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Figure 1, Cells Ectopically Expressing gsc Are Pluripotent and Recruit Neighboring Cells
(Top left) Schematic diagram of a 32-cell embryo highlighting blastomeres used for injections.
(Top right) Twinned tadpole resulting from microinjection into ventral blastomeres and the plane of section used in (A) are indicated.
(A) Nuclear staining (Hoechst 33258) of a secondary embryo (stage 34) resulting from coinjection of 0.2 ng of synthetic gsc mRNA and 20 mg/ml
FDA into two C4 blastomeres; only the gsc-induced secondary axis is shown in these oblique sections.
(Aâ) FDA labeling of the progeny of the injected blastomeres in the same embryo shown in (A). Neural tissue is largely unlabeled (e.g., auditory
vesicles), as is muscle in posterior areas (arrowheads in [Aâj). Labeled cells are found in all mesodermal tissues. Five embryos were serially sectioned
and analyzed in this way. Note that labeled cells are more concentrated in the anterior region of the secondary axis (arrow in [Aâj).
(B) Experimental design of a double labeling lineage tracing experiment in which the two C4 blastomeres were injected with rhodamine-dextran
and gsc mRNA and the two overlying 84 cells with FDA alone.
(C) Result of the experiment outlined in (B). The resulting secondary axis is labeled, with gsc-injected cells (red) contributing to notochord, muscle,
and head mesoderm, while the overlying cells are recruited (green) to form auditory vesicles, neural tissue, and muscle in the secondary axis.
Sections were 10 urn thick, explaining the yellow borders due to overlap of both types of cells. Six secondary axes were serially sectioned in this
way; only two had notochords, but all of them had green and red muscle cells in the secondary axes.
ap, animal pole: Av. auditory vesicle; d, dorsal; En, endoderm; Mu, muscle; Hm, head mesoderm; Ne, neural tissue; No, notochord; v, ventral;
vp, vegetal pole. Bar is 50 pm.
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Figure 2. Cells Ectopically Expressing gsc Migrate
to the Anterior of the Secondary Axis
(A) Embryo (stage 36) with secondary axis resulting
from a single C4 injection of 0.2 ng of
gsc mRNA.
(6) Same embryo as in (A), after visualization of
coinjected CG lineage tracer. Note that labeled
cells contribute mostly to the anterior of the
secondary axis (arrowheads), while the posterior
of the secondary axis is mostly unlabeled,
as is the posterior of the main axis.
(C) Control embryo injected with CG alone,
showing labeling of lateral plate mesoderm,
muscle, and epidermis as typically found in C4
lineage-traced blastomeres (Dale and Slack,
1987; Moody, 1987). Embryos injected with
dgsc mRNA into C4 blastomeres did not develop
secondary axes or ectopic muscle, but
sometimes showed less lateral plate labeling
for unknown reasons. A total of four experiments
were carried out (n = 23).
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Figure 3. gsc mRNA Injection Enhances Involution in Ventral Blastomeres
CG and either dgsc (left) or gsc mRNA (right) (0.2 ng) were coinjected
into one C4 blastomere of 32-tell embryos and fixed at the early neurula
stage (stage 14). Embryos are shown in ventral view with the
blastopore at the bottom. The embryos were not cleared, but were
analyzed instead in 70% ethanol where they remained opaque and
predominantly showed the noninvoluted external tissue layer. Note
that in the gsc-injected embryo, the labeled mesoderm has involuted
almost completely, with only a few cells near the blastopore remaining
on the surface.
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Figure 4. gsc mRNA Injection Induces Rapid Cell Movements in the Early Gastrula
gsc mRNA was injected into two ventral vegetal blastomeres (0.5 ng each) of 16-cell stage embryos. One uninjected control embryo (control, upper
right) and five experimental embryos were time lapse-recorded from the early gastrula ([A]-[C]) until the mid-neurula (D) stage using epiillumination.
Single frames taken at the indicated time points are represented.
(A) The position of the primary dorsal lip (1â) is indicated.
(B) The position of secondary lips at the ventral side (2â) of all injected embryos is indicated.
(C)Tracings of movement of individual cells of darker pigmentation filmed between time points A and C are indicated by the length of the arrows.
Note that ectopic cell movement can be seen at the ventral side of injected embryos.
(D) Mid-neurula embryos. The formation of a secondary axis in the anterior is indicated by the double arrows. All injected embryos developed a
secondary axis in this experiment, but two embryos turned such that they are viewed from the posterior (anus). A single arrow indicates the anterior
of the dorsal midline in the control embryo. The initial position of the blastopore is indicated by a bar.
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Figure 5. gsc Acts in the Organizer Proper
(A and B) CG and either Xwnf-8 (A) or gsc mRNA (B) were coinjected into a D4 blastomere of 32cell embryos. Tadpoles (stage 34) are shown from
the front (A) or the side (B). Note in (A) that Xwnf-8 has induced a twinned head, while labeled injected cells remain in the deep endoderm (n =
16). Note in (B) that gsc-injected cells form part of the secondary axis, where they concentrate in the anterior (n = 17).
(C) Embryo injected with 50 pg of pCMVgsc plasmid DNA into each of two ventral vegetal blastomeres at the E-cell stage.
(0) An uninjected embryo at the same stage. Note that the total length of the embryo in (C) is much shortened. Microinjection of DNA into 8- to
16-tell stage embryos produced secondary axes with an average frequency of 28% (two experiments, n = 107). Only one tadpole developed
complete anterior structures (eye and cement gland; shown in [Cl).
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Figure 6. gsc mRNA Injection in Prospective Organizer Cells Changes the Motile Behavior of Involuting Cells Leading to Changes in Cell Fate
(A) Schematic depiction of dorsal mesoderm movements in Xenopus gastrulation. At the early gastrula stage, the dorsal mesoderm consists of two
distinct regions: the migratory deep zone cells (indicated by the filled areas) that crawl along the blastocoel roof and the involuting marginal zone
cells (hatched sections) that undergo intercalation movements. At late gastrula, cells of the involuting marginal zone undergo intercalation, elongating
the anteroposterior axis of the embryo. At the neurula stage, migratory deep zone cells have involuted furthest and differentiate into head mesenthyme
(stippled) and endoderm (filled), part of which folds back ventroposteriorly, while cells of the involuting marginal zone differentiate into
chordamesoderm (muscle and notochord). For simplicity, only the movements of the dorsal mesendoderm are indicated, and the positions of yolky
endoderm cells and of the archenteron are not shown.
(B and Bâ) Early gastrula (stage 10.5) in dorsal view, injected into one Cl blastomere either with CG tracer alone (B) or gsc mRNA (0.1 ng) and
CG (B). Note that gsc-injected cells are concentrated at the leading edge.
(C and C3 Same embryos as in (B), but in side view.
(D and Dâ) Mid-gastrula embryos (stage 12) in side view, control and gsc injected, respectively. Note that in the control embryo, the entire length
of the dorsal involuting tissue contains labeled Cl progeny while gsc-injected cells are restricted to the leading edge.
(E and E) Tadpole embryos (stage 34) injected with CG and either dgsc or gsc, respectively. Note that gsc-injected cells contribute less to somites
and notochord and more to head mesenchyme and endoderm in the resulting tadpole. Tadpoles were scored for the frequency of occurrence of
notochord labeling, which decreases as cells occupy a more anterior position. At this stage we observed 44%, 39%, and 14% notochord labeling
in CG controls (n = 99) dgsc-injected embryos (n = 33) and gsc-injected embryos (n = 66) respectively. Data are pooled from five experiments
(0.07-0.1 ng of mRNA injected). The position of the dorsal blastopore is indicated by arrowheads. He, head endoderm; Hm, head mesenchyme;
No, notochord; So, somites.
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Figure 7. gsc Affects Cell Movements in
Groups of Cells but Not in Individual Cells
(A and B) Deep zone cells from the ventral (A)
or the dorsal (B) side of early gastrula embryos
were dissociated, seeded on a fibronectincoated
dish (200 @ml), and recorded by timelapse
microscopy. A single frame of the recordings
is shown. Note that both cell
populations are spread and extend filopodia
with which they move. Dorsal and ventral cells,
as well as gsc-injected ventral cells. failed to
show any differences in their ability to move on
fibronectin-covered plates in these time-lapse
recordings.
(C and D) A 16cell stage embryo was injected
into each vegetal blastomere with gsc mRNA
(0.5 ng each). At the late blsstula stage (stage
9) a ventral marginal zone explant was prepared
and recorded by time-lapse microscopy
using epiillumination. Single frames soon after
explantation (C) and after incubation for 3 hr
(D) are shown. Animal pole is at the top. Note
the flat and bright cells that lie on top of each
other and have migrated toward the animal
pole in (D). In the time-lapse film, these cells
can be seen to slide on each other, resembling
shingles on a roof. The ventral side was assigned
by inclination and Nile bluesulfate labeling
before first cleavage (Steward and Gerhart,
1990).
(E and F) Embryos at the l&cell stage were
coinjected into each vegetal blastomere with
fluorescent lineage tracer (FDA) and either 0.5
ng of Agsc (E) or gsc mRNA (F). Ventral marginal
zone explants were prepared from early
gastrula embryos and scored after 2.5 hr incubation
for migration of labeled cells in an epifluorescence
microscope. Owing to injection into
vegetal blastomeres, the animal cap cells remain
unlabeled, while most of the label is in
mesodermal and endodermal cells. Note extensive
covering of the animal cap tissue by
fluorescent gsc-injected cells in (F). In three
experiments that have been scored blind by
an uninformed observer, 53% (n = 32) of the
gsc-injected explants underwent mesodermal
cell movement compared with 9% (n = 24) in
Agsc-injected explants.
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