XB-ART-1775Dev Biol June 15, 2005; 282 (2): 442-54.
Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases.
BMP signaling performs multiple important roles during early embryogenesis. Signaling through the BMP pathway is mediated by different BMP ligands expressed in partially overlapping temporal and spatial patterns. Assignment of different BMP-dependent activities to the individual ligands has relied on the patterns of expression of the various BMP genes. Temporal analysis of BMP signaling prior to and during gastrulation was performed using glucocorticoid-controlled Smad proteins. Overexpression of the BMP-specific Smad1 and Smad5 revealed that suppression of Spemann''s organizer formation in Xenopus embryos can only take place by activating the BMP pathway prior to the onset of gastrulation. Blocking BMP signaling with the inhibitory Smad, Smad6, results in dorsalized embryos or secondary axis induction, only when activated up to early gastrula stages. BMP2 efficiently represses organizer-specific transcription from the midblastula transition onwards while BMP4 is unable to prevent the early activation of organizer-specific genes. Manipulation of the BMP pathway during mid/late gastrula affects mesodermal patterning with no external phenotypic effects. These observations suggest that the malformations resulting from inhibition or promotion of organizer formation, ventralized or dorsalized, respectively, are the result of a very early BMP function, through its antagonism of organizer formation. This function is apparently fulfilled by BMP2 and only at its latest phase by BMP4. Subsequently, BMP functions in the patterning of the mesoderm with no apparent phenotypic effects.
PubMed ID: 15950609
Article link: Dev Biol
Genes referenced: acta4 actc1 actl6a bmp2 bmp4 gsc hist1h4d muc2 myod1 ncam1 not otx2 smad1 smad6 smad6.2 wnt8a
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|Fig. 1. Timed activation or repression of the BMP signaling pathway during early embryogenesis. Embryos were injected with mRNA encoding the Smad1/GR (A-F), Smad5/GR (G-L), or Smad6/GR (M-R) proteins. The embryos were exposed to dex at the stages marked and incubated to st. 29-31 for phenotypic analysis.|
|Fig. 3. Secondary axis induction by BMP signaling inhibition as a function of time. Embryos were injected ventrally with Smad6/GR mRNA to induce the formation of secondary axes (A�F). The fusion protein was activated by dex at several developmental stages and the embryos were incubated until st. 27 for analysis. Identification of the secondary axes was achieved by in situ hybridization with probes for N-CAM (neural tube), muscle actin (somites), and XCG1 (cement gland). The numbers summarize the percentage of embryos with secondary axes and the sample size.|
|Fig. 4. Temporal effect of BMP signaling on explanted dorsal marginal zones. Embryos were injected with Smad1/GR mRNA and activated by dex treatment at different developmental stages (A�D). At the onset of gastrulation, the dorsal marginal zone were explanted and incubated to st. 30.|
|Fig. 5. Inhibition of organizer-specific gene expression by BMP signaling as a function of time. Embryos were injected with Smad1/GR mRNA and treated with dex at the specified stages. Four hours after the dex treatment was initiated, the embryos were fixed and processed for in situ hybridization with gsc (A�F), Otx2 (G�L), and Xnot2 (M�R) specific probes. The numbers summarize the percentage of embryos resembling the representative embryo and the sample size.|
|Fig. 6. BMP signaling performs a mesodermal patterning role during gastrulation. Embryos were injected with Smad1/GR (A,B,E,F) or Smad6/GR mRNA (C,D,G,H). The Smad1/GR-injected embryos were exposed to dex at st. 11 and fixed at st. 11.5 and the Smad6/GR-injected embryos were treated with dex at st. 11.5 and fixed at st. 12�12.5. The embryos were processed for in situ hybridization with Xwnt8 (A�D) or MyoD (E�H) specific probes to determine changes in their patterns of expression. The regions exhibiting changes from the normal expression patterns are marked by dashed lines.|
|Fig. 7. Temporal pattern of BMP2 and BMP4 expression. RNA was prepared from normal embryos at different stages. The expression of BMP2 and BMP4 in the different samples was determined by RT-PCR with specific primers. The level of histone H4 expression was determined as a loading control.|
|Fig. 8. BMP2 and BMP4 regulate organizer-specific expression at different times during embryogenesis. Embryos were injected with BMP2 (B,E,H,K) and BMP4 (C,F,I,L) mRNA and subjected to in situ hybridization analysis with gsc (A�F) and Otx2 (G�L) specific probes at stage 9.5 (A�C, G�I) and stage 10.5 (D�F, J�L).|