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Localized maternal determinants control the formation of dorsal axial structures in Xenopus embryos. To examine the spatial distribution of dorsal determinants, we injected cytoplasm from various regions of the egg and 16-cell embryo into the ventral vegetal cells of a 16-cell recipient embryo. Cortical cytoplasm from the egg vegetal surface induced the formation of a secondary dorsal axis in 53% of recipients. In contrast, animal cortical, equatorial cortical and vegetal deepcytoplasm never induced secondary axis formation. We also compared the axis-inducing ability of animal versus vegetal dorsal cortical cytoplasm from 16-cell embryos. Significantly more dorsalizing activity was found in vegetal dorsal cytoplasm compared to animal dorsal cytoplasm at this stage. Previous work has shown that UV irradiation of the vegetal surface of either prophase I oocytes, or fertilized eggs, leads to the development of embryos that lack dorsal structures. Egg vegetal cortical cytoplasm was capable of restoring the dorsal axis of 16-cell recipient embryos derived from UV-irradiated oocytes or fertilized eggs. We also tested the axis inducing ability of cytoplasm obtained when UV-irradiated oocytes and eggs were treated as donors of cytoplasm. While vegetal cortical cytoplasm from UV-irradiated fertilized eggs retains its dorsalizing activity, cytoplasm obtained from eggs, UV irradiated as oocytes, does not. The eggvegetal cortex provides a suitable source for the isolation of maternal dorsal determinants. In addition, since UV irradiation of the oocyte vegetal surface destroys the dorsalizing activity of transferred cytoplasm, UV can be used to further restrict possible candidates for such determinants.
Fig. 1. Secondary axis formation by injection of dorsal cortical
cytoplasm. All 16-cell stage embryos are viewed laterally with
ventral blastomeres evident by the accumulation of pigment.
(A) Cytoplasm was drawn from the cortex of two dorsal vegetal
blastomeres and microinjected (hatched arrow) into two ventral
vegetal blastomeres. Recipient embryos were capable of
developing a secondary axis. (B) Controls in which ventral
cortical cytoplasm was transferred developed normally. D, dorsal;
V, ventral; stippled regions, location of putative dorsal activity;
hatched arrows, transfer of cytoplasm.
Fig. 2. Tailbud embryos with a secondary
axis resulting from the injection of dorsal
cortical cytoplasm. (A) Embryo with
secondary axis, indicated by arrowheads.
The DAI value of the secondary axis is 1.
Bar, 1 mm. (B) Section of embryo that
has a secondary spinal cord. Primary and
induced secondary notochords (n, nâ),
neural tubes (nt, ntâ) and sets of somites
(s, sâ) are indicated. t, tail; Bar, 100 mm.
(C) Embryo with complete secondary axis
including well formed head structures.
The DAI value of the secondary axis is 5.
Bar, 1 mm. (D) Control embryo that was
injected with ventral cytoplasm appears
normal. Since no secondary axis is
observed, the DAI value of the induced
axis is 0. Bar, 1 mm.
Fig. 3. Superior dorsalizing activity in dorsal vegetal blastomeres
compared to dorsal animal blastomeres. (A) To compare animal
versus vegetal dorsal cytoplasm, one donor embryo was used as a
source of cytoplasm for each pair of recipient embryos. Cortical
cytoplasm from two dorsal animal blastomeres was microinjected
(hatched upper arrow) into the two ventral vegetal blastomeres of
a recipient embryo. Cortical cytoplasm from the two dorsal
vegetal blastomeres was microinjected (hatched lower arrow) into
the two ventral vegetal blastomeres of another recipient. The pair
of recipients was allowed to develop to tailbud stage and the
degree of secondary dorsal axis formation was expressed as a DAI
value. (B) Secondary axis formation in paired recipients of animal
and vegetal dorsal cytoplasm. DAI of the vegetal dorsal recipient
is plotted on the horizontal axis and that of the animal dorsal
recipient is plotted on the vertical axis. Note that each point
represents a pair of recipients that received cytoplasm from the
same donor embyro. The 12 points that lie on the origin (no
secondary dorsal axis in either recipient) are not shown. The
straight line denotes the location of points that would be observed
if cytoplasm from vegetal and animal dorsal blastomeres had
equivalent dorsalizing activity. Most of the points lie below the
line and closer to the horizontal axis, and thus the dorsalizing
activity of vegetal dorsal cytoplasm is significantly superior to
that of animal dorsal cytoplasm (P < 0.01, Wilcoxon Signed Rank
Test, paired, twotailed).
Fig. 4. Dorsalizing activity from UV-irradiated prophase
I oocytes versus UV-irradiated fertilized eggs. (A) UVirradiated
fully grown prophase I oocytes were matured,
prick activated, and used as donors of vegetal cortical
cytoplasm. Cytoplasm was transferred to two ventral
vegetal blastomeres of a 16-cell embryo (hatched arrow).
Recipients of cytoplasm did not develop a secondary
dorsal axis and thus appeared normal. A subset of
oocytes (lower curved arrow) was matured, implanted
into the body cavity of a host female and fertilized. These
embryos developed with dorsoanterior deficiencies.
(B) UV irradiated fertilized eggs were used as donors of
vegetal cortical cytoplasm. Cytoplasm was transferred to
two ventral vegetal blastomeres of a 16-cell embryo
(hatched arrow). Recipients developed a secondary dorsal
axis. Sibling donor embryos (upper curved arrow)
developed dorsoanterior deficiencies. V, ventral
blastomeres; D, dorsal blastomeres; hatched arrow,
transfer of cytoplasm; stippled regions, location of
putative dorsal activity.
Fig. 5. Rescue of dorsal axis formation in embryos derived
from UV-treated oocytes and UV-fertilized eggs. (A) The
vegetal surface of prophase I oocytes was exposed to UV
light prior to maturation, transfer and fertilization. At the
16-cell stage, the ventral vegetal blastomeres were
microinjected with cortical cytoplasm from the vegetal pole
of a donor egg (hatched arrow). The donor was previously
UV-treated after fertilization. Many of the recipient
embryos developed normally. Uninjected embryos (lower
curved arrow) developed with dorsoanterior deficiencies.
(B) UV-irradiated fertilized eggs were grown to 16-cell
stage and used as recipients. Injection of vegetal cortical
cytoplasm (hatched arrow) rescues dorsal development and
many embryos develop with dorsal axial structures.
Uninjected sibling embryos (upper curved arrow)
developed dorsoanterior deficiencies. Note that
pigmentation differences shown in the animal hemisphere
of recipient 16-cell embryos are due to the sperm entry
point and do not reflect dorsoventral polarity. V, ventral
blastomeres; D, dorsal blastomeres; stippled regions,
location of putative dorsal activity; hatched arrow, injection
of cytoplasm.
