Vodicka MA and Gerhart JC (1995), Blastomere derivation and domains of gene expre...

XB-ART-19069

Development 1995 Nov 01;12111:3505-18.

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Blastomere derivation and domains of gene expression in the Spemann Organizer of Xenopus laevis.

Vodicka MA , Gerhart JC .

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Spemann's Organizer, located in the dorsal marginal zone of the amphibian gastrula, induces and differentiates dorsal axial structures characteristic of this and other vertebrates. To trace the cellular origins of the Xenopus Organizer, we labelled dorsal blastomeres of three of the four tiers (A, B and C) of the 32-cell embryo with green, red and blue fluorescent lineage tracers. A strong vegetalward displacement of labelled clones occurs between the late blastula and early gastrula stages but clones mix only slightly at their borders. The typical early gastrula Organizer is composed of approximately 10% A1 progeny in its animalmost region, 70% B1 progeny in the central region, and 20% C1 progeny in vegetal and deep regions. Variability in the composition of the early gastrula Organizer results from variability in the position of early cleavage planes and in pregastrulation movements. As the Organizer involutes during gastrulation, forming dorsal axial mesoderm, clonal boundaries are greatly dispersed by cell intermixing. Within a clone, deep cells are displaced and intermixed more than superficial cells. Variability in the distribution of progeny in the dorsal axial mesoderm of the late gastrula results mostly from variable intermixing of cells during gastrulation. Experiments to perturb later developmental events by molecular or embryonic manipulations at an early stage must take this variability into account along with the majority distributions of the fate map. Within the early gastrula Organizer, the genes Xbra, goosecoid, noggin and xNR3 are expressed differently in the animal-vegetal and superficial-deep dimensions. In situ hybridization and lineage labelling define distinct regions of the dorsal marginal zone. By the end of gastrulation, dorsal axial mesoderm cells derived from the Organizer have altered their expression of the genes Xbra, goosecoid, noggin and xNR3. At a given stage, a cell's position in the embryo rather than its lineage may be more important in determining which genes it will express.

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Species referenced: Xenopus laevis
Genes referenced: gsc nodal3.1 nodal3.2 nog tbxt

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	Fig. 1. 32-cell blastomere designations and dorsal injections. Dorsal is to the right. (A) Diagram of one-half of a stage 6 embryo indicating the tiers A-D, animal to vegetal, and the blastomere nomenclature of Nakamura and Kishiyama (1971). (B) Schematic diagram of dorsal lineage label injections at the 32-cell stage. (C) Parasagittal section of a stage 6 embryo in which A1 was injected with FDA, B1 with TxRDA and C1 with CBDA. Scale bar is 160 mm.
	Fig. 2. The gastrula fates of the cleavage stage dorsal blastomeres. (A-C) Early gastrula, stage 10-; (D-F) late gastrula, stage 12. (A,D) Sagittal sections; (B,E, and C,F) parasagittal sections 65 and 130 mm, from the midline, respectively. The green (FDA) cells are A1 progeny; the red (TxRDA) cells are B1 progeny. The blue (CBDA) cells are C1 progeny. Dorsal is to the right. The dorsal lip is indicated by an arrow in A-C. Scale bar is 160 mm.
	Fig. 3. Progeny locations of dorsal blastomeres at early (stage 10-) and late (stage 12) gastrula. (A,B) Stage 6-10 fate maps; (C,D) stage 6-12 fate maps. (A,C) Modal maps; (B,D) statistical maps. Green indicates regions populated by A1 progeny; red indicates regions populated by B1 progeny and blue indicates regions populated by C1 progeny. Location of progeny and their ultimate fates are described in text for Figs 2, 3. Scatter in B is due to variation between embryos; scatter in D primarily is due to cell mixing. Dorsal is to the right. Data include 30 sections for stage 10 and 18 for stage 12. See text for details on construction of fate maps.
	Fig. 4. Comparison of the locations of dorsal and ventral cleavage stage blastomere progeny at pregastrula and gastrula stages. Late blastula, stage 9 dorsally injected embryo (A). Early gastrula stage 10-, ventrally injected (B) and dorsally injected (C). Late gastrula, stage 12, ventrally injected (D-E) and dorsally injected (F-G). (E,G) Close-ups of the involuted ventral and dorsal mesoderm from D and F, respectively. Green (FDA) cells are descendants of A1 (A,C,F,G) and A4 (B,D,E); red cells (TxRDA) are descendants from B1 (A,C,F,G) and B4 (B,D,E); blue cells (CBDA) are descendants from C1 (A,C,F,G) and C4 (B,D,E). All sections are parasagittal, approximately 65 mm from the midline; dorsal is to the right, except in E and G, dorsal is at the top with anterior to the left. The dorsal lip is indicated by an arrow in C. Scale bar for A-C,D,F is 220 mm; scale bar for E, G is 97 mm.
	Fig. 5. Animal blastomere injections. Early gastrula, stage 10-, parasagittal section, 65 mm from the midline. Green (FDA) cells are progeny of A4; blue (CBDA) cells are progeny of A3; and red (TxRDA) cells are progeny of A1. B is a close-up of the animal cap region in A. Scale bar is 220 mm in A and 97 mm in B.
	Fig. 6. Tailbud fates of dorsal blastomeres. A transverse section of the right side of the head of a stage 35/36 tailbud. The brain is indicated by short arrow, the notochord by curved short arrow and the eye by long arrow. Green (FDA) cells are descendants from A1; red cells (TxRDA) are descendants from B1; and blue cells (CBDA) are descendants from C1. Scale bar is 80 mm.
	Fig. 7. Stage 10 Xbra, xNR3, noggin and goosecoid expression. (A) Xbra in situ hybridization; (B) corresponding lineage label; (C) another Xbra in situ hybridization showing staining in the superficial layers and in the ventral marginal zone; (D) xNR3 in situ hybridization; (E) corresponding lineage label; (F) another xNR3 in situ hybridization demonstrating that intense staining remains restricted to the superficial layer; (G) nog in situ hybridization; (H) corresponding lineage label; (I) another section from same embryo where lineage label does not interfere with in situ hybridization signal and the full extent of nog expression is visible; (J) gsc in situ hybridization; (K) corresponding lineage label. All photographs are of dorsal marginal zone of 20 mm parasagittal sections from stage 10 embryos. Green (FDA) cells are descendants of A1; red (TxRDA) cells are descendants of B1; and blue (CBDA) cells are descendants of C1. Dorsal is to the right and the dorsal lip is indicated by an arrow or arrowhead in each frame. Scale bar is 220 mm.
	Fig. 8. Stage 12 Xbra, xNR3, noggin and goosecoid expression. (A) Xbra in situ hybridization; (B) corresponding lineage label; (C) xNR3 in situ hybridization with arrow indicating single positive staining cell; (D) corresponding lineage label; (E) nog in situ hybridization; (F) lineage label of contra-lateral section (because we sometimes encountered incompatibilities between lineage label and hybridization signal, we used one section for detecting nog expression on the unlabelled half of the embryo and a contralateral section for lineage analysis); (G) nog in situ hybridization, albino embryo: neural plate and chordal mesoderm staining both visible; (H) gsc in situ hybridization; (I) corresponding lineage label: only TxRDA label survived procedure. All 20 mm parasagittal sections. Green (FDA) cells are descendants of A1; red (TxRDA) cells are descendants of B1; and blue (CBDA) cells are descendants of C1. Dorsal is at the top with anterior to the left. Scale bar is 220 mm in A,B,E,F,H,I; 187 mm in C and D; 172 mm in G.
	Fig. 9. Gene expression summary. Schematic section diagrams of stage 10 dorsal marginal zone (A) and stage 12 dorsal half of an embryo (B). Xbra expression is represented by dark blue-purple triangles; nog expression by magenta circles; gsc expression by gold rectangles and xNR3 expression by cyan squares. The dotted line in the uninvoluted marginal zone is to indicate the approximate boundary between anterior and posterior organizing influences (J. S. Zoltewicz and J. C. Gerhart, personal communication).