Lef1 regulates caveolin expression and caveolin dependent endocytosis, a process necessary for Wnt5a/Ror2 signaling during Xenopus gastrulation

Abstract

The activation of distinct branches of the Wnt signaling network is essential for regulating early vertebrate development. Activation of the canonical Wnt/β-catenin pathway stimulates expression of β-catenin-Lef/Tcf regulated Wnt target genes and a regulatory network giving rise to the formation of the Spemann organizer. Non-canonical pathways, by contrast, mainly regulate cell polarization and migration, in particular convergent extension movements of the trunk mesoderm during gastrulation. By transcriptome analyses, we found caveolin1, caveolin3 and cavin1 to be regulated by Lef1 in the involuting mesoderm of Xenopus embryos at gastrula stages. We show that caveolins and caveolin dependent endocytosis are necessary for proper gastrulation, most likely by interfering with Wnt5a/Ror2 signaling. Wnt5a regulates the subcellular localization of receptor complexes, including Ror2 homodimers, Ror2/Fzd7 and Ror2/dsh heterodimers in an endocytosis dependent manner. Live-cell imaging revealed endocytosis of Ror2/caveolin1 complexes. In Xenopus explants, in the presence of Wnt5a, these receptor clusters remain stable exclusively at the basolateral side, suggesting that endocytosis of non-canonical Wnt/receptor complexes preferentially takes place at the apical membrane. In support of this blocking endocytosis with inhibitors prevents the effects of Wnt5a. Thus, target genes of Lef1 interfere with Wnt5a/Ror2 signaling to coordinate gastrulation movements.

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. 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 M2. 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.