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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01178

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Determination of embryonic polarity in a regulative system: evidence for endogenous inhibitors acting sequentially during primitive streak formation in the chick embryo

Federica Bertocchini, Isaac Skromne^*, Lewis Wolpert and Claudio D. Stern

Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK

View larger version (118K): [in a new window]   Fig. 1. Spontaneous initiation of axis formation in isolated embryo fragments. (A-D) When a pre-primitive streak stage embryo is cut in half (separating anterior and posterior halves, A), each half forms a primitive streak. The primitive streak forming in the anterior half expresses brachyury (B,B') and the organizer marker chordin (C,C'), and a normal hypoblast expressing crescent develops and moves away from the new primitive streak (D,D'). (B',C',D') Transverse sections at the levels shown in the adjacent whole mounts. (E-J) Time-course of expression of genes associated with primitive streak formation in the anterior half after cutting. None of the genes is detected 3 hours (E) or 6 hours (F) after cutting; chick Vg1 is the first gene to be expressed, which appears on one side (arrow) of the posterior edge in the isolated anterior half 9 hours after cutting (G) and is still detectable at 12 hours (H, arrow). At this time, Nodal also appears (I, arrow), while the hypoblast, which expresses FGF8, starts to coalesce into a layer (J). In this and all subsequent figures, the probe(s) used for in situ hybridisation are indicated in the lower left-hand corner of each panel. Yellow. epiblasts; green, extra-embryonic area opaca; red, marginal zone; blue, hypoblast; white, endoblast; brown, primitive streak. The original posterior end is shown to the bottom of each panel in all cases.

View larger version (98K): [in a new window]   Fig. 2. Evidence for an inhibitor of primitive streak formation. (A,B) When a pellet of chick Vg1-expressing cells is implanted into the lateral margin of a chick embryo (A), a single primitive streak often forms, arising from the site of grafting and inhibiting the original streak (B). (C-H) Timed implantation of two chick Vg1 pellets (C) allow estimation of the speed of travel of the inhibitor. When implanted 0 hours (D) and 4 hours (E) after the first, the second pellet also induces a primitive streak. When implanted 6 hours (F,H) after the first, the second pellet does not induce a primitive streak. When two axes form, the tips of both primitive streaks express Chordin (E,G), the body of the primitive streak expresses brachyury (D,E) and the hypoblast (expressing Hex) is displaced along the axis of each streak (G,H). p, posterior.

View larger version (41K): [in a new window]   Fig. 3. Vg1 can rescue the ability of the anterior half of an older embryo to form an axis. When an embryo at stage 3 is cut in half, a Vg1 pellet implanted into the isolated anterior half (A) can induce the formation of a primitive streak in this half (B).

View larger version (70K): [in a new window]   Fig. 4. When misexpressed with Vg1, both Chordin and Nodal can bypass the inhibition. A pellet of Vg1 was grafted into the lateral margin of a pre-streak stage embryo; 6 hours later, a second pellet of Vg1 and a pellet of either Chordin or Nodal were implanted together into the opposite side (A). In both cases (B,C) primitive streaks arise from both sides of the embryo.

View larger version (106K): [in a new window]   Fig. 5. A role for FGF in primitive streak formation. In gain-of-function experiments (A-C), misexpression of FGF4 (or FGF8, not shown) initiates the formation of an ectopic primitive streak in intact embryos at pre-streak stages (A,B) as well as at stage 3 (C). The induced axis (arrows) expresses brachyury (A,C) and chordin (B). Loss-of-function experiments: when beads soaked in SU5402 are grafted in the posterior area pellucida (D), primitive streak formation is inhibited (F) or the streak displaced away from the beads (G). By contrast, embryos grafted with control beads develop normally (E).

View larger version (76K): [in a new window]   Fig. 6. Epistasis between FGF, Vg1 and Chordin. When a Vg1 pellet is implanted laterally and an FGF-coated bead grafted on the opposite side 6 hours later (A), FGF induces a primitive streak that bypasses the inhibitor (B). FGF signalling is required for induction of an axis by Vg1: misexpression of Vg1 together with SU5402 (C) does not yield an ectopic axis (D). When Vg1 is implanted laterally, followed 6 hours later by FGF8 beads and a pellet of Chordin-secreting cells on the opposite side (E), the combination of FGF8 and Chordin can bypass the inhibition (F). FGF8 and Chordin can also induce an axis in intact embryos (G,H) [in this case Chordin increases the frequency of ectopic axes with respect to FGF8 misexpressed alone (see text for details)].

View larger version (23K): [in a new window]   Fig. 7. Cell interactions during primitive streak formation. The diagrams show four successive stages in the development of the primitive streak, illustrating the sequential signalling steps and the three proposed inhibitory activities. At stage X, chick Wnt8C is expressed all around the marginal zone and chick Vg1 in its posterior part (PMZ; red), while FGF is expressed in Koller's sickle (lilac) and in the islands of hypoblast (not shown). Vg1 induces an inhibitor (shown as a yellow gradient) that travels through the embryo. At stage XII, the hypoblast (blue) starts to form a layer and secretes the Nodal antagonist Cerberus, while the combined action of Vg1 and Wnt8C induce expression of Nodal in the adjacent epiblast (hatched pattern). As the primitive streak (hatched) starts to form at stage 2 (under the influence of Nodal and Chordin, with additional input from FGF secreted from the adjacent tissues), it expresses Chordin and Nodal. At stage 3 (mid-primitive streak stage) the streak (black) expresses the inhibitor Lefty (green) which further blocks Nodal signalling. Extra-embryonic tissues are shown in white. Note the three sequential inhibitions: an early inhibitor induced by Vg1, followed by Cerberus emitted by the hypoblast and finally Lefty from the primitive streak itself.