Jones CM et al. (1996), Bone morphogenetic protein-4 (BMP-4) acts durin...

XB-ART-18273

Development 1996 May 01;1225:1545-54.

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Bone morphogenetic protein-4 (BMP-4) acts during gastrula stages to cause ventralization of Xenopus embryos.

Jones CM , Dale L , Hogan BL , Wright CV , Smith JC .

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Injection of RNA encoding BMP-4 into the early Xenopus embryo suppresses formation of dorsal and anterior cell types. To understand this phenomenon, it is necessary to know the stage at which BMP-4 acts. In this paper, we present three lines of evidence showing that BMP-4 misexpression has no effect on the initial steps of mesoderm induction, either dorsal or ventral, but instead causes ventralization during gastrulation. Firstly, activation of organizer-specific genes such as goosecoid, Xnot, pintallavis and noggin occurs normally in embryos injected with BMP-4 RNA, but transcript levels are then rapidly down-regulated as gastrulation proceeds. Similarly, BMP-4 does not affect the initial activation of goosecoid by activin in animal caps, but expression then declines precipitously. Secondly, embryos made ventral by injection with BMP-4 RNA cannot be rescued by grafts of Spemann's organizer at gastrula stages. Such embryos therefore differ from those made ventral by UV-irradiation, where the defect occurs early and rescue can be effected by the organizer. Finally, the dorsalizing effects of the organizer, and of the candidate dorsalizing signal noggin, both of which exert their effects during gastrulation, can be counteracted by BMP-4. Together, these experiments demonstrate that BMP-4 can act during gastrulation both to promote ventral mesoderm differentiation and to attenuate dorsalizing signals derived from the organizer.

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Species referenced: Xenopus
Genes referenced: actl6a bmp4 foxa4 gsc nog not
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	Fig. 1. BMP-4 represses dorsal-specific gene expression during gastrula stages. (A) Embryos were injected at the 1-cell stage with RNA encoding BMP-4. RNA was prepared from a large batch of embryos at each of the indicated stages and analysed for expression of the various markers by RNAase protection. The organizer-specific genes goosecoid, Xnot, noggin and pintallavis are all induced in BMP-4-injected embryos, but expression of goosecoid, Xnot and noggin is subsequently suppressed. In contrast, Xhox-3 expression is precociously increased under the influence of BMP-4 in the same embryos. C, control injected embryos; B, BMP-4 RNA injected embryos. (B) Overexpression of BMP-4 under the control of the cytoskeletal actin promoter has no effect on the expression of goosecoid, but Xhox-3 levels are increased in late gastrulae. See Table 1 for details of CSKA:BMP-4 injections. C, control injected embryos; B, CSKA:BMP-4-injected embryos. EF1-a serves as a control for RNA integrity in all lanes.
	Fig. 2. BMP-4 mediated repression of goosecoid in activin-induced animal caps. Embryos were injected with BMP-4 RNA and caps were isolated from injected or control embryos at blastula stages and treated with activin, or used as controls. Stage 10 samples demonstrate that in animal caps, initial induction of goosecoid by activin is largely unaffected by the presence of BMP-4. By stage 11.5, the actions of BMP-4 repress goosecoid expression, which becomes completely eliminated by stage 16. Simultaneously, BMP-4 induces a dramatic increase in the levels of Xhox-3, which is maintained well into neurula stages (stage 16). EF1-a demonstrates equal RNA loading among samples at each stage.
	Fig. 3. Effects of a wild-type organizer grafted into BMP-4-injected embryos. (A) Morphological appearance of BMP-4-injected embryos. (B) Normal embryo following implantation of an organizer at the early gastrula stage. Note the primary (1°) and secondary (2°) axes. (C) BMP-4-injected embryos following implantation of a normal organizer. Control grafted embryos are induced to form secondary dorsal axes, while grafted embryos expressing BMP-4 are indistinguishable from BMP-4-injected embryos that have not received a transplant.
	Fig. 4. Histological analysis of control (A,C,E) and BMP-4-injected (B,D,F) embryos that have received fluorescently labelled organizer transplants. Differential interference contrast optics reveals the general architecture of representative samples of control (A) and BMP-4-injected (B) host embryos. Lineage tracing analysis of grafted organizers demonstrates that, in control embryos (C), the fluorescent organizer tissue contributes mostly to axial mesoderm in the secondary axis. In contrast, no axial mesoderm is formed in BMP-4- injected hosts (D) and the organizer itself becomes ventralized and differentiates into tissues indistinguishable from those forming in the ventralized host. DAPI staining shows overall nuclear staining in each section (E,F).
	Fig. 5. Immunolocalization of notochord using antibody MZ15 in control embryos (A) and BMP-4-injected embryos into which a wildtype organizer has been grafted (B). Grafted embryos (B) are representative cases in which organizer tissue was isolated at stage 10, cultured to stage 12 and then transplanted into a stage 10 BMP-4- injected host. Approximately 95% (n=56) of all organizers transplanted at either stage 10, 11 or 12 become ventralized and contain no MZ15 immunopositive cells. The positive case displaying weak notochord staining was one of two such embryos from twenty hosts receiving transplants after culture to stage 12. not, notochord.
	Fig. 6. Muscle differentiation in control and BMP-4-injected VMZ explants juxtaposed to wild-type DMZs. VMZs derived from control (A-C) or from BMP-4-injected (D-F) early gastrulae were juxtaposed with fluorescently labelled DMZs from wild-type embryos. In each case, the DMZ tissue is marked by fluorescence (A,D) and forms characteristic tissues which include notochord (arrow, use as a reference point in each panel) and muscle. 12/101 antibody staining localises muscle tissue in each explant conjugate (B,E). In control explants (B), substantial muscle differentiation is detected in tissues arising from the VMZ (not marked by fluorescence). In the case of recombinants where the VMZ overexpresses BMP-4 (E), muscle is only formed in the DMZ itself, and never in the BMP-4-injected VMZ. DAPI staining (C,F) illuminates nuclei throughout each section.
	Fig. 7. Zygotically expressed BMP-4 attenuates noggin-induced dorsalization of ventral mesoderm. Ventral marginal zones derived from control (A,D), CSKA:Noggin (B,E), or CSKA:Noggin/CSKA:BMP-4 injected (C,F) embryos were cultured to stage 17 (A-C) or stage 35 (D-F). Noggin induces extensive morphogenetic movements (B), compared with controls (A) or with tissues expressing both noggin and BMP- 4 (C). Histological analysis (D-F) demonstrates that noggin (E) induces large muscle blocks (mus) while controls (D) form loosely packed mesenchyme (mes). When BMP-4 is over-expressed simultaneously with noggin (F), no muscle differentiation is observed and the tissue resembles controls, although they always remain more compact. ym, yolk mass.
	Fig. 8. BMP-4 blocks noggin-induced differentiation of muscle in explants of ventral marginal zone tissue. VMZs were isolated from early gastrulae (stage 10) previously injected with either CSKA:Noggin or both CSKA:Noggin and CSKA:BMP-4 together. They were then cultured until tailbud stages and subsequently assayed for the presence of muscle-specific actin transcripts by RNAase protection. This probe also detects cytoskeletal actin, which serves as a control.