Microinjection of hBgn mRNA induces dorsalization and blocks the ventralizing activity of BMP4 in Xenopus embryos. (A–C) hBgn mRNA induced secondary axes. Embryos at the four- to eight-cell stage were microinjected once in the ventral vegetal side with synthetic mRNA. (B) When 500 pg of hBgn was injected, 60% of the embryos had double axis (N=51), similar to the results obtained when (C) 5 pg of chordin was injected (65%, N=23). (D–F) hBgn mRNA increased the expression of the dorsal marker MyoD. Albino embryos were injected four times animally at the four-cell stage with (D) lacZ (500 pg/blastomere), (E) hBgn (500 pg/blastomere) and (F) chordin (75pg/blastomere). The embryos were cultivated until stage 13 and MyoD expression was analyzed by in situ hybridization. The expansion of the MyoD domain of expression after injection of hBgn was observed in four out of six embryos. (G) hBgn dorsalized VMZ explants. Embryos were microinjected twice in the ventral side at the four-cell stage and VMZ was isolated at stage 10 and cultivated until sibling embryos reached stage 26. The expression of the dorsal marker α-actin and the loading control EF-1α was detected by RT–PCR. Whole embryos were used as an indication of the level of expression in sibling embryos (WE). (H–M) hBgn blocks the ventralizing activity of BMP4. Embryos were microinjected four times animally with synthetic mRNA for (I) BMP4, (J) BMP4 and hBgn, (K) BMP4 and lacZ, (L) ca-BR and (M) ca-BR and hBgn. The number of ventralized embryos (DAI⩽4) for each combination of mRNAs was BMP4 (10/13), BMP4/lacZ (18/21), BMP4/hBgn (5/30), ca-BR (13/13), ca-BR/lacZ (13/14) and ca-BR/hBgn (19/21). The total amounts of mRNA used for these experiments were 1.5 ng hBgn and lacZ, and 250 pg BMP4 and ca-BR. (N, O) Xolloid inactivates hBgn. Embryos at the four- to eight-cell stage were microinjected once in the ventral vegetal side with synthetic mRNA for (N) hBgn (63% double axes, N=53) and (O) hBgn (500 pg) and Xld (250 pg, 0%, N=41).

Bgn makes Chd a better BMP4 antagonist. (A) Chd interacts with hBgn-flag. Chd-myc and hBgn-flag were incubated and pulled down using α-Nchd antibody. The amount of hBgn-flag precipitated was visualized by α-flag Western blot. The hBgn-flag used was not treated (lane 2) or treated (lane 3) with C_ABC. For reference, hBgn inputs untreated and treated with C_ABC were loaded (lanes 4 and 5, respectively). Note that Chd precipitates the Bgn core protein from untreated and treated inputs, and the difference in the efficiency of the interaction could be explained by the higher amounts of Bgn core protein present in the inputs when the samples were treated with C_ABC (compare lanes 4 and 5). (B) Chd binds BMP4 more efficiently in the presence of hBgn-flag. Upper panel: Chd baculovirus protein was incubated with BMP4 (10 nM) in the presence of hBgn (5, 10 and 20 nM), and the amount of BMP4 bound to Chd was determined by immunoprecipitation with α-Nchd followed by α-BMP4 immunoblot. Lower panel: The amount of Chd precipitated is revealed by α-myc Western blot. (C) Bgn increases Chd anti-BMP4 activity in C2C12 cells. AP induction assay was performed as in Figure 3D. In this case, the effect of 25 nM hBgn-flag in the presence of different Chd concentrations was tested. At suboptimal concentrations of Chd (25 nM), Bgn cooperated with Chd in blocking BMP4 activity. ^*P<0.05 when the effect of Bgn and Chd (25 nM) was compared to the effect of Chd (25 nM) alone. (D) hBgn-flag increases the ability of Chd and Tsg to compete for BMP4 binding to a soluble receptor. BMP4 (2 nM) was incubated with 10 nM BR-Fc (lane 2) in the presence of 10 nM Chd (lane 3), 10 nM Tsg (lane 4), Chd and Tsg (lane 5), and Chd/Tsg plus 2.5 nM (lane 6) or 5.0 nM hBgn-flag (lane 7). The receptor was precipitated with protein-A-agarose and the amount of BMP4 bound was determined by α-BMP4 Western blot (upper panel). Western blot using α-human IgG against BR-Fc was performed as loading control (lower panel). (E) hBgn-flag increases the anti-BMP4 activity of Chd/Tsg complexes in C2C12 cells. The ability of Chd, Tsg and Bgn proteins to inhibit BMP4 activity was tested in C2C12 cells. The graphic shows the fold of inhibition obtained by each protein alone or a mixture of them. Fold of inhibition indicates the times that each protein decreases the induction of AP by 2 nM BMP4 alone. The maximal level of induction obtained by BMP4 alone (inset) was used as reference to calculate the fold of inhibition. It was shown that 10 nM Chd, 20 nM Tsg and 20 nM hBgn have no effect when added separately. Chd and Tsg together inhibit AP induction 3.8-fold as expected from their known cooperative effect. When hBgn (5 nM) was added, an even stronger inhibition was observed (8.9 times inhibition). When higher amounts of hBgn were used (20 nM), the effect of Bgn on Chd/Tsg complexes was lost. ^*P<0.05 when the effect of Chd/Tsg/Bgn was compared to the effect of Chd/Tsg. (F) Bgn siRNA reduces the inhibition of BMP4 by Chd/Tsg complexes. Wild-type C2C12 cells or cells treated with siNeg or siBgn were induced with BMP4 in the absence or presence of 5 nM Chd and 10 nM Tsg. After 2 days, the levels of AP were measured. Note that 5 nM Chd and 10 nM Tsg protein inhibit BMP4 activity approximately 2.7 times (wild-type and siNeg cells) and this effect is reduced approximately 1.54 times in cells treated with siBgn. ^*P<0.05 when siBgn effect is compared to wild-type and siNeg cells.

Bgn synthesis and expression during Xenopus development. (A) Gel filtration profile of ³⁵S-labeled PGs from late blastula stage embryos. Embryos labeled with ³⁵S-sulfate were homogenized at stage 9, homogenates were enriched in PGs by ion exchange chromatography and the labeled extracts were directly loaded in a gel filtration column (white circles) or after treatment with nitrous acid to degrade HS chains (red circles) or after incubation with C_ABC to degrade CS and DS chains (blue circles). Note that approximately 5–10% of the radioactivity incorporated into GAGs is resistant to nitrous acid treatment, suggesting that they could correspond to CS/DS PGs (arrow). (B) SDS–PAGE analysis of ³⁵S-labeled PGs from late blastula stage embryos. The same samples analyzed above were separated in a 4–15% gradient SDS–PAGE after incubation with Hase (lane 2), C_ABC (lane 3) or C_AC (lane 4), and then were developed by fluorography. The synthesis of a CSPG with an apparent molecular mass between 200 and 250 kDa is observed (brackets). (C) RT–PCR analysis of the expression of xBgn at different developmental stages. chordin (chd) and siamois (sia) were used as references to detect early zygotic transcription and ODC was used as a loading control. (D–I) xBgn and chordin expression was analyzed by in situ hybridization in paraplast sections. Sections of embryos at (D, E) stage 9, (F, G) 10 and (H, I) 28 were analyzed with (D, F, H) xBgn, (E) xBgn sense or (G, I) chordin probes. e: epidermis; n: notochord; nt: neural tube; s: somites; vbi: ventral blood islands.

Bgn binds BMP4 directly. (A) hBgn-flag binds BMP4. Upper panel: Western blot analysis of BMP4 (10 nM) bound to hBgn-flag (10 and 30 nM, lanes 3 and 4, respectively) after immunoprecipitation of hBgn-flag using anti-flag antibody. As a positive control, 40 nM Tsg-flag was used (lanes 5 and 6). Lower panel: To measure the recovery of hBgn-flag or Tsg after immunoprecipitation, the same membrane was probed with α-flag antibody. (B) BMP2 partially competes for the binding of BMP4 to Bgn. Co-immunoprecipitation under the same conditions as in (A) in the presence of a seven-fold excess of BMP2, FGF, Activin or TGF-β1. Note that only BMP2 was able to partially compete for the binding of BMP4 (lane 3). Upper panel: α-BMP4 blot; lower panel: α-flag blot as loading control. (C) Bgn directly binds BMP4. hBgn-flag (300 nM) or Tsg (100 nM) was incubated with BMP4 (20 nM), and the complexes formed were covalently crosslinked using disuccinimidyl suberate (DSS) as described (Larraín et al, 2000). The samples were separated in a 4–15% gradient gel and developed using α-BMP4 antibody. Note the presence of a band of nearly 90 kDa that could correspond to a complex between a dimer of BMP4 and one molecule of hBgn-flag (lane 3). Tsg and BMP4 formed a complex as expected (lane 5; Oelgeschläger et al, 2000). (D) High levels of hBgn-flag inhibit alkaline phosphatase (AP) induction by BMP4. AP was measured after 2 days of incubation of C2C12 cells in the presence of 2 nM BMP4. AP induction decreased when increasing amounts of hBgn-flag (100–400 nM) were added. Similar effects were obtained with 50 nM Chd. ^*P<0.05 or ^**P<0.01 compared with AP induction by BMP4 in the absence of any BMP4 antagonist. (E) Effect of Bgn siRNA on BMP4 activity. C2C12 cells were transfected with a negative control for siRNA (siNeg) or a mixture of three siRNA for mouse Bgn (siBgn). After 2 days of transfection, cells were incubated with 2 nM BMP4. AP was measured as described above. Inset: Levels of Bgn and GAPDH mRNA were measured by RT–PCR in wild-type cells (lane 1), and cells transfected with siNeg control (lane 2) or siBgn (lane 3). ^*P<0.05 compared to wild-type or siNeg-treated cells.

hBgn requires Chd to induce double axes in Xenopus embryos. (A–E) Secondary axes induced by hBgn require Chd. Embryos were injected at the four-cell stage into the B-tier of each dorsal blastomere with 8 ng of (C) chdMO (Oelgeschläger et al, 2003a), (D) nogMO (Kuroda et al, 2004) or (E) coMO followed by one ventral microinjection in the vegetal side of hBgn (500 pg) synthetic mRNA. The number of double axes induced by each combination of mRNA and morpholinos was bgn (44/77), bgn+chdMO (0/68), bgn+nogMO (36/80) and bgn+coMO (34/53). The insets in panels C–E show the phenotypes obtained by microinjection of the different morpholinos. (F–J) bgnMO and chdMO cooperate in the ventralization of Xenopus embryos. Embryos were injected four times animally at the four-cell stage with (G) bgnMO (8 ng/blastomere), (H) chdMO (1.0 ng/blastomere), (I) chdMO+bgnMO and (J) chdMO+coMO. Development was stopped at stage 20 and in situ hybridization for sizzled was performed. Eight out of 13 embryos showed an expanded domain of szl expression after microinjection of chdMO and bgnMO together. None of the embryos injected with each morpholino alone or with chdMO and coMO have an expansion of the szl domain (N=7–11). (K) Control embryos (lane 1), injected with chdMO (lane 2) or with chdMO and bgnMO (lane 3) were cultured up to stage 25 and analyzed by RT–PCR for different molecular markers. (L) RT–PCR analysis of szl to show rescue of the effect of bgnMO. Embryos were injected with chdMO (lane 1), chdMO and bgnMO (lane 2) and chdMO, bgnMO and hBgn synthetic mRNA (lane 3).