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The cement gland is a simple secretory organ that marks the anterior-most dorsal ectoderm in Xenopus embryos. In this study, we examine the timing of cement gland induction and the cell interactions that contribute to cement gland formation. Firstly, we show that the outer ectodermal layer, from which the cement gland arises, becomes specified as cement gland by mid-gastrula. Curiously, at early gastrula, the inner layer of the dorsal ectoderm, which does not contribute to the mature cement gland, is strongly and transiently specified as cement gland. Secondly, we show that the mid-gastrula dorsoanterior yolky endoderm, which comes to underlie the cement gland primordium, is a potent inducer of cement gland formation and patterning. The cement gland itself has an anteroposterior pattern, with the gene XA expressed only posteriorly. Dorsoanterior yolky endoderm greatly enhances formation of large, patterned cement glands in partially induced anterodorsal ectoderm, but is unable to induce cement gland in naive animal caps. Neural tissue is induced less frequently than cement gland by the dorsoanterior yolky endoderm, suggesting that the endoderm induces cement gland directly. Thirdly, we demonstrate that the ventralectoderm adjacent to the cement gland attenuates cement gland differentiation late during gastrulation. The more distant ventralmesendoderm is also a potent inhibitor of cement gland formation. These are the first data showing that normal ventral tissues can inhibit cement gland differentiation and suggest that cement gland size and position may be partly regulated by negative signals. Previous work has shown that cement gland can be induced by neural plate and by dorsal mesoderm. Together, these data suggest that cement gland induction is a complex process regulated by multiple positive and negative cell interactions.
Fig. 8. In situ hybridization with neural markers. Stage 11 aDE explants alone (a,d,g), stage
11 aDE + DAYE conjugates (b,e,h) and stage 26 controls (c,f,i) were probed with
digoxygenin-UTP labeled N-CAM (a-c), b-Tubulin (d-f) or XCG (control, g-i) antisense
RNA probes. Scale bar, 200 mm.
Fig. 2. In situ hybridization of cultured explants and tailbud stage control embryos. Antisense RNA probes were digoxygenin-UTP-labeled (dark blue). (a-c) Stage 10 ventral animal caps analyzed for XCG expression; (d-f) stage 10.5 dorsal ectoderm, underlain by mesendoderm and analyzed for XCG expression; (g-i) stage 11.5 dorsal ectoderm analyzed for XCG expression; (j-l) stage 10 animal caps analyzed for XK81 expression; (m-o) stage 10 animal caps analyzed for UVS-2 expression. Control embryos, photographed at the same magnification as explants, are shown in the right column. Black arrowheads show examples of cement gland staining, white arrowhead shows hatching gland and white arrow shows XK81 staining. VE, ventralectoderm; DE, dorsal ectoderm. The fourth column shows tailbud stage control embryos. Scale bar, 200 um.
Fig. 4. Cement gland patterning. Double label in situ hybridization was carried out on stage 26 control embryos using digoxygenin labeled XCG (in red) and fluorescein-labeled XA (in dark blue) antisense RNA probes (a). Single in situ hybridization was carried out on stage 26 embryos using digoxygenin-labeled XA and XK81 probes (b). (a) Anterior view of a stage 26 embryo showing XCG transcripts in red, XA transcripts in blue. (b) Medial longitudinal sections of the heads of XA-(left) and XK81- (right) stained embryos. Black arrowheads indicate cement gland; white arrowhead indicates hatching gland; small white arrows indicate the epidermis, e, that surrounds the cement gland. Scale bar in a, 200 mm; embryos in b are at twice that magnification.
Fig. 6. Double staining in situ hybridization. Explants and stage 26 control embryos were probed with digoxygenin labeled XCG (in red) and fluorescein labeled XA (in dark blue) antisense RNA probes. (a,b) Stage 10 dorsal animal caps; (c) stage 10 animal cap conjugated to stage 11 DAYE; (d) stage 10 animal cap conjugated to stage 12 DAYE; (e) stage 11 aDE; (f) stage 11 aDE conjugated to stage 11 DAYE; (g) stage 11 aDE conjugated to stage 12 DAYE; (h) stage 11 aDE conjugated to stage 11 VYE; (i) stage 11 VE; (j) stage 11 VE conjugated to stage 11 DAYE; (k) stage 12 aDE; (l) stage 12 aDE conjugated to stage 12 DAYE. Control embryo is shown for comparison in Fig. 4 at same scale as explants. Scale bar, 200 mm, as for Fig. 4.
