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Zygotic expression of the BMP-4 gene in Xenopus embryos is regulated by an auto-regulatory loop. Since AP-1 is known as a mediator of auto-regulatory loops both in the case of the Drosophila dpp and the mammalian TGF-beta genes, we have analysed the potential of Xenopus c-Jun (AP-1) as a mediator of BMP-4 expression during Xenopus development. RNA injection experiments revealed that both heteromeric c-Fos/c-Jun and homodimeric c-Jun/c-Jun strongly activate BMP-4 transcription, whereas BMP signaling was found to activate the Xenopus c-Jun gene only at a rather low extent. In addition, the lack of zygotic c-Jun transcripts until the end of gastrulation should exclude a role of AP-1 in the activation and the early expression of BMP-4 during gastrulation in vivo. However, at later stages of Xenopus development, we find a spatial overlap of c-Jun and BMP-4 transcripts which suggests that AP-1 might serve as an additional activatory component for the auto-regulation of BMP-4. Promoter/reporter and gel mobility shift assays demonstrate multiple responsive sites for AP-1 in the 5'' flanking region and two in the second intron of the BMP-4 gene. We further demonstrate that AP-1 acts independently of Xvent-2 which has recently been shown to mediate the early expression of BMP-4 in gastrula stage embryos.
Fig. 3. c-Jun (AP-1) leads to ectopic BMP-4 expression and to embryos showing a posteriorized phenotype. In situ whole-mount hybridizations demonstrate dorsal activation of BMP-4 upon injection of 500 pg AP-1 RNA (B) or 1 ng c-Jun (C) into both dorsal blastomeres of four-cell stage embryos; an uninjected control is shown in (A). While c-Jun transcripts are not detected within gastrula stage embryos (D), injection of 400 pg BMP-4 RNA into both dorsal blastomeres of four-cell stage embryos leads to a weak but distinct activation of the c-Jun gene (E). Injection of 1.5 ng c-Jun RNA into dorsal blastomeres suppresses head formation and causes a posteriorized phenotype (insert shows a control embryo at reduced size) (F). All embryos are photographed with dorsal side (top) and ventral side (bottom).
Fig. 2. Xenopus c-Jun expression during embryogenesis. (A) Temporal expression of c-Jun was analyzed with total RNA from embryos of different developmental stages (Nieuwkoop and Faber, 1975) by RT-PCR using oligonucleotides 5′-GGC AGA AAG GAA GCG TAT GAG-3′ as forward and 5′-CTG CTG TGT TAA CAT TAG CTC AC-3′ as reverse primer, repectively. Histone H4 was used as internal control. The last lane refers to a control assay lacking RNA. Spatial expression of Xenopus c-Jun was analyzed by whole-mount in situ hybridizations. (B) Four-cell, eight-cell, morula and gastrula stage embryos. Note the weak staining being most probably due to maternal transcripts. (C) Neurula stage embryos from dorsal and posterior (left) or from ventral and anterior (right). (D) Stage 22. c-Jun expression is observed at the posterior and lateral region at the lateral border of the neural plate but not within the dorsal midline. Anterior staining is within the neural plate. (E) Stages 25 and 27. (F) Stage 31/32. c-Jun transcripts are found within trunk neural crest cells, otic vesicle, the eye and head mesenchyme. (G) BMP-4 whole-mount in situ hybridization of embryos at later developmental stages. Note the similarities to the c-Jun expression pattern.