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.
Image published in: Suzuki A and Hemmati-Brivanlou A (2000)
Copyright © 2000. Image reproduced with permission of the Publisher, Elsevier B. V.
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