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Figure 1.
Isolation of Genes that Activate the Expression of Posterior Region–Specific Genes
(A) Expression screening strategy. A gastrula expression library was divided into 100 fractions of 1000 clones each, and mRNA was synthesized from each pool by in vitro transcription. The synthetic RNA was injected into animal poles of two-cell stage embryos, and ectodermal explants were isolated at blastula stages. The positive pools were identified by analyzing the expression of posterior-specific genes at neurula stages by RT-PCR.
(B) Differing amounts of 20CHG11 RNA (0–5 ng) were injected into animal poles of two-cell embryos (lanes 3–6). Ectodermal explants were isolated at blastula stages and subjected to RT-PCR analysis at neurula stages (stage 15). −RT indicates sibling control embryos processed without reverse transcriptase. Histone was used as a loading control.
(C) 20CHG11 induces the ventral mesodermal marker, Xhox3, but not other regional markers such as Xwnt-8 and chordin, whose expression is localized to the ventral and dorsal sides of embryo, respectively. Ectodermal explants injected with 20CHG11 RNA were processed for RT-PCR assay at early gastrula stages (stage 11.5).
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Figure 2.
Structure and Expression of Xenopus E2F (xE2F)
(A) Alignment of amino acid sequences between xE2F and human E2F-3 (hE2F-3). The sequences were aligned by DNASTAR MegAlign software (DNASTAR Inc.). Identical amino acids and gaps are shown as dots and dashes, respectively. The amino acid number is shown at left. Functional domains are indicated by double-headed arrows.
(B) Amino acid identity of xE2F to the human E2F family. The amino acid identity is shown by percentage.
(C) Expression of the xE2F gene during early Xenopus embryogenesis. Expression of xE2F was detected by RT-PCR. ODC was used for a loading control; the stage 32 embryos processed without reverse transcriptase in the RT-PCR reaction are indicated as −RT.
(D) Spatial expression pattern of xE2F transcripts in Xenopus embryos. (a) Vegetal view of a stage 10.5 embryo; dorsal is at top. (b) Dorsal view of a stage 14 embryo; anterior is at top. (c) Lateral view of a stage 25 embryo; dorsal is at top and anterior is at left. (d) Dorsal view of (c); anterior is at top. (e) Lateral view of stage 30; anterior is at right.
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Figure 3.
Overexpression of xE2F Affects Dorsoanterior Structures
(A) Two different doses of xE2F RNA (2.5 or 5.0 ng) were injected into all four animal pole blastomeres of 8-cell embryos (a–f). The injected embryos show relatively small eyes at a low dose (b) and lack both eyes and cement glands at a high dose (c and f). Injection of xE2F(1–361) RNA (5.0 ng) did not affect normal development (d). Uninjected embryos are shown as a control (a and e). The anterior region of an xE2F RNA–injected embryo (f) is compared to that of an uninjected embryo (e). Eyes (eye) and cement gland (cg) are indicated. UI, uninjected; E2(low), 2.5 ng of xE2F; E2(high), 5.0 ng of xE2F; E2(1-361), 5.0 ng of xE2F(1-361). (g–i) Effect of xE2F:GR on the anterior–posterior axis of Xenopus embryos. Either xE2F:GR or xE2F(1–88):GR RNA (2.5 ng each) was injected into animal poles of four-cell embryos. The injected embryos were developed to late blastula stages, and then cultured with or without 20 μM DEX. xE2F:GR affects normal development when it is stimulated by DEX treatment (h). Even after DEX treatment, xE2F(1-88):GR does not affect normal development (i). (j and k) xE2F:GR is not able to affect normal development when activated at neurula stages. Embryos injected with xE2F:GR RNA (2.5 ng) were treated with DEX at gastrula (stage 11, j) and neurula (stage 14, k) stages. E2GR/−, xE2F:GR without DEX treatment; E2GR/+, xE2F:GR with DEX treatment; E2(1-88)GR/+, xE2F(1-88):GR with DEX treatment.
(B) Schematic structure of xE2F mutants and glucocorticoid receptor fusion proteins. Black and dark gray boxes indicate the DNA-binding domain (DBD) and transactivation domain (TAD), respectively. Light gray boxes indicate the hormone-binding domain of the glucocorticoid receptor (GRHBD).
(C) xE2F(1-361) is not able to induce the expression of HoxB9. xE2F or xE2F(1-361) RNA (5 ng each) were injected into animal poles of two-cell embryos, and ectodermal explants were processed for RT-PCR analysis.
(D) Conditional activation of ectopic xE2F activity. Ectodermal explants were prepared from embryos injected with xE2F:GR RNA or xE2F(1-88):GR (2.5 ng each) at blastula stages (stage 9) and cultured in the absence or presence of 20 μM DEX. The expression of HoxB9 was detected by RT-PCR analysis at gastrula stages. Embryo and −RT indicate sibling control embryos with or without reverse transcriptase.
(E) Activation of xE2F:GR at neurula stages is able to induce the expression of HoxB9. Ectodermal explants expressing xE2F:GR were isolated at blastula stage and cultured in the presence of DEX at stages indicated. The expression of the HoxB9 gene was detected by RT-PCR 2 hr after the DEX treatment.
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Figure 4.
Analysis of Embryos Overexpressing xE2F by Whole-Mount In Situ Hybridization
xE2F RNA was injected into animal poles of eight-cell albino (B) or wild-type embryos (C) and developed to tailbud stages. The expression of HoxB9 was detected in both control and injected embryos by whole-mount in situ hybridization. Anterior is to the left. In (A) and (B), the dorsal view of embryos is shown in the bottom. Arrows indicate position of eyes. Arrowheads indicate ectopic expression sites of HoxB9 in the brain of xE2F-injected embryos. Overexpression of xE2F leads to expansion of HoxB9 expression that is normally restricted in the posterior spinal cord. The ectopic expression of the HoxB9 gene in the brain that is adjacent to eyes is clearly seen in (C). (D–G) Embryos injected with xE2F:GR RNA (2.5 ng) were developed to late blastula stages and then cultured in the presence (E and G) or absence (D and F) of 20 μM DEX. The expression of HoxB9 (D and E) and Otx (F and G) was detected at early neurula stages by whole-mount in situ hybridization. Dorsal views of injected embryos are shown with the anterior side at the top. Location of the blastopore is indicated as bp.
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Figure 5.
xE2F Does Not Require Cell–Cell Communication or De Novo Protein Synthesis to Induce Target Gene Expression
(A) xE2F does not require cell–cell communication within ectoderm to activate target gene expression. Ectodermal explants injected with xE2F:GR RNA (2.5 ng) were isolated at late blastula stages and dissociated into single cells. The ectodermal cells were transferred into DEX-containing medium and were kept dissociated for about 3 hr (Diss.). Gene expression was detected by RT-PCR analysis. Explants cultured without dissociation are indicated as Intact.
(B) Induction of target gene expression by xE2F does not require de novo protein synthesis. xE2F:GR RNA (2.5 ng) was injected at the two-cell stage, and ectodermal explants were isolated at blastula stages. Explants were treated with CHX for 30 min and transferred into medium containing both CHX and DEX to activate xE2F:GR. The expression of HoxB9 was detected by RT-PCR analysis after 3 hr of DEX treatment.
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Figure 6.
xE2F:EnR Acts as a Dominant-Negative xE2F
(A) Schematic representation of the xE2F:EnR construct. The DNA-binding domain (DBD) of wild-type xE2F is indicated as a black box. A dark gray box indicates the transactivation domain (TAD). Light gray boxes indicate the repressor domain from the Drosophila Engrailed gene.
(B) xE2F:EnR suppresses the expression of HoxB9 induced by wild-type xE2F in ectoderm. xE2F RNA (0.3 ng) was injected with increasing amounts of xE2F:EnR RNA (30–125 pg) into the animal poles of two-cell embryos (lanes 5–7). The explants were isolated at blastula stages, and the expression of HoxB9 was detected by RT-PCR at neurula stages. Overexpression of the EnR domain alone does not affect the expression of HoxB9 (lane 9).
(C) Suppression of xE2F function by xE2F:EnR (dnxE2F) is rescued by increasing amounts of xE2F. The induction of HoxB9 expression by 0.15 ng of xE2F RNA is suppressed by coinjection of 30 pg of dnxE2F. This suppression is rescued by injection of 0.6 ng xE2F RNA.
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Figure 7.
Inhibition of xE2F Function Affects the Development of Ventral and Posterior Structures
Uninjected embryos are shown in (A) as a control. The dnxE2F RNA (120 pg) was injected into the ventral or dorsal side of four-cell stage embryos (B and C, respectively), and the injected embryos were developed to tadpole stages. The injected embryos showed defects in trunk and posterior regions, but not in anterior regions. The effect of dnxE2F on normal development was partially rescued by coinjection of wild-type xE2F RNA (0.5 ng) (D and E). Cont, control; DN/ven, dnxE2F RNA into ventral side; DN/dor, dnxE2F RNA into dorsal side; DN+WT/ven, dnxE2F RNA with wild-type xE2F RNA into ventral side; DN+WT/dor, dnxE2F RNA with wild-type xE2F RNA into dorsal side. (F–H) Histological section of control and dnxE2F-injected embryos. ne, neural tube; m, muscle; no, notochord. (I and J) Overexpression of dnxE2F affects the expression of the spinal cord marker HoxB9. dnxE2F RNA (120 pg) was injected into the dorsal side of four-cell stage wild-type embryos and subjected to whole-mount in situ hybridization with an antisense HoxB9 probe at tadpole stages. Anterior side of embryos is to the left. Cont, control; DN/dor, dnxE2F RNA into dorsal side. (K) Inhibition of Hox gene expression by dnxE2F. Embryos injected dorsally with dnxE2F RNA (120 pg) were developed to tailbud stage (stage 25), and two embryos each were subjected to RT-PCR analysis. (L) Inhibition of xE2F function in marginal zone explants dorsalizes ventral mesoderm. dnxE2F RNA (120 pg) was injected into the ventral side of four-cell stage embryos, and dorsal and ventral marginal zone (DMZ and VMZ) explants were excised at early gastrula stages. The explants were subjected to RT-PCR analysis at midgastrula (stage 11) and tailbud (stage 27) stages. DN, dnxE2F RNA. (M) Wild-type xE2F rescues dorsalization of ventral mesoderm caused by dnxE2F. dnxE2F RNA (120 pg) was injected with increasing amounts of wild-type xE2F RNA (2.5 ng and 5.0 ng in lanes 6 and 7, respectively). DMZ and VMZ explants are isolated at early gastrula stages and subjected to RT-PCR at midgastrula stages. MZ, marginal zone; D, dorsal; V, ventral. (N) xE2F affects mesoderm formation induced by growth factors. Xenopus embryos were injected either with 60 pg (lanes 5 and 8) or 120 pg (lanes 6, 9, and 11) dnxE2F RNA into animal poles of two-cell stage embryos. Ectodermal explants from blastulae were treated with activin (supplied as oocyte supernatant; see Experimental Procedures) or FGF (100 ng/ml) and subjected to RT-PCR analysis at early gastrula stages. For BMP4 treatments, BMP4 RNA (0.5 ng) was coinjected with dnxE2F RNA.
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e2f3 (E2F transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.
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e2f3 (E2F transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 14, dorsal view, anterior up.
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e2f3 (E2F transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
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e2f3 (E2F transcription factor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior right, dorsal up.
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