Figure 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 the right.
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 b-catenin (0.5 ng RNA
per embryo) is not inhibited by coinjected E1A or p300N. Ectopic
activation of goosecoid expression by b-catenin was inhibited significantly
by E1A, as expected (data not shown), because goosecoid
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-17a and Mixer expression assayed by quantitative RT-PCR. In this and all of the following RT-PCR results, EF-1a 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.
Figure 2. Inhibition of mesoderm, endoderm, and epidermis formation
in animal pole explants. A, Inhibition of mesoderm markers induced by
FGF examined at stage 10.5. WE is the positive control for RT-PCR,
using the cDNA from whole embryos at the equivalent stage. RNA
injected per embryo: 0.1 ng for E1A, RG, and DL; 3 ng for p300N. bFGF
concentration is 50 ng/ml. B, Inhibition of mesoderm and endoderm
markers induced by activin examined at stage 10.5. mix1 is a homeobox
gene expressed in both mesoderm and endoderm. Endodermin induction
by activin was examined at stage 35. The RNA amount that was injected
is the same as in A. Activin concentration: 5 ng/ml (for mesoderm
induction) and 50 ng/ml (for endoderm induction). C, Inhibition of early
BMP-4 target genes induced by an activated ALK2, a BMP-4 receptor.
RNA injected per embryo: 0.05 ng for E1A, RG, and DL; 3 ng for p300N;
0.2 ng for ALK2. D, A lack of inhibition of Xwnt-8 or b-catenin signaling.
RNA injected per embryo is the same as in A, except 10 pg for Xwnt-8 and
0.1 ng for b-catenin. E, E1A or p300N inhibition of mesodermal and
endodermal gene expression was rescued by p300(dl10), which has wildtype
p300 function but is refractory to E1A inhibition (Eckner et al., 1994;
Lee et al., 1996). RNA injected per embryo is the same as in A, except 1
ng for p300(dl10).
Figure 3. Neuralization without anteroposterior patterning in animal
pole explants. A, Induction of neural markers was examined at stage 20 by
RT-PCR. Note the concomitant suppression of cytokeratin, an epidermal
marker. RNA injected per embryo: 5 pg for E1A, RG, and DL; 3 ng for
p300N; 1 ng for chordin (chd). Note that neurogenin was not induced by
chordin at this stage, as expected from published results (see Discussion;
Lamb et al., 1993; Papalopulu and Kintner, 1996). Similar results were
obtained when noggin was used in place of chordin (data not shown). B,
E1A or p300N induction of neural markers and suppression of cytokeratin
could be rescued by p300(dl10), coinjected at 1 ng of RNA per embryo.
Figure 4. Rescue of early neural development in UV-ventralized embryos.
RNA injected per embryo: 20 pg for E1A and RG, 3 ng for p300N,
and 150 pg for chordin. In situ hybridization and RT-PCR were performed
on embryos at stage 14. A, Rescue of zic-r1 expression by E1A and
chordin, but not by RG2 or b-galactosidase (used as a lineage-tracing
marker). Note that the expression of zic-r1 overlaps with X-gal staining in
E1A or chordin (chd)-injected embryos. B, Rescue of neural markers
examined by RT-PCR.
Figure 5. Neural gene induction in ventral marginal explants (prospective
ventral mesoderm) and vegetal pole explants (prospective
endoderm). RNA injected per embryo: 20 pg for E1A, RG, and DL; 3 ng
for p300N. A, Induction of neural markers and suppression of a mesoderm
marker in ventral marginal explants examined at stage 20 by RT-PCR.
a-Globin is a hematopoietic (ventral mesoderm) marker. B, Induction of
neural markers in vegetal explants examined at stage 20 by RT-PCR. Note
the concomitant suppression of endoderm markers (see Fig. 1E).