First published online 20 August 2003
doi: 10.1242/dev.00698
Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis
Tom D. Bunney1,*,
Albertus H. De Boer1 and
Michael Levin2,
1 Vrije Universiteit, Faculty of Earth and Life Sciences, Department of
Developmental Genetics, Section Molecular Plant Physiology and Biophysics, De
Boelelaan 1085, 1081 HV, Amsterdam, The Netherlands
2 Department of Cytokine Biology, The Forsyth Institute, 140 The Fenway, and
Department of Developmental and Craniofacial Biology, Harvard School of Dental
Medicine, Boston, MA 02115, USA
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Fig. 1. Fusicoccin (FC) induces heterotaxia. Systemic exposure of Xenopus
embryos to FC between fertilization and stage 14, or microinjection of FC into
dorsal blastomeres at the four-cell stage, resulted in heterotaxia relative to
control embryos (A). Exposed embryos exhibited cardiac inversions (B), heart
and gut inversions (C), gut inversions (D), and complete mirror image
inversions (E). All embryos are shown in ventral views (thus the embryo's
right is the reader's left), with anterior toward the top. Red, yellow and
green arrows indicate the asymmetry of the heart, gut and gall bladder,
respectively.
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Fig. 2. Biochemistry of the FC receptor in Xenopus tissue. Xenopus
laevis embryos possess a Fusicoccin-binding site whose properties differ
from that of the classical higher plant Fusicoccin receptor. (A) Saturation
analysis of specific binding of
[3H]9'-nor-fusicoccin-8'-alcohol to plasma membranes of
barley roots (black circle) and embryo cytoplasmic protein extract (white
circle). Data was fitted using the equation describing a rectangular
hyperbola. The resulting coefficients gave rise to values for the
KD and Bmax, which are referred to in the main text. The
data are expressed as the means±s.e.m. of a representative experiment.
(B) Competitive binding experiments with
[3H]9'-nor-fusicoccin-8'-alcohol and unlabelled
Fusicoccin A. Barley root plasma membranes were incubated with 1 nM
radioligand (black circle) and embryo cytoplasmic protein extract was
incubated with 4 nM radioligand (white circle). The data was fitted using the
equation describing a sigmoidal curve of 3 parameters (Sigmaplot, SPSS
scientific).
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Fig. 3. 14-3-3 protein misexpression randomizes the localization of XNR1
expression. Control embryos examined for the expression of XNR1 by in
situ hybridization exhibit the normal left-sided signal (whole mount in A;
section in B). By contrast, embryos receiving injections of 14-3-3E
mRNA at the one-cell stage often exhibit bilateral XNR1 expression
(whole mount in C; section in D). Red arrowheads indicate expression; yellow
arrowheads indicate lack of expression.
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Fig. 4. Localization of 14-3-3 proteins during early development. Embryos were
fixed, sectioned and processed for immunohistochemistry with 14-3-3 protein
antibodies. (A-D) 14-3-3Z protein. (A) Unfertilized embryos sectioned parallel
to the animal-vegetal (AV) axis display signal in the vegetal-most two-thirds
of the embryo. (B) By the two-cell stage, the staining is much reduced in
intensity. (C) By the four-cell stage, staining is almost completely absent.
(D) At stage 10, weak staining is seen throughout the endodermal yolk mass of
the gastrulating embryo. (E-J) 14-3-3E protein. (E) By contrast to 14-3-3Z,
the 14-3-3E signal is seen in a coherent spot in the center of the
unfertilized embryo. (F) By the two-cell stage, signal can be detected in only
one of the two blastomeres. (G) At the four-cell stage, signal is seen in the
right blastomeres. (H) During gastrulation, a strong signal is detected in the
endodermal yolk mass. To check the mRNA construct as well as the antibody,
embryos injected immediately after fertilization with 14-3-3E mRNA were
processed for immunohistochemistry. A strong 14-3-3E signal can be detected
throughout the embryo at the two- and four-cell stages (I,J). Red arrowheads
indicate protein localization; yellow arrowheads indicate lack of signal.
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Fig. 5. Ectopic Fusicoccin abolishes the asymmetry of 14-3-3E protein localization.
(A,B) Control embryos exhibit 14-3-3E protein localization in only one
blastomere. (C,D) By contrast, exposure to Fusicoccin abolishes the asymmetry
and results in localization in both blastomeres. Sections in panels A and C
were taken perpendicular to the animal-vegetal (AV) axis. Sections in panels B
and D were taken parallel to the AV axis. Red arrowheads indicate
localization; yellow arrowheads indicate lack of signal. Green line in panel B
indicates cleavage plane between the blastomeres.
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Fig. 6. mRNA localization of 14-3-3 family during early development. A
representative set of embryonic stages are shown for each mRNA. Maternal mRNA
for 14-3-3 isoform T is localized to the animal pole of the unfertilized egg
(data not shown), and to embryonic blastomeres at the two-cell stage (A;
parallel to AV axis). (B) This pattern is observed in most blastomeres during
the first few cleavages (perpendicular to the AV axis). The mRNA is located
near the cell cortex. (C) 14-3-3T is later expressed throughout the nervous
system, including very strong expression in the head and somites, and a thin
border at the caudal end of the embryo, up to the developing anus. (D) 14-3-3
isoform Z is detected in the cortex of cleaving cells (four-cell stage embryo
sectioned perpendicular to AV axis). During subsequent cleavages, transcripts
are also detected in the animal half of vegetal cells (E) and in an unusual
horseshoe pattern in cleavage-stage embryos (F). 14-3-3E mRNA is
present throughout the animal pole of the unfertilized egg (data not shown).
It becomes localized to one of the two blastomeres at the first cell division
(G). This pattern is maintained at the four-cell stage (H). Sections of G and
H, shown in I and J, respectively, confirm the pattern. Note that
14-3-3E mRNA at the two-cell stage is located in the central
cytoplasm and not in the cell cortex (compare panel I with panels D and B).
Red arrowheads indicate presence of mRNA; yellow arrowheads indicate lack of
signal.
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Fig. 7. A model of 14-3-3 signaling in LR asymmetry in normal and perturbed
embryos. Our results suggest the following model. (A) In unmanipulated
embryos, endogenous localization machinery ensures that only one cell of a
two-cell embryo contains 14-3-3E protein. This protein interacts with an
unknown target (see Discussion for probable candidates) whose activation on
one side of the midline feeds into the pathway of asymmetric genes. (B) When
14-3-3E protein is misexpressed by the injection of 14-3-3E mRNA
immediately after fertilization, excess 14-3-3E protein overwhelms the
localization machinery and is present in both cells at the first cleavage.
This subsequently provides identical signal to the L and R sides, resulting in
a randomization of asymmetry. (C) Exposure to FC in the medium abolishes the
asymmetric localization of 14-3-3E (and induces heterotaxia as in B) by
competing for its binding with the endogenous localization mechanism. (D)
Injection of NR-P peptide abolishes the asymmetry by interfering with the
one-sided binding of 14-3-3E to its downstream target.
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© The Company of Biologists Ltd 2003
