Gamer LW et al. (1999), A novel BMP expressed in developing mouse limb,...

XB-ART-13379

Dev Biol 1999 Apr 01;2081:222-32. doi: 10.1006/dbio.1998.9191.

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A novel BMP expressed in developing mouse limb, spinal cord, and tail bud is a potent mesoderm inducer in Xenopus embryos.

Gamer LW , Wolfman NM , Celeste AJ , Hattersley G , Hewick R , Rosen V .

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The bone morphogenetic proteins (BMPs) play critical roles in patterning the early embryo and in the development of many organs and tissues. We have identified a new member of this multifunctional gene family, BMP-11, which is most closely related to GDF-8/myostatin. During mouse embryogenesis, BMP-11 is first detected at 9.5 dpc in the tail bud with expression becoming stronger as development proceeds. At 10.0 dpc, BMP-11 is expressed in the distal and posterior region of the limb bud and later localizes to the mesenchyme between the skeletal elements. BMP-11 is also expressed in the developing nervous system, in the dorsal root ganglia, and dorsal lateral region of the spinal cord. To assess the biological activity of BMP-11, we tested the protein in the Xenopus ectodermal explant (animal cap) assay. BMP-11 induced axial mesodermal tissue (muscle and notochord) in a dose-dependent fashion. At higher concentrations, BMP-11 also induced neural tissue. Interestingly, the activin antagonist, follistatin, but not noggin, an antagonist of BMPs 2 and 4, inhibited BMP-11 activity on animal caps. Our data suggest that in Xenopus embryos, BMP-11 acts more like activin, inducing dorsal mesoderm and neural tissue, and less like other family members such as BMPs 2, 4, and 7, which are ventralizing and anti-neuralizing signals. Taken together, these data suggest that during vertebrate embryogenesis, BMP-11 plays a unique role in patterning both mesodermal and neural tissues.

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Species referenced: Xenopus
Genes referenced: acta4 actc1 actl6a fst gdf11.1 gdf5 mix1 mstn.1 ncam1 nog otx2 tbxt twist1

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	FIG. 3. Morphological and histological analysis of control and BMP-11 treated animal pole explants. Blastula stage (8 –9) animal pole explants were treated with 200 ng/ml of recombinant human BMP-11 and cultured to neurula stage for morphology (A, B) and tadpole stage for histology (C, D). (A) Animal caps cultured in media alone remain rounded. (B) Animal caps cultured in BMP-11 protein (200 ng/ml) undergo a dramatic elongation indicative of mesoderm induction. (C) Histological section through a control explant reveals formation of atypical epidermis (ep). (D) Histological section through a BMP-11 treated explant reveals differentiated blocks of muscle (m), notochord (no), and neural tissue (nt).
	FIG. 4. Analysis of gene expression induced in animal cap explants by BMP-11 protein. (A) Animal caps were explanted from stage 8–9 embryos and treated with increasing doses of recombinant human BMP-11 (10–1000 ng/ml) or buffer alone (untreated) until sibling embryos reached late neurula (stage 22). Total RNA from pools of 10 animal caps was used as the template for cDNA synthesis and the indicated markers were then assayed for by RT-PCR. BMP-11 induced the mesodermal markers Xbra and muscle actin (m. actin) at all doses tested. At higher doses (50–1000 ng/ml), BMP-11 induced the pan-neural marker NCAM. EF1-a serves as a loading and reverse transcription control while the embryo and -RT are additional positive and negative controls. (B) Animal caps were explanted from stage 9 embryos and treated for 2 h in buffer alone (untreated), activin (2 ng/ml), FGF (50 ng/ml), or BMP-11 (20 ng/ml) and collected at gastrula (stage 10.5). Low doses of activin and BMP-11 induced the expression of Mix.1, while FGF did not. All factors tested induced mesoderm as seen by the expression of Xbra.
	FIG. 5. Follistatin but not noggin inhibits BMP-11 activity. Blastula stage animal pole explants were treated with 50 ng/ml BMP-11 protein alone (A, E) or in combination with 1000 ng/ml mouse noggin protein (B) or 500 ng/ml human follistatin protein (F) until late neurula (stage 22). (A) BMP-11 induces morphogenetic movements in animal caps. (B)A20-fold molar excess of noggin does not inhibit BMP-11 activity. (C) RT-PCR analysis shows that noggin is unable to block BMP-11 induction of muscle actin (m. actin). (D) Recombinant mouse noggin induces the neural marker, NCAM, indicating the protein has the predicted activity in our assay. (E) BMP-11 protein induces elongation in animal caps. (F) Follistatin completely inhibits the activity of BMP-11 at a 10-fold molar excess. (G) RT-PCR analysis shows that follistatin fully blocks muscle actin induction by BMP-11. EF1-a is a loading and reverse transcription control and untreated animal caps serve as a negative control.
	FIG. 6. BMP-11 induces region-specific neural markers and modifies the neural tissue induced by noggin. Blastula stage animal pole explants were treated with BMP-11 (50 ng/ml), noggin (1000 ng/ml), or a combination of both factors until early neurula (stage 18) and analyzed for regional neural marker expression by RT-PCR. BMP-11 induces the neural crest marker, Xtwist (Xtwi), and the spinal cord marker, Hox B9, but not the forebrain marker, otx2. Noggin alone induces only otx2 expression. In combination, BMP-11 dramatically reduces the amount of otx2 induced by noggin and allows continued expression of Xtwi and Hox B9. EF1-a serves as a loading and reverse transcription control.