XB-ART-4814Development October 1, 2003; 130 (20): 4847-58.
Fusicoccin signaling reveals 14-3-3 protein function as a novel step in left-right patterning during amphibian embryogenesis.
To gain insight into the molecular mechanisms underlying the control of morphogenetic signals by H+ flux during embryogenesis, we tested Fusicoccin-A (FC), a compound produced by the fungus Fusicoccum amygdali Del. In plant cells, FC complexes with 14-3-3 proteins to activate H+ pumping across the plasma membrane. It has long been thought that FC acts on higher plants only; here, we show that exposing frog embryos to FC during early development specifically results in randomization of the asymmetry of the left-right (LR) axis (heterotaxia). Biochemical and molecular-genetic evidence is presented that 14-3-3-family proteins are an obligate component of Xenopus FC receptors and that perturbation of 14-3-3 protein function results in heterotaxia. The subcellular localization of 14-3-3 mRNAs and proteins reveals novel cytoplasmic destinations, and a left-right asymmetry at the first cell division. Using gain-of-function and loss-of-function experiments, we show that 14-3-3E protein is likely to be an endogenous and extremely early aspect of LR patterning. These data highlight a striking conservation of signaling pathways across kingdoms, suggest common mechanisms of polarity establishment between C. elegans and vertebrate embryos, and uncover a novel entry point into the pathway of left-right asymmetry determination.
PubMed ID: 12930777
Article link: Development
Genes referenced: nodal1 ywhae ywhaz ywhaq ywhah
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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.|
|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.|
|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.|
|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.|
|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.|
|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.|