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Fig. 1. Three-dimensional views of normal frog development. Normal embryos from gastrulation stages are presented in mid-sagittal view. Internal events of gastrulation are clearly visible, including blastopore groove formation (A), archenteron formation (B), archenteron elongation (C) and archenteron inflation (D-F). Mesendoderm migration over the blastocoel roof initiates on the dorsal side (A), and spreads laterally (B) and ventrally (C) before meeting at the animal pole. In a corresponding manner, the blastocoel shrinks and shifts ventrally. (G-I) Three embryos with very similar blastopores, but with significantly different degrees of archenteron elongation. bc, blastocoel; ac, animal cap; dbpl, dorsal blastopore lip; cleft, cleft of Brachet; vbpl, ventral blastopore lip; arc, archenteron; mm, mesodermal mantle; v, ventral; d, dorsal.
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Fig. 12. Archenteron inflation by fluid transfer from the blastocoel. (A-D) Cavities between the blastocoel and the archenteron were visualized with SIM (sagittal section, A), in cleaved embryos (B), and with MRI (sagittal section of intact embryo, C). (D) Sagittal view of the SIM dataset from A rendered to visualize internal spaces, highlighting (red arrowheads) the cavities connecting the blastocoel and archenteron. (E) Transverse volume render of the SIM dataset from A. Cavities are highlighted in red in the tracings shown in d′ and e′. An animation of this volume rendering is presented in Movie 8 in supplementary material.
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Fig. 2. Metrics for quantifying gastrulation. (A) Blastopore closure: ratio of the blastoporal surface area (yellow) to the surface area of the vegetal hemisphere (green) of the embryo. (B) Convergent extension: length-to-width ratio of the Xnot domain (red outline). (C) Spherical approximation to the embryonic surface visualized by reprojecting the 2D image into 3D. White lines in B and C indicate the dorsal midline. (D) Archenteron measurement: distance in degrees between the dorsal lip of the blastopore (blue line) and the anterior limit of the archenteron (white line); degrees of arc between the anterior limit of the Xnot domain (pink line) and the anterior limit of the archenteron (white line); degrees of arc between the dorsal mesendodermal mantle (orange line) and the center of the blastopore. (E) Archenteron inflation: area of the archenteron at the mid-sagittal plane divided by the area of the embryo at that plane.
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Fig. 8. Kinetics of blastopore closure in Xdd1-injected embryos. (A) Kinetics of closure for the embryos shown in Fig. 7A-D. (B) The mean kinetics of closure for eight independent blastopore closure experiments (n=20 control, 12 ventral, 11 dorsal, 16 circumferential). Within each day, and on average, we observed increasingly severe defects in blastopore closure in ventrally, dorsally and circumferentially injected embryos.
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Fig. 3. Effects of Xdd1 on convergent extension and blastopore closure. (A) Convergent extension measurements at mid-gastrulation. (B) Convergent extension measurements at mid-gastrulation (***P<0.001; four experiments; n=113 control, 109 dorsal, 70 circumferential). (C) Blastopore closure measurements at mid-gastrula stage (P<0.001, four experiments; n=113 control, 70 circumferential). (D) Plot of convergent extension versus blastopore closure. Each point repesents one embryo.
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Fig. 4. Marginal zone internalization in Xdd1-expressing embryos. (A) Mid-sagittal plane of SIM dataset of a control embryo. (a′) Section from the embryo shown in A, 30° off mid-sagittal. (B) Mid-sagittal plane of SIM dataset of a dorsally injected Xdd1 embryo. (b′) Section from the embryo shown in B, 30° off from mid-sagittal. (C) Mid-sagittal view of a control embryo hybridized to Xnot (yellow arrowhead indicates the anterior limit of Xnot staining; green arrowheads indicate blastopore lips). (D) Mid-sagittal cleavage of a circumferentially injected Xdd1 embryo hybridized to Xnot. Ridge of internalized tissue (red arrowheads) is Xnot positive.
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Fig. 5. Marginal zone internalization without blastopore closure in Xdd1-expressing embryos. Optical flow analysis of control (A; see Movie 2 in supplementary material) and Xdd1-injected (B; see Movie 3 in supplementary material) embryos. As indicated in the key in panel C, color denotes the direction of flow in a′ and b′ (e.g. yellow lines indicate dorsal movement; blue lines indicate ventral movement). Opposed flow (e.g. yellow meeting blue) represents internalization and is highlighted with white boxes. Cell tracings of Xdd1-injected embryos (D,d′) also reveal the internalization of cells in both the marginal zone and yolk plug.
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Fig. 6. Xdsh is required in ventral/lateral and dorsal tissues for blastopore closure. Plot of blastopore closure in embryos expressing Xdd1 in ventral, dorsal, or all four blastomeres (circumferential) at the four-cell stage (***P<0.001, n=36 control, 30 each dorsal and ventral, 21 circumferential). Note the morphology of the blastoporal boundary in each condition; the affected side is visibly less taut in each case (red arrowheads).
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Fig. 7. Time-lapse analysis of blastopore closure in Xdd1-injected embryos. (A-D) Still frames from time-lapse movies (see Movies 4-7 in supplementary material); control (A), ventral (B), dorsal (C) and circumferential (D) embryos. Ventral, dorsal and circumferential embryos all show defects in closure. Measurements for blastopore size over time are shown in Fig. 8.
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Fig. 9. Archenteron elongation requires Xdsh function during early but not late gastrula stages. Archenteron elongation is disrupted in dorsally and circumferentially injected embryos at mid-gastrula (A) (***P<0.001; n=36 control, 30 dorsal, 21 circumferential) and late-gastrula (***P<0.001; n=38 control, 52 dorsal, 23 circumferential) stages. The fraction of the archenteron (red arrowhead at anterior limit) overlain by the Xnot domain (yellow arrowhead) is very different between control and experimental embryos, demonstrating that the lengthening of the archenteron is proceeding in the absence of convergent extension. (C) Archenteron anterior expansion is not statistically different between Xdd1-injected embryos, at late gastrula stages.
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Fig. 10. Late archenteron formation in Xdd1-injected embryos. (A) Sagittal view of a tailbud stage control embryo imaged using SIM. (a′) Tranverse view of the embryo shown in A. (B) Sagittal view of a tailbud stage Xdd-1 injected embryo imaged using SIM. (b′) Tranverse view of the embryo shown in B. Despite severe disruption of convergent extension in the dorsal axis, the archenteron forms and elongates to cover the entire head-to-tail length.
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Fig. 11. Effects of Xdd1 on archenteron inflation. (A) Archenteron volume is reduced significantly in both dorsally and circumferentially injected embryos (***P<0.001, n=38 control, 50 dorsal, 24 circumferential). In contrast to archenteron length, archenteron volume is significantly reduced in circumferentially injected embryos compared with in dorsally injected embryos (**P<0.01). (B) A plot of archenteron inflation against archenteron elongation shows no obvious correlation between the two processes (each point represents a single embryo).
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Fig. 13. Dissociability of archenteron elongation and blastopore closure. (A) Plot of blastopore closure versus archenteron elongation for 97 normal embryos of various gastrula stages. (B,C) Embryos with highly similar archenterons and very different blastopores, indicated in A with pink points. (D,E) Embryos with highly different archenterons and very similar blastopores, indicated in A with purple points. The anterior limit of the archenteron is indicated with a red arrowhead. Nieuwkoop and Faber standard stages are presented for reference (Nieuwkoop and Faber, 1994), with the size of the circles approximately corresponding to the predicted variability.
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Fig. 14. Uncoupling of blastopore closure and archenteron elongation. (A,a′) Embryos fixed at mid (A) and late (a′) gastrula stages are plotted to show the combined progress in both convergent extension and blastopore closure. (B,b′) Embryos fixed at mid (B) and late (b′) gastrula stages are plotted to show the combined progress in both blastopore closure and archenteron elongation. Xdd1-injected embryos make essentially no progress in convergent extension between the two time points, but progress significantly in both blastopore closure and archenteron elongation.
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