Wallkamm V et al. (2016), Regulation of distinct branches of the non-cano...

XB-ART-52271

BMC Biol 2016 Jul 05;14:55. doi: 10.1186/s12915-016-0278-x.

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Regulation of distinct branches of the non-canonical Wnt-signaling network in Xenopus dorsal marginal zone explants.

Wallkamm V , Rahm K , Schmoll J , Kaufmann LT , Brinkmann E , Schunk J , Kraft B , Wedlich D , Gradl D .

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BACKGROUND: A tight regulation of the Wnt-signaling network, activated by 19 Wnt molecules and numerous receptors and co-receptors, is required for the establishment of a complex organism. Different branches of this Wnt-signaling network, including the canonical Wnt/β-catenin and the non-canonical Wnt/PCP, Wnt/Ror2 and Wnt/Ca(2+) pathways, are assigned to distinct developmental processes and are triggered by certain ligand/receptor complexes. The Wnt-signaling molecules are closely related and it is still on debate whether the information for activating a specific branch is encoded by specific sequence motifs within a particular Wnt protein. The model organism Xenopus offers tools to distinguish between Wnt-signaling molecules activating distinct branches of the network. RESULTS: We created chimeric Wnt8a/Wnt11 molecules and could demonstrate that the C-terminal part (containing the BS2) of Wnt8a is responsible for secondary axis formation. Chimeric Wnt11/Wnt5a molecules revealed that the N-terminus with the elements PS3-1 and PS3-2 defines Wnt11 specificity, while elements PS3-1, PS3-2 and PS3-3 are required for Wnt5a specificity. Furthermore, we used Xenopus dorsal marginal zone explants to identify non-canonical Wnt target genes regulated by the Wnt5a branch and the Wnt11 branch. We found that pbk was specifically regulated by Wnt5a and rab11fip5 by Wnt11. Overexpression of these target genes phenocopied the overexpression of their regulators, confirming the distinct roles of Wnt11 and Wnt5a triggered signaling pathways. Furthermore, knock-down of pbk was able to restore convergent extension movements in Wnt5a morphants. CONCLUSIONS: The N-terminal part of non-canonical Wnt proteins decides whether the Wnt5a or the Wnt11 branch of the Wnt-signaling network gets activated. The different non-canonical Wnt branches not only regulate cellular behavior, but, surprisingly, also regulate the expression of different target genes. One of these target genes, pbk, seems to be the relevant target gene executing Wnt5a-mediated regulation of convergent extension movements.

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Species referenced: Xenopus
Genes referenced: chrd ctnnb1 pbk rab11fip5 wnt11 wnt11b wnt5a wnt8a
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	Fig. 1. xWnt11 impairs elongation, xWnt5a impairs constriction. a Representative phenotypes of DMZ explants. The overexpression of xWnt5a results in broader, less elongated explants; the overexpression of xWnt11 inhibits elongation. Elongation and constriction were determined by phenotypically analyzing the length (red line) and width (blue line) of the outgrowth. Control explants show a protrusion with length > > width. For explants with inhibited elongation (xWnt11), hardly any protrusion can be detected. In explants showing failures in constriction, the protrusion is broader at the expense of length (xWnt5A). Constriction of explants that did not elongate could not be determined. b Quantification of elongation. xWnt5a has no influence on elongation, whereas xWnt11 inhibits elongation in a dose-dependent manner. c Quantification of constriction. xWnt5a inhibits constriction in a dose-dependent manner, xWnt11 has no significant influence on constriction of the explants. Shown is the frequency of the indicated phenotypes. The superimposed error bars illustrate the variation between N independent experiments (biological replicates). In each experiment, the absolute frequency of the indicated phenotypes was normalized to the control siblings. In total, 69 explants of the uninjected controls could be analyzed, 60 of which elongated and could be evaluated for constriction. For 50 pg xWnt11 this means that n = 36 explants were analyzed for “relative elongation”, but only n = 10 explants could be considered to analyze relative constriction. N: number of biological replicates; n: number of analyzed explants. *** P < 0.001 according to Fisher’s exact test; Bars: 200 μm
	Fig. 2. Summarizing scheme of Wnt5a/Wnt11 chimeras and their effects
	Fig. 3. Analysis of chimera pair 3 and 4 in dorsal marginal zone (DMZ) explants. a Representative phenotypes of DMZ explants of embryos injected with the indicated mRNAs of chimera pair 3. Wnt5a/Wnt11 Chimera 3.1 inhibits constriction, whereas Wnt11/Wnt5a chimera 3.2 influences constriction and elongation. b Quantification of elongation. c Quantification of constriction. d Representative phenotypes of DMZ explants of embryos injected with the indicated mRNAs of chimera pair 4. Wnt5a/Wnt11 chimera 4.1 does not disturb convergent extension movements, whereas Wnt11/Wnt5a chimera 4.2 inhibits constriction. e Quantification of elongation. f Quantification of constriction. Shown is the frequency of the indicated phenotypes. The superimposed error bars illustrate the variation between N independent experiments. In each experiment, the absolute frequency of the indicated phenotypes was normalized to the control siblings. N: number of biological replicates, n: number of analyzed explants, * P < 0.001, P < 0.01, * P < 0.05 according to Fisher’s exact test, Bars: 200 μm
	Fig. 4. Rescue experiments chimera. To test whether the chimeric constructs can compensate for the loss of xWnt11 and xWnt5a, 200 pg of Wnt5a/Wnt11 chimera 3.1 and Wnt11/Wnt5a 3.2 were co-injected with 2.5 pmol of an xWnt11 (Wnt11 Mo) and xWnt5a (Wnt5a Mo) morpholino (Mo) antisense oligonucleotide. a To determine the effect of the Wnt11 Mo, we counted the fraction of stage 12 embryos with open blastopore (blastopore defects). To test whether the chimeric constructs can compensate for the loss of xWnt5a we calculated (b) relative elongation and (c) relative constriction of dorsal marginal zone explants. Shown is the frequency of the indicated phenotypes. The superimposed error bars illustrate the variation between N independent experiments. N: number of biological replicates, n: number of analyzed explants, * P < 0.001, P < 0.01, * P < 0.05 according to the χ2 test (a) and Fisher’s exact test (b, c)
	Fig. 5. pbk and rab11fip5 are specific non-canonical Wnt target genes. a Dorsal marginal zone (DMZ) explants were dissected at stage 10.25 and cultivated until siblings reached stage 12. Total RNA was isolated from 30 DMZ explants. Samples with an RNA integrity number value > 8 were analyzed in a microarray or nanostring analysis. b Differential regulation of six spots on the microarray representing three putative xWnt5a target genes. Shown is the average fold change of biological triplicates, the indicated P value is relative to the control morphant-injected explants, ** P < 0.01, * P < 0.05. c Differential regulation of four spots on the microarray representing three putative xWnt11 target genes. Shown is the average fold change of biological triplicates (d). Reevaluation of the six putative non-canonical Wnt target genes by nanostring analysis. Only pbk and rab11fip5 were found to be differentially regulated. Shown is the average fold change of biological triplicates. ** P < 0.01 according to one-sample t test
	Fig. 6. The N-terminal part of Wnt11 is required to suppress rab11fip5 expression. 200 pg of the indicated Wnt mRNA were co-injected with the lineage tracer Dextran-FITC into one blastomere of two-cell stage embryos. a At the neurula stage, rab11fip5 expression was determined by RNA in situ hybridization. Asterisks mark the injected site. b The quantification of the phenotype. Shown is the absolute frequency of the indicated phenotypes. The superimposed error bars illustrate the variation between N biological replicates, n: number of embryos. * P < 0.05, *** P < 0.001 according to the χ2 test
	Fig. 7. Pbk and rab11fip5 interfere with convergent extension movements. a Representative whole mount in situ hybridization for chordin of stage 12 embryos injected dorsal equatorially at the 4-cell stage with the indicated mRNAs. The dorsal overexpression of pbk and rab11fip5 results in a shorter and broader chordin expression. Bars: 500 μm. b Quantification of chordin phenotypes. * P < 0.001 according to χ2 significance test (c). Phenotypes of dorsal marginal zone (DMZ) explants derived from pbk and rab11fip5 overexpressing embryos. Overexpression of pbk leads to broader elongated explants and overexpression of rab11fip5 results in an inhibition of elongation. Bars: 200 μm. d Quantification of elongation. Pbk does not interfere with elongation but rab11fip5 significantly inhibits elongation. e Quantification of constriction. Pbk overexpressing DMZ explants fail to constrict whereas rab11fip5 does not affect constriction. Shown is the frequency of the indicated phenotypes. In each experiment, the absolute frequency of the indicated phenotypes was normalized to the control siblings. The superimposed error bars illustrate the variation between N independent experiments. P < 0.01, *** P < 0.001 according to Fisher’s exact test. N: number biological replicates, n: number of analyzed embryos/explants
	Fig. 8. Epistasis experiments. Injection of antisense morpholinos specific for xWnt5a (Wnt5aMo), xWnt11 (Wnt11Mo), pbk (pbkMo), and rab11fip5 (rabMo) impaired convergent extension movements as seen as mild phenotype by a shortened and broad chordin expression or a strong phenotype by a blurred chordin expression or a staining at the borders of the non-closing blastoporus. a Shows representative dorsal marginal zone explants. b Quantification of the epistasis experiments revealed that knock-down of pbk can compensate for the loss of xWnt5a, but (c) knock-down of rab11fip5 cannot compensate for the loss of xWnt11. Shown is the absolute frequency of the indicated phenotypes. The superimposed error bars illustrate the variation between N biological replicates. P < 0.01, * P < 0.001 according to the χ2 test, n: number of analyzed embryos

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