Li S et al. (2008), Retinoid signaling can repress blastula Wnt sig...

XB-ART-37671

Differentiation 2008 Oct 01;768:897-907. doi: 10.1111/j.1432-0436.2008.00269.x.

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Retinoid signaling can repress blastula Wnt signaling and impair dorsal development in Xenopus embryo.

Li S , Lou X , Wang J , Liu B , Ma L , Su Z , Ding X .

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Vitamin A derivatives (retinoids) are actively involved during vertebrate embryogenesis. However, exogenous retinoids have also long been known as potent teratogens. The defects caused by retinoid treatment are complex. Here, we provided evidence that RAR-mediated retinoid signaling can repress Xenopus blastula Wnt signaling and impair dorsal development. Exogenous retinoic acid (RA) could antagonize the dorsalizing effects of lithium chloride-mediated Wnt activation in blastula embryos. The Wnt-responsive reporter gene transgenesis and luciferase assay showed that excess RA can repress the Wnt signaling in blastula embryos. In addition, the downstream target genes of the Wnt signaling that direct embryonic dorsal development, were also down-regulated in the RA-treated embryos. Mechanically, RA did not interfere with the stability of beta-catenin, but promoted its nuclear accumulation. The inverse agonist of retinoic acid receptors (RAR) rescued the Wnt signaling repression by RA and relieved the RA-induced nuclear accumulation of beta-catenin. Our results explain one of the reasons for the complicated teratogenic effects of retinoids and shed light on the endogenous way of interactions between two developmentally important signaling pathways.

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Species referenced: Xenopus
Genes referenced: acvr1 ag1 chrd ctnnb1 en1 fos gsc hoxb1 myf5 ncam1 nodal5 nodal6 nrp1 otx2 rab40b shh sia1 szl tbxt ventx1 ventx1.2
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	Fig. 1 Early exposure of RA impairs dorsal development in Xenopus embryo. (A) The embryos treated at the 32-cell stage with 0.3M NaCl for 10 min developed normally at stage 32. (B) The embryos treated at the 32-cell stage or the stage 9 by 1 mM RA displayed phenotypes with various severity. Embryos (E, H, M–Q) were treated at the 32-cell stage with 1 mM RA for 1 hr, and DMSO treated embryos (C, F, I–L, and Q) served as controls. Embryos (D and G) were treated with 1 mM RA for 1 hr at stage 12.5. Embryos in (C) and (F) displayed normal patterns of muscle and neural staining by 12/101 and Xen1 antibodies, whereas embryos in (E) and (H) showed very weak staining of both antibodies. The embryos (D and G) treated at stage 12.5 retained most of the body structures. Embryos in (I) and (M) were in situ hybridized with Chordin probe. RA treatment dramatically decreased Chordin expression in the gastrula embryo. Embryos in (J) and (N) were in situ hybridized with Sizzled probe. RA treatment did not change Sizzled expression in gastrula embryos. In situ hybridization with Xbra (K and O) in stage 15 embryos showed that posterior Xbra expression was obviously attenuated, meanwhile the midline expression of Xbra was also abolished in the same RA-treated embryos (L and P). RT-PCR showed the gene expression profile in stage 12 embryos (Q).
	Fig. 2 Retinoic acid (RA) can antagonize the dorsalizing effects of LiCl when embryos were treated at cleavage stage. (A) Embryos treated at the 32-cell stage with 0.3 M NaCl for 10 min underwent normal development at the stage 32. The embryos in (B–D and G–I) were treated with 0.3 M LiCl for 10 min, 1 mM RA for 1 hr or sequential 0.3 M LiCl for 10 min and 1 mM RA for 1 hr as indicated and cultured for the same period as the embryos in (A). (B) The embryos were dorsalanteriorized (85%–90%, n4400, 9oDAIo10) when exposed to LiCl at the 32-cell stage. (C) Exposure to RA at the 32-cell stage severely disrupted the Xenopus embryos, with shortened body axis, complete loss of anterior structures (480%, n4400). (D) Sequential treatment by LiCl and RA largely recovered the embryonic body structures like cement gland (white arrowhead). (E) The graph showed the percentage of embryos with different size of cement glands. (F) Reverse transcription-PCR showed the effects of LiCl and RA treatment on gene expression in stage 13 embryos. (G) The embryos were severely ventroposteriorized (75%, n 5 71) when exposed to LiCl at stage 10. (H) Exposure to RA at the stage 10 also severely destroyed the Xenopus embryonic structures (82%, n 5 100). (I) LiCl and RA treatment at stage 10 led to shorter body axis and more disrupted anterior structures and defective posterior neural tube closure (100%, n 5 72).
	Fig. 3 Retinoid signaling can repress Wnt signaling in blastula embryos. (A) The transgenic embryos using the p-LEF7-fos-GFP Wntresponsive reporter gene were treated with 0.1 M NaCl for 20 min at the 32-cell stage and probed with GFP antisense RNA probe. The stage 8.5 embryo showed normal signals in a patch of dorsal cells. (B) A good portion of transgenic embryos treated with 0.1 M LiCl for 20 min at the 32-cell stage displayed enhanced GFP expression expanding the normal domain. (C) When treated with 1 mM retinoic acid (RA) for 20 min at the 32-cell stage, the number of the embryos expressing GFP greatly decreased. (D) The diagram shows the statistical data for the transgenic embryos expressing GFP after different treatments (composite data from three independent experiments). LiCl treatment in (E, F, H, L, J, and N) was 0.3 M at 32-cell stage for 10 min. NaCl treatment in (E, F, G, and K) was 0.3 M at 32-cell stage for 10 min. RA treatment (E, F, I, J, M, and N) was 1 mM for 1 hr at 32-cell stage. (E) The luciferase activities of Super Top Flash were enhanced after LiCl treatment and attenuated upon RA treatment. RA treatment also repressed LiCl-mediated activation. However, the treatments did not affect the luciferase activities of Super Fop Flash. (F) Downstream genes of Wnt signaling in stage 8.5 was activated by LiCl treatment (lane 3) and attenuated by 1 mM RA treatment (lane 4). Sequential treatment by LiCl and RA compromised the change by the LiCl or RA treatment alone (lane 5). (G–N) Embryos at stage 8.5 were in situ hybridized with Chodin. (G and H) Embryos were shown from dorsal views. (K–N) Embryos were shown from animal pole views. NaCl-treated embryos showed a patch of staining co-localizing with blastula Wnt activity (compare G and A), and a slice of signal can be seen from animal pole view (arrowhead in K). LiCl treatment expanded Chordin expression to the entire marginal zone (H) and across the whole animal pole (arrowheads in L). RA-treated embryos showed weakened expression of Chordin from both views (I and M). Sequential treatment with LiCl and RA rescued the upregulation of Chordin by LiCl (dotted area in both J and N). (O) The percentage of embryos with different treatment, displayed different level of Chordin expression.
	Fig. 4 Excess retinoic acid (RA) increases the nuclear b-catenin accumulation without affecting its stability. (A–E) All the embryos were treated at the 32-cell stage with 0.1M NaCl, 0.1M LiCl, 1 mM RA or both treatments for 1 hr, and collected at the stage 8.5 for further analysis. (A) LiCl treatment markedly decreased the phosphorylated b-catenin at stage 8.5 (lanes 2 and 4), whereas RA treatment did not affect this effect. (B) LiCl treatment resulted in increases in both the cyotplasmic and the nuclear b-catenin (lanes 2 and 6). Although RA treatment did not affect the cytoplasmic b-catenin (lane 3), the nuclear b-catenin increased (lane 7). Co-treatment with RA could not interfere with the LiCl-mediated b-catenin stability increase (lane 4). (C–E) All the embryos were immunostained with the b-catenin antibody and placed dorsal to the right. (C) The NaCltreated embryo showed the normal pattern of the b-catenin nuclear accumulation, a patch of dorsal cells (dotted area), and the red arrow points to a cell with the nuclear accumulation of b-catenin. (D) LiCl dramatically induced the ectopical nuclear accumulation of b-catenin (see white arrowheads outside the dotted area). (E) RA treatment also increased the ectopical nuclear accumulation of b-catenin (see white arrowheads outside the dotted area).
	Fig. 5 The repression of Wnt signaling by retinoic acid (RA) is mediated by RARs. (A–M) Embryos were treated at 32-cell stage with 1 mM RA, 10 mM AGN193109 (AGN), or both together for 1 hr, DMSO-treated embryos served as controls. (A–D) Black arrowheads indicated the eyes, whereas black arrows pointed to the cement glands. (A) The embryos treated with DMSO developed normally. (B) The body structures of RA-treated embryo were severely destroyed (480%, n4400). (C) When co-treated with 1 mM AGN, the RA-treated embryos regained AP axis (470% n4250). (D) When co-treated with 10 mM AGN, the RA-treated embryos almost fully re-established AP axis, with re-occurrence of eyes (black arrowhead) (465%, n4250). (E) AGN treatment increased RA-induced Top Flash luciferase activity reduction around the stage 9.5, with Fop Flash unaffected. (F–K) Embryos were in situ hybridized with Chordin. (F–G) Embryos were shown form dorsal view. (I–K) Embryos were shown from animal pole view. Chordin expression reduction in RA-treated embryos was rescued by AGN treatment. (L) Wnt-responsive genes down-regulated by RA were recovered by AGN co-treatment. (M) AGN treatment relieved the RA-induced nuclear accumulation of b-catenin.
	Fig. 6 AGN193109 (AGN)’s effects on Wnt signaling. Embryos were treated with 10 mM AGN at 32-cell stage for 1 hr (B–D, F and H). The control embryos were treated with DMSO at 32-cell stage for 1 hr (A, C, D, E and G). (B) A typical AGN-treated embryo exhibited shortened body axis, defective tail outgrowth and a bit increased head structures. (C) AGN treatment could increase blastula Wnt signaling, which could be off-set by co-treatment with 1 mM retinoic acid (RA). (D) Nuclear translocation of b-catenin was not much affected. (E–H) Embryos were in situ hybridized with Chordin. (E and F) Embryos were shown form dorsal view. (G and H) Embryos were shown from animal pole view. Chordin expression was not increased by AGN treatment (compare E and F, G and H)