Fig. 4

Roles of the yolk cell, the marginal mesoderm and FGF-R signalling in A-P patterning of the epiblast. Embryos are oriented with the animal pole to the top. (A-C) Transplantation of the yolk cell (YC) in the animal pole region. (A) Schematic representation of the experiment. The recombinants were fixed when the sibling reached 80%-epiboly and examined for otx2 (B) or hoxa-1 (C) expression by in situ hybridization. In the recombinant, otx2 expression domain is reduced into a small patch (arrowhead) in between both YC, while the donor YC induces hoxa-1 expression (red arrows) in the animal pole region of the host. Dotted lines in B indicate the blastoderm margin on both sides. (D-G) Injection of mRNAs encoding dominant-negative FGF receptor (XFD) (E,G) or control (HAVnot) mRNAs (D,F). The gastrula embryos injected at the 1-cell stage were hybridized with otx2 (D,E) or hoxa-1 (F,G) probe, followed by anti-Ntl staining (light brown). Dorsal views are shown. The otx2 expression domain shifts toward the blastoderm margin in XFD-injected embryos. Probably due to an uneven distribution of injected RNAs, the effects of RNA injection were sometimes limited. The posterior shift in the expression boundary is always correlated with the loss of the mesoderm in the blastoderm margin (a loss of the Ntl staining is indicated by a pair of red arrowheads in E). The XFD injection greatly reduces hoxa-1 expression (G). Control RNA injection does not affect these gene expressions in the embryos (D,F). (H-J) Transplantation of the shield into XFD-injected early gastrula (6 h). (H) Schematic representation of the experiment. (I) Lateral view of the host embryo (late gastrula, 10 h) stained with otx2 probe and anti-Ntl antibody. Ectopic (to the left, arrowhead) as well as endogenous (to the right) expression have no clear posterior boundary but extend near the margin. (J) Cross section of the ventral part of the host shown in I. Ectopic expression is induced around the donor-derived notochord (positive for Ntl staining, red arrowhead). (K-M) Transplantation of the ventral-marginal cells into the animal-pole region of the host blastula. (K) Schematic representation of the experiment. The transplanted embryos (late gastrula, 10 h) were stained with otx2 (L, oblique animal-pole view) or hoxa-1 probe (M, animal-pole view) and then the donor cells stained. otx2 expression is suppressed while hoxa-1 expression is induced (arrowheads) around the transplanted donor cells (brown). Scale bars, 100 μm.