September 1, 1983;
Axis determination in eggs of Xenopus laevis: a critical period before first cleavage, identified by the common effects of cold, pressure and ultraviolet irradiation.
Exposure of eggs of Xenopus laevis to a temperature of 1.0 degree C for 4 min or a pressure of 8000 psi for 5 min in a critical period before first cleavage
results in embryos exhibiting a reduction and loss of structures of the body axis. The deficiencies occur in a craniocaudal progression which is dose dependent. In the extreme, totally axis-deficient embryos with radial symmetry are formed. Maximum sensitivity to cold and pressure occurs at 0.6 of the time from fertilization to first cleavage
and extends from approximately 0.4 to 0.8, the period between pronuclear contact and mitosis, and the approximate period of gray crescent
formation. The effects of cold and pressure resemble those previously reported for uv irradiation in that (1) the types of axis-deficient embryos produced are morphologically indistinguishable; (2) sensitivity in all cases ends before 0.8; (3) cold and uv effects, although not those of pressure, can be prevented by cotreatment with D2O; and (4) impaired eggs can be rescued by oblique orientation. We interpret these results as follows: during the 0.4-0.8 period the egg
reorganizes its contents in a manner critical for subsequent development of the embryonic body axis. The reorganization process involves cytoskeletal elements, some of which are sensitive to cold, pressure, and uv, and protected by D2O. Rescue by oblique orientation can be understood as the result of a gravity-driven reorganization of the egg''s contents, supplanting the normal mechanochemical process impaired in treated eggs.
[+] show captions
FIG. 1. Axis-deficient embryos from cold-, pressure-, and uv-treated eggs. Panel A: control stage 44 tadpole; panel B: IAD grade 4 embryo
at control stage 46 produced by pressure treatment (8000 psi, 5 min); only a trace of somites is seen (view is from dorsal side); panels C
through G: IAD grade 1 through grade 5 embryos, respectively, at control stage 44, produced by cold (lower embryo) and uv irradiation
(upper embryo). uv dose was varied from 1.3 to 2.7 X ld ergs/mm*. Standard cold treatment was employed. All embryos except those of
panel B are from eggs from a single frog and were fertilized simultaneously. Grade 5 embryos (panel G) are oriented with the original
blastopore leftward. cg, cement gland; e, eye(s); g, gut; o, otic vesicle(s); s, somites; y, yolk mass. Bar length: 1 mm.
FIG. 2. Morphology of totally axis-deficient (grade 5) embryos. Panel A: median section of embryo from uv-irradiated egg (control stage
41); panel B: external view of embryo from cold treated egg (control stage 41); panel C: median section of embryo from cold treated egg
(control stage 41); panel D: median section of radially symmetric gastrula from uv-irradiated egg, fixed just after circular blastopore lip
formation (control stage 11 l/2). Sections were cut randomly in a plane which included the original animal pole-blastopore axis. Orientation
is with blastopore leftward in all cases. blc, blastocoel; blp, bastopore lip; e, erythrocyte-like cells; ect, ectoderm; end, endoderm; ce, ciliated
epithelium; m, mesenchyme cells; mes, mesoderm; y, yolk-filled cells. Bar length: 0.5 mm.
FIG. 3. Dose response to cold and pressure treatments. Each point
represents IAD measured at 0.60-0.65. Panel A: eggs were exposed
for 4 min at various temperatures and then warmed slowly to 15”C,
at which temperature they were kept until first cleavage. The procedure
for slow warming is described under Materials and Methods. Panel
B: eggs were exposed to 5.0°C and then slowly warmed to 15°C until
first cleavage. Panel C: eggs were exposed to 5.O”C for 5 min and
either immediately warmed to the temperatures shown (0) or warmed
slowly over a period of lo-15 min to those temperatures (V). At first
cleavage all eggs were transferred to 19°C. Panel D: eggs were treated
at various pressures for 5 min at 1822’C, data from four independent
experiments are combined. Survival ranged from 60-100% in cold
experiments to 30-952 in pressure experiments. Each point represents
at least 10 embryos.
FIG. 4. Stage dependence of survival following cold and pressure
treatment. Panel A: Cold treatment: eggs were exposed to 1.0 f 0.3”C
for 4 min, slowly warmed to 15°C and kept at 15°C until first cleavage.
Time points, each with an average of 38 eggs, were taken every 3 min.
Control survival was 98%) and control time to first cleavage was 105
min. Panel B: pressure treatment: eggs were exposed to 8000 psi for
4 min. Time points, each with an average of 50 eggs, were taken every
6 min. Control survival was 95% and time to first cleavage was 115
min. For both experiments embryos were scored for survival at control
FIG. 5. Stage dependence of cold, pressure and uv effects on axis
determination. Panel A: cold treatment, low dose: eggs were exposed
to 1.0 i 0.3”C for 4 min and then immediately warmed to 17°C until
first cleavage. Survival was from 80 to loo%, except during mitosis
(0.73-0.79 and 1.13-1.19) when it decreased to 40-50s. Panel B: cold
treatment, high dose: eggs were exposed as in low dose, but warmed
slowly to 15°C and maintained at 15°C until first cleavage. Survial
data for this experiment are shown in Fig. 4A. Panel C: pressure
treatment, low dose (m): eggs were exposed to 8000 psi for 4 min at
18-20°C. Survival data for this experiment are shown in Fig. 4B.
Pressure treatment, high dose (Cl): eggs were exposed to 9000 psi for
5 min at 19-21°C. Survival ranged from 4 to 68%. Panel D: uv-irradiation:
eggs received either 1.3 X 10’ ergs/mm’ in 3 min (A), or 2.7
X lo* ergs/mm’ in 6 min (A) at times shown. Survival was 95-100%.
In all of the above experiments, control IAD was from 0.0-0.2, and
time to first cleavage was from 105-115 min. An average of 30 to 60
eggs per time point were treated, but due to stage-specific lethality,
the actual number of storable embryos per time point ranged from
3 to 60.
FIG. 6. Prevention of axis deficiency by cotreatment with I&O. Each
data point represents a comparison of DpO-cotreated eggs (+D&)
with eggs treated simultaneously and equivalently in 20% MR (-D&L
uv irradiation (0): eggs were pretreated in D20 for 3 min, irradiated
in DzO (l-3 x 10’ ergs/mm’ in 2-6 min) during the 0.3-0.4 period, and
removed from D20 after lo-min total exposure. Cold treatment (A):
eggs were pretreated in DzO for 3 min, transferred to Da0 at l.O”C
at the time 0.60-0.65 for 4 min, allowed to warm to 10°C in D20 (3
min), transferred to 20% MR at 10°C and allowed to warm slowly
to 15°C. Thus eggs were exposed to temperatures of less than 10°C
only in the presence of DaO. Pressure treatment (0): eggs were pretreated
in D20 for 4 min, exposed to 8000 psi for 4 min at 0.60-0.65
in DaO, and removed from 40 after a total of 10 min. Survival following
D20 cotreatment with both cold and pressure was TO%, whereas survival
was 30% for eggs treated with Da0 only, and ‘70 and 50%, respectively,
for eggs treated with cold and pressure only. Survival following
D&/uv cotreatment was 85-90% compared to 95% for uvirradiated
eggs. All experiments were carried out at 18-19°C. Data
points represent comparisons of groups of 15-30 embryos. A total of
seven separate “v-irradiation, three separate cold, and three separate
pressure experiments are represented. The dashed line is that line
expected if Da0 had no effect. The solid line is a visual fit to the data
for cold and uv.