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We report the cloning of a cDNA encoding a Xenopus laevis Ets-type transcription factor. This new Xenopus gene belongs to the PEA3 subfamily of Ets proteins and shows the highest degree of sequence similarity to the mouse and human ER81 genes. The Xenopus ER81 gene (XER81) is transcribed in the embryo after mid blastula transition (MBT) and three transcripts of 3, 4 and 6 kb are detected throughout embryogenesis. XER81 mRNA is localized in the animal pole of the late blastula stage and higher levels of XER81 transcripts are detected in the marginal zone at the onset of gastrulation. In later embryogenesis XER81 transcripts are found in neural crest cells, eyes, otic vesicles and pronephros. The transcription of XER81 can be stimulated by bFGF and eFGF in animal and vegetal cap explants. Expression of the dominant negative FGF receptor mutant in animal caps and embryos blocks XER81 transcription, arguing that the expression of this Ets gene requires active FGF signaling. The spatial overlap of eFGF and XER81 expression domains supports the idea that XER81 transcription could be a marker for regions with active FGF signaling in the embryo.
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10096063
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Fig. 3. Spatial expression of XER81 during normal development. Embryos were hybridized with a digoxigenin-labeled XER81 antisense RNA probe. (A) show different gastrula stages in a vegetal view, (Aʹ′ʹ′) in a lateral view. Arrows indicate the dorsal blastopore lip. (D) and (Dʹ′) show a neurulaembryo in a lateral and anterior view, a: anterior, p: posterior. In (E) and (F) a distinct pattern is visible in the eyes (e), ear vesicles (ev), brain, branchial arches (ba), tailtip and the pronephric duct (pd).
Fig. 4. Spatial expression of XER81 during gastrula stages. Vibratome sections (100 mm thick) were hybridized with a digoxigenin-labeled XER81 antisense RNA probe. With the onset of gastrulation (A) XER81 mRNA is predominantly found in the animal cap. At stage 10 + expression appears in the dorsal marginal zone (B), at stage 10.5 in the ventral marginal zone (C). XER81 expression increases ventrally until it is equally strong in the total marginal zone at stage 11 (D). As gastrulation proceeds, expression of XER81 decreases in the animal cap and increases in the marginal zone (E, F). Arrows denote the dorsal blastopore lip.
Fig. 6. Expression of XER81 requires FGF signaling. (B) 500 pg of dominant-negative FGF receptor (XFD) mRNA were injected radially in the marginal zone at the 4 cell stage. The embryos were cultured until stage 10.5, fixed in Memfa and hybridized with a digoxigenin-labeled XER81 antisense RNA probe. XFD blocks the expression of XER81 at the sites of injection. (A) Unperturbed XER81 staining in the marginal zone is seen in uninjected control embryos. (C) Experimental design of the animal cap experiment. 200 pg of full length FGF receptor (XFR) or 400 pg of XFD were injected into the four animal blastomeres at the 4 cell stage. Animal caps of injected and not injected embryos were dissected at stage 8.5 and cultured in the presence of bFGF until stage 10.5. The explants were transferred to bFGF-free medium and cultured until stage 14. RNA was isolated and RT-PCR was performed to analyze the expression of XER81, Xbra as a marker and EF-1a as a loading control. 1/17 embryo- or 0.2 animal cap equivalents were used for each PCR reaction. (D) bFGF induces the expression of XER81 and, as expected, of Xbra (lanes 2, 3), but not in the presence of dominant-negative FGF receptor (lanes 4, 5). Addition of the full-length FGF receptor rescues the inducing activity of FGF in the presence of XFD (lane 6).
Fig. 7. Expression of XER81myc in Xenopus embryos. (A) Embryos were injected with either 600 pg of CMTXER81 DNA or 2 ng of XER81myc mRNA and proteins were isolated at stage 10.5. SDS-PAGE was performed with one embryo equivalent from DNA injected or two equivalents from mRNA injected or not injected control embryos. Western blotting was done with the anti-myc antibody 9E10. XER81myc protein of the expected size of 71 kDa was detected. (B) Albino embryos were injected with 500 pg of XER81myc mRNA and whole-mount immunostaining was performed with the anti-myc antibody at stage 10.5. XER81myc protein was detected at the site of injection (B, a). Higher magnification of cleared embryos revealed a strong XER81 signal in the nuclei and weaker staining in the cytoplasm (B, b). (C) Embryos were injected with 2 ng of XER81 mRNA and whole-mount in situ hybridization was performed at stage 10.5 with antisense mRNA probes for Xbra and Xcad-2. Overexpression of XER81 did not change the expression pattern of Xbra (b) and Xcad-2 (d) compared to uninjected control embryos (a, c). Bar in B corresponds to 25 mm.
Fig. 2. XER81 is expressed maternally and zygotically and expresses three messages. Embryos were harvested at the indicated developmental stage (NF,
Nieuwkoop and Faber, 1967), and 1.5 embryo equivalents total RNA were used for Northern blotting. Histone H4 mRNA was used as a loading control.
Three XER81-specific transcripts of 3, 4 and 6 kb are detectable. The 3 kb transcript is strongest in pre-MBT stages (egg and stage 6). Expression of the 4 and
6 kb transcripts peaks at neurula stages (stages 13-23).
Fig. 5. XER81 is inducible by FGF in animal and vegetal caps. (A) Experimental design of the animal cap experiment. Animal caps were dissected at stage 8.5 and cultured in the presence of bFGF or activin until stage 15. (B) bFGF clearly induced XER81 expression (B, lane 3) whereas induction by activin was weaker in animal cap explants (B, lane 4). (C) Experimental design of the vegetal cap experiment. 10 pg of eFGF or 100 pg of XFR (wild type FGF receptor) were injected into two vegetal blastomeres at the 2-cell stage. Vegetal caps were dissected at stage 9 and cultured until stage 11. (D) eFGF induced the XER81 expression in vegetal cap explants (D, lane 3). This effect was enhanced by coexpression of the full length FGF receptor and eFGF (D, lane 4). RNA of animal and vegetal explants was isolated and RT-PCR was performed to analyze the expression of XER81, Xbra as a marker and EF-1a as a loading control. Equivalents of 1/17 whole embryos, 0.2 animal caps or 0.5 vegetal caps were used in each PCR reaction.
