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Fig. 7. Suppression of Xhip function by antisense-morpholino injection resulted in loss of lens and olfactory placode formation. Whole mount in situ hybridization of Xhip-morpholino (a, f, j, n) and control-morpholino (e, i, m, q) injected embryos at NF stage 36. (a, f, j, n) Lateral anterior view of the non-injected side. (c, e) Frontal view, injected side to the right. (d, h, l) Horizontal vibratome sections of the corresponding embryos. In control-morpholino (5 pmol) injected embryos LacZ mRNA was co-injected as a lineage tracer (light blue). (a) Expression of Krox-20, En-2 and Emx2 was detected simultaneously. (b, c, d) Xhip-morpholino injection into one cell of a two cell-stage embryo (2.5 pmol/blastomere) did suppress the expression of Emx2 in the olfactory placode and branchial arches (74%, n = 31; red arrows), and that of Xpitx3 in the lens ectoderm (65%, n = 40; red arrow in g, h). (k, l) The expression level of Rx1 in the remaining eye cup was not altered, although the morphology of the eye cup was perturbed and the retinal pigment epithelium was not formed (68%, n = 38; d, h, l). (n) Xhip-morpholino injection did not suppress otic vesicle formation as shown by Pax2 expression, but RPE formation (100%, n = 47; red arrows in o, p). (r) Increasing amounts of mHip1 mRNA (50 or 100 pg) injected together with Xhip morpholino rescued the formation of lens and olfactory placodes. (s) Summary diagram of interactions of Hip, Hh, Wnt-8, Fgf-8 and activin. The arrows do not suggest direct interactions; for details, see Discussion. Abbreviations: ba, branchial arches; ey, eye; is, injected site; le, lens; mhb, midbrainindbrain boundary; nis, non-injected side; olp, olfactory placode; op, otic vesicle; os, optic stalk; r3/5, rhombomere 3/5; ret, retina; rpe, retinal pigment epithelium; tel, telencephalon. |
