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Fig. 1. (A) Consensus points between the traditional and revised views of amphibian axial patterning. Both views concur that the egg (A) is radially symmetric about its animal/vegetal (An/Vg) axis, and that (B) fertilization establishes two axes, by initiating cortical rotation (CR). The sperm entry point (SEP) organizes the cytoskeleton for the rotation, which results in the animal hemisphere being lightly colored on the side opposite the SEP after CR. The SEP serves as one pole of an axis and on the opposite side of the embryo, organizers and signaling centers will form later in development. This axis is disputed by the two views. In the traditional view, it is the ventral-to-dorsal axis but in the revised view, it is the caudal-to-rostral axis. The second axis established by CR is the left/right (L/R) axis. The left side of the embryo is shown in all panels. (C) Both views agree on the arrangement of the germ layers. Ectoderm (blue) maps to the animal region, mesoderm (red) maps to the equatorial region and endoderm (yellow) maps to the vegetal region.
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Fig. 2. The traditional axis orientation and fate maps, and model of mesoderm patterning. (A) The traditional view holds that CR establishes the dorsal/ventral (D/V) axis across the equator of the embryo. The midline bisecting the lightly pigmented side is the future dorsal midline (DML) of the embryo. (B, C) A traditional model of mesodermal patterning, the 4-signal model, which is used to explain axial patterning. The R/C axis is not assigned in the traditional view until neurulation. Four signals pattern the mesoderm along the D/V axis. Signal 1 from the vegetal cells induces the overlying cells to form ventral mesoderm in the marginal zone. Signal 2 from the dorsal vegetal cells induces dorsal mesoderm/Spemann's organizer (SO) in the overlying dorsal marginal zone (DMZ). Signal 3 from the DMZ converts ventral mesoderm to form somites and cells slightly further away to form pronephros. Signal 3 does not reach the ventral marginal zone (VMZ), which forms blood. BMP antagonists from Spemann's organizer carry out this function, known as orsalization Ventral mesoderm (vm) in the ventral and lateral marginal zone emit signal 4, which limits the size of Spemann's organizer. Signal 4 is believed to be BMP4. As a result of these four signals, the embryo is patterned as shown in D. D. A highly schematic diagram of the traditional fate map of the gastrula. The mesoderm in the marginal zone is arranged in a dorsal-to-ventral projection running from Spemann's organizer in the DMZ to blood in the VMZ. Notochord (N) is dorsalmost, with somites (S) adjacent, followed by the pronephros (P). Blood (B) occupies the ventral marginal zone. Ectoderm is divided into prospective epidermal (epi) and neural fields.
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Fig. 3. The revised axis orientation and fate maps, and nomenclature for the embryo. (A) The revised view holds that CR establishes the rostral/caudal (R/C) axis across the horizontal axis of the embryo. The D/V axis is reassigned to the animal/vegetal axis. The rostral midline (RML) forms opposite the SEP. (B) Mesoderm in the marginal zone is arranged with dorsal mesoderm (notochord in brick, somites in red) situated animally and ventral mesoderm (the lateral plate in pink includes head, heart, pronephros, blood and more) situated vegetally. The rostral end of each tissue or germ layer is indicated by either a blue or white asterisk. The caudal end of each tissue or germ layer is indicated by a yellow asterisk. All rostral tissues are situated near the prime meridian and all caudal tissues are distal to the prime meridian. (C) The revised nomenclature for the embryo. The DMZ occupies the animal marginal zone, while the VMZ occupies the vegetal marginal zone. The rostral marginal zone (RMZ) includes Spemann's organizer and the rostral lip of the blastopore, called the upper lip by Spemann. The caudal marginal zone (CMZ) occupies the opposite side and includes the caudal lip of the blastopore, called the lower lip by Spemann. The prime meridian (pm) equals the rostral midline and bisects Spemann's organizer.
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Fig. 4. The prime meridian is specified to form the head. Embryos were dissected at st. 8 into fragments from the animal pole to the vegetal pole that either include or exclude the prime meridian. The fragments were cultured to approximately st. 28, unless otherwise specified, and immunostained for notochord and somites, except for the final image in panel F, which shows an unstained fragment at control st. 36. The fragments are diagrammed from a vegetal pole view. All fragments that include the prime meridian form a head and those that exclude the prime meridian do not. (A) A 70fragment (70 UL) centered on the prime meridian forms a head with notochord and somites, while the corresponding 290fragment without a prime meridian forms a properly patterned trunk/tail body plan without a head. (C) In a second experiment, embryos were cut along symmetrical meridians at decreasing distance from the prime meridian. The resulting body plans show that both dorsal and ventral tissues are truncated, and that the closer cuts are made to the sperm entry meridian, the more caudally complete is the body plan. In panel F, the head that forms from the 90upper lip fragment is shown at st. 28 and 36. The head is fairly complete but is missing the gill pouches. Abbreviations: LL, lower lip; UL, upper lip.
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Fig. 5. Separation of dorsal and ventral blastomere pairs at the four-cell stage results in rostral (head + trunk) and caudal (trunk + tail) embryo fragments. (A) The 4-cell embryo, depicted in the traditional view. The embryo consists of dorsal and ventral blastomere pairs, which are separated from one another, and cultured in isolation. The results of Kageura and Yamana (B + C) and Cooke and Webber (D + E) are shown. Both groups observed trunk + tail body plans from the ventral blastomere pair (B + D) and head + trunk body plans from the dorsal blastomere pair.
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Fig. 6. Re-analysis of Keller's gastrula-to-tailbud fate map demonstrates a rostral-to-caudal progression running from the prime meridian to the sperm entry point. The pertinent blastomeres, C14, are indicated in different colors on the diagram of a st. 6 embryo in A. C1 is orange; C2 is red; C3 is purple and C4 is blue. These four blastomeres form much of the marginal zone of an early gastrula, the stage mapped by Keller. In panel B, the lines of cells Keller marked are indicated by arrows of different shades of the four colors used for the st. 6 embryo in panel A, except that the grey, yellow and orange lines all descend primarily from blastomere C1. The polarity of the marked lines of cell is indicated by the arrows. The arrowheads mark the prospective ventral mesoderm, which crawls along the roof of the blastocoel before differentiating as ventral mesoderm. In panel C, the emerging dorsal midline, which forms as the blastopore (bp) closes over the yolk plug, is indicated by a dotted line. The blastopore has crossed the vegetal pole but remains open. Large dotted and dashed lines indicate the emerging dorsal and ventral midlines of the body plan, respectively. In panel D, the blastopore has closed near the former lower lip and the dorsal midline, indicated by the dotted line, is much longer than the ventral midline because dorsal mesoderm and neural tissues are elongating due to strong convergence extension movements. The arrowheads in the ventral mesoderm are deleted to simplify the diagrams. In panel E, the contributions from the marginal zone to the ventral blood islands (vbi) are arranged in a rostral-to-caudal progression from the C1-to-C4 blastomeres. The approximate position of the first 3 head somites is shown. They descend from cells derived from blastomeres C1 and C2. The formation of somites is complex and the diagram is meant to indicate only rostral/caudal contributions, not dorsal/ventral somite origins. In panel F, the rostral/caudal contributions from the four C blastomeres are shown in the somites of a st. 41 tadpole. Cells from the marginal zone contribute to somites in a rostral-to-caudal progression that corresponds to the order C1, C2, C3 and finally C4. See text for further explanation.
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Fig. 7. Reinterpretation of the role of organizer-derived BMP antagonists and Spemann and Mangold's grafting experiment. (A) Experimental design for lineage tracing the fate of normal and noggin-expressing C4 blastomere progeny. (B) St. 6 embryos were injected with lineage tracer (black cells, embryo on left) or tracer plus noggin mRNA (black cells, embryo on right) into one C4 blastomere and cultured to st. 280. Normal C4 progeny populate caudal structures, both dorsal (somites) and ventral, much of which will end up in the tail of the tadpole by st. 41. C4 progeny expressing noggin ectopically form a secondary axis and contribute to both dorsal and ventral tissues. However, labeled cells form rostral levels of the secondary axis and continue into the tail, because of mediolateral intercalation. The presence of cells at rostral positions of the secondary axis indicates that cells have been incorporated into axial structures precociously, and hence are ostralizedrather than orsalized (C) Formation of an ectopic, secondary axis following an organizer-derived graft. C4 progeny exposed to an organizer graft form axial structures precociously, and start a second site of mediolateral intercalation (Lane et al., 2004). Thus, two sets of trunk structures are initiated and merge into a single tail, but only the endogenous axis forms a head.
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Fig. 8. Three chordate fate maps. In the ascidian fate map (A), and the amphioxus fate map (B), the R/C axis runs across the equator from the notochord field (brick red) to the muscle/somite field (red). Ventral mesoderm in shown in pink, and neural tissue is shown in blue. In the Xenopus fate map (C), this dimension has historically been called D/V, but new mapping data indicates it is the previously unassigned R/C axis. Similarities in the three maps are discussed in the text.
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