Puzik K , Tonnier V , Opper I , Eckert A , Zhou L , Kratzer MC , Noble FL , Nienhaus GU , Gradl D .

GR1620 6/1 Deutsche Forschungsgemeinschaft (German Research Foundation), CRC 1324 Deutsche Forschungsgemeinschaft (German Research Foundation), STN Helmholtz Association

             Wnt signaling pathway

	Figure 1. Lef1 regulates Wnt5a clustering and the expression of caveolin family members. (a) Knock-down of Lef1 reduces the formation of Wnt5a clusters at the plasma membrane of DMZ cells. Both dorsal blastomeres of Xenopus 4-cell stage embryos were injected with 150 pg Wnt5aeGFP mRNA together with the membrane marker GAP43mCherry and 1 pmol of the Lef1 specific antisense morpholinos (LefMo) or 500 pg dominant negative Lef1 mRNA (Lef∆HMG). The arrows indicate some Wnt5a clusters located at the plasma membrane, arrowheads point to cytoplasmically located Wnt5a clusters. (b) Downregulation of Lef1 reduces the presence of Wnt5a clusters at the plasma membrane. Shown are the relative membrane localization according to Manders et al.53 and the p values according to Student’s t-test. The images were processed using the Coloc 2 plugin in ImageJ which allows calculation of the Manders coefficient M253. This coefficient indicates colocalization of Wnt5aeGFP clusters and the red coloured membrane. (c) In transfected HEK293 cells, Lef1∆HMG and wild-type Lef1 do not significantly regulate the non-canonical Wnt reporter ATF2Luc. (d) In Wnt5a stimulated cells (cotransfected with Wnt5aeGFP), Lef1∆HMG represses ATF2Luc activity. Wild-type Lef1, instead, activates the ATF2Luc reporter. p values of at least nine independent transfections were determined according to Student’s t-test. e) Real-time RT-PCR confirmed that the expression of cavin1, caveolin1 and caveolin3 are downregulated in Lef1 morphants and partially restored in morphants co-injected with 500 pg Lef1 mRNA. Shown are mean values and standard error of four biological replicates.
	Figure 2. The caveolar core components caveolin1, caveolin3 and cavin1 are necessary for convergent extension movements. Antisense morpholino oligonucleotides directed against caveolin1 (cav1Mo), caveolin3 (cav3Mo) and cavin1 (cavin1Mo) were injected in the marginal zone of both dorsal blastomeres of 4-cell stage embryos. Embryos were fixed at stage 14 and analyzed for localization of the axial mesodermal marker gene chordin. (a) In control embryos, chordin is expressed in a long and narrow strip along the dorsal midline. Depletion of caveolar proteins resulted in mislocalization of chordin expression. In severe cases, chordin expression remained at the dorsal part of the unclosed blastopore. The phenotype was classified as mild when the chordin expression was bent or short and broad. (b) Quantification of the phenotypes in the morphants resulting from the injection of 4 pmol cav1Mo, 4 pmol cav3Mo and 0.4 pmol cavin1Mo, respectively. (c) Caveolin1 and cavin1 patially restore chordin expression in cav1 morphants. 4 pmol cav1Mo were coinjected with 500 pg caveolin1 and cavin1 mRNA, respectively. (d) Epistasis experiments with Wnt5a morpholino (5aMo, 0.4 pmol), caveolin1 morpholino (cav1Mo, 0.25 pmol) and caveolin1 mRNA (500 pg) revealed additive effects of the morpholinos. Furthermore, caveolin1 mRNA partially restored gastrulation in Wnt5a morphants. P values according to a χ2 test, n: number of embryos. (e) Realtime PCR of animal cap explants revealed that the expression of the Wnt5a target gene PAPC depends on caveolin1 expression. Shown are the mean values, standard errors and p values according to Student’s t-test of four independent experiments.
	Figure 3. Endocytosis is necessary for convergent extension movements. Wild-type embryos were incubated from stage 9 to stage 14 in MBSH containing the indicated concentration of genistein (a) or chlorpromazine (b). (c) Quantification of the chordin expression revealed that genistein blocked convergent extension movements even at low doses (50 µM). At high doses (250 µM), almost all embryos showed the severe phenotype with chordin being expressed at the dorsal side of the open blastoporus, whereas 60% of the DMSO-treated siblings expressed chordin in a wild-type pattern. (d) Quantification of the chordin expression revealed that 50 µg/ml chlorpromazine induced a mild mislocalization of chordin expression in more than 60% of the embryos. Doubling of the dose to 100 µg/ml increased the frequency of chordin mislocalization to more than 70%. e) Simultaneous treatment with both inhibitors revealed additive effects. Quantification of chordin mislocalization revealed that both inhibitors together inhibit convergent extension movements even at doses when one inhibitor alone has only mild effects. n: number of embryos, p-values according to χ2 test. n.s. not significant,
	Figure 4. Wnt5a induces redistribution of Ror2 homodimers and Ror2/dsh heterodimers. The subcellular localization of fluorophore tagged Ror2 homodimers (a) and Ror2/dsh heterodimers (b) was analyzed by a bimolecular fluorescence complementation assay (BiFC) in transiently transfected Xenopus tissue culture cells. The relative grey values show the intensity profiles at the positions marked by yellow bars. (c,d) The membrane/cytoplasm values were calculated in the following way: membrane/cytoplasm = (intensity at the membrane – intensity outside)/(intensity inside – intensity outside). Shown are the mean values and standard errors of at least 399 profiles of more than 60 cells from at least ten independent experiments. Median and dispersion (25–75% range) are shown as box-and-whiskers plots in Supplementary Fig. 5b. (d) The effect of endocytosis inhibitors chlorpromazine and genistein on the subcellular distribution of Ror2/dsh heterodimers. Shown are the mean values and standard errors of at least 280 profiles of more than 40 cells in at least seven independent experiments. p values according to Student’s t-test, n.s.: not significant. Median and dispersion are shown in Supplementary Fig. 5c.
	Figure 5. Wnt5a induces disappearance of Ror2/Ror2 dimers at the apical membrane. (a) Animal cap explants were analyzed for the localization of total Ror2 (Ror2mCherry, red channel) and Ror2 homodimers (splitYFP signal, green channel) in the absence (−Wnt5a) and presence (+Wnt5a) of co-injected Wnt5a and in the presence of 50 µg chlorpromazine. Ten optical sections of 1 µm thickness allowed us to visualize localization of the signals in the z-dimension. The white bar in the close-ups of the yz-plane marks the z-coordinate shown in the xy-images. (b) xy-planes at three different z-values illustrate the exclusively basal and basolateral localization of Ror2/Ror2 dimers in Wnt5a co-injected explants. (c) To quantify the z-distribution of the fluorescence signals, at least 11 explants (each with 4 to 6 different cells) derived from 3 independent experiments were analyzed. Individual fluorescence spots were selected in the maximum intensity projections, and the images were analyzed section by section for visibility of these spots. Shown is the fraction of fluorescence spots found in the xy-images as a function of the z-coordinate from basal (0 µm) to apical (9 µm). These data were calculated for each cell by dividing the number of fluorescence spots of a particular xy-plane by the number of spots in all planes. Shown are mean values and standard deviations from at least 11 explants. It is apparent that the BiFC signal (Ror2 homodimers) gets shifted to the basal side in the presence of Wnt5a; chlorpromazine and genistein revert this effect. (d) Paraffine sections of early gastrula stages revealed that Ror2 homodimers are located at the entire lateral site of animal cap cells in the absence of Wnt5a. In the presence of Wnt5a, however, the dimers are found exclusively at the basal side.
	Figure 6. Caveolin regulates internalization of Wnt5a/Ror2 clusters. (a) Colocalization of Ror2mCherry/Wnt5aeGFP clusters in animal cap explants of caveolin1 and caveolin3 morphants and caveolin1 and caveolin3 overexpressing embryos. (b,c) Quantification of the clusters. More than 80% of the clusters are located at the plasma membrane. Overexpression of caveolin1 slightly increases the frequency of membrane localization. (c) Average number of intracellular clusters per cell. p values according to Students t-test. (d) The BiFC signal of Ror2/dsh dimers partially colocalizes with the red fluorescence of caveolin1mGarnet in transfected XTC-cells, both, at the plasma membrane and in the cytoplasm (arrowheads in the close-ups). (e) Single particle tracking using widefield fluorescence microscopy indicates that cav1eGFP (blue trajectory) and Ror2mcherry (red trajectory) co-migrate in the cytoplasm (arrowheads) and leave the membrane together and continue co-migrating in the cytoplasm (arrows).
	Figure 7. The Lef1 targets of the caveolin family are required for Wnt5a induced receptor complex internalization. (a) Lef1 regulates the expression of the caveolar core components caveolin1, caveolin3 and cavin1. (b) Wnt5a induces rapid clustering of non-canonical receptor complexes and subsequently their internalization via clathrin and caveolin dependend mechanisms, (c) Apical internalization of receptor clusters results in a symmetry break of polarized cells with membrane bound receptor clusters at the basal membrane.
	S1. a) Both dorsal blastomeres of Xenopus 4-cell stage embryos were injected with either 4 pmol of the control morpholino (control) or the xLef1 specific morpholino (LefMO). DMZ cells were explanted at the onset of gastrulation and mRNA was isolated when siblings reached late gastrulation (stage 12). Purified mRNA probes (RIN>8) where analyzed on an ATLAS Microarray. The TOP-100 of the differentially regulated genes include the caveolar core components caveolin1 (cav1), caveolin3 (cav3), and cavin1. The total list of the TOP-100 target genes is found in Supplementary Table 1. b) Expression of the caveolar core components during early Xenopus development revealed that cavin1 and caveolin1 (cav1) are co-expressed in the notochord from early tailbud stage on. Caveolin3 (cav3), instead, is expressed in a salt and pepper pattern in multiciliated cells of the epidermis. At gastrula stages, no local accumulation of the caveolin3 mRNA was detectable. c) Lef1 (LefMO) were injected into one blastomere of Xenopus two- cell stage embryos. The asterisks mark the injected site. In situ hybridization revealed that 10 out of 29 of the Lef1 morphants embryos displayed markedly reduced expression of caveolin3 in multiciliated cells of the skin. In Lef1 morphant embryos co-injected with XLef1 mRNA (500 pg) only 2 out of 20 embryos showed reduced cav3 expression.
	S2. Caveolin1, caveolin3 and cavin1 are necessary for convergent extension movements. a) Sequences of caveolin1 (cav1), caveolin3 (cav3) and cavin1 morpholinos and their target mRNAs. The AUG start codon is indicated in bold. b) Dose response curve of the phenotypes to increasing amounts of the caveolin3, caveolin1 and cavin1 specific antisense morpholinos indicates that the severity of chordin mislocalization increases with the morpholino dose. c) Cavin1 and caveolin1 patially restore chordin expression in cavin1 morphants. 1 pmol cavin1MO was co-injected with 500 pg cavin1 and caveolin1 mRNA, respectively. p values according to chi-square test. d) In transfected HEK293 cells, caveolin1 and caveolin3 significantly activate the non-canonical Wnt reporter ATF2Luc. e) However, in Wnt5a stimulated cells (co-transfected with Wnt5aeGFP), caveolin1 and caveolin3 have no effect. p values of at least nine independent transfections were determined according to Student's t-test. f) Lef1 specific morpholinos were injected into the dorsal marginal zone of 4- cell stage embryos and co-injected with caveolin1 mRNA and a cocktail of caveolin1, caveolin3 and cavin1 mRNA (50 pg each) and analyzed at stage 14 for the localization of the axial mesodermal marker gene chordin. n.s. not significant according to chi-square test.
	S3. Endocytosis is necessary for convergent extension movements. a) To determine the time-window where caveolin-dependent endocytosis is relevant for gastrulation, we incubated the embryos with 150 microM genistein for different periods from late blastula (stage 9) until early neurula (stage 14) and analyzed the localization of chordin expression and the defects in blastopore closure. Quantification of blastopore defects (b) and chordin localization (c) revealed that the longer the inhibitor is present, the more severe are the gastrulation defects. Significant effects were only observed when the inhibitor was added at stage 9. p- values according to chi-square test., n: number of embryos. d) RT-PCR revealed that the caveolae-specific endocytosis inhibitor genistein activates the expression of the Wnt5a target gene pbk in transfected XTC cells. Shown are mean values and SEM from three independent experiments. p-values according to Student's t-test.
	S4. a) Drift corrected snapshots of Supplementary Movie 1. Scale bar, 5 microm. b) The yellow region of interest (ROI) of frame 120s was analyzed using Fiji software. A segmented line with a width of 2 pixels was drawn along the membrane, and the intensity profile was fitted with a superposition of 5 Gaussian functions representing the 5 intensity peaks indicated by numbers in the ROI. The fitted widths (full widths at half maximum, FWHM) are shown in the table on the right.
	S5. BiFC assay. a) Bimolecular fluorescence complementation (BiFC) assay on animal cap explants revealed that Ror2 forms homodimers and heterodimerizes with Fzd7. The N-terminus and C-terminus of YFP (YN and YC) fused to Ror2 or Fzd7 do not form a fluorescent protein. However, co-injection of YN and YC -tagged receptor molecules resulted in the constitution of a fluorescent protein located in the membrane, indicating that the receptor molecules form dimers. Homodimerization of Ror2 depends on the extracellular CRD region because the splitYFP constructs fused to deletion mutant Ror2 CRD fail to reconstitute a fluorescent protein. b) Median and dispersion (25-75% range) are shown as box-and-whiskers plots to illustrate the membrane localization of total Ror2 as well as Ror2/Ror2 and Ror2/dsh dimers in the absence and presence of Wnt5a. While receptor dimers are less prominently found at the membrane in the presence of Wnt5a, the localization of total Ror2 remains almost unchanged. c) Box-and-whiskers plots show that the effects of Wnt5a is partially reverted in the presence of endocytosis inhibitors. The blue dashed line indicates the median of Ror2/dsh in the absence of Wnt5a, the red dashed line in the presence of Wnt5a.
	S6. Wnt5a induces disappearance of Fzd7/Ror2 dimers at the apical membrane. a) Animal cap explants were analyzed for localization of total Ror2 (Ror2mCherry, red channel), Ror2 homodimers (splitYFP signal, green channel) and Fzd7/Ror2 dimers (splitYFP signal, green channel) in the absence (-Wnt5a) and presence (+Wnt5a) of co-injected Wnt5a and in the presence of 50 microM genistein. Ten optical sections of 1 microm thickness allowed us to visualize localization of the signals in the z- dimension. The white bar in the close-ups of the yz-plane marks the z-coordinate shown in the xy-images. b) Selected xy-planes at three different z-values illustrate the exclusively basal and baso-lateral localization of Fzd7/ Ror2 dimers in Wnt5a co-injected explants. c) Quantification of the z-distribution of the fluorescence signals, as described in the legend of Figure 5. A total of more than 3,500 splitYFP fluorescence spots and more than 4500 mCherry fluorescence spots were counted in optical planes of 14 explants derived from four independent experiments. Shown is the fraction of fluorescence spots in the xy-plane as a function of the z-coordinate, from basal (0 microm) to apical (9 microm). Obviously, in the presence of Wnt5a, the splitYFP signal (Fzd7/Ror2) is shifted to the basal side.

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