Vonica A et al. (2020), Apcdd1 is a dual BMP/Wnt inhibitor in the devel...

XB-ART-56905

Dev Biol 2020 Aug 01;4641:71-87. doi: 10.1016/j.ydbio.2020.03.015.

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Apcdd1 is a dual BMP/Wnt inhibitor in the developing nervous system and skin.

Vonica A , Bhat N , Phan K , Guo J , Iancu L , Weber JA , Karger A , Cain JW , Wang ECE , DeStefano GM , O'Donnell-Luria AH , Christiano AM , Riley B , Butler SJ , Luria V .

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Animal development and homeostasis depend on precise temporal and spatial intercellular signaling. Components shared between signaling pathways, generally thought to decrease specificity, paradoxically can also provide a solution to pathway coordination. Here we show that the Bone Morphogenetic Protein (BMP) and Wnt signaling pathways share Apcdd1 as a common inhibitor and that Apcdd1 is a taxon-restricted gene with novel domains and signaling functions. Previously, we showed that Apcdd1 inhibits Wnt signaling (Shimomura et al., 2010), here we find that Apcdd1 potently inhibits BMP signaling in body axis formation and neural differentiation in chicken, frog, zebrafish. Furthermore, we find that Apcdd1 has an evolutionarily novel protein domain. Our results from experiments and modeling suggest that Apcdd1 may coordinate the outputs of two signaling pathways that are central to animal development and human disease.

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Species referenced: Xenopus laevis
Genes referenced: acvr1b apcdd1 babam2 bmp2 bmp7.1 bmpr2 chrd gsc nodal sox2 szl
GO keywords: embryo development [+]
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	Fig. 1. Expression of Xenopus apcdd1. A-T. RNA in situ hybridization for Xenopus apcdd1. Maternal expression at 8 cell stage (A, B) is localized in the animal pole and is frequently unevenly distributed. In late blastulas (stage 9, D-F), the expression is animal and marginal (arrows in F, blastocoel in outlined in F). At the start of gastrulation (stage 10, G-I) apcdd1 RNA is present in animal and marginal cells, including the dorsal lip with the Spemann organizer (arrow in H, I, blastocoel is outlined in I). In neurula embryos (stage 18, K-N) and tailbud (stage 22), expression is limited to diencephalon and midbrain precursors (J, K, M, N, Q), and the tailbud (K, M, O, P, Q). Arrowhead in the transversal section (R) indicates ependymal precursors. In tadpoles (stage 30, S) expression closely mirrors that in mouse embryos. D – diencephalon; M – midbrain; Ov – otic vesicle; T – tailbud; A – anus. Bracket in S indicates the branchial arches. For control in situ hybridization (C: 8-cell stage; T: tadpole), the RNA probe used was LacZ. The scale bar in A is 0.3 mm (all embryos are shown at the same scale). At least 10 Xenopus embryos were examined for each time point.
	Fig. 2. Apcdd1 regulates expression of both positive and negative targets of BMP signaling in Xenopus embryos. In situ hybridization of morpholino-depleted (A1MO), or Apcdd1 overexpressing embryos. Embryos were injected at the 4-cell stage dorsally (A-C, double in situ hybridization for chordin and LacZ RNA as injection marker, in red), or 1 cell stage (D–R). Control embryos were injected solely with LacZ RNA (1 ng). A-F. Spemann organizer genes: chordin is not affected by apcdd1 depletion (B) (n = 18 embryos), but inhibited by apcdd1 RNA (A1 RNA) overexpression (n = 23 embryos), consistent with the inhibition of the maternal, dorsal Wnt pathway; gsc is unaffected by either treatment (n = 15 and n = 12 embryos, respectively, E, F). G-L. The ventral marker and BMP target sizzled is expanded by Apcdd1 depletion (n = 23, I, J), and restored by expression of a MO-resistant apcdd1 RNA (n = 21 embryos, K, L). M-R. The early neural marker and negative BMP target sox2 is inhibited by Apcdd1 depletion (n = 27 embryos, O, P, arrows in O indicate a hole in the ventral expression pattern), and partially restored by expression of Apcdd1 RNA (n = 17 embryos, Q, R).
	Fig. 3. APCDD1 inhibits BMP signaling in cells and tissues. A-B. The activation of BMP and Wnt pathways was measured at single-cell level, using NIH 3T3 mouse fibroblast cells cultured in the presence of BMP and Wnt ligands. Cells transfected with human APCDD1 and a fluorescence reporter (displayed in red) have low levels of both pSmad1 (green in A) and β-catenin (green in B), while neighboring cells that are not transfected have higher levels of both effectors. Nuclei are visualized with DAPI (blue). C. The immunofluorescence levels of pSmad1 and β-catenin were measured relative to the background (relative expression, Y axis), in the presence (0) or absence (1) of one or both ligands (Bmp2, Wnt3A) or of APCDD1 (n = 40 cells per condition). The outputs of BMP and Wnt signaling are inhibited by APCDD1 (p < 0.001 in Bonferroni-adjusted 2-way heteroscedastic t-test comparisons). D. Effect of Xenopus Apcdd1 (XA1) modulation on BMP-induced transcriptional activity in Xenopus animal injections. Apcdd1 depletion increases transcription induced by exogenous BMP2 RNA, while overexpression of apcdd1 RNA reduces transcription from both the BRE reporter induced by exogenous BMP2, and of the Vent promoter induced by endogenous BMP ligands. ∗∗∗p < 0.001 in Student’s t-test. Embryos were collected at stage 10.5, four embryos per assay, in triplicate, and each experiment was repeated a minimum of 3 times. E-I. The phenotype of XA1 depletion on the dorsal side is rescued most effectively by simultaneous BMP7 depletion (quantification in Fig. S1E). Embryos injected at the 4-cell stage on the dorsal side were collected at stage 36. The dorso-anterior index (DAI) values (Kao and Elinson, 1988) are: E – DAI5 (normal), F – DAI2, G – DAI2, H – DAI3, I – DAI4. For D-I, at least 10 Xenopus embryos, in three different experiments, were examined for each time point and experimental condition. J. Human APCDD1 specifically reduces the protein level of type I BMP receptor BMPRIA, but not the type II receptor BMPR2 and nodal type I receptor ACVR1B. Beta-galactosidase serves as loading control. Xenopus embryos were injected in the animal poles. K. APCDD1 interacts directly with BMPRIA, but not with BMPR2, in CHO cells. APCDD1 and BMPRIA coprecipitated in both APCDD1 (center panel) and BMPRIA (right panel) immunoprecipitation. BMPR2 coprecipitated with BMPRIA (right panel), but not with APCDD1 (center panel).

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