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Fig. 1. Distribution of Yap1 protein during tadpole tail regeneration. Tadpole tails were amputated at st41/42 and subjected to whole-mount immunofluorescent staining. (A)–(I) Yap1 and Sarcomeric Myosin heavy chain (MyHC) co-immunofluorescent staining. (J)–(R) Yap1 and acetylated Tubulin (acTub) co-immunofluorescent staining. Muscles (white arrow) and spinal cord initiated regeneration at 3 dpa (days post amputation). Yap1 protein was broadly distributed in the regenerating tail. Nerve axons appear as linear and branched acTub staining signals, and the punctate acTub signals in the tail samples at 0 and 1 dpa were ciliated cells in the skin ((J)–(O)). (S)–(U) Immunofluorescent staining of phosphorylated Yap1 (pYap1), the inactive form of Yap1. pYap1 was preferentially distributed in the tip of the intact notochord (S) and regenerating notochord (U). Red line indicates the amputation plane. Scale bar=500 µm.
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Fig. 2. Tail regeneration is impaired by dnYap or dnTead4 induction. (A) and (B) Transgene expression was induced by heat shock. Tadpoles were subjected to heat shock at st41/42, and GFP reporter expression was observed 3 h later in the dnYap (dnY) and the dnTead4 (dnT4), but not in the wild type (WT, heat-shocked) tadpole. The dnY and dnT4 tadpoles could be distinguished from WT by lens-specific tdTomato fluorescence under control of the gamma crystallin promoter. Inset is a high-power view of a tdTomato-positive lens. (C) Experimental schedule. Tadpoles at st41/42 were heat shocked 3 h prior to amputation, and observed at 7 dpa (days post amputation). (D)–(F) Regenerated tails were observed after fixation and bleaching. While WT tadpoles had regenerated an almost complete tail at 7 dpa (D), the dnYap and dnTead4 tadpoles did not. The pictures show the “severe defect†phenotype ((E) and (F)). Red line indicates the amputation plane. (G) Measured length of the regenerated tail (the average length). The regenerated tail length in dnYap and dnTead4 Tg was significantly shorter than that in WT (double asterisks: p<0.01, Welch’s t-test). Error bar indicates s.e.m. (H) Percentage of WT, dnYap, and dnTead4 tadpoles displaying varying degrees of the regenerative response, from “severe defect†(least regenerative) to “regenerated†(most regenerative) after the heat shock procedure described in (C). The degree of regeneration was markedly reduced in the dnYap1 and dnTead4 Tg tadpoles. Scale bar=500 µm.
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Fig. 3. dnYap and dnTead4 expressions affect cell differentiation, cell proliferation, and cell death in tail regeneration. The regenerated tail of WT and Tg tadpoles was subjected to whole-mount immunofluorescent staining at 7 dpa (days post amputation). (A) Cartoon of the regenerating tadpole tail. Red line indicates the amputation plane. Photographs in (B)–(J) show the caudal region marked by the blue square, and those in (K)–(M) show a more restricted tail region marked by the green square. (B)–(D) MyHC immunostaining showed clustered myocytes that had already initiated regeneration in the WT tail (heat-shocked). In contrast, only fibrous myocytes were seen in the dnYap and dnTead4 Tg tadpoles. Inset indicates the high-power view of the white-boxed region. (E)–(G) acTub immunostaining marked an appropriately elongated nerve axon in the WT and shortened nerve axons in the dnYap and dnTead4 Tg regenerating tail. (H)–(J) pH3 immunostaining, which indicates mitotic cells, showed that cell proliferation in the regenerating tail was reduced in the dnYap and dnTead4 Tg tadpoles. (K)–(M) acCas3 immunostaining, indicating apoptotic cells. High-power view of the tip of the regenerating tail showed very few apoptotic cells in the regenerating WT tail. In contrast, ectopic apoptosis was frequently observed in the regenerating tail of dnYap and dnTead4 Tg tadpoles. Scale bar=100 µm in (K)–(M) and 500 µm in (B)–(J).
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Fig. 4. Yap and Tead4 are cell-autonomously required for the cell survival of neural progenitors in the spinal cord. (A) Experimental schedule for labeling neural progenitors at the single-cell level by IR-LEGO. (B) Cartoon of the regenerating tail. Green line represents the spinal cord, and light orange region represents the notochord. Red line indicates the amputation plane. Blue square marks the photographed region shown in C–F, and the red cross indicates the point targeted by IR-REGO. (C)–(F) A single neural progenitor cell in the spinal cord was induced to express GFP or dnTead4 tagged with GFP (dnT4-GFP) by infrared laser irradiation at 2 dpa (days post amputation). White arrow indicates the irradiated site. Insets indicate high-power views of the fluorescent images of the irradiated cell and its descendants. (C) A single cell was induced to express GFP by infrared laser irradiation. (D) and (E). Another example showing active cell division. Two GFP-positive cells appeared 1 day after irradiation, suggesting cell that division occurred before the GFP expression was detected (D) and the cell number was increased the next day (E). (F) A single cell was induced to express dnTead4 tagged with GFP by the same infrared laser irradiation as in (C)–(E). The dnTead4 expression cell-autonomously affected cell survival and mitosis. Note that the GFP-positive cell has a crumpled appearance, and looks like a dying cell. (G) Average number of GFP-expressing cells after each irradiation. The induced dominant-negative proteins significantly reduced the cell number in dnYap Tg 2 days and in dnTead4 Tg 1 and 2 days after irradiation. Error bar indicates s.e.m. Scale bar in C–F=100 µm and in inset=10 µm. Single and double asterisks indicate statistically significant differences between WT and Tg tadpoles at p<0.05 and p<0.01 (Welch’s t-test), respectively.
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Fig. 5. Tead4 contributes to position-dependent growth control. Experimental schedule. The tail of st52 tadpoles was amputated 3 h after the first heat shock. Tadpoles were heat shocked again at 3 dpa. Note that the observation of resultant tail regeneration at 7 dpa is far earlier than the onset of metamorphic tail regression that normally starts at st62 (Nieuwkoop and Faber, 1994). (B) and (C). WT (heat shocked) regenerated tail 7 days after amputation at the proximal (B) and distal (C) level (50% and 25% of the distance from the tail tip to the cloaca, respectively). The growth rate in the tail amputated at the proximal level was faster than that in the tail amputated at the distal level during the same period. (D) and (E) dnYap Tg regenerated tail 7 days after amputation at the proximal (D) and distal (E) level. (F) and (G) dnTead4 Tg regenerated tail 7 days after amputation at the proximal (F) and distal (G) level. Tail regeneration in the dnYap and dnTead4 Tg tadpoles was impaired and shortened after both proximal and distal amputation. Red line indicates the amputation plane. Scale bar (B–G)=1 mm. (H) Scatter graph of the regenerated tail (the average length). The difference between the proximal and distal regenerated tail length was severely decreased in the dnYap and dnTead4 Tg tadpoles. Horizontal bars indicate average length of regenerated tail. Single and double asterisks indicate statistically significant differences at p<0.05 and p<0.01 (Welch’s t-test) between the distal and proximal growth, respectively. Minus mark indicates no significant difference (Welch’s t-test). Error bar indicates s.e.m.
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Fig. S1. Systemic distribution of Yap1 protein at the tailbud and tadpole stages.
A. Cartoon of the Xenopus tadpole tail, which is composed of various types of tissues. B-G. Whole-mount immunofluorescent staining at the tailbud stage (B, D, F) and early tadpole stage (C, E, G). B, C. Negative control without primary antibody. D, E. Immunostaining for Yap1. Yap1 protein was distributed throughout the whole body. F, G. Immunostaining for phosphorylated Yap1 (pYap1). The anti-pYap1 antibody recognizes the phosphorylated serine in a conserved motif of the Yap1 protein. This phosphorylation sequesters Yap1 from the nucleus and inactivates the transcription of Yap1’s target genes. B, D, F. st38 embryos. C, E, G. st46 tadpoles. Scale bar = 1 mm.
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Fig. S2. Neurogenesis in the regenerating tail.
pH3 immunofluorescent staining (red) on cross sections counterstained with DAPI (blue). A. Cartoon of a transverse section of the Xenopus tadpole tail. The dorsal side is up. Orange square shows the spinal cord region photographed in B-D. B. Intact spinal cord at st52. C. Spinal cord in the stump at 5 dpa (days post amputation). D. Spinal cord in the regenerating tail at 5 dpa. Scale bar = 10 µm.
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Fig. S3. Map of the heat shock-inducible dominant-negative Yap and Tead4 transgenes. A dominant-negative form of Yap or Tead4 was inserted under the hsp70 promoter. GFP was used as the indicator for transgene expression with 2A peptide. tdTomato under control of the γ-crystalline promoter was included to select for transgenic individuals. The γ-crystallin-tdTomato cassette was inserted in the reverse and forward direction in the hsp70-dnYap and hsp70-dnTead4 cassette, respectively.
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Fig. S4. Yap1 and Tead4 are strongly required for developmental growth of the tadpole body at stage 41/42 but are less required for tail elongation.
Tadpole body length and tail length were measured 7 days after heat shock, as indicated in Fig. 2C without amputation. A. WT and dnYap Tg tadpoles 7 days after heat shock. The body length and tail length were measured as the distance from the rostral edge of the head to the cloaca (red bracket) and the distance from the cloaca to the tail tip (yellow bracket), respectively. B. WT and dnTead4 Tg tadpoles at 7 days after heat shock. C. Body length 7 days after heat shock (the average length). The body length of the dnYap and dnTead4 Tg tadpoles was significantly reduced. Error bar indicates s.e.m. D. Tail length 7 days after heat shock (the average length). Yap1 and Tead4 inhibition had less of an effect on tail elongation. E. Comparison of body lengths among WT siblings heat-shocked at st41/42, dnYap Tg tadpoles without heat shock and dnTead4 Tg tadpoles without heat shock at 7 days after st41/42. In dnYap and dnTead4, no inhibitory effect on tadpole growth was observed without heat shock. Error bar indicates s.e.m. Scale bar = 1 mm in A, B. Single and double asterisks indicate a statistically significant difference between WT and Tg tadpoles at p<0.05 and p<0.01 (Welch’s t-test), respectively
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Fig. S5. Yap1 and Tead4 are required for apoptosis inhibition.
Stage 41/42 tadpoles were heat shocked, fixed 7 days later, as shown in Fig. 2C without tail amputation, and subjected to whole-mount immunofluorescent staining. A. Cartoon of the immunostained tadpole. Orange square indicates the photographed region shown in B-D. B-D. Active Caspase3 (acCas3) staining indicating apoptotic cells. Apoptosis was slightly increased in dnYap Tg and more markedly increased in dnTead4 Tg tadpoles. E. qPCR for birc5.1 (survivin). bicr5.1 expression was significantly reduced in the dnYap and dnTead4 Tg tadpoles. cDNA was synthesized from the total RNA extracted from whole tadpoles 2 days after heat shock. Single asterisk indicates a statistically significant difference at p<0.05 (Welch’s t-test). Error bar indicates s.e.m.
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Fig. S6. Distinct inhibitory effects on tail regeneration by a Wnt/β-catenin antagonist (Dkk1) or Fgf signal inhibitor (SU5402).
A. Experimental schedule. To inhibit Wnt/β-catenin signaling, st52 Dkk1 tadpoles were heat-shocked 3 hours before tail amputation. The tadpoles were heat-shocked again at 3 dpa. After heat shock, the induction of Dkk1 was confirmed by GFP fluorescence (data not shown; Dkk1 was tagged with GFP. See Materials and Methods.). To inhibit Fgf signaling, 5 μl of 100 μM SU5402 or control DMSO was injected into tadpoles 3 hours before tail amputation. The same injection was repeated at 3 dpa. B, C. WT regenerated tail at 7 days after amputation at the proximal (B) and distal (C) levels (50% and 25% of the distance from the tail tip to the cloaca, respectively). The growth rate in the tail amputated at the proximal level was faster than that in the tail amputated at the distal level during the same period. D, E. Dkk1 Tg regenerated tails 7 days after amputation at the proximal (D) and distal (E) levels. F. Scatter graph of the regenerated tail lengths. Tail regeneration in Dkk1 tadpoles was almost completely blocked at both the proximal and distal amputation levels, as represented by the flat slope of Dkk1 close to “0â€. This graph is distinct from the same analysis in dnYap or dnTead4 tadpoles (Fig. 5H), in which the slope was steeper, especially in the dnTead4 tadpoles, and the regenerated tail was longer than 1 mm, suggesting that regeneration itself was not inhibited. Error bar indicates s.e.m. (WT: 7.35 ± 0.19 mm for proximal amputation and 4.33 ± 0.30 mm for distal, Dkk1: 0.50 ± 0.04 mm for proximal and 0.25 ± 0.02 mm for distal, n=12 and 13 for WT proximal and distal, n=14 and 12 for Dkk1 proximal and distal, respectively). G, H. Regenerated tails of DMSO-treated tadpoles 7 days after amputation at the proximal (G) and distal (H) levels. Growth rate in the tail amputated at the proximal level was faster than that in the tail amputated at the distal level during the same period. I, J. Regenerated tails of SU5402 (SU)-treated tadpoles 7 days after amputation at the proximal (I) and distal (J) levels. K. Measured length of the regenerated tail (average length). Under the condition of Fgf inhibition, tail regeneration was reduced, but position-dependent differential growth represented by the slope of SU5402 was similar to that of the control line DMSO. Error bar indicates s.e.m. (DMSO control: 7.27 ± 0.10 mm for proximal amputation and 4.54 ± 0.19 mm for distal, SU5402: 6.22 ± 0.19 mm for proximal and 3.55 ± 0.28 mm for distal, n=7 and 7 for DMSO control proximal and distal, n=7 and 6 for SU5402 proximal and distal, respectively). Red line indicates the amputation plane. Scale bar (B-E, G-J) = 1 mm. Single and double asterisks indicate statistically significant differences at p<0.05 and p<0.01 (Welch’s t-test), respectively. Horizontal bars in the graph indicate average length of regenerated tails.
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