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Both activin-like signaling and Notch signaling play fundamental roles during early development. Activin-like signaling is involved in mesodermal induction and can induce a broad range of mesodermal genes and tissues from prospective ectodermal cells (animal caps). On the other hand, Notch signaling plays important roles when multipotent precursor cells achieve a specific cell fate. However, the relationship between these two signal pathways is not well understood. Here, we show that activin A induces Delta-1, Delta-2 and Notch expression and then activates Notch signaling in animal caps. Also, in vivo, ectopic activin-like signaling induced the ectopic expression of Delta-1 and Delta-2, whereas inhibition of activin-like signaling abolished the expression of Delta-1 and Delta-2. Furthermore, we show that MyoD, which is myogenic gene induced by activin A, can induce Delta-1 expression. However, MyoD had no effect on Notch expression, and inhibited Delta-2 expression. These results indicated that activin A induces Delta-1, Delta-2 and Notch by different cascades. We conclude that Notch signaling is activated when activin-like signaling induces various tissues from homogenous undifferentiated cells.
Fig. 1. Time course analysis of Delta-1, Delta-2, Notch and ESR-1 mRNA expression induced by activin A by real-time RT-PCR. RNA samples were derived from stage-9 animal caps which were treated with 0.1, 1.0, 10, and 100 ng/ml activin A for 1 hour, and then cultured for 3, 5 and 9 hours. The expression levels of Delta-1 (A), Delta-2 (B), Notch (C) and ESR-1 (D) mRNA were quantified by real-time RT-PCR. The relative "stimulation fold" was calculated as the individual expression in activin A-treated animal caps relative to that in untreated animal caps at each developmental stage. The efficiency of cDNA synthesis from mRNA was assessed on the basis of real-time RT-PCR for ODC. The results represent the mean from three or four independent experiments, and error bars indicate the SEM.
Fig. 2. The effect of Activin-like signaling on Delta-2 expression in vivo. The expression pattern of Delta-2 at stage 11 was examined by whole mount in situ hybridization. (A,B) Uninjected control embryos. (C,C',D) Embryos were injected with 500 pg of ALK4 dn mRNA into one blastomere at the 2- or 4-cell stage dorsally (C) or ventrally (C'). The injection of ALK4 dn mRNA suppressed the expression of Delta-2 on the injection side (black arrow). (E,E',F) Embryos were injected with 200 pg of ARI mRNA into one blastomere at the 2- or 4-cell stage dorsally (E) or ventrally (E'). The injection of ARI mRNA increased the expression of Delta-2 on the injection side (red arrowhead). Vegetal views, dorsal at top (A,C,C',E,E'). Lateral views, animal pole at top (B,D,F).
Fig. 3. The expression levels of Delta-1, Delta-2, Notch and ESR-1 in MyoD-injected animal caps. Animal caps were dissected from stage 9 embryos which were injected with 300 pg of MyoD mRNA into animal pole at 2-cell stage, and then cultured for 3 hours. The expression levels of Delta- 1, Delta-2, Notch and ESR-1 mRNA were quantified by real-time RT-PCR. The relative stimulation fold was calculated as the individual expression in MyoD-injected animal caps relative to that in untreated animal caps. The efficiency of cDNA synthesis from mRNA was assessed on the basis of real-time RT-PCR for ODC. The results represent the mean from three independent experiments, and error bars indicate the SEM.
Fig. 4. The effects of MyoD injection on Delta-1 and Delta-2 expression in vivo. Embryos were injected with 1.0 ng MyoD mRNA together with 100 pg of lacZ mRNA as lineage tracer into one blastomere at the 2-cell stage. The expression pattern of Delta-1 (A) (lateral view) and Delta-2 (B) (vegetal view) at stage 11 was examined by whole mount in situ hybridization.
