Fig. 6. GATA4 does not induce cardiac tissue precociously, and acts both non-cell- and cell-autonomously to induce cardiomyocytes. Cardiac actin-GFP transgenic embryos were injected with GATA4 (A-C), or with GATA4 together with rhodamine-dextran as a lineage tracer (G,H), in one blastomere at either the four- or eight-cell stage. Temporally correct cardiac differentiation was documented by observation of GFP. The cardiac actin promoter is active in both skeletal and cardiac muscle tissue. GATA4 does not induce skeletal muscle (Fig. 1), and it also fails to induce GFP at stages 17 (A) and 24 (B), when the transgene is active in skeletal muscle of sibling control embryos (D,E). GFP activity in GATA4-injected explants (C) is only detected when it is also present in the heart of sibling embryos (F). In G and H injected tissue was detected by rhodamine fluorescence (red); overlap with GFP is evident (yellow). Most GFP fluorescence is distinct from the rhodamine signal, which demonstrates the non-cell-autonomous action of GATA4. However, the areas of overlap also suggest cell-autonomous action of GATA4. (F) Stage 40 control sibling embryo showing cardiac-GFP expression in the heart (h) and somites (s). (I-K) Embryos were injected with GATA4 together with Cerberus in one blastomere at the four- or eight-cell stage, and biotinylated dextran was co-injected as a lineage tracer. MLC2 expression at stage 38 was detected by in situ hybridisation (pale blue in J), and the injected part of explant is revealed in whole mount (I) or on sections (K) as magenta staining. Overlap of the two colours creates a purple signal (arrow), which indicates a cell-autonomous action of GATA4. Embryos are shown with the anterior end to the left. (L) Cell-cell interactions are not required for GATA4 action until at least stage 16. Animal pole explants injected with GATA4-GR were dispersed in Ca²⁺/Mg²⁺-free medium after excision. GATA4 was subsequently activated by dexamethasone. Single-cell suspensions were cultured with constant gentle agitation to prevent spontaneous formation of cell contacts. Cells were re-aggregated at an indicated stage by the addition of Ca²⁺ and pelleting, and were cultured until sibling controls reached stage 37/38. Note that viability of cells kept in suspension until stage 16 is low, as revealed by poor EF1 recovery; nevertheless, cardiac markers were still detectable.

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