Nakamura M et al. (2020), The AP-1 transcription factor JunB functions in...

XB-ART-56554

Biochem Biophys Res Commun 2020 Feb 19;5224:990-995. doi: 10.1016/j.bbrc.2019.11.060.

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The AP-1 transcription factor JunB functions in Xenopus tail regeneration by positively regulating cell proliferation.

Nakamura M , Yoshida H , Takahashi E , Wlizla M , Takebayashi-Suzuki K , Horb ME , Suzuki A .

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Xenopus tropicalis tadpoles can regenerate an amputated tail, including spinal cord, muscle and notochord, through cell proliferation and differentiation. However, the molecular mechanisms that regulate cell proliferation during tail regeneration are largely unknown. Here we show that JunB plays an important role in tail regeneration by regulating cell proliferation. The expression of junb is rapidly activated and sustained during tail regeneration. Knockout (KO) of junb causes a delay in tail regeneration and tissue differentiation. In junb KO tadpoles, cell proliferation is prevented before tissue differentiation. Furthermore, TGF-β signaling, which is activated just after tail amputation, regulates the induction and maintenance of junb expression. These findings demonstrate that JunB, a downstream component of TGF-β signaling, works as a positive regulator of cell proliferation during Xenopus tail regeneration.

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Species referenced: Xenopus tropicalis
Genes referenced: hpse junb myod1 pam rps18 shh sox2
GO keywords: cell proliferation [+]

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	Fig. 1. junb is expressed during tail regeneration of X. tropicalis tadpoles. WISH analysis was performed using regenerating tadpoles at 0, 0.25, 0.5, 1, 2, 4, 6, 12, 24, 48 and 72 hpa. The expression of junb is shown in blue/purple. Black arrowheads indicate the amputation plane. Scale bar: 200 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 2. Knockout of junb prevents tail regeneration. (A) Summary of phenotypes in regenerating tadpoles at 72 hpa. The tadpoles were classified into 4 types: normal tail regeneration; weakly delayed tail regeneration; moderately delayed tail regeneration; and severely delayed tail regeneration. (B) junb KO sg 1 + sg 2 tadpoles show considerably delayed tail regeneration. tyrosinase KO was used as the control. (C) Lengths of regenerating tails. (D) WISH analysis of sox2, myod1 and shh in tyrosinase KO and junb KO tadpoles. Black arrowheads indicate amputation plane. Scale bar: 200 μm. ***P < 0.001, Student’s t-test.
	Fig. 3. Knockout of junb reduces cell proliferation. (A) Whole-mount immunostaining of junb KO tadpoles with pH3 antibody at 36 and 48 hpa. (B) Quantification of pH3 positive cells in the region of regenerating tail, excluding the fin. The number of pH3 positive cells was divided by individual area, and normalized against control samples. White arrowheads indicate the amputation plane. Scale bar: 200 μm. **P < 0.01, Student’s t-test.
	Fig. 4. The expression of junb is downregulated by TGF-β signaling inhibition. Tadpoles were treated with a medium containing 12.5 μM SB-505124 or DMSO (Control) from 1 h before tail amputation and cultured until 1, 2 and 6 hpa. (A) WISH analysis of junb in SB-treated tadpoles. N ≥ 29 for each sample. (B) qPCR analysis of junb in SB-treated tadpoles. The expression of junb was normalized against rps18 expression, and then against control samples. Scale bar: 50 μm. P < 0.01, * <0.001, Student’s t-test.
	Supplementary Fig. 1. Genotyping of junb KO sg 1 + sg 2 tadpoles. (A) Schematic drawing of junb structure and junb sgRNA target sites. Bars, untranslated region; box, coding region; TAD, transactivation domain; DBD, DNA-binding domain; LZD, leucine zipper domain. (B) Sequencing analysis of junb mutations in tyrosinase KO (n = 5) and junb KO (n = 5) tadpoles. Target sites of sg 1 and sg 2 are highlighted in green, and blue boxes indicate the PAM sequence. Deleted sequences are highlighted with red dashes. Insertions and substitutions are shown in blue and red letters, respectively. Mutation types were categorized as wild-type, in-frame, and out-of-frame. (C) Summary of mutation types as in panel B. Sequencing analysis of TA cloning demonstrates that all of alleles contain mutations in both sg 1 and sg 2 target sites. (D) Percentage of mutation types of junb KO sg 1 + sg 2 as in panel B.
	Supplementary Fig. 2. The delay in tail regeneration in junb KO is rescued by junb mRNA. (A) Co-injection of junb mRNA (250 pg) with junb sg 1 + sg 2 rescues the junb KO phenotype. (B) Lengths of regenerating tails. Black arrowheads indicate the amputation plane. Scale bar: 200 μm. ***P < 0.001, Student's t-test.
	Supplementary Fig. 3. Compound heterozygous junb mutants show a delay in tail regeneration. (A) Schematic drawing of junb structure and junb sgRNA target sites. (B) Compound heterozygous junb mutants (Mut/Mut) show considerable delay in tail regeneration compared to wild-type (WT/WT, not sibling) tadpoles. WT/WT tadpoles were used as the control. (C) Lengths of regenerating tails. Black arrowheads indicate the amputation plane. Scale bar: 200 μm. ***P < 0.001, Student's t-test.

References [+] :

Adams, H+ pump-dependent changes in membrane voltage are an early mechanism necessary and sufficient to induce Xenopus tail regeneration. 2007, Pubmed, Xenbase