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Interactions between Wnt and Vg1 signalling pathways initiate primitive streak formation in the chick embryo

Isaac Skromne^* and Claudio D. Stern^,

Department of Genetics and Development, Columbia University, 701 West 168th Street, New York, NY 10032, USA
^* Present address: Department of Molecular Biology, Moffett Building, Princeton University, Princeton, NJ 08544, USA
Present address: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (111K): [in a new window]   Fig. 1. Comparison of the inducing ability of cVg1, Wnt1 and cVg1+Wnt1 in the area pellucida of stage X-XIII embryos. Cells secreting the factors indicated on the left were grafted into the anterior third of the area pellucida of host embryos, and these were cultured for 24 or 36 hours (right-hand column) before in situ hybridisation (purple) for the genes indicated at the top. Anti-Myc (top row) or anti-HA (middle and bottom rows) immunohistochemistry (brown) was used to identify the cell aggregates and confirm protein synthesis. Red arrows indicate ectopic expression of the gene in the host. The probe used to detect expression of cVg1 in the embryo also hybridises with the cVg1-transfected COS cells in the top and bottom rows of the first column.

View larger version (109K): [in a new window]   Fig. 2. Wnt signalling in the early chick embryo. (A) Expression of Wnt8C at stage XII. Expression is seen all around the marginal zone (MZ) and is strongest posteriorly. Some expression is also seen in the peripheral area opaca. (B) Expression of Wnt5a at stage XIII. Weak expression is detected throughout the outer half of the area opaca (AO), but none in the area pellucida or marginal zone (MZ). (C) Expression of Wnt11 at stage XII. Weak expression is detected throughout the epiblast and hypoblast of the area pellucida and in the epiblast and deep layers of the marginal zone and area opaca. (D) Wnt3a is not expressed at stage XIII or at any other stage that precedes primitive streak formation. Its expression starts to be detected by stages 3+-4 (not shown).

View larger version (150K): [in a new window]   Fig. 3. Expression of cLef1. (A) At stage XI, cLef1 is expressed in Koller’s sickle (ks) and posterior marginal zone (which is partly obscured by the overlying germ wall, see section shown in I). (B) At stage XIII, cLef1 expression continues in the posterior marginal zone and Koller’s sickle. (C) By stage 2+, cLef1 transcripts are restricted to the primitive streak. (D) At stage 3+, expression is restricted to the posterior two-thirds of the primitive streak. (E) In stage 4 embryos, expression accompanies the emerging lateral mesoderm (see section shown in J). F. By stage 4+, cLef1 expression is strong in the primitive ridges and in mesoderm emerging from the streak. (G) At stage 6, cLef1 transcripts are found in the forming paraxial mesoderm and primitive streak. (H) At stage 11, cLef1 expression is seen in the remnants of the streak, the anterior segmental plates and the rostral half of the forming somite. cLef1 is rapidly downregulated from the somites after their formation, but in older (more rostral) somites it appears in the caudal half. cLef1 is also expressed in the heart and brain (see Kengaku et al., 1998). (I) Sagittal section of the embryo in A at the level indicated, showing expression of cLef1 in Koller’s sickle (ks) and in the epiblast of the posterior marginal zone. (J) Transverse section of the stage 4 embryo shown in E at the level indicated, showing expression in the primitive streak and lateral mesoderm.

View larger version (103K): [in a new window]   Fig. 4. Regulation of intracellular ß-catenin and induction of cLef1 by cVg1+Wnt1. (A-D) Subcellular localisation of ß-catenin. In all panels, the green signal corresponds to ß-catenin immunofluorescence and the red signal to propidium-iodide stained nuclei. (A) Throughout the area pellucida, most of the immunoreactivity is concentrated at cell membranes. (B) After grafting cVg1-secreting cells (position outlined by broken line), downregulation of ß-catenin expression is seen near the graft (outlined). (C) A graft of Wnt1-secreting cells (outlined) increases the level of ß-catenin throughout the cell; where it overlaps with the nucleus, the signal appears yellow. (D) When a pellet of Wnt1-secreting cells (indicated on top of left outline) is grafted together with a pellet of cVg1-secreting cells (indicated on top of right outline), the pattern seen is a combination of those in B and C. ß-catenin is upregulated in the proximity of the Wnt1 cells and downregulated near the cVg1 cells. Scale bar: 25 µm for A-D; 5 µm for E-H. Expression of cLef1 after transplantation of cells secreting cVg1 (E,F), Wnt1 (G) or cVg1+Wnt1 (H) in the anterior marginal zone (E) or area pellucida (F-H). (E) cVg1 induces cLef1 when misexpressed in the anterior marginal zone (arrow). Note that induction of cLef1 is restricted to the marginal zone and area opaca. In the area pellucida, neither cVg1 (F) nor Wnt1 (G) induces cLef1, but a combination of the two factors does (arrow, H).

View larger version (116K): [in a new window]   Fig. 5. The broad-spectrum Wnt antagonist Fz-N8 impairs primitive streak formation in stage X (A,B) but not stage XIII (C,D) embryos. (A,B) When cell aggregates secreting Fz-N8 are grafted in the posterior border of the area pellucida of stage X embryos, primitive streak formation is inhibited (A) or seriously impaired (B). After 15 hours’ incubation, cBra (A) and chordin (B) expression are strongly downregulated (white arrowheads, compare with C,D). (C,D) Stage XIII embryos receiving four Fz-N8-secreting cells in the posterior border of the area pellucida develop a normal primitive streak, expressing cBra (C) and chordin (D). (Stage X embryos receiving mock-transfected cell aggregates develop a normal primitive streak identical to those in C,D.) Black outlines indicate the position of the grafted cell aggregates; black arrowheads indicate cBra and chordin expression.

View larger version (81K): [in a new window]   Fig. 6. The competence of the marginal zone to respond to cVg1+Wnt is lost at the primitive streak stage. Cell aggregates secreting cVg1 were grafted alone (A-F) or in combination with Wnt1-secreting cells (G,H) into the anterior margin of stage 2-3 host embryos. After 6 (A-D) or 24 (E-H) hours of incubation, embryos stained for cVg1 (A), cLef1 (B), cNodal (C), cFGF-8 (D), chordin (E,G) or cBra (F,H) by in situ hybridisation. Anti-Myc (A-F) or anti-HA (G,H) immunohistochemistry identifies the cell aggregate and confirms synthesis of the factor. Both cVg1 (A-F) and cVg1+Wnt1 (G,H) are unable to induce any of the markers tested (the dark colour in the grafts in panels A and B is nonspecific).

View larger version (40K): [in a new window]   Fig. 7. Distribution of Vg1 and various Wnt signalling pathway components in amphibian and avian pre-gastrula embryos. (A) Lateral view (animal pole up) of a stage 9 (Nieuwkoop and Faber, 1967) Xenopus embryo. Vg1 protein is localised to the vegetal pole of the embryo (yellow; Tannahill and Melton, 1989), whereas ß-catenin is concentrated in the nucleus of prospective dorsal cells (red; Schneider et al., 1996), even though cLef1/TCF3 is expressed throughout the embryo (blue; Molenaar et al., 1996; Molenaar et al., 1998). Wnt11 transcripts (green; Ku and Melton, 1993) are restricted to the marginal zone, in a dorsal-to-ventral gradient. (B) Ventral view of a stage X (Eyal-Giladi and Kochav, 1976) chick embryo (anterior to the top). Wnt8C expression in the marginal zone describes a gradient highest posteriorly (purple; Fig. 2A; Hume and Dodd, 1993), where cVg1 (yellow; Seleiro et al., 1996; Shah et al., 1997) and cLef1 (blue, Fig. 3) transcripts are also detected. In contrast to Xenopus, chick Wnt11 is ubiquitously expressed at low levels throughout the embryo (green, Fig. 2C). In both species, the region where Vg1 and Wnt activities overlap (the dorsal-vegetal part in Xenopus and the posterior marginal zone in the chick embryo) is where the organiser-inducing centre resides (Bachvarova et al., 1998; Harland and Gerhart, 1997; Heasman, 1997; Moon and Kimelman, 1998).