|
Figure 1
Inhibitory interaction between follistatin and BMPs. (A) Phenotypes of Xenopus embryos dorsally coinjected with BMP mRNA and follistatin mRNA. BMP-4 mRNA (200 pg), either alone (b) or with 100 pg of follistatin mRNA (d), and BMP-7 mRNA (500 pg), either alone (c) or with 50 pg of follistatin mRNA (e), were injected into the equatorial region of the two dorsal blastomeres at the four-cell stage as described (28). In contrast, equatorial regions of two ventral blastomeres at the four-cell stage were injected with follistatin mRNA (60 pg), either alone (f), with 500 pg of BMP-4 mRNA (g), or with 2 ng of BMP-7 mRNA (h). Embryos were evaluated and photos were taken at the tadpole stage. (a) Uninjected embryo. (B) Expression of molecular markers in dorsal and ventral marginal explants. Embryos were either uninjected (lane 1, 6, 7, 11, and 12) or injected with the mRNAs indicated on the top of each lane into the equatorial region of two dorsal (lane 2–5) or ventral blastomeres (lane 8–10) at the four-cell stage. The amounts of mRNA for dorsal injection were 200 pg of BMP-4 (lane 2), 200 pg of BMP-4 plus 200 pg of follistatin (lane 3), 500 pg of BMP-7 (lane 4), and 500 pg of BMP-7 plus 50 pg of follistatin (lane 5). For ventral injection, 60 pg of follistatin mRNA, 500 pg of BMP-4 mRNA, and 2 ng of BMP-7 mRNA were used. Lane 12 shows the expression of markers in whole embryos and lane 6 and 11 show the control reactions with no RT step. Dorsal or ventral marginal zones were excised and explanted at the early gastrula stage and incubated until sibling embryos reached stages 11 and 36.
|
|
Figure 2
Failure of follistatin to block intracellular BMP signaling. (A) Phenotype of ventralized embryos by the dorsal injection of mRNA for constitutively active BMP-signaling receptor, with or without follistatin mRNA. Fifty picograms of mRNA for a constitutively activated form of Xenopus ActRI (CA-ALK2) was injected dorsally with (b) or without (a) 200 pg of follistatin mRNA as described in the legend for Fig. 1. (B) Expression of molecular markers in dorsal marginal explants of embryos that were uninjected (lane 1), injected with 500 pg of mRNA for CA-ALK2 alone (lane 2), and coinjected with 500 pg of CA-ALK2 and 200 pg of follistatin (lane 3). Dorsal mRNA injection and RT-PCR analysis were performed as described in Fig. 1. Coinjection of follistatin mRNA did not reduce the effect of CA-ALK2 overexpression for both dorsal (goosecoid) and ventral (Xvent1, αT1 globin) markers. Similar results were obtained in the experiments using CA-ALK3 (data not shown).
|
|
Figure 3
Direct binding of follistatin to immobilized BMPs. (A) BMP-4, activin A, and TGF-β1 were immobilized on the sensor chip surface. The immobilization levels of BMP-4, activin A, and TGF-β1 were 2904, 2543, and 2681 resonance units, respectively. FS-288, at a concentration of 5 μg/ml, was injected over these surfaces. (B) FS-288 (807 resonance units) was immobilized on the sensor chip surface. (a) BMP-4 (1 μg/ml) was injected over the surface. (b) BMP-4 (1 μg/ml) and FS-288 (0.5 μg/ml) were incubated for 30 min at room temperature. The mixture was injected over the surface. (c) Mixture of BMP-4 (1 μg/ml) with FS-288 (2.5 μg/ml) was incubated and injected as described above. Excess soluble FS-288 almost completely abolished the binding activity on the FS-288 surface. (C) FS-288 and sBMPR, at the concentration of 5 μg/ml and 20 μg/ml, respectively, were injected over the surface of immobilized BMP-4, separately. (D) BMP-4, BMP-4/7, and BMP-7 were immobilized on the sensor chip surface. The immobilization levels of these ligands were 622, 768, and 697 resonance units, respectively. FS-288 at 5 μg/ml was injected over these surfaces.
|
|
Figure 4
Diagonal SDS/PAGE analysis of FS-288-BMP-4 complex. (A) For comparison of molecular mass, a mixture of FS-288 and BMP-4 was analyzed by 2D electrophoresis. Although FS-288 (32 kDa) tends to shift to higher molecular mass region (>60 kDa) on first dimension electrophoresis probably by self aggregation, a major band of BMP-4 shows the estimated molecular mass of dimeric form (30 kDa). FS-288 was detected as a 43-kDa protein, and BMP-4 was detected as 16- and 18-kDa proteins, which were generated because of difference of glycosylation, under reducing condition. (B) Cross-linked FS-288-BMP-4 complex was analyzed. Proteins were separated by 2D electrophoresis and subjected to Western blotting. At least, four-shifted bands (arrowheads) of BMP-4, which were not seen in A, were detected. Relative molecular mass is indicated horizontally for nonreducing, vertically for reducing condition.
|
|
Figure 5
Different competitive mechanisms of two BMP-4 antagonists. (A) BMP-4, chordin, and FS-288, at the concentration of 10 μg/ml, 2 μg/ml, 20 μg/ml, respectively, were injected over the surface immobilized sBMPR, separately. For the competition assay, a mixture of BMP-4 with chordin or FS-288 was injected. (B) FS-288 was injected after the injection of BMP-4 over the sBMPR-immobilized surface by using of the coinjection method. The concentrations of BMP-4 and FS-288 were 10 μg/ml and 5 μg/ml, respectively. Both proteins were injected at a flow rate of 10 μl/min for 300 s.
|
|
Figure 6
Possible inhibitory mechanisms of the BMP action by follistatin and chordin. Chordin inhibits the binding of BMP-4 to its type I receptor (sBMPR). On the other hand, follistatin can bind to sBMPR through BMP-4, forming a trimeric complex.
|
