Onichtchouk D et al. (1996), The Xvent-2 homeobox gene is part of the BMP-4 ...

XB-ART-17628

Development 1996 Oct 01;12210:3045-53. doi: 10.1242/dev.122.10.3045.

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The Xvent-2 homeobox gene is part of the BMP-4 signalling pathway controlling [correction of controling] dorsoventral patterning of Xenopus mesoderm.

Onichtchouk D , Gawantka V , Dosch R , Delius H , Hirschfeld K , Blumenstock C , Niehrs C .

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We describe a novel Xenopus homeobox gene, Xvent-2, which together with the previously identified homeobox gene Xvent-1, defines a novel class of homeobox genes. vent genes are related by sequence homology, expression pattern and gain-of-function phenotype. Evidence is presented for a role of Xvent-2 in the BMP-4 pathway involved in dorsoventral patterning of mesoderm. (1) Xvent-2 is expressed in regions that also express BMP-4. (2) Xvent-2 and BMP-4 interact in a positive feedback loop. (3) Xvent-2 ventralizes dorsal mesoderm in a dose-dependent manner resulting in phenoytpes ranging from microcephaly to Bauchstück pieces, as does BMP-4. (4) Like BMP-4 and gsc, Xvent-2 and gsc are able to interact in a crossregulatory loop to suppress each other. (5) Microinjection of Xvent-2 mRNA can rescue dorsalization by a dominant-negative BMP-4 receptor. The results suggest that Xvent-2 functions in the BMP-4 signalling pathway that antagonizes the Spemann organizer.

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Species referenced: Xenopus
Genes referenced: actc1 actl6a bmp4 evx1 gsc h4c4 not tbx2 ventx1.2 ventx2 ventx2.2 wnt8a

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	Fig. 1. Xvent-2 and Xvent-1 are members of a new class of homeobox genes. (A) Deduced amino acid sequence of Xvent-2 protein. The homeodomain is underlined. (B) Sequence alignment of the Xvent-2 homeodomain with other homeoprotein sequences. Amino acids identical to those of Xvent-2 are indicated by bars, and are expressed as % homology. Xvent-1 (Gawantka et al., 1995); Lbe (Jagla et al., 1994); HBox-2.8 (Belleville et al., 1992); pS6 (Fjose et al., 1988); Mox-1 (Candia et al., 1992).
	Fig. 2. Northern blot and expression profile of Xvent-2. (A) A northern blot of stage 13 poly(A+) mRNA (PolyA+) and in vitro transcribed Xvent-2 mRNA was probed with Xvent-2. Sizes of markers and the band observed in PolyA+ are indicated in kb on the right and left, respectively. (B) RT-PCR analysis of Xvent-2 expression at the embryonic stages indicated. Top, Xvent- 2; bottom, Histone H4, for normalization.
	Fig. 3. Spatial expression of Xvent-2. Xvent-2 expression was analyzed by in situ hybridisation of whole-mount (A,D,E,H) and sagittally cut (B,G) Xenopus embryos. For comparison, whole-mount in situ hybridisations of Xvent-1 are shown (C,F). The dorsal blastopore lip is indicated by the arrowhead. (A) Stage 10 (early gastrula) embryo shown in vegetal view. The dorsal expression boundary of Xvent-2 is indicated by dashed lines. (B) Stage 10 (early gastrula) embryo shown in lateral view. (C) Stage 10 (early gastrula) embryo shown in vegetal view. Note that the dorsal expression boundary of Xvent-1 (dashed lines) is more lateral than that of Xvent- 2 (A). (D-F) Stage 13 (early neurula) embryos shown in dorsal (D) and posterior view (E,F). Note that the expression of Xvent-2 comprises the whole circumblastoporal colar (E) while the expression domain of Xvent-1 (F) is more ventral. (G) Stage 13 (early neurula) embryo para-sagittally cut and shown in lateral view. Note two staining somites (arrows). (H) Stage 30 (tailbud) embryo in lateral view. a, anterior; an, animal pole; ba, branchial arches; ey, eye; lp, lateral plate mesoderm; p, posterior; pr, proctodeum; so, somites; ve, vegetal pole.
	Fig. 4. Interaction of Xvent-2 with Xvent-1 and gsc. All embryos were analyzed at the gastrula stage (stage 10-11) by whole-mount in situ hybridisation and are shown in vegetal view. (A) UV-ventralized embryo probed for Xvent-2 expression. (B) LiCl-dorsalized embryo probed for Xvent-2 expression. (C) Embryo microinjected into two opposite blastomeres at the 4-cell stage with 50 pg gsc mRNA each and probed for Xvent-2 expression. Note the two domains lacking Xvent-2 expression (arrowheads). (D) Embryo microinjected with 0.1 ng Xvent-1 mRNA each into two opposite blastomeres at the 4-cell stage, treated with LiCl and probed for Xvent-2 expression. Note the two opposite domains of induced expression (arrowheads). (E) Embryo microinjected with 1.5 ng DXvent-2 mRNA (Co) each into two opposite blastomeres at the 4-cell stage, treated with LiCl and probed for gsc expression. gsc is radially expressed. (F) Embryo microinjected with 1.5 ng Xvent-2 mRNA each into two opposite blastomeres at the 4-cell stage, treated with LiCl and probed for gsc expression. Note the two domains lacking gsc expression (arrowheads).
	Fig. 5. BMP-4 is necessary and sufficient for expression of Xvent-2 and Xvent-1. Wild-type embryos or treated embryos (as indicated on the top) were probed at the early gastrula stage for Xvent-1 or Xvent-2 expression as indicated on the left. (A,D) Wild-type (wt) embryos. (B,E) Embryos were microinjected with 0.6 ng BMP-4 mRNA each into two opposite blastomeres at the 4-cell stage and treated with LiCl. Note the two opposite domains of induced expression (arrowheads). (C,F) Embryos were microinjected with 1 ng each of dominantnegative BMP-4 receptor mRNA (DmTFR11) into four blastomeres of 4-cell stage embryos. Expression of both Xvent-1 and Xvent-2 is repressed.
	Fig. 6. Xvent-2 mRNA microinjection causes axial defects in a dosedependent manner. Phenotype of embryos microinjected at the 4- cell stage into four blastomeres with 1.6 ng per blastomere DXvent-2 mRNA (Co, top embryo) or with the indicated amount of Xvent-2 mRNA (bottom three embryos).
	Fig. 7. Xvent-2 mRNA microinjection ventralizes dorsal mesoderm. Embryos were either uninjected (DMZ, VMZ, -RT), microinjected with 1.6 ng DXvent-2 (Co), or microinjected with increasing amounts of Xvent-2 mRNA (indicated on top in ng mRNA per blastomere) into the equatorial region of four blastomeres at the 4-cell stage. Dorsal (DMZ) or ventral marginal zones (VMZ) as indicated on top were explanted at the early gastrula stage and incubated until sibling embryos reached stage 19. Total RNA was isolated and analyzed by RT-PCR assays for expression of gsc (Cho et al., 1991), Xnot (von Dassow et al., 1993; Gont et al., 1993), cardiac actin (m.actin) (Mohun et al., 1984), Xwnt-8 (Christian and Moon, 1993), BMP-4 (Dale et al., 1992; Jones et al., 1992), Xhox3 (Ruiz i Altaba and Melton, 1989), Xvent-1 (Gawantka et al., 1995) and Histone H4, as indicated. -RT, uninjected control DMZ sample without reverse transcription.
	Fig. 8. Microinjection of Xvent-2 mRNA rescues dorsalization by a dominant-negative BMP-4 receptor. 4-cell stage embryos were microinjected into two ventral blastomeres with (A) 0.2 ng mRNA encoding a dominant-negative BMP-4 receptor (DmTFR11; Suzuki et al., 1994), or (B) a mixture of 0.2 ng DmTFR11 and 1.5 ng Xvent- 2 mRNA. White and black arrowheads in (A) point to primary and secondary embryonic axes, respectively.
	Fig. 9. Cell fate changes induced by Xvent-2 mRNA injection. 32-cell stage embryos were coinjected into individual blastomeres (indicated at the upper right in each part) with colloidal gold-BSA and either 0.8 ng DXvent-2 mRNA (control, left column) or 0.8 ng Xvent-2 mRNA (right column). Embryos were fixed at the tadpole stage and processed for silver staining to visualize microinjected cells. (A,B) Embryos were injected into C4 blastomeres. Embryos injected with Xvent-2 mRNA show the normal lateral plate and somitic fate of descendants. (C,D) Embryos were injected into B1 blastomeres. Embryos injected with Xvent-2 mRNA specifically fail to populate the notochord. 0% (n=31) of embryos injected with Xvent-2 showed more than ten labeled notochord cells, in contrast to 83% (n=12) of controlinjected embryos. Notochord (no) in the control is the column of labeled thin vertical lines. Note labeled cells in a position ventral to the notochord (closed arrowheads in D), possibly corresponding to immature notochord cells. Note also the absence of welldifferentiated neurons in the brain (open arrowheads in D). (E,F) Embryos were injected into C1 blastomeres. In embryos injected with Xvent-2 mRNA more labeled descendants populate somitic mesoderm. Head mesodermal cells were generally populated to a lesser degree than in control embryos and labeled cells did not line up with the anatomical feature of the arches, appearing more clustered instead (arrowheads in F). hm, head mesoderm; lp, lateral plate; ne, neural tissue; no, notochord; so, somites.