Fig. 6

Effects of overexpression of pitx2. (A) Induction of ectopic cement gland and mesenchymal tissue by microinjection of pitx2 RNA into ventral blastomeres. Uninjected stage 34 embryo (top) and two siblings injected with 300 pg of pitx2b RNA into one ventral cell at the four-cell stage. Lumps of tissue containing pigmented and secretory cement gland tissue (cg) are visible in ventral/posterior regions. (B) Stage 41 embryos injected with 200 pg of pitx2b into the two dorsal vegetal cells at the eight-cell stage (uninjected embryo at top). Anterior and dorsal axial structures are absent or greatly reduced, while some additional tail-like structures have formed. (C) Embryos exposed to ultraviolet irradiation at the one-cell stage. Injection of 200 pg of pitx2c RNA into one cell at the four-cell stage induced formation of a cement gland and tail structures but did not rescue dorsal axial structures. The lower embryo was irradiated but not injected. (D) Animal cap explants prepared from stage 9 embryos and cultured until stage 20. The cap on the right was prepared from an embryo injected at the four-cell stage with 50 pg of pitx2c RNA into the animal pole of each blastomere. Extensive cement gland tissue was induced by pitx2c, and a mass of large, yolky, nonadhesive cells (y) is also present. A control explant, which has developed epithelial characteristics, is shown on the left. (E–L) pitx2b RNA (200 pg) was injected into one or two blastomeres at the four- or eight-cell stage and embryos were fixed for whole-mount in situ hybridization at stage 10.5 or 11. (E, F) chordin expression in the organizer region of uninjected (E) and pitx2b-injected (F) embryos at stage 10.5. chordin expression is strongly reduced following injection of pitx2 into the two dorsal vegetal blastomeres at the eight-cell stage. (G, H) Vegetal views of xbra expression in the presumptive mesoderm of an uninjected (G) and pitx2b-injected (H) embryo at stage 10.5. Following injection of pitx2b RNA into the two dorsal vegetal blastomeres at the eight-cell stage, half the Xbra expression has been abolished. (I, J) Lateral views of sox-17β expression in the endoderm of an uninjected (I) and a pitx2-injected (J) embryo at stage 11. sox-17β is expressed ectopically following injection of pitx2 into one blastomere at the four-cell stage. (K, L) Vegetal views of goosecoid expression in uninjected embryo (K) or following injection of pitx2 RNA into the two ventral vegetal blastomeres at the eight-cell stage. Ectopic patches of goosecoid expression are visible in the vegetal and marginal regions.