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Role of Goosecoid, Xnot and Wnt antagonists in the maintenance of the notochord genetic programme in Xenopus gastrulae

Laboratoire de Génétique et Physiologie du Développement, Institut de Biologie du Développement de Marseille, CNRS-INSERM-Université de la Méditerrannée-AP de Marseille, Campus de Luminy Case 907, F-13288 Marseille Cedex 9, France
^* Present address: UMR7009, Laboratoire de Biologie du Développement, CNRS-UPMC, Observatoire Océanologique, F-06230 Villefranche-sur-mer, France

View larger version (64K): [in a new window]   Fig. 1. Effect of ventral tBR injection on whole embryos and explanted ventral marginal zones. (A-C) Uninjected embryos. (D-F) Embryos injected ventrally with tBR RNA. (A,D) Dorsal view of tadpoles. (B,E) Transverse sections through tadpoles. (C,F) Immunostaining of the notochord with the monoclonal antibody MZ15 (the otic vesicle is also labelled with MZ15). (G-I) Ventral marginal zone explants differentiate into notochord in the absence of BMP signals. Dorsal (G) or ventral (H,I) marginal zones were isolated at early gastrula stage from uninjected embryos (G,H) or embryos injected ventrally with tBR RNA (I). Explants were cultured until stage 23, and immunostained with MZ15.

View larger version (93K): [in a new window]   Fig. 2. Local repression of BMP and canonical Wnt signalling leads to notochord formation. (A) Lateral view of an embryo injected ventrally with tBR and Lef1HMG RNA. (B) Cross-section through the embryo shown in A. (C) Position of injection for D-G. (D,E) Lateral view of cleared embryos injected with the indicated reagents. (F,G) Cross-sections through embryos injected with the indicated reagents. ß-galactosidase activity was revealed in blue at stage 32.

View larger version (91K): [in a new window]   Fig. 3. Expression of organiser genes is initiated on the ventral side when BMP signals are ectopically blocked, but is not maintained by the late gastrula stage. (A,E,I,M,Q,U) Vegetal view of uninjected mid-gastrulae (stage 11). (B,F,J,N,R,V) Vegetal view of mid-gastrulae injected ventrally with tBR RNA and lacZ RNA as lineage tracer. (C,G,K,O,S,W) Dorsal-vegetal view of uninjected late gastrulae (stage 13). (D,H,L,P,T,X) Dorsal-vegetal view of late gastrulae injected ventrally with tBR RNA. The identity of the probes used is indicated on the left of each row of panels. Note that the expression of all the genes tested is lost at the late gastrula stage, with the exception of chordin (D). ß-galactosidase activity is shown in red, gene expression in purple.

View larger version (39K): [in a new window]   Fig. 4. gsc and Xnot complement inhibition of BMP signals to promote notochord formation. (A-C) Tadpoles injected ventrally with tBR and gsc RNA. (D-F) Embryos injected ventrally with tBR and Xnot RNA. (A,D) External views of injected tadpole. (B,E) Transverse sections through tadpole larvae. (C,F) Lateral views of cleared injected embryos after immunostaining with MZ15.

View larger version (84K): [in a new window]   Fig. 5. Xnot acts as a transcriptional repressor. (A) Schematic of constructs. The homeodomain is shown in blue. The repressor domain (enR) of engrailed is indicated by a dotted box. The VP16 activator domain is represented by a hatched box. (B,C) Cross-sections through stage 32 embryos, which were co-injected ventrally at the four-cell stage with RNA for tBR and either (B) enRXnotHD (25pg) or (C) VP16XnotHD (100pg). (D) Sequence comparison of Xnot (amino acids 26-48) and the active repressing domain (eh-1) of engrailed. Identical amino acids are in dark blue. Conserved amino acids are in light blue. (E-G) Immunostaining with MZ15 of DMZ explants from (E) uninjected embryos, or from (F) embryos injected with VP16XnotHD RNA (200pg) or (G) VP16XnotHD (200pg) plus wild-type Xnot (100pg) RNA.

View larger version (91K): [in a new window]   Fig. 6. Comparative analysis of the expression of Xnot, gsc and frzb during the course of gastrulation. Embryos at the indicated stages were bisected along the dorsoventral axis, and processed for whole-mount in situ hybridisation analyses with the indicated probes. Dorsal is towards the right.

View larger version (66K): [in a new window]   Fig. 7. gsc is able to act in vegetal blastomeres, while Xnot acts exclusively in the marginal zone. (A) Experimental scheme. tBR RNA was injected into ventral blastomeres of four-cell embryos. When embryos reached the 32-cell stage, gsc or Xnot RNA was injected into B4, C4 or D4 blastomeres together with lacZ RNA. (B-G) Cleared tailbud-stage embryos, injected with the indicated combination of factors/blastomeres, and showing the position of the X-gal staining (blue) and of the immunoreactivity for MZ15 (red).

View larger version (114K): [in a new window]   Fig. 8. Regulatory interactions between Xnot, gsc and frzb during notochord formation. Embryos at the indicated stages (left) were injected with the indicated combination of factors (lower right corner of each panel) and processed for in situ hybridisation with the indicated probes (upper right corner of each panel). (A-H) Vegetal views. (I-L) Dorsal or dorso-lateral views. (M-T) Embryos were bisected along the dorsoventral axis before being processed for whole-mount in situ hybridisation. Injected cells are revealed by their ß-galactosidase activity (red). Gene expression is shown in purple.

View larger version (30K): [in a new window]   Fig. 9. A model for notochord formation. BMP signals are blocked in dorsal mesoderm in both somitic (yellow) and notochord (orange) territories. Wnt signals are also blocked in the notochord region, allowing maintenance of Xnot expression. This state is maintained by the antagonistic action of Frzb, secreted from the prechordal plate (red). frzb expression is positively regulated by Gsc.