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Development
2011 Dec 01;13824:5345-56. doi: 10.1242/dev.068908.
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Novel functions of Noggin proteins: inhibition of Activin/Nodal and Wnt signaling.
Bayramov AV
,
Eroshkin FM
,
Martynova NY
,
Ermakova GV
,
Solovieva EA
,
Zaraisky AG
.
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The secreted protein Noggin1 is an embryonic inducer that can sequester TGFβ cytokines of the BMP family with extremely high affinity. Owing to this function, ectopic Noggin1 can induce formation of the headless secondary body axis in Xenopus embryos. Here, we show that Noggin1 and its homolog Noggin2 can also bind, albeit less effectively, to ActivinB, Nodal/Xnrs and XWnt8, inactivation of which, together with BMP, is essential for the head induction. In support of this, we show that both Noggin proteins, if ectopically produced in sufficient concentrations in Xenopus embryo, can induce a secondary head, including the forebrain. During normal development, however, Noggin1 mRNA is translated in the presumptive forebrain with low efficiency, which provides the sufficient protein concentration for only its BMP-antagonizing function. By contrast, Noggin2, which is produced in cells of the anterior margin of the neural plate at a higher concentration, also protects the developing forebrain from inhibition by ActivinB and XWnt8 signaling. Thus, besides revealing of novel functions of Noggin proteins, our findings demonstrate that specification of the forebrain requires isolation of its cells from BMP, Activin/Nodal and Wnt signaling not only during gastrulation but also at post-gastrulation stages.
Fig. 1. Wild-type Noggin2 mRNA elicits effects distinct from those induced by wild-type Noggin1 mRNA. (A,B) Ventral injections of Noggin1δ5 mRNA induce secondary axes (A), whereas similar injections of Noggin2 mRNA resulted in mushroom-shaped embryos (B). (C-F) Whole-mount in situ hybridization of the control (left in each photo) and the Noggin2 mRNA-injected (right) embryos demonstrates strong upregulation of the neural (C,D) marker genes, and inhibition of epidermal (E) and muscle (F) marker genes. (G) Ventral injections of Noggin2 mRNA induce formation of secondary heads with cyclopic eyes. (H) The forebrain marker XBF1 is expressed in the secondary heads of embryos injected by Noggin2 mRNA. (I-I′) The embryo of a transgenic line expressing RFP in muscles has reduced muscle differentiation on the left side where a secondary head with cyclopic eye was induced by injection of the mixture of Noggin2 mRNA and FLD tracer. The same embryo under white light (I) and as an overlay of white light, red and green fluorescent images (I′, left side; I′, right side). (J-K′) Noggin2 (J,J′) but not Noggin1 mRNA (K-K′) inhibits (arrows) XBra expression in blastopore marginal zone. Embryos at stage 10.5 are shown from the vegetal pole.
Fig. 2. Noggin1 and Noggin2 can bind TGFβ and Wnt ligands. (A) Selected mRNA and proteins used in the present study. (B,C) Comparison of translation capacities of MycNoggin1δ5 and MycNoggin2δ5 mRNA (B) or MycNoggin2δ5, SynMycNoggin1 and SynMycNoggin2 mRNA (C) injected in two-cell embryos at the indicated concentrations. Five embryos of each type were collected at stage 10 in 50 μl of lysis buffer and Noggin proteins were revealed by western blotting with anti-Myc antibody either in 1/5 (B) or in 1/125 (C) aliquots of this volume. Here and below, α-tubulin was detected with anti-tubulin antibodies (DM1A, Sigma, final dilution 1:10,000) as a loading control. (D) qRT-PCR analysis of endogenous Noggin1 and Noggin2 mRNA in the anterior neural fold explants of stage 15 embryos. (E,F) Only endogenous Noggin2 (lane 2), but not Noggin1 (lane 1), was detected in the anterior neural fold explants of stage 15 embryos by antibodies specific to Noggin1 and to Noggin2 (E), despite these antibodies demonstrating similar affinities to exogenous Noggin1 (lane 3) and Noggin2 (lane 4) translated from injected SynNoggin1 and SynNoggin2 mRNA (F). In the last case, a mixture of antibodies to both Noggin proteins was used. (G,H) Both Noggin1 and Noggin2 (Ng1 and Ng2) translated from SynMycNoggin1 and SynMycNoggin2 mRNA co-precipitate with Flag-tagged BMP4, ADMP, Activin, Xnr2, Xnr4 and XWnt8. In case of Noggin1 translated from MycNoggin1δ5mRNA (wtNg1), only precipitation with BMP was detected. No precipitation of Noggin proteins was revealed with Flag-tagged Zyxin (negative control). (I) Deletion of the clip-domain sharply reduce ability of Noggin proteins (δNg1, δNg2) to bind BMP4 but much more poorly influences the binding of Noggin to all non-BMP TGFβ ligands tested and to XWnt8.
Fig. 4. Noggin proteins translated from mRNA with consensus Kozak (SynNoggins) site can influence developmental processes regulated by Nodal/Xnrs and Wnt. (A,A′) Injection of Noggin2 mRNA (10 pg/embryo) elicits inhibition of Xbra expression in 100% of embryos (black arrows). (B,B′) Injections of Noggin1 mRNA (10 pg/embryo) resulted in inhibition of Xbra expression in 40% of embryos (black arrows). (C,C′) Injections of Noggin2 mRNA (10 pg/embryo) do not induce goosecoid expression in areas exactly corresponding to the injection sites. A very weak goosecoid expression can be seen just at the periphery of the injected areas, where the concentration of Noggin2 presumably drops down to the level insufficient for effective inhibition of Xnrs but still enough to antagonize BMP (see deviation between red and white arrows indicating maximums of goosecoid expression and FLD tracer signal, respectively). (D,D′) Injections of Noggin1 mRNA (10 pg/embryo) induce a weak goosecoid expression. Areas of ectopic goosecoid expression exactly correspond to the injection sites (red and white arrows coincide). (E) Ventral injections of SynNoggin1 mRNA (5 pg/embryo) induce development of the head-containing secondary axes with cyclopic eyes, the result indicating simultaneous inhibition of BMP, Nodal/Xnrs and Wnt signaling. (F,F′) Consistent with the ability of Noggin2 to inhibit Wnt, dorsal injections of Noggin2 mRNA (5 pg/embryo) elicit enlargement of the forebrain rudiment marked by XBF1 expression. (G) The control 5-day old tadpole as it is seen from the dorsal side. (H,I) Dorsal injections of Noggin2 or Noggin1 mRNA (5 pg/embryo) elicit cyclopic phenotype. (J) Injections of δclipNoggin2 mRNA elicit enlargement of eyes, an effect consistent with the ability of this mutant to inhibit Wnt signaling. (K,L) Dorsal injections of δclipNoggin2 or δclipNoggin1 mRNA cyclopic phenotype. Anterior view.
Fig. 5. δclip-domain mutants of Noggin proteins suppress Wnt signaling. (A,A′) Ventral injections of XWnt8 mRNA (3 pg/embryo) elicit development of the head-containing secondary axes due to ectopic induction of the Nieuwkoop center at the blastula stage. (B-C′) Co-injection of δclipNoggin1 or δclipNoggin2 mRNA (50 pg/embryo) with XWnt8 resulted in suppression of secondary axes.
Fig. 6. The effects of inhibition of Noggin2 mRNA translation by MO. (A,A′) Head of the tadpole injected with Noggin2 MO into right side has severe malformations of the righteye, nasal placode and forebrain. Dorsal view, anterior side upwards. (B) Head of the control tadpole. (C,C′) The embryo injected with Noggin2 MO into the right side has a reduced expression of the forebrain marker XBF1 (arrows) in the injected cells at midneurula stage. Anterior view, dorsal side upwards. (D) Normal expression pattern of XBF1 in embryo injected with mis-Noggin2 MO. (E,E′) The embryo injected with Noggin2 MO on the right side has a reduced expression of Pax6 (arrows) in the injected cells at the tailbud stage. Anterior view, dorsal side upwards. (F) Normal expression pattern of Pax6 in the embryo injected with the misNoggin2 MO. (G,H) Embryos injected with Noggin2 MO and rescued by co-injection of Noggin2 mRNA. Brackets indicate the distance (h) from the beginning of the dorsal hatching gland (dhg) to the dorsal margin of the cement gland (cg) (see inset photo in H for details). Anterior view, dorsal towards the top. (I) Mean values of ‘h’ in embryos injected with the indicated MO and mRNAs. Error bars indicate s.d., n, number of embryos. Statistical significance of the difference between mean values was confirmed by independent two-sample Student’s t-test for unequal sample sizes, unequal variance.
Fig. 7. Rescue of Noggin2 MO morphants by Wnt-, BMP- and Activin/Nodal-specific inhibitors. (A) Plasmids expressing EGFP, Dkk1, tALK4 and tBR under the control of the Xenopus tropicalis Noggin2 promoter. (B) Analysis of pNog2-EGFP expression in Xenopus laevis embryos by qRT-PCR. Each of the dorsal animal blastomeres of eight-cell embryos was injected in the animal corners with 2 nl of pNog2-EGFP (4 ng/μl). Embryos were collected at the indicated stages in batches of 10 in triplicate and qRT-PCR was performed on RNAase-free DNAase pre-treated extracted RNA using primers specific to EGFP and ODC as references. (C,C′) A typical stage 26 embryo expressing injected pNog2-EGFP in the forebrain. Anterior view, dorsal side upwards. (D-F′) Expression domains of XBF1 (framed by yellow line on D) in forebrains of embryos injected by the indicated mixtures of MO, plasmids and FLD. The integrated density of XBF1 in situ hybridization signal was measured using the ImageJ program. (G) Statistical analysis of integrated density of in situ hybridization signal within XBF1 expression domain of stage 26 embryos injected with the indicated mixtures of MO and plasmids. The analysis was performed using independent two-sample Student’s t-test for unequal sample sizes, unequal variance. Broken lines separate values that are most significantly different from one another. Errors bars represent s.d. See supplementary material Table S2 for original data.
Fig. 8. The inhibition of Activin signaling at the anterior margin of the neural plate is essential for the forebrain development. (A-F) Expression patterns of ActivinB and Noggin2 in the forebrain rudiment revealed by the whole-mount in situ hybridization. (A) At the midneurula stage, Noggin2 is specifically expressed in cells of the anterior neural fold. Anterior view of the whole embryo, dorsal side upwards. Red asterisk marks the median of anterior neural fold. (B) The area of embryonic halves (outlined) shown in C-E. (C) Expression of ActivinB and Noggin2 on halves of the same midneurula embryo. ActivinB is not expressed in the triangular region of the neurectoderm, between the broken red line and asterisk, in which Noggin2 is expressed. (C′) The same hemi-section as in C, but combined together to form the whole embryo and shown from the anterior side. (D) Expression of the telencephalic marker XBF1 and Noggin2 on halves of the same midneurula embryo. XBF1 and Noggin2 are expressed in the same cells at the anterior margin of neural plate. (E) Expression of ActivinB and Noggin2 on bilateral halves of the same tailbudembryo. ActivinB is not expressed in the telencephalon rudiment (tel) in which Noggin2 is expressed. (E′) Expression of ActivinB and Noggin2 on halves shown in E but further sectioned along the broken line indicated in E; dorsal view. (F) The expression patterns of ActivinB, XBF1 and Noggin2 in the midneurula embryo; anterior view, dorsal side upwards. (G-K) Inhibition of ActivinB by Noggin2 is necessary for normal development of the forebrain. (G) The double cassette and control vectors used for targeting of ActivinB expression to the forebrain rudiment under the control of Xanf1 promoter. (H-H′) In contrast to the transgenic embryo bearing control vector CardKate (upper row), the embryo containing double-cassette vector XanfActB-CardKate (bottom row) has reduced eyes and telencephalon (marked by XBF1 expression). H and H′, side views; H′, anterior view of the same pair of embryos. (I,J) Examples of embryos injected into the presumptive head region by ActivinB mRNA (0.5 pg/embryo) have smaller eyes (arrows) reflecting the smaller forebrains. Co-injection with ActivinB mRNA of Noggin2 mRNA (3 pg/embryo) resulted in partial rescuing of normal phenotype. See control embryos in supplementary material Fig. S6E. (K) Average eye sizes (d) of embryos injected with indicated mRNAs. Statistical significance of the difference between mean values was confirmed by Student’s t-test. Error bars show s.d.
