Nakamura M et al. (2024), Injury-induced cooperation of InhibinβA and Jun...

XB-ART-60563

Sci Rep 2024 Feb 14;141:3679. doi: 10.1038/s41598-024-54280-w.

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Injury-induced cooperation of InhibinβA and JunB is essential for cell proliferation in Xenopus tadpole tail regeneration.

Nakamura M , Kyoda T , Yoshida H , Takebayashi-Suzuki K , Koike R , Takahashi E , Moriyama Y , Wlizla M , Horb ME , Suzuki A .

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In animal species that have the capability of regenerating tissues and limbs, cell proliferation is enhanced after wound healing and is essential for the reconstruction of injured tissue. Although the ability to induce cell proliferation is a common feature of such species, the molecular mechanisms that regulate the transition from wound healing to regenerative cell proliferation remain unclear. Here, we show that upon injury, InhibinβA and JunB cooperatively function for this transition during Xenopus tadpole tail regeneration. We found that the expression of inhibin subunit beta A (inhba) and junB proto-oncogene (junb) is induced by injury-activated TGF-β/Smad and MEK/ERK signaling in regenerating tails. Similarly to junb knockout (KO) tadpoles, inhba KO tadpoles show a delay in tail regeneration, and inhba/junb double KO (DKO) tadpoles exhibit severe impairment of tail regeneration compared with either inhba KO or junb KO tadpoles. Importantly, this impairment is associated with a significant reduction of cell proliferation in regenerating tissue. Moreover, JunB regulates tail regeneration via FGF signaling, while InhibinβA likely acts through different mechanisms. These results demonstrate that the cooperation of injury-induced InhibinβA and JunB is critical for regenerative cell proliferation, which is necessary for re-outgrowth of regenerating Xenopus tadpole tails.

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Species referenced: Xenopus tropicalis
Genes referenced: inhba inhbb junb

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	Figure 1. The expression of inhba and junb is regulated by injury-activated signaling during wound healing. qRT-PCR analysis of inhba (a) and junb (b) expression in DMSO (control), SB-505124 (SB), and PD0325901 (PD)-treated tails. The regenerating tails were isolated at 1 and 2 hpa for the expression of junb and inhba, respectively. The data were normalized against expression of rps18, and then by the value of DMSO. P < 0.05, *P < 0.01.
	Figure 2. Spatiotemporal expression pattern of inhba during Xenopus tail regeneration. Whole-mount in situ hybridization of inhba in uncut tails and in regenerating tails at 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48 and 72 hpa. Negative control using a sense probe shows no signals. Blue/purple signals indicate the expression of inhba. Black arrowheads indicate amputation sites. Scale bar, 200 μm.
	Figure 3. InhibinβA is required for tail regeneration. (a) Schematic drawing of sgRNA target sites (sg 1 and sg 2) in the inhba locus. Grey boxes, untranslated regions; orange boxes, coding regions; arrows, single-guide RNA target sites; bar, intron region. (b) Representative phenotypes of tyr KO (control), inhba KO sg 1, inhba KO sg 2, and inhba KO sg 1 + sg 2 tadpoles at 72 hpa. (c) The lengths of regenerating tails in KO tadpoles at 72 hpa. (d) Summary of phenotypes in KO tadpoles at 72 hpa. On the basis of the lengths of regenerating tails at 72 hpa, tadpoles were classified into three phenotypic groups (normal regeneration, weakly delayed regeneration, or severely delayed regeneration). Black arrowheads indicate amputation sites. Scale bar, 200 μm. NS, not significant; ***P < 0.001.
	Figure 4. InhibinβA and JunB cooperate for regenerative outgrowth/cell proliferation. (a) Representative phenotypes of tyr KO (control), inhba KO, junb KO, and inhba/junb DKO tadpoles at 72 hpa. (b) The lengths of regenerating tails in KO and DKO tadpoles at 72 hpa. (c) Representative immunofluorescent images of pH3 staining (white dots) in KO and DKO tadpoles at 36 hpa. Whole-mount immunostaining was performed with the pH3 antibody; immunostaining without the pH3 antibody was used as a negative control (no 1st Ab). (d) Relative number of proliferating cells at 36 hpa. The number of pH3-positive cells was divided by the corresponding area. All values were normalized against the value of tyr KO. Black and white arrowheads indicate amputation sites. Scale bars, 200 μm. P < 0.05, P < 0.01, **P < 0.001.
	Figure 5. JunB regulates regenerative outgrowth/cell proliferation via FGF signaling. (a) Relative expression of fgf20 and tbxt in inhba KO and junb KO tadpoles at 36 hpa. The data were normalized against the expression of rps18, and then by the value of tyr KO. (b) Representative phenotypes of tyr KO (control), junb KO, junb KO + junb mRNA, and junb KO + fgf20 mRNA tadpoles at 72 hpa. (c) The lengths of regenerating tails in KO tadpoles at 72 hpa. (d) Summary of phenotypes in KO tadpoles at 72 hpa. On the basis of the lengths of regenerating tails at 72 hpa, tadpoles were classified into three phenotypic groups (normal regeneration, weakly delayed regeneration, or severely delayed regeneration). (e) Representative immunofluorescent images of pH3 staining (white dots) in tyr KO (control), junb KO, junb KO + junb mRNA, and junb KO + fgf20 mRNA tadpoles at 36 hpa. (f) Relative number of proliferating cells at 36 hpa. The number of pH3-positive cells was divided by the corresponding area. All values were normalized against the value of tyr KO. Black and white arrowheads indicate amputation sites. Scale bars, 200 μm. NS, not significant; P < 0.05, P < 0.01, **P < 0.001.
	Figure 6. FGF signaling is required for regenerative outgrowth/cell proliferation. (a) Schematic of the experimental plan. Tadpoles were incubated in water containing 0.0375% DMSO (control) or 15 μM SU5402 (SU) from 24 hpa and analyzed at 72 hpa for regenerating tail length and proliferating cells. (b) Representative phenotypes of DMSO and SU-treated tadpoles at 72 hpa. (c) The lengths of regenerating tails at 72 hpa. (d) Relative number of proliferating cells at 72 hpa. The number of pH3-positive cells was divided by the corresponding area. In (b) and (c), the photograph and length of regenerating tails were obtained after whole-mount immunostaining, which involves bleaching and methanol treatments. Due to these treatments, the measured regenerating tail length is relatively shorter than in other experiments. All values were normalized against the value of DMSO-treated tadpoles. Black arrowheads indicate amputation sites. Scale bar, 200 μm. P < 0.01, *P < 0.001.
	Figure 7. A model for the transition from wound healing to regenerative cell proliferation mediated by InhibinβA and JunB. Upon tail amputation, Tgfβ1/TGF-β receptor (TGF-βR) and MEK independently phosphorylate/activate Smad2/3 (pSmad2/3) and ERK (pERK), respectively. These injury-activated signals coordinately induce the expression of inhba and junb during the wound healing phase. After wound healing, InhibinβA and JunB cooperate to initiate outgrowth and cell proliferation. Mechanistically, JunB promotes tail regeneration, at least in part, via Fgf20/FGF receptor (FGFR) signaling; InhibinβA/Activin receptor (ActR) signaling may act either in parallel with or downstream of Fgf20/FGFR signaling (see Discussion).

References [+] :

Andrecht, Cell cycle promoting activity of JunB through cyclin A activation. 2002, Pubmed

	Figure 1. The expression of inhba and junb is regulated by injury-activated signaling during wound healing. qRT-PCR analysis of inhba (a) and junb (b) expression in DMSO (control), SB-505124 (SB), and PD0325901 (PD)-treated tails. The regenerating tails were isolated at 1 and 2 hpa for the expression of junb and inhba, respectively. The data were normalized against expression of rps18, and then by the value of DMSO. P < 0.05, *P < 0.01.
	Figure 2. Spatiotemporal expression pattern of inhba during Xenopus tail regeneration. Whole-mount in situ hybridization of inhba in uncut tails and in regenerating tails at 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48 and 72 hpa. Negative control using a sense probe shows no signals. Blue/purple signals indicate the expression of inhba. Black arrowheads indicate amputation sites. Scale bar, 200 μm.
	Figure 3. InhibinβA is required for tail regeneration. (a) Schematic drawing of sgRNA target sites (sg 1 and sg 2) in the inhba locus. Grey boxes, untranslated regions; orange boxes, coding regions; arrows, single-guide RNA target sites; bar, intron region. (b) Representative phenotypes of tyr KO (control), inhba KO sg 1, inhba KO sg 2, and inhba KO sg 1 + sg 2 tadpoles at 72 hpa. (c) The lengths of regenerating tails in KO tadpoles at 72 hpa. (d) Summary of phenotypes in KO tadpoles at 72 hpa. On the basis of the lengths of regenerating tails at 72 hpa, tadpoles were classified into three phenotypic groups (normal regeneration, weakly delayed regeneration, or severely delayed regeneration). Black arrowheads indicate amputation sites. Scale bar, 200 μm. NS, not significant; ***P < 0.001.
	Figure 4. InhibinβA and JunB cooperate for regenerative outgrowth/cell proliferation. (a) Representative phenotypes of tyr KO (control), inhba KO, junb KO, and inhba/junb DKO tadpoles at 72 hpa. (b) The lengths of regenerating tails in KO and DKO tadpoles at 72 hpa. (c) Representative immunofluorescent images of pH3 staining (white dots) in KO and DKO tadpoles at 36 hpa. Whole-mount immunostaining was performed with the pH3 antibody; immunostaining without the pH3 antibody was used as a negative control (no 1st Ab). (d) Relative number of proliferating cells at 36 hpa. The number of pH3-positive cells was divided by the corresponding area. All values were normalized against the value of tyr KO. Black and white arrowheads indicate amputation sites. Scale bars, 200 μm. P < 0.05, P < 0.01, **P < 0.001.
	Figure 5. JunB regulates regenerative outgrowth/cell proliferation via FGF signaling. (a) Relative expression of fgf20 and tbxt in inhba KO and junb KO tadpoles at 36 hpa. The data were normalized against the expression of rps18, and then by the value of tyr KO. (b) Representative phenotypes of tyr KO (control), junb KO, junb KO + junb mRNA, and junb KO + fgf20 mRNA tadpoles at 72 hpa. (c) The lengths of regenerating tails in KO tadpoles at 72 hpa. (d) Summary of phenotypes in KO tadpoles at 72 hpa. On the basis of the lengths of regenerating tails at 72 hpa, tadpoles were classified into three phenotypic groups (normal regeneration, weakly delayed regeneration, or severely delayed regeneration). (e) Representative immunofluorescent images of pH3 staining (white dots) in tyr KO (control), junb KO, junb KO + junb mRNA, and junb KO + fgf20 mRNA tadpoles at 36 hpa. (f) Relative number of proliferating cells at 36 hpa. The number of pH3-positive cells was divided by the corresponding area. All values were normalized against the value of tyr KO. Black and white arrowheads indicate amputation sites. Scale bars, 200 μm. NS, not significant; P < 0.05, P < 0.01, **P < 0.001.
	Figure 6. FGF signaling is required for regenerative outgrowth/cell proliferation. (a) Schematic of the experimental plan. Tadpoles were incubated in water containing 0.0375% DMSO (control) or 15 μM SU5402 (SU) from 24 hpa and analyzed at 72 hpa for regenerating tail length and proliferating cells. (b) Representative phenotypes of DMSO and SU-treated tadpoles at 72 hpa. (c) The lengths of regenerating tails at 72 hpa. (d) Relative number of proliferating cells at 72 hpa. The number of pH3-positive cells was divided by the corresponding area. In (b) and (c), the photograph and length of regenerating tails were obtained after whole-mount immunostaining, which involves bleaching and methanol treatments. Due to these treatments, the measured regenerating tail length is relatively shorter than in other experiments. All values were normalized against the value of DMSO-treated tadpoles. Black arrowheads indicate amputation sites. Scale bar, 200 μm. P < 0.01, *P < 0.001.
	Figure 7. A model for the transition from wound healing to regenerative cell proliferation mediated by InhibinβA and JunB. Upon tail amputation, Tgfβ1/TGF-β receptor (TGF-βR) and MEK independently phosphorylate/activate Smad2/3 (pSmad2/3) and ERK (pERK), respectively. These injury-activated signals coordinately induce the expression of inhba and junb during the wound healing phase. After wound healing, InhibinβA and JunB cooperate to initiate outgrowth and cell proliferation. Mechanistically, JunB promotes tail regeneration, at least in part, via Fgf20/FGF receptor (FGFR) signaling; InhibinβA/Activin receptor (ActR) signaling may act either in parallel with or downstream of Fgf20/FGFR signaling (see Discussion).