September 1, 2010;
The role and regulation of GDF11 in Smad2 activation during tailbud formation in the Xenopus embryo.
A key role for phosphorylation of Smad2
by TGFβ superfamily ligands in the axial patterning of early embryos is well established. The regulation and role of Smad2
signaling in post-neurula
embryonic patterning, however, is less well understood. While a variety of TGFβ superfamily ligands are implicated in various stages of anterior
patterning, the ligand GDF11
has been shown to have a particular role in post-gastrula
patterning in the mouse. Mouse GDF11
is specifically localized to the developing tail
and is essential for normal posterior
axial patterning. Mature GDF11
ligand is inhibited by its own prodomain, and extracellular proteolysis of this prodomain is thought to be necessary for GDF11
activity. The contribution of this proteolytic regulatory mechanism to Smad activation during embryogenesis in vivo, and to the development of posterior
pattern, has not been characterized. We investigate here the role of Xenopus GDF11
in the activation of Smad2
during the development of tailbud
-stage embryos, and the role of this activation in larval development. We also demonstrate that the activity of BMP-1
-like proteases is necessary for the normal GDF11
-dependent activation of Smad2
phosphorylation during post-gastrula
development. These data demonstrate that GDF11
has a central role in the activation of Smad2
phosphorylation in tailbud stage
Xenopus embryos, and provide the first evidence that BMP-1
-mediated prodomain cleavage
is important for activation of GDF11
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References [+] :
Fig. 1. p-Smad2 expression in stage 26 embryos. (A) Embryos treated with 100 μM SB-431542 or 0.2% DMSO from stage 13 were dissected into regions 1–4 (head, anterior dorsal trunk, medial dorsal trunk, and posterior dorsal trunk/tailbud, respectively) as indicated on the schematic and Western blotted for p-Smad2. Actin serves as a loading control. (B) Stage 26 embryos treated with SB-431542 or DMSO as in (A) were immunostained with anti-p-Smad2. Tailbuds of stained embryos were photographed without clearing (left column) or with clearing (right column, DMSO only; cleared SB-431542 tailbuds are not shown, as they were almost impossible to photograph due to lack of staining and contrast). tb, tailbud; nt, posterior neural tube.
Fig. 2. p-Smad2 immunostaining at later tailbud stages. (A) Tailbud-stage embryos were treated with 100 μM SB-431542 or 0.2% DMSO for 22 h prior to harvesting at stage 29 or 36. Embryos were stained with anti-p-Smad2 and cleared. (B) Stage 34 embryos stained for p-Smad2 were cleared to reveal specific structures in the head (top) and tail (bottom). Note that some bilateral structures can be seen twice in cleared heads. br, brain; e, eye; ov, otic vesicle; ba, branchial arches; m, mouth; tb, tailbud; f, fin; nt, posterior neural tube; pw, posterior wall of tailbud; cnh, chordoneural hinge.
Fig. 3. Expression of Xenopus GDF11. (A) Sequence of Xenopus GDF11 protein. The following conserved regions are present: putative signal sequence (red), TGFβ propeptide domain (blue), TGFβ mature domain (green). Cleavage sites for BMP-1/Tolloid protease (GD, purple) and furin protease (RSRR, orange) are also indicated. (B) Temporal expression profile of xGDF11. RT-PCR was performed on whole embryo samples harvested at indicated stages. xGDF11 RT-PCR was performed on both +RT and −RT samples. Ornithine decarboxylase (ODC) was used as a loading control. (C) In situ hybridization for xGDF11 from stages 13–37. In stages 20–37, anterior is to the left, dorsal to the top. bp, blastopore; tb, tailbud; br, brain; ba, branchial arches; nc, neurenteric canal; pw, posterior wall of tailbud; cnh, chordoneural hinge.
Fig. 4. GDF11 knockdown results in loss of p-Smad2 at tailbud stages. (A) Confirmation of xGDF11 morpholino oligos. Top panel: embryos were injected with 50 pg xGDF11-3flag mRNA along with 50 ng of tMO or scr-tMO and harvested at stage 10+ (uninjected controls were stage 11) for Western blotting against the flag epitope. Actin was used as a loading control. Bottom panel: embryos injected with 20 ng spMO or cont-MO were harvested at stage 25 for RT-PCR using primers spanning the splice site targeted by spMO. Elongation factor 1a (EF1a) was used as a loading control. (B) Effect of xGDF11 knockdown on p-Smad2 at stage 26/27. Embryos injected with 50 ng xGDF11 tMO or scr-tMO were dissected into regions 1 (head, anterior dorsal trunk, medial dorsal trunk, and posterior dorsal trunk/tailbud, respectively) as indicated in the schematic and Western blotted for p-Smad2. we, whole embryo. Actin was used throughout as a loading control. (C) Effect of xGDF11 knockdown on p-Smad2 and p-Smad1 at stage 34. Embryos injected with 40 ng xGDF11 tMO or cont-MO were dissected into regions 1 as indicated in the schematic and Western blotted for p-Smad2 (top) and p-Smad1 (bottom). (D) xGDF11 knockdown eliminates endogenous p-Smad2 staining in multiple tissues at stage 25. Embryos were injected with 60 ng xGDF11 tMO, 60 ng xGDF11 scr-tMO, 20 ng xGDF11 spMO, or 20 ng xGDF11 scr-spMO and immunostained at stage 25 with anti-p-Smad2. br, brain; e, eye; ba, branchial arches; m, mouth; tb, tailbud.
Fig. 5. Effect of xGDF11 knockdown on embryonic phenotype. (A) Phenotype of stage 41 embryos injected with 60 ng tMO versus scr-tMO (left), and of stage 40 embryos injected with 20 ng spMO or cont-MO (center), and stage 40 embryos treated with 100 mM SB-431542 or 0.1% DMSO at stage 13. (B) Cell proliferation was measured by phospho-histone H3 staining of tails from stage 40 embryos treated with 60 ng xGDF11 tMO (right) or 60 ng xGDF11 scr-tMO (left). Higher magnification of each tail tip is shown at bottom. Data shown are typical of three separate experiments, a similar reduction in proliferation with xGDF11 knockdown was seen when measured using BrdU incorporation (not shown).
gdf11.1 ( growth differtiation factor 11, gene 1 ) gene expression in Xenopus laevis embryos, NF stage 20, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
gdf11.1 ( growth differtiation factor 11, gene 1 ) gene expression in Xenopus laevis embryos, NF stage 28, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
gdf11.1 ( growth differtiation factor 11, gene 1 ) gene expression in Xenopus laevis embryos, NF stage 31, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
gdf11.1 ( growth differtiation factor 11, gene 1 ) gene expression in Xenopus laevis embryos, NF stage 37, as assayed by in situ hybridization, lateral view, anterior left, dorsal up.
Identification of a novel pool of extracellular pro-myostatin in skeletal muscle.