Fig. 1. Inhibition of p300/CBP by E1A, E1A–RG2, and p300N interferes with mesoderm and endoderm development.A, Phenotypes of embryos injected dorsally at four-cell stage with E1A, RG2, DL120–140 (DL), and p300N RNAs, shown at stage 25. E1A, DL120–140, and p300N-injected embryos cleaved and developed normally up through the late blastula stage (stage 9) but began to exhibit severe gastrulation abnormality from the early gastrula stage (stage 10.5). A failure of blastopore closure was observed in many of the injected embryos (stage 13). Most of injected embryos displayed a lack of any axial development at stage 25. The RG2-injected embryo is phenotypically identical to normal controls. RNA injected per embryo: 0.1 ng for E1A, RG2, and DL120–140; 2 ng for p300N. Note that because p300N mRNA and protein sizes are each approximately seven times of those for E1A, the efficiency of synthesis and the local concentration of p300N protein are likely to be much smaller than those of E1A in the embryo if the same amount of RNA were to be injected. B, Sagittal sections of a RG2 (top) and an E1A-injected embryo (bottom) at stage 25. Note that the RG2-injected embryo exhibits normal development, whereas the E1A-injected one lacks any axial tissues, such as the notochord and muscles. Axial tissues are absent in all sections (data not shown). C, Inhibition of Xbra expression examined at stage 10.5 by in situ hybridization. Note that the control (CON) and RG2-injected embryo exhibit a uniform circumferential expression pattern, whereas expression is abolished on the injected side (either dorsal or ventral) in E1A, DL120–140, or p300N-injected embryos. Dorsal is to theright. RNA injected per embryo: 20 pg for E1A, RG2, and DL120–140; 500 pg for p300N. D, Lack of inhibition of Xnr3 expression by in situ hybridization analysis, examined at stage 10.5. Note that the endogenous Xnr3 expression is not blocked by dorsally injected E1A, and an ectopic induction of Xnr3 by ventrally injected β-catenin (0.5 ng RNA per embryo) is not inhibited by coinjected E1A or p300N. Ectopic activation ofgoosecoid expression by β-catenin was inhibited significantly by E1A, as expected (data not shown), becausegoosecoid expression depends on mesoderm-inducing signals (Watabe et al., 1995). Dorsal is to the right. RNA amount injected is the same as in C.E, Inhibition of Xsox-17α and Mixer expression assayed by quantative RT-PCR. In this and all of the following RT-PCR results, EF-1α is used as a control for the template amount. The RNA amount injected is the same as in A. F, Immunoblot of stage 9 embryo extracts to examine protein levels of E1A, E1A–RG2 (RG2), and E1A–DL120–140 (DL) from injected synthetic RNAs. CON, Uninjected control extract.