Chang C and Hemmati-Brivanlou A (1999), Xenopus GDF6, a new antagonist of noggin and a ...

XB-ART-12762

Development 1999 Aug 01;12615:3347-57. doi: 10.1242/dev.126.15.3347.

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Xenopus GDF6, a new antagonist of noggin and a partner of BMPs.

Chang C , Hemmati-Brivanlou A .

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In Xenopus, ectodermal cell fates are determined by antagonistic interaction between the BMP subfamily of TGF-(beta) ligands and the organizer-specific secreted factors (e.g. noggin, chordin and follistatin). Inhibition of BMP function by these factors can convert cells from an epidermal to a neural cell fate. In this study, we report that GDF6, a new member of the Xenopus TGF-(beta) family, can function in antagonistic interaction with neural inducers. GDF6 induces epidermis and inhibits neural tissue in dissociated cells, and this activity is blocked by the presence of noggin. We demonstrate that GDF6 binds directly to the neural inducer noggin. Furthermore, we find that GDF6 and BMP2 can form heterodimers and the process seems to require cotranslation of the proteins in the same cells. In normal embryos, GDF6 and BMP2 are coexpressed in several places, including the edge of the neural plate at early neurula stages, suggesting that GDF6 may synergize with BMPs to regulate patterning of the ectoderm. Our data show for the first time that noggin can bind directly to and inhibit another TGF-(beta) family member: GDF6. In addition, BMP and GDF6 heterodimers may play an important role in vivo to regulate cell fate determination and patterning.

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Species referenced: Xenopus
Genes referenced: bmp2 bmp4 bmp7.1 bmp7.2 chrd fst gdf1 gdf5 gdf6 gdf7 krt12.4 myc ncam1 nog tbxt ventx1.2 wnt8a zic3

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	Fig. 1. Xenopus GDF6 is a close homolog of GDFs and BMPs. (A) Sequence of the Xenopus GDF6 protein. The signal peptide is underlined and the proteolytic processing site is boxed. (B) GDF6 is highly conserved between species. Alignment of Xenopus GDF6 mature region with that of GDF6, GDF5 and GDF7 from other species. Xenopus GDF6 is more than 90% identical to mouse GDF6. (C) GDF6 is homologous to BMPs. Alignment of the mature region of Xenopus GDF6 with that of Xenopus BMPs. GDF6 is about 50% identical to BMP2, BMP4 and BMP7 in their mature regions, while it is less homologous to activin and other TGF-bs. GenBank accession number for Xenopus GDF6 is AF155125
	Fig. 2. GDF6 is downregulated by neural inducers at gastrula stages at the RNA level and is efficiently secreted from oocytes. (A) GDF6, but not BMP4, transcripts are significantly downregulated by neural inducers noggin and follistatin at late gastrula stages (stage 11-12). Embryos were injected at the 2-cell stage with 0.2 ng noggin or 2 ng follistatin RNA (lanes 2 and 3, respectively). Animal caps were dissected at blastula stage and incubated to late gastrula stage before they were processed for RT-PCR assay. Lane 1 is from uninjected control embryos, while lanes 4 and 5 are from whole embryos with (lane 5) or without (lane 4) reverse transcriptase during RT reaction. (B) GDF6 is secreted from Xenopus oocytes. Oocytes were injected with RNAs encoding GDF6 (lane 2) or a myc-tagged GDF6 (G6myc, lane 3). The oocyte culturing medium was collected 2 days later and analyzed on 10% SDS-PAGE. While uninjected oocytes did not secret any proteins at a significant level (lane 1), the supernatant from oocytes injected with GDF6 and G6myc contains two bands, corresponding to the processed mature and the pro-region of the protein.
	Fig. 3. Early zygotic expression of GDF6 is restricted to the animal cap. (A) GDF6 is expressed weakly after the midblastula transition. The expression increases significantly around the mid-gastrula stages and maintains its level until at least tailbud stages. (B) GDF6 expression is restricted to the animal caps of early gastrula embryos. Early gastrula stage (stage 10+) embryos were dissected into animal and vegetal (lanes 1 and 2), dorsal marginal zone and ventral marginal zone (DMZ and VMZ respectively, lanes 3 and 4) regions. The dissection scheme is shown at the left. RT-PCR method was used to assay for region-specific markers (Chordin is a dorsal and Xvent1 is a ventral marker, Xwnt8 is expressed ventral-laterally, Vg1 is a marker for vegetal pole and Xbra, Xenopus brachyury, is a panmesoderm marker) as well as GDF6 expression. GDF6 is expressed only in the animal caps at this early gastrula stage. (C) GDF6 transcripts are downregulated in the dorsal ectoderm at early gastrula stages. Stage 10 embryos were dissected into dorsal and ventral animal caps (scheme shown at the left). RT-PCR was used to assay for dorsal animal (lane 1) and ventral animal pole (lane 2) markers. Zic3 is a dorsal-specific marker, while Vent1 is a ventral marker. Xbra is a pan-mesodermal marker. Unlike BMP4, which is uniform at this stage in animal caps, GDF6 is downregulated in the dorsal animal region (compare lane 1 with lane 2).
	Fig. 4. Spatial expression of GDF6 during early Xenopus development. (A) GDF6 is expressed at the border of the dorsal and ventral ectoderm around stages 11 to 12. (B,C) At neurula stages, GDF6 is expressed at the edges of the neural plate, as well as in two patches in the anterior neural plate. (D-F) At late neurula stages 18- 19, GDF6 is distributed in a salt-and-pepper pattern in the neural folds (inset of panel E), possibly representing the premigratory neural crest cells. In addition, GDF6 is expressed in olfactory placode and in the eye field. (G,H) After the neural tube closure, GDF6 occupies the dorsal midline of the neural tube, again in a dotted fashion. The expression in the olfactory placode persists, and the staining in the eye field becomes localized to the dorsal part. (I-L) At tailbud stages, GDF6 is seen at the dorsal hindbrain and the dorsal midline of the spinal cord. It is also expressed in the dorsal retina of the developing eyes. Embryos are viewed from dorsal side in A, B, D, E, H, J and K, with anterior end to the left. In C, F and G, the embryos are viewed head on from anterior end. Embryos in I and J are viewed from lateral side with anterior end to the left
	Fig. 5. GDF6 inhibits neural tissue and induces epidermis in dissociated animal caps, and its activity is blocked by noggin. (A) Animal caps from stage 9 embryos were dissociated in Ca2+- and Mg2+-free medium for 4 hours before reaggregated and cultured to neurula stages. Lane 1 is from the intact caps, which expresses the epidermal marker epidermal keratin and does not express neural genes such as NCAM and OtxA. Lanes 2-5 are animal caps dissociated for 4 hours. Lanes 3-5 contain conditioned oocyte medium collected from uninjected control oocytes (lane 3), BMP2-injected (lane 4) and GDF6-injected (lane 5) oocytes. BMP2 and GDF6 convert the cells from a neural to an epidermal fate. Lanes 6 and 7 are whole embryo control, with (lane 7) or without (lane 6) reverse transcriptase during the RT reaction. (B) Noggin blocks epidermal inducing activity of GDF6. Lane 1 is from intact animal caps, while lanes 2 to 4 are from cells dissociated for 4 hours. GDF6-conditioned medium is present in samples in lanes 3 and 4, and noggin protein is present in samples on lane 4 during cell dissociation.
	Fig. 6. (A) GDF6 binds specifically to noggin. Lanes 1 and 2 are markers for labeled GDF6 and BMP2, respectively. Lanes 3 and 4 are the two proteins incubated only with the protein G beads in the absence of noggin-Fc. Lanes 5 to 10 are the two proteins coincubated with noggin-Fc and protein G beads. Lanes 7 and 8 include unlabeled GDF6, while lanes 9 and 10 include BMP4 as specific competitor for noggin binding. Odd-numbered lanes contain labeled GDF6 protein, and even-numbered lanes contain labeled BMP2 protein. Binding of GDF6 or BMP2 to noggin can be competed away by unlabeled BMP4 or GDF6 ligands. (B) BMP2, but not activin or Vg1, can compete with GDF6 for noggin binding. Lanes 1 to 4 are markers for secreted TGF-b ligands activin, Vg1, BMP2 and GDF6. Lanes 5-8 contain noggin-Fc with labeled GDF6, and lanes 6-8 include binding competitors activin, Vg1 and BMP2, respectively. (C) GDF6 binds to noggin with an affinity closer to BMP2 than to BMP7. Lane 1 is binding of 35S-labeled GDF6 to noggin-Fc in the absence of any competitors, while the rest of the lanes are samples containing 1:1 (lanes 2, 6 and 10), 3:1 (lanes 3, 7 and 11), 5:1 (lanes 4, 8 and 12) and 10:1 (lanes 5, 9 and 13) ratio of unlabeled GDF6 (lanes 2-5), BMP2 (lanes 6-9) or BMP7 (lanes 10-13) to labeled GDF6. Binding of 35S-labeled GDF6 to noggin is competed away by the presence of 3- to 5-fold of unlabeled GDF6 or BMP2, while BMP7 cannot compete for GDF6 binding even at 10-fold excess.
	Fig. 7. Heterodimer formation between GDF6 and BMP2. Conditioned oocyte supernatant was precipitated with anti-myc antibody. The antibody precipitates G6myc, but not BMP2 protein (compare lanes 2 and 1). However when the two genes are coexpressed in the oocytes, BMP2 can be coimmunoprecipitated with G6myc, suggesting heterodimer formation between the two proteins (lane 3). Lanes 4 and 5 are markers for BMP2 and G6myc, respectively.