PMC

BMP15 protein blocks the epidermal-inducing activity of BMP4 protein. Top, schematic of intact versus dissociated ectodermal explants exposed to BMP4 and BMP15 purified recombinant protein. RT, reverse transcription. Bottom right, BMP15 reduces the induction ability of BMP4 in intact explants. Bottom left, BMP15 inhibits the ability of BMP4 protein to induce epidermis and to inhibit neural fate. ODC, ornithine decarboxylase.

BMP15 physically interacts with BMP4. A, schematic of the experimental design. B, immunoprecipitation of BMP15, BMP4, and activin from animal halves of frog embryos. The first three rows show Western blots of crude lysates showing expression of BMP15 in embryos injected with different constructs. The last row shows an immunoprecipitation using the anti-Myc antibody followed by blotting with the hemagglutinin (HA) antibody. BMP15 can immunoprecipitate BMP4. IB, immunoblot; CL, crude lysate; IP, immunoprecipitation.

BMP15 induces the formation of ectopic heads. A, schematic shows sites of injections in an eight-cell stage embryo, targeting the two ventral-vegetal blastomeres. B, control uninjected embryo stage 28. C–E, phenotype of embryos injected with 100 pg of hBMP15 mRNA into ventral vegetal blastomeres displaying induction of ectopic heads and cement glands. The red arrows point to the primary head and cement gland of the tail-bud, and green arrows point to the secondary induced ectopic heads. In both cases arrowheads point to the heads, and arrows point to cement glands.

BMP15 dominant negative mutant elicits strong ventralization phenotype. A, the BMP15-Y235C dominant-negative mutant. Tyrosine 235 is mutated to a cysteine in the pre-pro domain. B, schematic of animal pole injection of both blastomeres at the two-cell stage. C, control uninjected embryos. D, G, and I, phenotype of embryos injected with BMP15-Y235C alone, completely lacking head structures. E and F, phenotype of embryos injected with BMP15 wild type (BMP15 wild type (WT)), displaying the reverse phenotype with embryos lacking trunk and tail structures and displaying duplicated heads (arrows in F) and cement glands tissue (arrows in E). H, rescue by coinjection of equal amounts of BMP15-Y235C with BMP15 wild type RNA restores the normal phenotype. J and K, BRE-luciferase reporter assay. J, BMP15 wild type inhibits BRE. BMP15-Y235C does not inhibit BRE. K, coexpression of BMP4 with BMP15-WT, and BMP15-Y235C reactivates BRE.

BMP15 inhibits Wnt signaling. A, BMP15 reduces the expression of Wnt responsive genes. Experiments setting are the same as shown in A. Ectodermal explants expressing either 50 pg of Wnt8 or 100 pg of xBMP15 or both were submitted to reverse transcription (RT)-PCR to evaluate the level of expression of Xnr3 and Siamois. st., stage. ODC, ornithine decarboxylase. B, schematic of experimental setting for measurement of Wnt-responsive element TOP-FLASH. Embryos were injected at two cell stages, with 50 pg of Wnt8 or 200 pg of β-catenin T2 alone or together with different concentrations of BMP15. Luciferase assays were performed at early gastrula stage. C, BMP15 inhibits Wnt8-induced transcription from TOP-FLASH. D, BMP15 does not affect the activity of β-catenin T2. This was not because of the ratio of BMP15 to β-catenin, as a 10-fold increase in BMP15 still did not lead to inhibition (data not shown).

BMP15 is a direct neural inducer. A, schematic of experimental design. 100 pg of hBMP15 mRNA was injected into both blastomeres of two-cell-stage embryos in the animal pole, and ectodermal explants were isolated at blastula stage and cultured until early gastrula (stage 10.5) and late neurula (stage 19) and submitted to reverse transcription (RT)-PCR for characterization of cell type-specific markers. B, hBMP15 induces the expression of the early neural marker Sox2 and inhibits the ventral ectodermal marker Msx1. This conversion to neural fate is direct, as neither the pan-mesodermal marker Xbra nor the endodermal marker Sox17α is induced. C, at neural groove stages (stage 19) hBMP15 induces the expression of the pan-neural marker Nrp1 and neural cell adhesion molecule (NCAM), the cement gland marker XAG, and the brain marker Otx, whereas the posterior neural (HOXB9), the mesoderm (muscle actin and globin), and endoderm (intestinal fatty acid-binding protein, IFABP) markers are not induced. ODC, ornithine decarboxylase.

BMP15 inhibits transcriptional response from BRE. A, schematic of experimental design. B, luciferase measurements of total embryo lysates from Xenopus embryos injected with the reporter plasmids BRE-Lux (20 pg). hBMP15 strongly inhibits the activation of the BRE in Xenopus embryos. C, ARE (25 pg) with different doses of hBMP15 mRNA and BMP4 (100 pg), activin (ACV, 100 pg) transcripts. BMP15 has no effect on the ARE. D, increasing amounts of hBMP15 mRNA (10, 50, 100, 250 pg) were injected into one blastomere of two-cell-stage frog embryos, and protein was extracted at the onset of gastrulation (stage 10) from 25 whole embryos/experimental condition. Protein extracts from early cleavage embryos (stage 6) were used as the negative control, and uninjected embryos at stage 10 were used as the positive control. Expression of hBMP15 in embryos strongly decreases Smad1 MH2 domain phosphorylation, demonstrating inhibition of the Smad1/5/8 pathway. BMP15 expression, on the other hand, had no effect on the Smad 2 MH2 phosphorylation, demonstrating the specificity of inhibition within the TGFβ signaling pathway. IB, immunoblot.

BMP15 expression in Xenopus embryos. A, alignment of the amino acid sequences for the Xenopus (xBMP15) human (hBMP15), and mouse (mBMP15) mature peptide. The mouse and Xenopus proteins show a homology of the 68 and 44%, respectively, with the human protein. B, schematic of a conventional TGFβ ligand, BMP4 (top), highlighting the prodomain (green), the mature domain (red), and the position of the seven signature cysteines. The fourth cysteines in the mature region (highlighted in red) are missing in BMP15 (bottom). C, temporal expression of BMP15 during Xenopus embryonic development, detected by reverse transcription-PCR. BMP15 is expressed maternally in the egg and maintained throughout development. Ornithine decarboxylase (ODC) was used as a loading control. St., stage. D–M, patterns of expression of xBMP15-RNA, as detected by whole-mount in situ hybridization. BMP15 expression is detected in the animal pole region of 4-cell stage embryo (D and E) and maintained in the prospective ectoderm at blastula stage (F and G). Expression then becomes ubiquitous within the ectoderm (H and I). At the neural tube closure, BMP15 is maintained in the nervous system (J and L), and at stage 26 the expression becomes exclusive to the anterior, including sensory organs and hindbrain (l). At tail-bud stage (), peak expression is maintained in the brain and eyes (K and M).

Loss-of-function of BMP15 by morpholino leads to loss of head structures and elevated Smad1 activation in vivo. A, sequence of morpholino designed specifically for interference with the synthesis of the Xenopus BMP15 (xBMP15) protein and used for loss-of-function experiments. Yellow highlights the sequence of the MO that targets the signal sequence of xBMP15 (but not hBMP15). The black arrow indicates the beginning of the open reading frame. B, schematic of injection sites. A total of 10 ng of BMP15-MO was injected in the animal pole of both blastomeres of two-cell-stage embryos, targeting the site of BMP15 expression. Phenotypes were evaluated at tail-bud stages. C, Western blot analysis shows the ability of the MO to block the production of xBMP15 protein (compare lane 3 to lane 4) but not hBMP15 (compare lanes 5 and 6). CO-MO, control morpholino. D, control-scrambled-MO (10 ng)-injected embryos showed normal phenotype. E and F, examples of BMP15-MO-injected embryos displaying anterior defects, with partial-to-complete loss of head structures and in 43%-to-complete ventralization phenotype (n = 105). This phenotype is consistent with excess BMP4 activity because of the removal of its inhibitor BMP15. G, the severe BMP15-MO phenotype can be rescued by co-injection with wild type xBMP15 mRNA. The large majority of the co-injected embryos display normal phenotypes (n = 52/55, 94.5%), providing a stringent control for specificity. 10 ng of BMP15-MO was co-injected with 100 pg of xBMP15 mRNA. H, BMP15-MO induces MH2 domain phosphorylation of Smad1 in Xenopus embryo. BMP15-MO (10 ng total) was injected in the animal pole of both blastomeres of 2-cell-stage embryos. Animal halves explants were dissected at blastula stage, cultured, and lysed at stage 10 and subjected to immunoblotting assay using the antibodies against the indicated targets, including anti-α-tubulin as a loading control. The results show an increase in the MH2 domain phosphorylation of SMAD1 in the BMP15-MO-injected explants (lane 3 and 4). As positive and negative control, lysates from stage (st.) 6 and 10 whole embryos were used.