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Fig. 1. Schematic representation of the sites of pricking for removal
of cytoplasm with or without the surface. Cytoplasm was removed at
the vegetal pole (VP) and sites dorsally 30° (D30°), dorsally 60°
(D60°), dorsolaterally 30° (DL30°), dorsolaterally 60° (DL60°),
laterally 30° (L30°), laterally 60° (L60°), ventrally 30° (V30°) and
ventrally 60° (V60°) off the vegetal pole. AP, the animal pole; VP,
the vegetal pole; D, the presumptive dorsal side; V, the presumptive
ventral side; SEP, sperm entry point.
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Fig. 2. Schematic illustration of the procedure for removal of small volumes of cytoplasm
together with the surface (A), removal of small volumes of cytoplasm without the surface
(B) and removal of large volumes of vegetal or dorsal cytoplasm (C). See text for details.
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Fig. 3. Top views of eggs from which cytoplasm with (A) or without
(C) the surface are being squeezed out of the fertilization membrane
into 6% Ficoll solution. Numbers on the top left of A and C indicate
the normalized times of the removal, and those on the top right
denote estimated volumes of extruded cytoplasm relative to the
entire egg volume. Numbers on the top right of B and D are the DAI
for the larvae, which were developed from the eggs shown in A and
C, respectively. The scale bar for A and C, 1 mm; B and D, 2 mm.
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Fig. 4. Two-day-old larvae that developed after
removal of cytoplasm along with the surface at the
vegetal pole at 0.1 (A), 0.3 (B) and 0.7 NT (C), those
that developed after removal of cytoplasm with the
surface at the site ventrally 30° off the vegetal pole at
0.2 NT (D), or at the site dorsally 60° off the vegetal
pole at 0.8 NT (E) and those that developed after
removal of cytoplasm without the surface at the
vegetal pole at 0.3 NT (F). Numbers close to
individual larvae are the DAI scored for the larvae.
Numbers on the top right of individual panels
indicate the normalized time of cytoplasmic removal.
Scale bar, 2 mm.
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Fig. 5. Frequency of production of larvae with differently reduced or
enlarged dorsal structures after removal of a small volume of
cytoplasm along with the surface at the VP at the indicated times of
first cell cycle. Data for the effect of the removal at the indicated
times were obtained from 21 (0.1 NT), 78 (0.2 NT), 71 (0.3 NT), 44
(0.4 NT), 51 (0.5 NT), 24 (0.6 NT), 19 (0.7 NT) larvae from 8
females. Average DAI for 45 control larvae from the same females
was 5.0. Note that the frequency of production of larvae suffering
from severe dorsal reduction (DAI 0-1) changes remarkably: it
increased sharply after fertilization, reached its maximum at 0.3 NT
and decrease sharply after 0.3 NT. More than 240 larvae from intact
eggs from the 8 females were examined as the control for formation
of dorsal axial structures (the average DAI= 5.0).
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Fig. 6. Summary
of temporal
changes in the
effect on dorsal
development of
removal of
cytoplasm with
the surface at
different sites.
Average DAIs (y
axis) for larvae
from eggs
subjected to
removal of
cytoplasm
together with the
surface are
plotted against
the normalized
times (x axis) of
the removal. The
broken line in
each plot
indicates the level of normal dorsal development (DAI 5). The
nums at each point indicates the number of larvae scored. More
than 30 intact larvae were examined as the control for each
plotting (average DAI =5.0 for all plots). AP, the animal pole;
VP, the vegetal pole; D, the presumptive dorsal side; V, the
presumptive ventral side; SEP, the sperm entry point.
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Fig. 7. Summary of the effect on dorsal development of removal of
cytoplasm without the surface at different sites and at different times.
Average DAIs for larvae from eggs subjected to removal of
cytoplasm without the surface are plotted against the normalized
times of the removal. The broken line in each plot denotes the level
of normal dorsal development (DAI 5). The numbers at each point is
the number of larvae scored for obtaining the average DAI. More
than 30 intact larvae were examined as the control for each plotting
(average DAI =5.0 for all plottings). AP, the animal pole; VP, the
vegetal pole; D, the presumptive dorsal side; V, the presumptive
ventral side; SEP, the sperm entry point.
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Fig. 8. Cortical rotation in an egg from which cytoplasm was
removed along with the surface at the vegetal pole at 0.3 NT (A-C)
and the resulting two-day-old larva (D). Vegetal views at 0.4 NT (A)
and 0.95 NT (B) of the egg are presented. Arrowheads indicate the
point of extrusion for cytoplasmic removal. C is a computer
generated composit image fo A and B, showing the movement of
Nile blue spots. The pigment-free portion near the site of extrusion
was formed after the removal of cytoplasm with the surface,
suggesting that a cortical layer containing pigment granules was
removed by this surgery. Note that dorsal axial structures are heavily
reduced in the larva even though cortical rotation occurs normally.
Scale bars in panels A and D equal 0.5 mm.
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Fig. 9. 2-day-old larvae from a single spawning of
eggs that were irradiated with 254 nm light at
14,000 erg/mm2 and given cytoplasm from
extrusions formed at the vegetal pole at 0.3 NT (A)
and 0.6 NT (B) or no cytoplasm (C). Note that
dorsal axial structures are detectable only in larvae
that received cytoplasm from an extrusion formed at
0.3 NT (A). Numbers close to individual larvae are
their DAI score. Scale bar equals 1 mm.
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Fig. 10. Summary of the effect on dorsal development of removal of
large volumes of dorsal or vegetal egg cytoplasm by egg ligation at
varying times. Average DAIs for larvae from eggs subjected to the
removal are plotted against the normalized times of the removal. The
broken line in each plot depicts the level of normal dorsal development
(DAI 5). The number close to each point is the number of larvae scored
at that point. More than 30 intact larvae were examined as the control
for each plot(average DAI =5.0 for all plots). AP, the animal pole; VP,
the vegetal pole; D, the presumptive dorsal side; V, the presumptive
ventral side; SEP, the sperm entry point.
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Fig. 11. Illustration of presumed behavior of dorsal determinants during first cell cycle based on the present results. The scale indicates relative
times normalized with 0.0 as the time of fertilization and 1.0 as the time of the first cleavage. The darkness of the shaded portion indicate the
concentration of localized dorsal determinants. Broken lines indicate latitudes 30° and 60° off the vegetal pole. AP, animal pole; VP, vegetal
pole; SEP, sperm entry point.
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