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???displayArticle.abstract??? Lefty, antivin and related genes act in a feedback inhibition mechanism for nodal signaling at a number of stages of vertebrate embryogenesis. To analyze the function of the feedback inhibitor of nodal signaling, Xantivin in Xenopus embryos, we designed a morpholino antisense oligonucleotide (XatvMO) for this gene. XatvMO caused the expansion of mesodermal tissue and head defects. XatvMO-injected gastrulae showed up-regulated expression of the mesodermal markers Xbra, Xwnt8, Xnot, and Chordin, suggesting expansion of the trunk-tailorganizer. As expected, depletion of Xantivin also up-regulated nodal signaling as confirmed by the enhanced ectopic expression of Xantivin mRNA, a known target gene of nodal signaling. Furthermore, we investigated the relationship between Xantivin and the EGF-CFC gene FRL-1, which is a component of the nodal receptor. In animal cap assays, FRL-1 could not induce expression of nodal-responsive genes, but could up-regulate expression of these genes when FRL-1 was coinjected with a low dose of Xnr1; coinjection of Xantivin suppressed this up-regulation by FRL-1. We also found that Xantivin can rescue the caudalized phenotype induced by overexpression of FRL-1. Co-immunoprecipitation assays showed that Xantivin interacted with the EGF-CFC proteins, FRL-1 and cripto. Taken together, these results suggest that Xantivin opposes the activity of EGF-CFC genes and thereby antagonizes nodal signaling.
Fig. 1. XatvMO caused posteriorizing phenotypes. (A) XatvMO inhibited the translation of Xatv and this inhibition was dependent on the XatvMO target sequence. Xatv protein which has a HA-epitope tag, was detected using an anti-HA antibody. α-Actin served as the loading control. XatvHA mRNA ( Xatv with a HA epitope) and XatvHAmut mRNA (Xatv with no XatvMO target sequence) were injected with or without coinjection of XatvMO into oocytes. Oocyte lysates with uninjected controls were used for Western blotting. XatvMO inhibited the translation of XatvHA, but not of XatvHAmut mRNA. (B-F, I) XatvMO, StdMO, Xatv5misMO, anti-XleftyMO, or XatvMO was vegetally injected into each blastomere of 4-cell stage embryos, which were grown to tadpole stage. (B) Embryo injected with 20 ng XatvMO. (C) Embryo injected with 40 ng XatvMO. (D) Embryo injected with 40 ng XatvMO rescued by 2 pg Xatvmut mRNA. (E,F) Embryos injected with 40 ng StdMo (E) or Xatv5misMO (F) showed no developmental abnormality. (G) Embryo injected with 40 ng XatvMO rescued by 100 pg tAR1 mRNA. (H) pCS2-Xnr1 DNA (50 pg) was injected into dorsal vegetal blastomeres of 8-cell stage embryos, which showed a similar phenotype to the XatvMO-injected embryo. (I) Embryos injected with 2.5 ng anti-XleftyMO showed a caudalized phenotype.
Fig. 2. XatvMO-injected embryos have enlarged mesodermal tissues. (A) Diagrams of embryos injected with StdMO and XatvMO. Lines indicate the approximate position of sections shown in (BE). XatvMO (20 ng) or StdMO (40 ng) was injected into the vegetal region of 4-cell stage embryos. (B) Section of the head region of an embryo injected with XatvMO shows that forebrain and eye structures were reduced, and an expanded notochord had invaded anteriorly. (C) Section of the head region of an embryo injected with StdMO. (D) Section of the posterior region of an embryo injected with XatvMO shows an expanded notochord and muscle. (E) Section of the posterior region of an embryo injected with StdMO. (F,G) Injection of XatvMO caudalized the neuroectoderm. XatvMO (25 ng) or StdMO (25 ng) was coinjected with 250 pg β-gal mRNA into one blastomere of a 4-cell stage embryo, which was grown to early neurula to detect BF-1 (forebrain) and Krox20 (hindbrain) expression. (F) Embryos injected with XatvMO lost the expression of BF-1 but not of Krox20. (G) An embryo injected with StdMO marked with blue in the future forebrain and hindbrain, which was marked by the expression of BF-1 and Krox20, respectively. (H-K) XatvMO or StdMO was vegetally injected into each blastomere of a 4-cell stage embryo, which was grown to late neurula. (H) Immunostaining with monoclonal antibody Tor70, revealed that embryos injected with XatvMO had enlarged notochords. (I) Normal notochord of an embryo injected with StdMO specifically stained by Tor70. (J) The expanded expression of XmyoD was detected in an embryo injected with XatvMO. (K) The expression of XmyoD was detected in the somite of an embryo injected with StdMO.
Fig. 3. XatvMO disturbs the expression of mesodermal markers. (AL) 25 ng StdMO (A,C,E,G,I,K) or 25 ng XatvMO (B,D,F,H,J,L) were coinjected with 250 pg β-gal mRNA into (E-L) one or (A-D) two blastomeres of 4-cell stage embryos. The injected regions were visualized by red, indicating β-galactosidase activity. Injected embryos were grown to mid to late gastrulae for whole-mount in situ hybridization; signal is indicated by blue staining. Arrowheads indicate the XatvMO-injected region. (A,B) The expression of Xwnt8 (ventral view) and (C,D) Xbra (lateral view) was detected in the marginal zone, which was the future mesoderm of embryos injected with StdMO. However, the injection of XatvMO induced a dispersed expression into the animal region. (E-J) Dorsal view of late gastrulae ( Xbra) and mid gastrulae ( Xnot, Chordin). (E) Expression of Xbra, (G) Xnot and (I) Chordin was detected in the axial mesoderm of embryos injected with StdMO and was up-regulated in the XatvMO-injected region. (F) Xbra; (H) Xnot and (J) Chordin. (K,L) Vegetal view of mid gastrulae. The ectopic expression of Xatv mRNA was detected in the XatvMO-injected region (L), but not in the StdMOinjected region (K) .
Fig. 4. Xatv inhibits the activity of FRL-1. (A) Xatv inhibits nodal signaling enhanced by FRL- 1 in animal caps. mRNA was animally injected into each blastomere of 4-cell stage embryos, whose animal caps were dissected at stage 9 and harvested at stage 10.5. The animal cap assay showed that 1 ng FRL-1 or 5 pg Xnr1 did not induce the expression of Xbra, Chordin (chd), Mixer and Xnr1, but the coinjection of FRL-1 and Xnr1 did induce the expression of these genes. One nanogram of Xatv inhibited the expression of these nodal-responsive genes induced by the coinjection of 5 pg Xnr1 and 1 ng FRL-1. This result suggests that Xatv inhibits the activity of EGF-CFC genes to suppress nodal signaling. In addition, the synergistic induction with FRL-1 was not inhibited by FRL- 1 δEGF-like domain ( FRL-1 δE) but was inhibited by FRL-1 δCFC domain ( FRL-1 δC), suggesting it has a dominant-negative effect. (B-D) FRL-1, FRL-1 and Xatv, and FRL-1δC mRNA were injected into dorsal blastomeres of 4-cell stage embryos which were grown to tadpole stage. (B) FRL-1 (1 ng) induces caudalized phenotypes. (C) Xatv (2.5 pg) rescues the phenotypes caused by 1 ng FRL-1. (D) FRL-1δC (1 ng) induces the phenotype that is similar to overexpression of Xatv.
Fig. 5. Xatv interacts with EGF-CFC protein. mRNA was injected into each blastomere of 4-cell stage embryos, which were cultured until stage 10.5. The proteins were extracted from injected and uninjected embryos with lysis buffer and loaded along with 5% of the total samples that were used for immunoprecipitation (IP). (A) FRL-1 protein interacted with Xatv. Embryos injected with either 1 ng XatvHA, or 1 ng each of XatvHA and FRL-1-flag, as well as uninjected control embryos were harvested at stage 10.5, and their lysates were immunoprecipitated with an anti-flag antibody. XatvHA protein was not immunoprecipitated from embryos injected with XatvHA alone, but was precipitated from embryos coinjected with XatvHA and FRL-1 flag. (B) Cripto protein interacted with Xatv. Embryos injected with either 1 ng XatvHA or 1 ng each of XatvHA and Cripto-3flag, as well as uninjected embryos, were used for the immunoprecipitation assays. XatvHA protein was immunoprecipitated from embryos that were coinjected with XatvHA and Cripto- 3flag.
