Lee SY , Yoon J , Lee HS , Hwang YS , Cha SW , Jeong CH , Kim JI , Park JB , Lee JY , Kim S , Park MJ , Dong Z , Kim J .

27302C0115 NIEHS NIH HHS

	Figure 1. AP-1 functions similar to activin in dorsal mesoderm and neural gene expression in animal cap explants.(A–C) RT-PCR analysis of organizer genes (chordin, noggin and goosecoid) or late neural specific marker (N-CAM) in activin treated animal cap explants or in mRNAs encoding c-jun or/and c-fos injected animal cap explants. (C) Animal caps isolated from embryos injected with 1 ng of mRNAs encoding c-jun and c-fos was used for RT-RCR analysis. EF-1α, a loading control; -rt, control reaction without reverse transcriptase; cont, animal cap samples obtained from non-injected embryos; we, whole embryo as a positive control.
	Figure 2. AP-1 enhances promoter activity of activin response genes, but reduces that of BMP4-response gene.(A–C) Luciferase assay using animal caps (at stage 10.5 equivalent). Indicated mRNAs or expression plasmids were used: lane 1, Noggin, Goosecoid, or PV.1 promoter luciferase reporter gene; lane 2, co-injection with 1 ng of mRNAs encoding AP-1 (c-jun and c-fos). All values are averages of at least three independent experiments. RLU, Relative luciferase activity.
	Figure 3. AP-1 is required for activin signaling.(A) MO-Jun55/54 (20 ng of MO-jun55 and 54) specifically inhibit the translation of endogenous c-Jun. (B–C) Animal caps isolated from embryos injected with or without MO-Jun55/54 were cultured until stage 10.5 or stage 24. (B) The morpholino oligonucleotide Juns (MO-Jun55/54) inhibited morphological change induced by activin. (C) RT-PCR analysis of animal caps injected with the indicated MO-Jun55/54 or treated with activin (25 ng/ml). MO-Jun55/54 suppressed the markers induced by activin signaling (lane 3 and 4). (D–F) Animal caps isolated from embryos injected with or without MO-Jun55/54 and mRNA of Flag rat c-Jun were cultured in presence of activin until stage 10.5. (D) Inhibition of endogenous c-Jun and Flag rat c-Jun translation were confirmed by western blotting. (E) RT-PCR analysis of animal caps. Co-injection of rat c-Jun rescued the organizer genes suppressed by MO-Jun55/54. (F) The phenotype of animal caps inhibited by MO-Jun55/54 was rescued by co-injection of rat c-Jun mRNA. 20 embryos that had received a microinjection of indicated mRNA or MOs were used for animal cap isolation at the blastula stage. We presented the number of animal cap's phenotype for each group. EF-1α, a loading control; -rt, control reaction without reverse transcriptase; cont, animal caps sample dissected from non-injected embryos; we, whole embryo as a positive control; organizer genes, chordin, noggin and goosecoid: later dorsal mesoderm marker, Actin; neural marker, N-CAM.
	Figure 4. Physiological role of AP-1 in whole embryo.(A) Embryos were injected with 20 ng of MO-Jun54/55 into animal regions of one- or two-cell stage embryos and cultured until stage 40. c-Jun depleted embryos showed short axis and disrupted anterior structure. (B) Marginal zone of two dorsal blastomeres (DMZ) of four-cell stage embryos injected with 20 ng of MO-Jun54/55 was isolated and cultured until stage 11. Spemann organizer genes (noggin, goosdcoid and chordin) and mesoderm marker (Xbra) were investigated by RT-PCR analysis. The organizer genes expressed in DMZ were significantly reduced by depletion of c-Jun. ODC, a loading control; -RT, control reaction without reverse transcriptase; W.E., whole embryo as a positive control.
	Figure 5. AP-1c-Jun/c-Fos is involved in Smad3-mediated organizer gene expression.(A) (AP-1)4-luciferase assay using animal cap explants injected with Smad3 mRNA. Smad3 enhanced the AP-1 activity. (B–C) RT-PCR analysis of animal caps expressing the indicated mRNA of Smad3 or MO-Jun55/54. (B) Smad3 induced organizer genes (goosecoid, chordin and noggin), later dorsal mesoderm marker (Actin) and neural marker (NCAM). (C) MO-Jun55/54 suppressed Smad3-induced organizer genes (goosecoid andnoggin) at stage 10.5 and neural marker (NCAM) at stage 24. On the other hand, ventral mesoderm marker (GATA2) and non-neural marker (XK81) were increased by co-injection of MO-Jun55/54 and Smad3 mRNA. EF-1α, a loading control; -rt, control reaction without reverse transcriptase; cont, animal caps sample dissected from non-injected embryos; we, whole embryo as a positive control.
	Figure 6. Heterodimeric AP-1c-Jun/c-Fos directly regulates the transcription of goosecoid by physically interacting with Smad3, a mediator of activin signaling.(A) RT-PCR analysis of organizer genes in indicating mRNA injected animal caps explants (at stage 10.5 equivalent); lane 1, animal caps treated with activin (5 ng/ml); animal caps injected with 0.5 ng of AP-1c-Jun/c-Fos mRNA were incubated in the absence (lane 3) or presence (lane2) of activin (5 ng/ml). Activin and AP-1 have synergistic effect on goosecoid expression. (B) Relative band intensities of A shown in panel. Relative band intensities were analyzed by ImageJ software provided by NIH, USA. (C) Luciferase assay using animal caps (at stage 10.5 equivalent) expressing goosecoid promoter luciferase reporter genes with the indicated mRNAs. All values are averages of at least three independent experiments. Smad3 and AP-1 have synergistic effect on enhancement of the goosecoid promoter activity. (D–F) Chromatin immunoprecipitation (ChIP) assay from whole embryos (D) or embryos injected with AP-1 (flag-tagged rat c-jun and rat c-fos mRNAs) alone or together with Smad3 mRNAs (E & F). The presence of goosecoid promoter was detected by PCR using DNA samples obtained; after c-Jun antibody precipitation (D), Flag or Myc antibody precipitation (Flag Ab or Myc Ab) (E & F), IgG precipitation (normal IgG), and from cross-linked chromatin supernatant before immunoprecipitation (Input). MOCK was used as a control for the absence of DNA. (D) Endogenous c-Jun binds to the goosecoid promoter (lane 3). (E) Exogenous AP-1 binds to the goosecoid promoter (lane 4). (F) AP-1 and Smad3 cooperatively bind to goosecoid promoter (lane 8). (G & H) Immunoprecipitation assay from whole embryos injected with AP-1 (flag-tagged rat c-jun and rat c-fos mRNAs) alone or together with Smad3 mRNAs (at stage 10.5 equivalent). Cell extracts from embryos used in ChIP assay were co-immunoprecipitated (IP) with the indicated antibodies. (G) The physical interaction of c-Jun and c-Fos in embryo. (H) The physical interaction of AP-1 and Smad3 in embryo.
	Figure 7. Physical interaction of Smad3 and AP-1 is necessary for full activation of organizer genes, but not for Xbra.(A) Schematic representation of Smad3 constructs used in this study. (B) Immunoprecipitation assay of c-Jun and wild Smad3 or Smad3 mutants. Co-immunoprecipitation of Smad3 mutants and c-Jun was not seen (lane 2, 3). (C–F) Whole embryos injected with indicated mRNAs or isolated animal caps were cultured until stage 10.5 and stage 24. (C) Embryos injected with indicated mRNA were lysised and performed western blot with anti-Myc for confirmation of mRNA expression. Injected mRNA of Smad3 mutants and wild type was translated into proteins in a dose-dependent manner. (D) RT-PCR on animal caps expressing indicated mRNAs. Unlike wild Smad3 (lane 1–2), Smad3 mutants lost inducing activity of organizer genes and neural specific marker (lane 3–6): EF-1α, a loading control; -rt, control reaction without reverse transcriptase; cont, animal caps sample dissected from non-injected embryos; we, whole embryo as a positive control; organizer gene, chordin and goosecoid; pan-neural marker, N-CAM; ventral mesoderm marker, GATA2; epidermis marker (non-neural marker), XK81; pan-mesoderm marker, Xbra. (E) Embryos injected with indicated mRNAs (upper panel) were processed for whole mount in situ hybridization with indicated markers (left panel). (F) Embryos injected with indicated mRNAs (left panel) were processed for whole mount in situ hybridization with Xbra.

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