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Xenopus embryo serves as an ideal model for teratogenesis assays to examine the effects of any substances on the cellular processes critical for early development and adult tissue homeostasis. In our chemical library screening with frog embryo, capsaicin was found to repress the Wnt/β-catenin signaling. Depending on the stages at which embryos became exposed to capsaicin, it could disrupt formation of dorsal or posterior body axis of embryo, which is associated with inhibition of maternal or zygotic Wnt signal in early development. In agreement with these phenotypes, capsaicin suppressed the expression of Wnt target genes such as Siamois and Chordin in the organizer region of embryo and in Wnt signals-stimulated tissue explants. In addition, the cellular level of β-catenin, a key component of Wnt pathway, was down-regulated in capsaicin-treated embryonic cells. Unlike wild-type β-catenin, its non-phosphorylatable mutant in which serine and threonine residues phosphorylated by GSK3 are substituted with alanine was not destabilized by capsaicin, indicative of the effect of this chemical on the phosphorylation status of β-catenin. In support of this, capsaicin up-regulated the level of GSK3- or CK1-phosphorylated β-catenin, concomitantly lowering that of its de-phosphorylated version. Notably, capsaicin augmented the phosphorylation of a phosphatase, PP2A at tyrosine 307, suggesting its repression of the enzymatic activity of the phosphatase. Furthermore, capsaicin still enhanced β-catenin phosphorylation in cells treated with a GSK3 inhibitor, LiCl but not in those treated with a phosphatase inhibitor, okadaic acid. Together, these results indicate that capsaicin inhibits the patterning of the dorso-ventral and anterior-posterior body axes of embryo by repressing PP2A and thereby down-regulating the Wnt/β-catenin signaling.
Fig. 1. Morphological phenotypes of Xenopus early embryos exposed to capsaicin. (A,B) Embryos were exposed to DMSO (for control) or capsaicin (200 mM) from the four-cell stage
until the tadpole stages. (CeI) Embryos were grown in media containing capsaicin from the different time points to the tadpole stages as denoted in (C). (J,K) Anteriorization of
embryos exposed to capsaicin from the early gastrula stages to the tadpole stages. Arrows and arrowheads in (B,D-F) indicate the severe and mild defects in head structures,
respectively. All embryos are shown in lateral views with anterior to the right.
Fig. 2. Capsaicin inhibits the expression of Wnt target genes. (A) Dorsal marginal zone (DMZ) tissues were excised from the embryos that were cultured in the presence of capsaicin
or DMSO from the four-cell stage to stage 10.25 and then subjected to RT-PCR analysis. (B) Four-cell stage embryos were injected in the animal pole region with XWnt8 (50 pg) or
constitutively active (CA) LRP6 (1 ng) RNAs and then animal caps were dissected at stage 8.5 from the injected embryos, exposed to capsaicin until stage 10.5 and subsequently
analyzed by RT-PCR. eRT, control in the absence of reverse transcriptase (RT); WE, control whole embryo; Co AC, uninjected control animal caps. ODC serves as a loading control. ()
indicates the tissue explants without chemical treatments.
Fig. 3. Capsaicin affects the phosphorylation state of b-catenin. (AeD) Four-cell stage embryos were injected in the animal pole region with XWnt8 (50 pg) or combinations of mycb-
catenin(wt)(100 pg), myc-b-catenin(pt)(100 pg) and myc-Smad2 (200 pg) RNAs as indicated, and then animal cap explants were excised at stage 8.5, incubated to stage 10.5 in
media containing DMSO or capsaicin and harvested for western blotting. (E) Dorsal marginal zone (DMZ) or ventral marginal zone (VMZ) explants were dissected at stage 10 from
the DMSO or capsaicin-treated (from the four-cell stage) or untreated control embryos, cultured to stage 10.5 and subjected to western blotting. wt, wild-type; pt, point mutation.
( ) in (E) denotes VMZ explants with no chemical treatment. b-actin is a loading control.
Fig. 4. Capsaicin enhances the phosphorylation of b-catenin by down-regulating the activity of PP2A. (A,C) Four-cell stage embryos were injected animally with XWnt8 (50 pg) RNA
and treated or not with okadaic acid (100 nM) for 2 h just prior to animal cap dissection as indicated. Animal cap tissues isolated from the embryos were exposed to the increasing
doses of capsaicin (25, 50 mM) from stage 8.5 to stage 10.5 and then subjected to western blotting. Control in (C) denotes explants without okadaic acid treatment. (B) Uninjected
embryos were pretreated with LiCl (100 mM) for 30 min just before animal cap isolation, and the animal explants were excised, cultured in the presence of capsaicin (12.5, 25,
50 mM) and processed as carried out for (A,C). (D,E) Embryos were incubated for 30 min with LiCl (120 mM) at the 32-cell stage and then cultured to tadpole stages in media
containing DMSO or capsaicin (D) or harvested at stage 10 for in situ hybridization against Chordin (E). (F) Four-cell stage embryos were injected or not in the dorsal region with wt
PP2Ac (500 pg) or PP2Ac(Y307F)(500 pg) RNA, followed by 200 mM capsaicin treatment until untreated sibling embryos reach the tadpole stages. The percentages of phenotypes of
embryos: capsaicin, 85%, n ¼ 65; capsaicin þ PP2Ac(Y307F), 65%, n ¼ 75; capsaicin þ wt PP2Ac, 79%, n ¼ 70. Controls in (DeF) indicate no chemical treatments.
Supplementary Fig. 1. Morphological defects of capsaicin-treated embryos are rescued by co-expression of a non-phosphorylatable β-catenin but not by its wild-type version. Uninjected control embryos and embryos injected at the 4-cell stage dorsally with myc-β-catenin(wt)(20 pg) or myc-β-catenin(pt)(20 pg) RNA were cultured in the presence of capsaicin or DMSO to tadpole stages as indicated. All embryos are shown in lateral views with anterior to the left. The percentages of phenotypes of embryos: (A) 100%, n = 70; (B) 87%, n = 75; (C) 75%, n = 65; (D) 80%, n = 68.
Supplementary Fig. 2. Quantification of protein levels (normalized to β-actin) from three independent experiments for Fig. 4A. Error bars indicate the standard error (SE). Asterisks above the bars denote *p-value < 0.05 and **p-value < 0.01 compared to untreated or DMSO-treated control explants. Measurement of the intensities of Western blot bands was performed using ImageJ 1.40 g software. Statistical significance was determined using T-test using Microsoft Excel software. A p value of less than 0.05 was considered as significant
Supplementary Fig. 3. Okadaic acid causes anterior defects in early embryo. Embryos were incubated in media containing DMSO, capsaicin (200 μM) or okadaic acid (300 nM) from 2-cell stage to tadpole stages. All embryos are shown in lateral views with anterior to the left. Control, an embryo with no chemical treatment. The percentages of phenotypes of embryos: (A) 100%, n = 60; (B) 98%, n = 85; (C) 68%, n = 75; (D) 92%, n = 78.
