Ho DM , Yeo CY , Whitman M .

HD29468 NICHD NIH HHS , R01 HD029468 NICHD NIH HHS , 27302C0115 NIEHS NIH HHS

	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.

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