Yoon J et al. (2014), PV.1 induced by FGF-Xbra functions as a repress...

XB-ART-48510

BMB Rep 2014 Dec 01;4712:673-8. doi: 10.5483/bmbrep.2014.47.12.290.

Show Gene links Show Anatomy links

PV.1 induced by FGF-Xbra functions as a repressor of neurogenesis in Xenopus embryos.

Yoon J , Kim JH , Lee SY , Kim S , Park JB , Lee JY , Kim J .

???displayArticle.abstract???
During Xenopus early development, FGF signaling is involved in mesoderm formation and neurogenesis by modulating various signaling cascades. FGF-MAPK signaling induces Xbra expression, which maintains mesodermal fate through an autocatalytic-loop. Interestingly, previous reports have demonstrated that basic FGF (bFGF) treatment alone does not induce neurogenesis in ectodermal explants, even though FGF signaling inhibits BMP signaling via phosphorylation in Smad1 linker region. In addition, the overexpression of dominantnegative Xbra induces neurogenesis in ectodermal explants. However, the detailed mechanism underlying these phenomena has not yet been clarified. In this work, we showed that bFGF-Xbra signaling increased the PV.1 expression. DN-Xbra was found to decrease PV.1 expression, and the co-injection of PV.1 with DN-Xbra reduced neurogenesis in ectodermal explants. Furthermore, the knockdown of PV.1 induced neurogenesis in bFGF-treated ectodermal explants. Taken together, our results demonstrate that FGF-Xbra signaling induces PV.1 expression and that PV.1 functions as a neural repressor in the FGF-treated ectoderm.

???displayArticle.pubmedLink??? 24499677
???displayArticle.pmcLink??? PMC4345511
???displayArticle.link??? BMB Rep

Species referenced: Xenopus
Genes referenced: a2m acta4 actc1 actl6a chrd egr2 fgf2 gata2 hoxb9 krt12.4 mapk1 mixer ncam1 nog odc1 otx2 smad1 tbxt ventx1 zic3
???displayArticle.morpholinos??? ventx1.1 MO1

???attribute.lit??? ???displayArticles.show???

	Fig. 1. bFGF induces PV.1 expression. (A) Ectodermal explants were dissected at stage 8 and incubated in animal cap media containing bFGF (50 ng/ml) until stage 11 or 24. The relative gene expression was analyzed by RT-PCR. (B) Ectodermal explants were dissected at stage 8 and incubated in animal cap media containing either bFGF alone or bFGF and CHX (5 ng/ml) until stage 11. The relative gene expression was analyzed by RT-PCR. The graph indicates the band intensity of PV.1 (right panel). ODC, loading control; no RT, control reaction without reverse transcriptase; WE, whole-embryo positive control; Xbra, pan-mesoderm marker; chordin, dorsal mesoderm marker; PV.1, GATA2, and XVent-1a, ventral-specific markers; Zic3, early neural marker; NCAM, pan-neural marker; OTX2, anterior neural marker; HoxB9, posterior neural marker; Krox20, mid-brain marker; muscle actin (M. Actin), late dorsal mesoderm marker.
	Fig. 2. DN-Xbra reduces the expression of target genes downstream of BMP. (A) Xbra RNA (1 ng) was injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated until stage 11. The relative gene expression was analyzed by RT-PCR. (B) DNBR RNA (1 ng) and Xbra RNA (1 ng) were co-injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated until stage 11. The relative gene expression was analyzed by RT-PCR. (C) Xbra RNA (1 ng) was injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated in animal cap media containing CHX (5 ng/ml) until stage 11 or 24. The relative gene expression was analyzed by RT-PCR. The data shown represent the means ± the S.D.s from at least three independent experiments. The differences were considered significant at P＜ 0.05.
	Fig. 3. Xbra directly induces PV.1 expression. (A) Schematic representation of Xbra and DN-Xbra. Xbra and DN-Xbra have a DNA-binding domain (T-box), but DN-Xbra has an engrailed repressor domain instead of a transcriptional activation domain in the C-terminal region. (B) DN-Xbra RNA (2, 1, or 0.5 ng) was injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated until stage 11 or 24. The relative gene expression was analyzed by RT-PCR. (C) Smad1 RNA (2, 1, or 0.5 ng) and DN-Xbra RNA (2 ng) were co-injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated until stage 11 or 24. The relative gene expression was analyzed by RT-PCR. (D) DN-Xbra RNA (2 ng) was injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated in animal cap media containing CHX (5 ng/ml) until stage 11 or 24. The relative gene expression was analyzed by RT-PCT. Noggin, dorsal mesoderm marker; Mixer, Sox17b, and Edd, endoderm markers; Xk81, epidermis marker; globin, blood marker; CG13, cement glad marker. The data are shown as the means ± the S.D.s of the values of at least three independent experiments. Differences were considered significant at P＜ 0.05.
	Fig. 4. The knockdown of PV.1 induces neurogenesis in bFGF-treated ectodermal explants. (A) PV.1 RNA (0.5 ng) and DN-Xbra RNA (2 ng) were co-injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated until stage 11 or 24. The relative gene expression was analyzed by PCR. (B) Anti-sense morpholino oligos of PV.1 (20 ng) were injected at the one-cell stage. Ectodermal explants were dissected at stage 8 and incubated in animal cap media containing bFGF (50 ng/ml) until stage 11 or 24. The relative gene expression was analyzed by RT-PCR.

References [+] :

Amaya, Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. 1991, Pubmed, Xenbase