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The Xvent homeobox multigene family is essential for the patterning of the ventralmesoderm in Xenopus embryos. We have identified two novel members of this family, Xvent-1B and Xvent-2B, and have characterized their genomic structures. These two genes show a clustered organization and have probably arisen by gene duplication with subsequent inversion. Cis-regulatory elements within the promoters of both genes have been identified which contribute to their spatial activation. Xvent-2B is activated by BMP-2/4 in the absence of de novo protein synthesis, suggesting that this gene is a direct target of BMP-signalling. In contrast, Xvent-1B does not directly respond to BMP-2/4, but is activated by Xvent-2B. This activation is documented by Xvent-1B promoter/reporter studies, Xvent-2B overexpression and loss-of-function analysis using a dominant-negative Xvent-2 mutant. However, cycloheximide experiments reveal that Xvent-2B by itself is not sufficient to activate transcription of the Xvent-1B gene, but that there is a requirement for additional factor(s) being synthesized after midblastula transition.
Fig. 2. Spatial transcription patterns and phenotypic effects observed after ectopic expression of Xvent-1B and Xvent-2B. Transcription patterns were analyzed by whole mount in situ hybridization of Xenopus embryos using Xvent-1B (A) or Xvent-2B (E) antisense RNA. Developmental stages shown for Xvent-1B are: 10.0 (A), 12.5 (B) and 23 (C); Xvent-2B: 10.5 (E), 12.0 (F), 24 (G), 29 (H) and 35 (I). Note that at early stages Xvent-1B expression is restricted more ventrally and lacks anterior expression in the eyes at later stages. Over-expression experiments were carried out by microinjection of either Xvent-1B (D) or Xvent-2B RNA (J) into the dorsal blastomeres of four-cell stage embryos which were then cultured until controls had reached stage 30. Note the complete ventralization and loss of all anterior structures after ectopic expression of Xvent-1B as well as of Xvent-2B.
Fig. 5. Xvent-2B activates Xvent-lB but not vice versa. Xvent-1B (A,C) or Xvent-2B transcripts (B,D) have been analyzed by whole mount in situ hybridization after injection of 400 pg Xvent-2B RNA (C) or 400 pg Xvent-1B RNA (D) respectively. RNAs had been injected into the dorsal blastomeres of four-cell stage embryos. (A,B) Show uninjected control embryos. Note the activation of Xvent-1B in dorsal mesoderm.
Fig. 6. Effects of Xvent-2B but not of Xvent-1B can be overcome by Xvent-2 P(40). (A) Luciferase activity of -249 Xvent-1B/Luc construct after injection (20 pg) into both dorsal or ventral blastomeres of four- cell stage embryos. Co-injections have been performed with 500 pg Xvent-2 P(40) and/or 200 pg Xvent-2B RNA. (B) Phenotype of Xeno- pus embryos ventrally injected with 1 ng Xvent-2 P(40) RNA (B) or 1 ng Xvent-2 P(40) plus 200 pg of Xvent-1B RNA(C). Note that formation of double axis is completely abolished. Phenotype after dorsal injection of 200 pg Xvent-1B RNA (D) or 200 pg Xvent-1B plus 1 ng Xvent-2 P(40) RNA (E). Note, that Xvent-2 P(40) cannot prevent the ventralization caused by Xvent-1B.
Fig. 7. Interactions between BMP-4, Xvent-2B and Xvent-1B in whole embryos in the absence or presence of cycloheximide (CHX). Four-cell stage embryos have been injected with BMP-4 RNA (300 pg) or Xvent-2B RNA (400 pg) into both dorsal blastomeres. Cycloheximide treatment started at stage 7.5 and embryos were cultured until stage 10.5. RT-PCR was performed with total RNA and has been adjusted using histone H4 as an internal control.
