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FIG. 2. Fate of column 1 blastomeres. At st. 6, one of each of the column 1 blastomeres was injected with rhodamine dextran and one of
each of the column 4 blastomeres was injected with fluorescein dextran. (A) Diagram of the lineage labeling scheme. (B, C) Distributions
of rhodamine dextran and fluorescein dextran, respectively, at st. 41. Arrows in B refer to the positions of the sections shown below in E.
(D) Colocalization of both dextrans. The arrow marks anterior, ventral structures, which, by definition, are not dorsal structures. Essentially
all head structures originate from column 1 blastomeres. (E) Colocalization of column 1 and 4 progeny in sectioned, st. 41 embryo. Section
1 is posteriormost and section 6 is anteriormost. See text for discussion. The asterisk indicates the notochord/floor plate/central somite
domain, which is labeled throughout the embryo by column 1 blastomeres. Abbreviations used: cg, cement gland; ep, epidermis; fg, foregut;
h, heart; nt, neural tube/brain; ov, otic vesicle; pn, pronephros; rt, retina; s, somite.
FIG. 3. Surgical dissection of early gastrulae and assessment of surgical accuracy. (A) Diagram of the surgery at st. 101, viewed from the
vegetal pole, and nomenclature of the six fragment types to be analyzed by specification testing. The prime meridian is shown in red.
Incisions run along meridians from the animal to the vegetal pole, deep to the central axis, separating embryos into 90° and corresponding
270° sectors. ULS, upper lip sector; LtLS, lateral lip sector; LoLS, lower lip sector. (B) Double in situ hybridization at st. 101 for chordin
(blue), expressed at the upper lip, and Xwnt8 (purple), expressed in nonorganizer meridians of the marginal zone. (C) Northern blot analysis
of chordin and Xwnt8 as an indication of surgical accuracy. Total RNA was extracted from eight embryos or fragments; two embryo or
fragment equivalents were analyzed per lane. Intact controls (lane 1), 90° ULS (lane 2), 270° LoLS (lane 3), 90° LoLS (lane 4), and 270° ULS
(lane 5) were probed for chordin and Xwnt8. cytoskeletal actin was probed as a loading control.
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FIG. 1. Summary diagrams for two amphibian fate maps and diagrams of inversion of the marginal zone, including orientations of the
anterior/posterior and dorsal/ventral axes of the tadpole. Early embryos (A, B) or marginal zones (C–E) shown are oriented by convention
with their upper blastoporal lips on the lower right side. If this orientation could be held constant during morphogenesis by fixing the site
of blastopore closure (X) and the animal pole (1), the resulting embryo would be oriented as shown in F. Tracing the inversion of the
marginal zone through its morphogenetic movements should facilitate understanding how the prime meridian becomes the embryo’s
anterior midline. (A) Axolotl late blastula-to-tailbud fate map (Vogt, 1929), constructed by vital dye mapping technique. The dorsal/ventral
axis runs horizontally from the side on which the blastopore lip first forms (J, Spemann’s upper lip, Morgan’s dorsal lip) to the side on which
the lip forms last (Spemann’s lower lip, Morgan’s ventral lip). The notochord is shown in heavy stippling on the dorsal side. The first 10
somites are indicated by numbers, the remainder of the prospective somite field is faintly stippled (Sch). Translations for other
abbreviations include Eg, the limit of involution, and Spl, lateral plate mesoderm. In the animal region, the neural field is indicated by heavy
dashed lines and the epidermal field by faint dashed lines. (B) Revised fate map for Xenopus laevis mesoderm, early blastula-to-tadpole (st.
6–41), constructed by lineage labeling (Lane and Smith, 1999). The anterior/posterior axis runs horizontally from the upper lip to the lower
lip. The dorsal/ventral axis for the mesoderm (as well as the endoderm) runs from animal to vegetal. Two landmarks used by Vogt and Keller
are indicated on the new Xenopus map: X marks the approximate site at which the blastopore lips close at st. 13; 1 marks the animal pole,
which ultimately forms epidermis covering the heart. Notochord, N, and somites, S. (C) The marginal zone at st. 10. Anterior leading edge
mesoderm (which will give rise to the anterior blood islands, head mesoderm, heart mesoderm, and liver) has begun its migration toward
the animal pole, and dorsal mesoderm located just animal to the anterior leading edge mesoderm is displaced slightly toward the vegetal
pole. (D) The marginal zone at st. 11. The leading edge mesoderm (LEM) through all 360° is internalized and migrating toward the animal
pole, with anterior LEM advanced the farthest. Convergence within the prospective notochordal and somitic fields causes involution of the upper (dorsal) marginal zone and results in the blastopore constricting eccentrically in relation to the vegetal pole. (E) The marginal zone
at st. 12.5. Anterior LEM migrates past the animal pole, where it will settle to form the anterior blood islands, abi; head mesoderm, hd; and
heart, ht. Posterior LEM will give rise to the posterior blood islands, pbi, and other lateral plate derivatives, as well as the pronephros, pn,
which is intermediate mesoderm. Converging posterior, dorsal mesoderm (prospective notochord and somites) forms a circumblastoporal
collar, cc, around the closing blastopore, while posterior LEM cells are displaced from the blastopore. (F) St. 41 tadpole, oriented as if it had
been held in place during development. The solid green line, separating anterior from posterior, is placed at the approximate level of somite
7, which evidence indicates is the connecting point between the head and the trunk in most vertebrates. The solid purple line separates
dorsal from ventral. This series of diagrams illustrates how the prime meridian becomes the anterior midline during morphogenesis. A1 and
D1 blastomeres are labeled in B to indicate orientation.
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FIG. 2. Fate of column 1 blastomeres. At st. 6, one of each of the column 1 blastomeres was injected with rhodamine dextran and one of
each of the column 4 blastomeres was injected with fluorescein dextran. (A) Diagram of the lineage labeling scheme. (B, C) Distributions
of rhodamine dextran and fluorescein dextran, respectively, at st. 41. Arrows in B refer to the positions of the sections shown below in E.
(D) Colocalization of both dextrans. The arrow marks anterior, ventral structures, which, by definition, are not dorsal structures. Essentially
all head structures originate from column 1 blastomeres. (E) Colocalization of column 1 and 4 progeny in sectioned, st. 41 embryo. Section
1 is posteriormost and section 6 is anteriormost. See text for discussion. The asterisk indicates the notochord/floor plate/central somite
domain, which is labeled throughout the embryo by column 1 blastomeres. Abbreviations used: cg, cement gland; ep, epidermis; fg, foregut;
h, heart; nt, neural tube/brain; ov, otic vesicle; pn, pronephros; rt, retina; s, somite.
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FIG. 3. Surgical dissection of early gastrulae and assessment of surgical accuracy. (A) Diagram of the surgery at st. 101, viewed from the
vegetal pole, and nomenclature of the six fragment types to be analyzed by specification testing. The prime meridian is shown in red.
Incisions run along meridians from the animal to the vegetal pole, deep to the central axis, separating embryos into 90° and corresponding
270° sectors. ULS, upper lip sector; LtLS, lateral lip sector; LoLS, lower lip sector. (B) Double in situ hybridization at st. 101 for chordin
(blue), expressed at the upper lip, and Xwnt8 (purple), expressed in nonorganizer meridians of the marginal zone. (C) Northern blot analysis
of chordin and Xwnt8 as an indication of surgical accuracy. Total RNA was extracted from eight embryos or fragments; two embryo or
fragment equivalents were analyzed per lane. Intact controls (lane 1), 90° ULS (lane 2), 270° LoLS (lane 3), 90° LoLS (lane 4), and 270° ULS
(lane 5) were probed for chordin and Xwnt8. cytoskeletal actin was probed as a loading control.
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FIG. 4. Results of specification testing, evaluated by morphology.
Fragments generated by surgery at st. 101 were cultured to early
tadpole stage. For specimens with detectable axes, anterior is to the
right and dorsal is up. (A) Control embryo. (B) 90° ULS, with retina and
cement gland. (C) 270° LoLS, lacks apparent axial structures. The
concentrated pigment patch seen at one end of the specimen is not a
cement gland. (D) 270° LoLS, with axial structure. At higher magnification
(not shown), somites are seen in the region above the stripe of
pigmented cells (arrow). (E) 270° LtLS, with large open wound (black
arrow). The head includes two pigmented retinas (one indicated by
white arrow) and a cement gland. (F) 90° LtLS, with no apparent axial
structures. (G) 270° ULS, with wound in the region of the proctodeum.
(H) 90° LoLS, with no apparent axial structure.
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FIG. 5. Results of specification testing, evaluated by immunostaining for notochord (Tor 70) and muscle (12-101) differentiation, at late
tailbud stage. (A, E, H) Lateral views of control embryos and matched sets, made as diagrammed in Fig. 3A. The fragment that contains the
prime meridian is shown on the lower left and the matched fragment, which lacks a prime meridian, is shown on the lower right. (B) 90°
ULS, dorsal view. (C) 270° LoLS, no axial mesoderm. (D) 270° LoLS, with one somite file containing head and trunk somites, lateral view.
(F) 270° LtLS, dorsal view. (G) 90° LtLS, no axial mesoderm. The pigmented patch (bp) is the closed blastopore/proctodeum. (I) 270° ULS,
dorsal view. (J) 90° LoLS, no axial mesoderm. (K) Control embryo, dorsal view. hs, head somites; n, notochord; s, somites; ts, trunk somites.
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FIG. 6. Results of specification testing, evaluated for anterior marker genes by in situ hybridization. (A) Late neurula control, en2, dorsal
view. (B) 90° ULS expresses en2. (C) 270° LoLS does not express en2. (D) 270° LtLS expresses en2, dorsal view. (E) Late neurula controls
express cpl1, almost lateral (top) and dorsal (bottom) views. (F) Five 90° ULS express cpl1. (G) Five 270° LoLS do not express cpl1. (H) otx2
expression in st. 20 control (top), 270° LoLS (bottom left, negative), and 90° ULS (bottom right, positive). (I) Control st. 35 expresses Nkx2-5
in heart and pharyngeal arches. (J) 270° LoLS (left, negative) and 90° ULS (right, positive) expression of Nkx2-5. (K) XHex expression in
control st. 24 (top), 270° LoLS (bottom left, negative), and 90° ULS (bottom right, positive). (L) XHex expression in control st. 35. Expression
in vascular endothelial cells is marked by an arrow. (M). XHex expression in 270° LoLS (left, two domains positive) and 90° ULS (right,
presumably in the thyroid, t, and liver, l, as in the control embryo). cg, cement gland.
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FIG. 7. Stewart recombinates generate full-sized embryos containing two or zero prime meridians. (A) Diagram of surgical recombination
to generate full-sized embryos with either two prime meridians or no prime meridian. (B) Resulting embryos, stained for notochord and
somites. Embryos with two prime meridians (dorsal view) form two separate heads and two anterior notochords, each flanked by two
anterior somite files. The matching recombinate for this two-headed embryo formed no dorsal axial mesoderm. A control embryo (dorsal
view) contains a single head and notochord; two somite files flank the latter. Many recombinates that lacked a prime meridian formed a
bilateral body plan that contains either notochord and somites or only somites which span the midline. An example of the second result
is shown in lateral view. Yolky endoderm sometimes protrudes from the blastopore, as in this specimen. There is often a knob at the
anterior end, but this never forms a cement gland. These two anomalies appear at sites of contact between the embryo fragments and likely
result from slight abnormalities in healing.
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FIG. 8. Results from 70° ULS excision, stained for notochord and
somites. Anterior is to the right. (A) Lateral view of a 290° LoLS
that healed completely and formed the posterior body plan (top),
with notochord and somites arranged in bilateral symmetry, and a
control embryo (bottom). (B) Dorsal view of the same specimens.
(C) Lateral view of a 70° ULS. The cement gland (cg) and somitic
mesoderm (s) are indicated. The faint shadow between these two
structures is the notochord, which is below the focal plane. (D)
Dorsal view of a 290° LoLS that did not heal. Endoderm is exposed
at the surface between the two somite files.
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FIG. 9. Primary data adapted from Vogt’s axolotl fate map (Fig. 3
in Vogt, 1929; reproduced by permission of the publisher). Vogt’s
labels are indicated in red or blue, corresponding to the color of the
dye mark. Labels we added to facilitate discussion are shown in
black. (A) The dorsal/ventral distribution of the three germ layers,
illustrated schematically in a sagittal cross section of a tadpole.
Following the standards of classical embryology, derivatives of the
ectoderm are shown in blue, derivatives of the mesoderm in red,
and derivatives of the endoderm in yellow. Graded colors indicate
the dorsal/ventral distribution within a germ layer. Shades (colors
tinged with black) represent dorsal tissues and tints (colors mixed
with white) represent ventral tissues. For example, as dorsal
endoderm, the archenteron roof is shown in dark yellow, and as
ventral endoderm, the archenteron floor is shown in pale yellow.
(B) Vogt placed a series of alternating red and blue vital dye marks
along the prime meridian blastula (shown in lateral view, with the
prime meridian to the right). (C) The upper lip appears subsequently,
between marks 7 and 8 (gastrula shown in vegetal pole
view, with the prime meridian up). (D) At neural plate stage, marks
1–4 are visible in the neural groove in an anterior-to-posterior
sequence (neurula shown in dorsal view, anterior is left). (E) The
final distribution of the dye marks (tailbud shown in sagittal
section, anterior to the left). (F) To present the embryo in a
somewhat Cartesian mode, we straightened the tailbud in (E). This
emphasizes the ventral distribution of marks 8–11. The purple line
approximates the final distribution of the bottle cells that formed
the blastopore and thus separates dorsal from ventral endoderm.
See text for full explanation. Extensive morphogenesis must still
occur to generate a tadpole from a tailbud, but marks on the left
side of the green line in (F), including 3 and 11, will reside in the
tadpole’s head. As the tail organizer takes over production of the
axis, a tail that consists almost entirely of dorsal derivatives is
built. Thus, the tail is included in the dorsal territory in Fig. 1B.
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FIG. 10. Mediolateral intercalation within the notochord field
causes patterned cell mixing. The embryo as shown in Fig. 1B has
been rotated 90° so that the prime meridian is oriented toward the
reader. Solely for illustrative and heuristic purposes, the following
features listed below are shown as if they all occurred concurrently in
an embryo. These features are taken from numerous stages in development.
The features include st. 6 blastomeres and nomenclature,
shown in black; prospective fate map boundaries between tissues,
shown in gray; the approximate position of the vegetal alignment
zone that forms at st. 10.5, indicated as an arc of yellow, elongated
cells that spans the region of Spemannâs organizer; and the two
directions in which cells are recruited to undertake mediolateral
intercalation behavior (MIB) in shaved explants, shown on the left side
(lateral to medial in blue and vegetal to animal in purple), while six
cells are shown in white on the right. In (A), cells comprising the VAZ
have elongated and initiated MIB. In (B), cells 1â3 have been recruited
to undertake MIB in the pattern described for shaved explants by Shih
and Keller (1992), while cells 4â6 have not yet been recruited. See text
for discussion. It is important to realize that drawings are static
renditions of highly dynamic processes in the embryo. For example,
by the time cells 2 or 3 are recruited to undertake MIB, the upper
blastopore lip has involuted and moved significantly toward the
vegetal pole. By the time cells 5 or 6 are recruited, the blastopore
would be closed or nearly so. Throughout this time, the two
notochord/somite boundaries would be converging and the notochord
field would be elongating. Furthermore, we caution readers that the
position at which the vegetal alignment zone forms is known for the
early gastrula embryo (Lane and Keller, 1997), but its position in
relationship to the st. 6 blastomeres is currently under investigation.
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FIG. 11. Schematic representation of the distributions of the anteriormost and posteriormost tissues of the germ layers, in relation to the
prime meridian established by cortical rotation. The anteriormost tissues of the three germ layers arise from cells along the prime meridian,
whereas the posteriormost tissues of the three germ layers arise from cells on the opposite side of the embryo. A line (red) separating the
anteriormost and posteriormost tissues can be drawn. See text for explanation. Primitive endoderm (the archenteron) arises from the surface
cells of the vegetal hemisphere and the epithelial layer of the marginal zone. Mesoderm arises predominantly from the deep cells of the
marginal zone. Ectoderm arises from both the deep and the surface layers of the animal hemisphere. The direction of cortical rotation is
indicated.
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