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???displayArticle.abstract??? Bone morphogenetic protein 1 (BMP1) is a metalloprotease that ventralises dorsal mesoderm when overexpressed in early Xenopus embryos. Here we show that Xenopus BMP1 blocks the dorsalising activity of chordin, but not noggin or DeltaxBMPR, when coexpressed in the ventral marginal zone and degrades chordin protein in vitro. We also show that a dominant-negative mutation for XBMP1 (dnBMP1) dorsalises ventralmesoderm in vivo, and blocks degradation of chordin by both XBMP1 and Xolloid, a closely related Xenopus metalloprotease, in vitro. dnBMP1 does not dorsalise ventralmesoderm in UV-irradiated embryos, demonstrating that this activity is dependent upon a functional organiser--the natural source of chordin in Xenopus gastrulae. Our results suggest that XBMP1 may regulate the availability of chordin during vertebrate embryogenesis.
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10446267
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Fig. 3. dnXBMP1 and dnXld dorsalise Xenopus embryos. (A) Normal stage 40 embryos injected with 1.5 ng of dnbmp1 mRNA into a single ventralblastomere at the four cell stage. Many embryos injected with this mRNA are normal. (B) Partial secondary axes in stage 40 embryos injected with 1.5 ng of dnbmp1 mRNA into a single ventralblastomere at the four cell stage. (C and D) Complete secondary axes in stage 40 embryos injected with 1.5 ng of dnbmp1 mRNA into a single ventralblastomere at the four cell stage. (E) Section through control embryo showing internal organisation of mesodermal tissues (no notochord) and neural tube (nt). (F) Section through embryo in D showing duplicated secondary axis includes neural tube, notochord and somites. (G±L) Whole mount in situ hybridisation analysis of midgastrulae (stage 11.5-12) for chordin (G and J), xmyf5 (H and K) and xwnt8 (I and L). (G) Uninjected embryos showing dorsal expression of chordin (arrows). (H) Uninjected embryos showing dorsolateral expression of xmyf5. (I) Uninjected embryos showing ventral and lateral expression of xwnt8. (J) dnbmp1 injected embryos showing ventral expression (arrowheads) of chordin. (K) dnbmp1 injected embryos showing ventral expression of xmyf5 (arrowheads). (L) dnbmp1 injected embryos showing reduced expression xwnt8. Embryos in (G-L) were injected with 1.5 ng of dnbmp1 mRNA into a single ventralblastomere at the four cell stage.
Fig. 4. Dorsalisation of ventralmesoderm by dnBMP1 and dnXld. (A±C) Histological analysis of VMZs isolated from uninjected control (A), dnbmp1 injected
(B), and dnxld injected (C) embryos. 1.5 ng of mRNA was injected into each ventralblastomere at the four cell stage, VMZs were isolated at stage 10.5 and cultured until sibling controls had reached stage 40. Control VMZs (A) always differentiated ventral-type mesoderm (mesenchyme and blood) with a fluid filled vesicle (v), while dnbmp1 injected VMZs (B) always differentiated dorsal-type mesoderm, such as notochord (no) and muscle, and in some cases neural tissue (nt). Most dnxld injected VMZs differentiated ventral-type mesoderm, but 35% (C) differentiated muscle (mu). (D) RNase protection analysis confirmed that injected VMZs were dorsalised. Total RNA from stage 26 VMZs was hybridised with probes for either muscle-speci®c a-actin (m-actin) or blood-specific aT4-globin mRNAs. The a-actin probe also detects cytoskeletal-actin (c-actin) mRNA, which acts as a loading control, while levels of ornithine decarboxylase
(ODC) mRNA were determined as a loading control for aT4-globin. Lane 1, uninjected VMZs express aT4-globin mRNA but not a-actin mRNA; lane 2, dnBMP1 injected VMZs express a-actin mRNA but not aT4-globin mRNA; lane 3, dnxld injected VMZs express both a-actin and a-globin mRNAs. (E) RNase protection analysis shows that the dorsalising activity of dnBMP1 and dnXld is blocked by an excess of either XBMP1 or Xld. 250 pg of either dnbmp1
or dnxld mRNAs were injected into both ventral blastomeres at the four cell stage, both alone and in combination with 750 pg of either xbmp1 or xld mRNAs. VMZs were isolated at stage 10 and incubated until stage 26, when total RNA was prepared and hybridised with a probe for a-actin. Lane 1, control VMZs;
lane 2, dnbmp1 injected VMZs; lane 3, dnbmp1 and xbmp1 coinjected VMZs; lane 4, dnbmp1 and xld coinjected VMZs; lane 5, dnxld injected VMZs; lane 6, dnxld and xbmp1 coinjected VMZs; lane 7, dnxld and xld coinjected VMZs.
Fig. 5. dnBMP1 dorsalises animal caps. Whole mount in situ hybridisation
analysis of animal caps with probes for nrp1 and xa1. Embryos were
injected at the two cell stage with a total of 1.6 ng of either dnbmp1 or
dnxld mRNA, animal caps isolated from early gastrulae and incubated until
stage 26. (A) Uninjected caps do not express nrp1. (B) dnbmp1 injected
caps express nrp1 in a few cases. (C) dnxld injected caps do not express
nrp1. (D) Uninjected caps do not express xa1. (E) dnbmp1 injected caps
express xa1 in most cases. (F) dnxld injected caps do not express xa1 in a
few cases.
Fig. 6. dnBMP1 does not dorsalise UV-irradiated embryos. (A) Stage 45 UV-irradiated embryos showing the absence of a dorsal axis. (B) Stage 45 UV-irradiated embryos injected with dnbmp1 showing the absence of a dorsal axis. (C) Histological section showing that UV-irradiated embryos differentiate ventralmesoderm. (D) Histological section showing that UV irradiated embryos injected with dnbmp1 differentiate ventralmesoderm.(E) RNase protection analysis confirmed that UV-irradiated embryos were
not dorsalised by dnBMP1. Lane 1, control embryos express high levels of a-actin mRNA; lane 2, UV-irradiated embryos express low levels of a-actin mRNA; lane 3, UV-irradiated embryos injected with 1.5 ng of dnBMP1 mRNA express low levels of a-actin mRNA.
Fig. 7. dnBMP1 does not dorsalise UV-irradiated gastrulae. One nanogram of dnbmp1 mRNA was injected into a single blastomere at the four cell stage, either randomly (UV-irradiated embryos) or into a single ventralblastomere. Whole mount in situ hybridisation was performed on midgastrulae with probes for chordin (A-D), xmyf5 (E-H) and xwnt8 (I-L). (A) Control gastrulae showing localisation of chordin mRNA to the dorsal marginal zone. (B) dnbmp1 injected gastrulae showing increased expression of chordin mRNA in the dorsal marginal zone. (C) Control UV-irradiated gastrulae showing absence of chordin expression. (D) UV-irradiated gastrulae injected with dnbmp1 showing absence of chordin expression. (E) Control gastrulae showing localisation of xmyf5 mRNA to the dorsolateral marginal zone. (F) dnbmp1 injected gastrulae showing expansion of chordin expression domain into the ventral marginal zone (arrows). (G) Control UV-irradiated gastrulae showing absence of xmyf5 expression. (H) UV irradiated gastrulae injected with dnbmp1 showing absence of xmyf5 expression. (I) Control gastrulae showing localisation of xwnt8 mRNA to the ventral and lateral marginal zone. (J) dnbmp1 injected gastrulae showing reduction in xwnt8 expression (arrowheads). (K) Control UV-irradiated gastrulae showing expansion of xwnt8 expression into dorsal marginal zone. (L) UV-irradiated gastrulae injected with dnbmp1 showing continued
expression of xwnt8 throughout the marginal zone.
