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Int J Dev Biol
2013 Jan 01;5711-12:865-72. doi: 10.1387/ijdb.130163jk.
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AP-1(c-Jun/FosB) mediates xFoxD5b expression in Xenopus early developmental neurogenesis.
Yoon J
,
Kim JH
,
Lee OJ
,
Lee SY
,
Lee SH
,
Park JB
,
Lee JY
,
Kim SC
,
Kim J
.
???displayArticle.abstract??? AP-1 (activator protein-1) is composed of Jun and Fos proteins and functions in cell proliferation, apoptosis and differentiation. Previous studies have demonstrated that different AP-1 complexes participate in the determination of various cell fates. However, the role of different AP-1 complexes during early vertebrate development is not yet fully understood. In the present study, we demonstrate that AP-1(c-Jun/FosB) regulates neurogenesis via FoxD5b expression. We show that FoxD5 was induced by the inhibition of BMP and that FoxD5b plays roles in neurogenesis. Additionally, we show that the FoxD5b promoter region within -1336 and -1316 contains an AP-1 binding site, which is required for the transcriptional regulation of FoxD5b and is induced by the inhibition of BMP signaling in animal cap explants. Moreover, c-Jun, a component of AP-1, directly binds to the AP-1 binding site of the FoxD5b promoter and induces FoxD5b expression cooperatively with FosB, but not with c-Fos or Fra-1. Altogether, these results suggest that AP-1(c-Jun/FosB) may play a role in neurogenesis via the induction of FoxD5b expression during early vertebrate development.
Fig. 1. FoxD5b is induced by dominant-negative BMP receptor (DNBR) and promotes neurogenesis in animal cap explants. (A) DNBR RNA (1ng) was injected into the animal pole region at the one-cell stage. Animal caps were dissected at stage 8 and incubated until stage 10. RT-PCR was performed for the analysis of the expression of the indicated genes. (B) Embryos injected with DNBR mRNAs (1ng) were processed for whole mount in situ hybridization with anti-sense probe of FoxD5b at stage 11. (C) FoxD5b RNA (2 or 1 ng) was injected into the animal pole region at the one-cell stage. The animal caps were dissected at stage 8 and incubated until stage 10 or 24. qRT-PCR was performed for the analysis of the expression the indicated genes. *, p value < 0.05, **, p value < 0.01.
Fig.2. The AP-1 binding site has a critical role in dominant-negative BMP receptor (DNBR)-induced FoxD5b expression. (A) Schematic representation of FoxD5b serially truncated constructs and a point mutation of AP-1 and Vent-1 putative binding site constructs (B). Embryos were co-injected with the -1336 construct (20pg) and DNBR (1ng) at the one-cell stage and incubated until stage 10. Luciferase activity was measured as described in Methods. (C) Various truncated constructs (20pg) were either injected alone or co-injected with DNBR (1ng) at the one-cell stage. Animal cap explants were dissected at stage 8, incubated until stage 10 and then luciferase activity was measured. (D) The -1336, AP-1 or Vent-1 site mutants were either injected alone or co-injected with DNBR (1ng) at the one-cell stage. Animal caps were dissected at stage 8, and their luciferase activity was measured at stage 10. (E) A chromatin immunoprecipitation (ChIP) assay was performed using Xenopus embryos at stage 10. The presence of the FoxD5b promoter was detected by PCR in DNA samples obtained from anti-c-Jun antibody precipitation (c-Jun), normal IgG antibody precipitation (IgG), and cross-linked chromatin supernatant before immunoprecipitation (Input). RLU, relative luciferase activity. *, p value < 0.05, **, p value < 0.01; ***, p value < 0.001.
Fig. 3. Knockdown of c-Jun reduces FoxD5b and neural gene expression. (A) Wild-type -1336 was co-injected with DNBR (1ng) and c-Jun morpholino-oligos (54/55) (40ng), as indicated, at the one-cell stage. Animal cap explants were dissected at stage 8, and their luciferase activity was measured at stage 10. (B) DNBR (1ng) was co-injected with c-Jun morpholino-oligos (54/55) (40ng) or standard control morpholino-oligos (SC) (40ng) at the one-cell stage. Animal cap explants were dissected at stage 8 and incubated until stage 10. RT-PCR was performed for the analysis of FoxD5b expression. (C) DNBR (1ng) was injected alone or co-injected with 54/55 (40ng) at the one-cell stage. Animal cap explants were dissected at stage 8 and incubated until stage 24. RT-PCR was performed for the analysis of the expression of the indicated gene. RLU, relative luciferase activity. *, p value < 0.05, **, p value < 0.01; ***, p value < 0.001.
Fig. 4. An AP-1 complex composed of c-Jun and FosB induced FoxD5b expression. (A) AP-1 RNA (1ng), as indicated, was injected at the one-cell stage. Animal cap explants were dissected at stage 8 and incubated until stage 10. RT-PCR was performed for the analysis of FoxD5b expression. (B) The -1336 or -1336-mAP-1 construct was co-injected with AP-1c-Jun/FosB or DEPC-Q as indicated at the one-cell stage. Animal cap explants were dissected at stage 8, and their luciferase activity was measured at stage 10. (C) DNBR (0.5ng) and AP-1c-Jun/FosB (0.5ng) were co-injected at the one-cell stage as indicated. Animal cap explants were dissected at stage 8 and incubated until stage 10. RT-PCR was performed for the analysis of FoxD5b expression. (D) The -1336 construct was co-injected with DNBR (0.5ng) or AP-1c-Jun/FosB (0.5ng) as indicated at the one-cell stage. Animal cap explants were dissected at stage 8, and their luciferase activity was measured at stage 10. (E) AP-1 luc was co-injected with DNBR (2ng) or AP-1c-Jun/FosB (125pg) as indicated at the one-cell stage. Animal cap explants were dissected at stage 8, and their luciferase activity was measured at stage 10. (F) DNBR RNA (1ng) was injected into the animal pole region at the one-cell stage. Animal caps were dissected at stage 8 and incubated until stage 10. The western-blot assay was performed with specific anti-phospho c-Jun antibodies (#9261, Cell signaling, MA). (G) AP-1c-Jun/FosB mRNAs (1ng) were injected into the one blastomere at the 2 cell stage. Embryos were processed for whole mount in situ hybridization with anti-sense probe of FoxD5b at stage 10. (H) A chromatin immunoprecipitation (ChIP) assay was performed using Xenopus embryos at stage 10. The presence of the FoxD5b promoter was detected by PCR in DNA samples obtained from anti-Flag antibody precipitation (Flag), normal IgG antibody precipitation (IgG), and cross-linked chromatin supernatant before immunoprecipitation (Input). RLU, relative luciferase activity. *, p value < 0.05, **, p value < 0.01; ***, p value < 0.001.
