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Dev Dyn
2005 Jun 01;2332:418-29. doi: 10.1002/dvdy.20342.
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Establishment of mesodermal gene expression patterns in early Xenopus embryos: the role of repression.
Kurth T
,
Meissner S
,
Schäckel S
,
Steinbeisser H
.
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In Xenopus, activin-like signals are able to induce and pattern mesoderm in a concentration-dependent manner. Previous experiments demonstrated that discrete gene expression patterns can be formed in animal cap explants as a response to graded activin signals. We analyzed the spatiotemporal appearance of goosecoid (gsc), chordin (chd), and Xbrachyury (Xbra) mRNAs in whole Xenopus embryos ectopically expressing activin or BVg1. To discriminate between direct transcriptional regulation and indirect, protein synthesis-dependent effects of ectopic signals, we combined overexpression studies and cycloheximide treatment. Our experiments revealed long-range signaling of activin/BVg1, but the expression patterns of gsc, chd, and Xbra in response to activin/BVg1 indicated that repressors are essential to establish the proper expression of these genes. Analysis of endogenous gsc, chd, and Xbra transcript distribution in embryos treated with cycloheximide supported this concept. We, therefore, conclude that inhibition is fundamental during early embryonic patterning.
Figure 1. A-E: Expression profiles of mesodermal genes in the vicinity of endogenous and experimentally induced bottle cells. A: Expression of Xenopus brachyury (Xbra, blue) and chordin (chd, magenta) in early gastrulae. chd transcripts indicate the organizer region, which fits into the gap between the dorsal bottle cells (arrow) and the Xbra-positive mesodermal ring. B-E: Microinjection of activin mRNA causes the formation of an ectopic bottle cell field in the animal cap and the expression of chd (B), Xbra (C), and gsc (E) in characteristic spatial relationships to the ectopic bottle cells (schematically illustrated in D). Note the ldquo bridge rdquo of gsc-positive cells between the endogenous organizer and the ectopic gsc domain in E. F-H: Shifts of expression domains in regions of overlapping gradients of activin. F: Schematic drawing illustrating the theoretically postulated shifts of the domains I-III caused by different degrees of overlap of two signal gradients. A peripheral overlap leads to the formation of an additional domain II (1), a more extensive overlap to the expansion of domain I (2), and to the fusion of two bottle cell fields in the outer epithelium (3). G: Double-injections of activin mRNA into animal blastomeres of stage 6-6.5 embryos were performed at different distances. G1,2: Two complete Xbra-rings are formed. In the center of the animal cap, the two rings are fused. G3,4: Less distance between the injection sites causes the formation of a uniform Xbra-negative region encompassing both bottle cell fields. This region (corresponding now to region I) is surrounded by a single Xbra ldquo ring. rdquo The two bottle cell fields become fused under these conditions. H: Single injections at decreasing distances to the marginal zone (1). H2: At a distance, the ectopic Xbra domain forms a complete ring. The periphery of this ring (region III) fuses with the periphery of the endogenous Xbra ring (arrowheads) to form a Xbra-positive bridge. H3,4: More pronounced overlaps of endogenous and ectopic signal gradients cause partial (3) or complete (4) reduction of the endogenous Xbra expression, eventually causing an Omega-like Xbra pattern (4, compare with E). Arrowheads indicate the endogenous expression sites, and asterisks indicate the ectopic bottle cell fields, which sometimes can also be recognized by the accumulated brown pigment (C,G2,G4). I-M: Cell movements in embryos overexpressing activin in the animal cap. I: Activin mRNA was coinjected with fluorescein-dextran-amine (FDA), and animal caps were explanted at stage 8. K-M: They were cultured either alone (K) or in combination with an uninjected cap (L,M, the border between the two caps is roughly indicated by the dashed yellow line). At stage 10.5, they were fixed, embedded in plastic, and sectioned. FDA-positive epithelial cells undergo apical constriction (indicated by the curved arrows in K and M), and the stained cells underneath form a coherent mass. Arrowheads in K indicate some FDA-negative bottle cells (identified by their accumulated pigment) directly adjacent to the FDA-positive cell mass. M: In conjugates, only the injected cap forms bottle cells. N-P: Similar activin-induced distribution patterns of labeled cells appear in undissected embryos. N: Tangential section through an ectopic bottle cell field. Arrows indicate the bottle cell field at the center of the image; the asterisk indicates a region of constricted but FDA-negative cells. O,P: Cross-sections through bottle cells at the center (O) or at the periphery (P) of an ectopic bottle cell field. Note the FDA-negative bottle cells in P where cell borders were visualized with an antibody against beta -catenin. Scale bars = 100 mu m in K,M, 50 mu m in N-P.
Fig. 5. Spatiotemporal expression of chd, gsc, and Xbra in early control and cycloheximide (CHX)-treated embryos. A: In control stage (st) 9 blastulae, chd is expressed in a large area on the prospective dorsal side. B: At the onset of gastrulation, the organizer region is stained. D,E: Early gsc staining
is detected in the dorsal vegetal–marginal region (D) and later in the organizer (E). G,H: Xbra transcripts also appear first in the dorsal marginal zone (G) and later mark the mesodermal ring (H). C,F,I: These early gastrula patterns are schematically illustrated in C,F, and I, respectively (lateral views). K,O,R: In CHX-treated embryos, the early chd, gsc, and Xbra expression domains are similar to the control. L,M: At stage 10 , however, the chd pattern resembles an slightly expanded stage 9 pattern. L: Note for example the chd-positive vegetal cells. M: The lateral–ventral regions of the animal
cap remain chd-negative. P: In stage 10.5 embryos, gsc is strongly expressed in the dorsal marginal zone, weaker staining can be seen in lateroventral
regions (left, curved arrows; vegetal view) and in the animal cap (right; animal view). S,T: At the same time, Xbra is expressed dorsally in the same
region. It appear slightly expanded animalward when compared with the blastula pattern, but strong signal is still confined to the dorsal side of the
embryo. S: Weak signal can be seen in lateral and in animal regions. S,T: Vegetal and ventral areas seem to be devoid of Xbra transcripts. N,Q,U: The
CHX-“gastrula” patterns are schematically illustrated in N, Q, and U, respectively (lateral views; dark blue, strong expression, light blue, weaker expression). Arrowheads indicate the endogenous expression sites, the arrows in B,E,H indicate the dorsal lip. an, animal; do, dorsal; lat, lateral; veg, vegetal; ven, ventral.
