one cell of a four cell stage embryo inhibits expression of Xbra (b, b′; 72%, n = 25) and phosphorylation of MAPK (d, d′; 67%, n = 15). LacZ mRNA was co-injected as a lineage tracer (light blue). (C) (a, b, e) Second axis formation induced by Wnt-3a (a; 0.5 pg; 84% 2° axis; n = 57) or Wnt-8 mRNA (b; 5 pg; 78% 2° axis, n = 49). Co-injection of mHip1 (600 pg) did not block Wnt-3a (c; 71% 2° axis; n = 32), but Wnt-8 activities (d; 10% 2° axis, n = 39). RT-PCR analysis of animal caps at NF stage 10.5. (f) Induction of Xnr3 and siamois by Wnt-8 (50 pg), but not by Wnt-3a (e; 0.5 pg), was inhibited by mHip1. (D) Comparison of Xhip, Shh, Fgf-8, and Wnt-8 expression. (a, b, c) Lateral views, anterior to the left. (a′, a′, b′, b′) Transversal sections. Shh expression in the ventricular layer of the ventral diencephalon (arrow), while Xhip expression was detected near the sulcus limitans (arrow) in early tadpoles. (a′) Shh expression was found in the chorda dorsalis (red arrowhead) and floorplate (white arrowhead) and (b′) Xhip transcripts only dorsally to the floorplate (black arrowhead). (a, b, c′) Horizontal sections, anterior up. (a) Shh transcripts were detected in the posterior endothelium of the endodermal visceral pouches (black arrow) and Fgf-8 in the anterior endothelium (c′, black arrow), whereas (b) Xhip expression was detected in the adjacent mesenchyme of the branchial arches (arrow). (d, e, f, g) Anterior view of neurula stage embryos. (d) Cleared embryo. Shh transcripts in the chorda dorsalis (white arrow) and prechordal plate (black arrow). (e, f) Xhip expression in the preplacodal region (white arrowhead) limited by Fgf-8 transcripts in the anterior neural ridge (black arrowhead) and in a line outside the neural plate (red arrowhead). (g) Xhip in the lateral placodes is juxtaposed toward Wnt-8 positive cells (arrow, arrowhead). |
