TGF-beta signaling is required for multiple processes during Xenopus tail regeneration

Abstract

Xenopus tadpoles can fully regenerate all major tissue types following tail amputation. TGF-beta signaling plays essential roles in growth, repair, specification, and differentiation of tissues throughout development and adulthood. We examined the localization of key components of the TGF-beta signaling pathway during regeneration and characterized the effects of loss of TGF-beta signaling on multiple regenerative events. Phosphorylated Smad2 (p-Smad2) is initially restricted to the p63+ basal layer of the regenerative epithelium shortly after amputation, and is later found in multiple tissue types in the regeneration bud. TGF-beta ligands are also upregulated throughout regeneration. Treatment of amputated tails with SB-431542, a specific and reversible inhibitor of TGF-beta signaling, blocks tail regeneration at multiple points. Inhibition of TGF-beta signaling immediately following tail amputation reversibly prevents formation of a wound epithelium over the future regeneration bud. Even brief inhibition immediately following amputation is sufficient, however, to irreversibly block the establishment of structures and cell types that characterize regenerating tissue and to prevent the proper activation of BMP and ERK signaling pathways. Inhibition of TGF-beta signaling after regeneration has already commenced blocks cell proliferation in the regeneration bud. These data reveal several spatially and temporally distinct roles for TGF-beta signaling during regeneration: (1) wound epithelium formation, (2) establishment of regeneration bud structures and signaling cascades, and (3) regulation of cell proliferation.

FIGURE 1. Timecourse of phospho-Smad2 activation during tail regeneration

(A) p-Smad2 staining of regenerates harvested at 0, 1, 2, 4, 8, and 24 hpa, and at 2 and 3 dpa (B) 4 hpa and 2 dpa regenerates treated with 100 μM SB-431542 at 0 hpa and 24 hpa respectively show no p-Smad2 staining. Phenotypic differences in SB-431542-treated regenerates are discussed later in this work (see Figure 4). Arrowheads: amputation plane. (C) Regenerates stained for p-Smad2 at 0′, 15′, 30′, and 1 h post-amputation. A higher-magnification view of the boxed region of each image is shown below it. Arrows point to representative p-Smad2-positive nuclei.

FIGURE 2. Localization of phospho-Smad2 staining

(A) Regenerates treated with 0.2% DMSO or 100 μM SB-431542 at 0 hpa and fixed at 4 or 24 hpa were double stained with anti-p-Smad2 (green) and anti-p63 (red). DNA is stained in blue. A high-power confocal section through the regeneration bud area is shown. Note total overlap between p-Smad2 and p63 at 4 hpa but not at 24 hpa. White arrow marks a representative nucleus in the p-Smad2+/p63+ layer. Pink arrow marks an apical epidermal cell that does not express p-Smad2. (B) Low power confocal section of the DMSO-treated regenerate in (A), showing that p63+ cells in the fin (white arrowhead) do not express p-Smad2. (C) 3 dpa regenerates, treated with 0.2% DMSO or 100 μM SB-431542 at 2 dpa, were stained for p-Smad2 (green), phospho-Tyrosine (pY, red), and DNA (blue). arrow: apical epithelial cell; arrowhead: basal epithelial cell; bl: blastema; nt: neural tube; not: notochord. Scale bar: 50 μm.

FIGURE 3. Expression pattern of TGF-β ligands during tail regeneration

(A) Whole-mount in situ hybridization with xTGF-β2 antisense probe on regenerating tails at 0 hpa, 4 hpa, 8 hpa, 24 hpa, 2 dpa, and 3 dpa. Red arrows point to xTGF-β2 expression (blue staining) in the regeneration bud. (B) Phase-contrast image at higher magnification of the 24 hpa tail from (A), showing xTGF-β2 expression (red arrow) in the regeneration bud proper and not in the overlying epithelium (black arrow). (C) Whole-mount in situ hybridization with xTGF-β5 antisense probe on regenerating tails at 0 hpa, 2 hpa, 4 hpa, 24 hpa, 2 dpa, and 3 dpa, showing expression of xTGF-β5 (red arrows) as early as 2 hpa. (D) Phase contrast image at higher magnification of the 2 dpa sample from (C), showing xTGF-β5 expression (red arrows) in the regeneration bud proper but not in the overlying epithelium (black arrow). Arrowheads: amputation plane.

FIGURE 4. Inhibition of TGF-β signaling perturbs tail regeneration

(A) Amputated tadpoles were treated with 100 μM SB-431542 (bottom row) or 0.2% DMSO (top row) at 0 hpa, and photographed at 0 and 8 hpa. Note that an epithelial layer has formed in the DMSO control (arrow), but not in the SB-treated tail, where cellular material leaks from the wound (arrowhead). Tadpoles were stained in vivo with Nile Blue to improve resolution. (B) Regenerates were treated with 100 μM SB-431542 at 3 dpa and photographed at 6 dpa. Arrowheads mark the plane of amputation. (C) Regenerates were treated with 100μM SB-431542 from 0–8 hpa and photographed at 6 dpa. Compare to the DMSO control in (B). Arrow: healed epithelium.

FIGURE 5. Inhibition of p-Smad2 signaling reversibly prevents formation of an epithelial layer over the amputation site

The apical layer of the tadpole skin was labeled with 1 mg/ml sulfo-NHS-LC-biotin before amputation. (A) Regenerates were treated with 100μM SB-431542 or 0.2% DMSO at 0 hpa, fixed at 0, 1, 2, 4, and 8 hpa, and stained with streptavidin (red, apical epithelial layer) and anti-p63 (green, basal epithelial layer). Arrow: medial area showing no epithelial healing; arrowhead: fin showing normal epithelial healing. (B) Inhibition of epithelial healing is reversible. Regenerates were treated with 100 μM SB-431542 or 0.2% DMSO from 0–8 hpa, harvested at 2 dpa, and stained as above. Arrow: thickened epithelium seen at 2 dpa in the control but not the SB-431542-treated tadpole. Scale bar: 100 μm.

FIGURE 6. Early SB-431542 treatment blocks formation of regenerative structures at 48 hpa

Regenerates were treated from 0–8 hpa with 100 μM SB-431542 or 0.2% DMSO and harvested at 2 dpa. (A) H&E staining of sagittal sections (B) Xen-1 (neural tissue) staining (C) Tor70 (regenerating notochord) staining (D) double staining for Pax7 (red, muscle satellite cells) and DNA (blue). nt: neural tube, not: notochord, bl: blastema, sc: satellite cells. Arrowheads: amputation plane. Scale bar: 100 μm.

FIGURE 7. Early inhibition of TGF-β signaling prevents subsequent activation of the BMP signaling pathway in the regeneration bud

Regenerates treated with 100 μM SB-431542 or 0.2% DMSO from 0–8 hpa were harvested at 2 dpa and processed for the following markers: (A) immunofluorescence against phospho-Smad1 (p-Smad1, green), showing that p-Smad1 in the regeneration bud proper is eliminated by SB-431542 treatment. p-Smad1 (blue) in unamputated stage-matched tadpole tails was unaffected by SB-431542 (bottom row); (B) in situ hybridization with xMsx1 antisense probe (purple) –black arrow: xMsx1 expression in the dorsal blastema; (C) immunofluorescence for the mitotic marker phospho-Histone H3 (pH3, green). Total DNA in (A) and (C) is shown in blue. Arrowheads: amputation plane. Scale bar: 100 μM.

FIGURE 8. Early inhibition of TGF-β signaling results in missing or aberrant pERK1/2 activation in the regeneration bud

Immunofluorescence staining for phospho-ERK1/2 (red) and total DNA (blue). (A) 8 hpa untreated regenerates, showing pERK1/2 staining primarily in the wound epithelium (orange arrow). (B) 2 dpa regenerates, treated with 100 μM SB-431542 or 0.2% DMSO from 0–8 hpa. White arrow: blastemal p-ERK1/2 in controls. Green arrow: aberrant pERK1/2 in the epithelium of SB-431542-treated tadpoles. Arrowheads: amputation plane. Scale bar: 100 μM.

FIGURE 9. Effect of late TGF-β inhibition on proliferation in the regeneration bud

(A) Phospho-Histone H3 (pH3) expression. Regenerates were treated with 100 μM SB-431542 or 0.2% DMSO at 2 dpa and harvested at 3 dpa for immunofluorescence for anti-pH3 (green). Total DNA is shown in blue. (B) Quantitation of pH3+ nuclei in 3 dpa regenerates treated as in (A). Numbers of pH3+ nuclei in a 2000 μm² area of a confocal section were counted in the regeneration bud, fin, and somite. For each sample, a similarly located area was selected. N=7 for DMSO and 6 for SB, except for fin samples, where N=5 for both DMSO and SB. Samples were drawn from 3 separate experiments, except for fins (2 experiments). Error bars: ±1SD. *: p=0.00005. (C) Regenerates were treated with 100 μM SB-431542 or 0.2% DMSO for 9 hours total, including final 3 hours with nocodazole (NOC) arrest, and stained at 3 dpa for pH3 (green) and DNA (blue). nt:neural tube, not:notochord, bl:blastema. (D) BrdU incorporation assay. Regenerates were treated with 100 μM SB-431542 or 0.2% DMSO at 2 dpa and harvested at 4 dpa. BrdU was added to the media 6 hours after initiation of SB-431542 treatment. BrdU+ nuclei are labeled in red and total DNA in blue. BrdU incorporation in the control is increased at the regeneration bud (white arrow) but not rostral to the level of the amputation plane (orange arrow); in contrast, the SB-431542-treated tail does not show any obvious rostro-caudal differences in BrdU incorporation. Similarly, proliferation in the fin (green arrow) is not affected. White arrow: regeneration bud; orange arrow: tail rostral to amputation plane; green arrow: fin in regeneration bud. Scale bar: 100 μm. Arrowheads: amputation plane.