A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

<div><p>β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a ‘whole animal’ developmental framework to approach this question. Here we identify a C-terminally truncated <i>β-catenin</i> (<i>β-catenin4</i>), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and <i>in vitro</i> analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.</p></div

Proposed Model for Wnt/β-catenin activity modulation in planarian eyes.

<p>During planarian eye regeneration or maintenance, β-cat4 inhibits β-cat1/TCF-2 activity in photoreceptor cells, modulating Wnt signaling to an appropriate level to ensure correct differentiation of photoreceptors.</p

Plakoglobin and Neural Arm inhibit Wnt/β-catenin activity ‘in vitro’.

<p><b>(A)</b> Upper panel, schematic representation of Plakoglobin or β-catenin proteins, showing the protein interacting domains and the degree of homology of the N-terminal, central Arm repeat and C-terminal region between both proteins. Lower panel, activation of TOPflash reporter signal by β-catenin (S37A) and Plakoglobin in HEK293T cells. Plakoglobin showed limited capacity to activate reporter signal, and inhibited β-catenin (S37A) activity in a dose-dependent manner. <b>(B)</b> Upper panel, schematic representation of the exon composition of Arm and Neural Arm (in which exon 6 is skipped). Lower panel, activation of TOPflash reporter signal by Armadillo and Neural Arm in HEK293T cells. Neural Arm showed limited capacity to activate reporter signal and inhibited Arm activity in a dose dependent manner.</p

β-cat3 and 4 inhibit β-cat1 dependent Wnt signaling.

<p><b>(A)</b> Schematic of the four <i>β-catenin</i> homologs in <i>S</i>. <i>mediterranea</i>. β-cat1 and β-cat2 are structurally segregated. β-cat1 conserves the N-terminal GSK3 phosphorylation sites, the binding surface for Wnt signaling components in the central armadillo repeats, and the C-terminal transactivation domain. β-cat2 conserves the N-terminal α-catenin binding motif, the interacting platform for the cadherin complex in the central armadillo repeats but not for Wnt signaling elements, and the C-terminal PDZ domain. The newly identified β-cat3 and 4 conserve the GSK3 phosphorylation sites, the binding surface for Wnt signaling components and a partial conservation of the cell adhesion elements, whereas they have lost the C-terminal transactivation domain. (<b>B)</b> HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated (IP) with FLAG-M2 beads. Western blotting (IB) with anti-Myc revealed co-IP of β-cat3 and 4 following FLAG-M2 immunoprecipitation, indicating that both of them interact with TCF and β-Trcp. β-cat2 was analyzed as a negative control. <b>(C)</b> Localization of β-cat3 and 4 alone or co-expressed with Axin in transfected HeLa cells. In HeLa cells, β-cat3, which localized mainly in the nucleus alone, was recruited to the cytoplasm when co-transfected with Axin. β-cat4, which was more widely dispersed in the cytoplasm, was recruited by Axin and had a punctate distribution. Scale bar = 20 μm. <b>(D)</b> TOPflash reporter assay following co-transfection of HEK293T cells with β-cat1, β-cat2, β-cat3, β-cat4 and reporter plasmids.10 or 30 ng were transfected of each β-catenin. Just β-cat1 but not β-cat3 or 4 activated Wnt/β-catenin signaling. β-cat2 does not show Wnt reporter activity, as reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007030#pgen.1007030.ref040" target="_blank">40</a>]. <b>(E)</b> TOPflash reporter assay following co-transfection of HEK293T cells with β-cat1 (20 ng), β-cat2 (10, 20, 30 or 60 ng), β-cat3 (10, 20, 30 or 60 ng), β-cat4 (10, 20, 30 or 60 ng) and reporter plasmids. The co-transfection of β-cat3 or 4 inhibited β-cat1 reporter activation in a dosage dependent manner. Immunoblot analysis shows the protein expression level of β-cat1/2/3/4 in each line. <b>(F)</b> HEK293T cells were transfected with the indicated plasmids, and lysates were immunoprecipitated (IP) with FLAG-M2 beads. Western blot was performed with anti-Myc and anti-Flag antibodies. Upon co-transfection of β-cat1 and β-cat4, the amount of immunoprecipitated β-catenins by TCF was less than that during sole transfection of either, supporting that they compete with each other for TCF binding. The relative protein levels of precipitated β-catenins by TCF were quantified and normalized against total β-catenins in WCL (whole cell lysates). Quantitative results of relative binding of β-cat1 or β-cat4 alone with respect to β-cat1+β-cat4 derived from four independent experiments. β-cat1, 0.63±0.19; β-cat4, 0.51±0.14 (SD; n = 4). **p<0.05, ***p<0.001 (t test). Relative binding of β-cat1 or β-cat4 alone with respect to β-cat1+β-cat2 or β-cat4+β-cat2, respectively, was measured as a negative control. β-cat1, 1.11; β-cat4, 0.91 (n = 1). Blue asterisk indicates non-specific bands.</p

Possible Models for β-cat1 and β-cat4 transcriptional activity in planarians.

<p><b>(A)</b> β-cat4 could act as a dominant negative form of β-catenin, which is able to bind to TCF-2 but not to activate transcription, due to the missing C-terminal domain. <b>(B)</b> β-cat4 could be able to directly repress transcriptional targets through binding to TCF-2. The balance between β-cat1/TCF-2 transcriptional activation and β-cat4/TCF-2 transcriptional repression would determine the amount of photoreceptor cells. <b>(C)</b> Both β-cat1/TCF-2 and β-cat4/TCF-2 complexes could bind to different TCF responsive elements in the same promoter. The proper transcriptional regulation of photoreceptor targets is achieved by the combinatory action of both complexes.</p

<i>TCF-2</i> mediates <i>β-cat1</i> and <i>β-cat4</i> signaling.

<p><b>(A)</b> TCF-2 conserves the characteristic domains of <i>β-catenin</i> and DNA binding (HMG domain). <b>(B)</b> Expression of TCF-2 in the CNS and in the photoreceptors (yellow arrows in the magnification) <b>(C)</b> Live images and FISH of <i>opsin</i> (red) and <i>tph</i> (green) of <i>TCF-2</i> (RNAi) and <i>GFP</i> (RNAi) planarians at 7 days of regeneration, with the respective quantification of the <i>opsin+</i> and <i>tph+</i> cells per eye. <i>opsin+</i> cells in control R7d, 35.60±4.17 (SD; n = 10 eyes); <i>TCF-2</i> (RNAi) R7d, 50.8±4.21 (SD; n = 10 eyes). <i>tph+</i> cells in control R7d, 15.7±1.06 (SD; n = 10 eyes); <i>TCF-2</i> (RNAi) R7d, 20.70±2.06 (SD; n = 10 eyes). ***p<0.001 (t test). Anterior is to the top. <b>(D)</b> Double knockdown assay of <i>β-cat4</i> (RNAi) and <i>TCF-2</i> (RNAi). Live images and FISH of <i>opsin</i> (red) and <i>tph</i> (green) to show planarian regenerated eyes after the indicated RNAi treatment. The respective quantification of <i>opsin</i>+ and <i>tph</i>+ cells is shown. <i>opsin</i>+ cells in <i>GFP</i> (RNAi), 39.70±4.47 (SD; n = 10 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi), 21.60±4.48 (SD; n = 10 eyes); <i>TCF-2</i>;<i>GFP</i> (RNAi), 64.64±8.68 (SD; n = 10 eyes); <i>APC-1</i>;<i>β-cat4</i> (RNAi), 63.50±4.32 (SD; n = 12 eyes). <i>tph</i>+ cells in <i>GFP</i> (RNAi), 17.60±2.01 (SD; n = 10 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi), 12.89±2.15 (SD; n = 10 eyes); <i>TCF-2</i>;<i>GFP</i> (RNAi), 22.00±3.49 (SD; n = 10 eyes); <i>APC-1</i>;<i>β-cat4</i> (RNAi), 21.58±2.27 (SD; n = 12 eyes). **p<0.01, ***p<0.001 (t test). Scale bars, 250 μm (B), 50 μm (C, D).</p

<i>β-cat4</i> specifies photoreceptor cells through <i>β-cat1</i> inhibition.

<p><b>(A)</b> β-cat1 protein (green) localizes to the nucleus of photoreceptors (image corresponds to intact <i>S</i>. <i>polychroa</i>, syster species of <i>S</i>. <i>mediterranea</i>). β-cat4 protein (green) and <i>β-cat4</i> mRNA (red) colocalize in photoreceptor cells, where the protein is found in the nucleus (images correspond to intact <i>S</i>. <i>mediterranea</i>) <b>(B)</b> Eye phenotype of <i>β-cat1</i> (RNAi) animals at 15 days of regeneration. Live images and FISH of <i>opsin</i> (red) and <i>tph</i> (green). <i>β-cat1</i> (RNAi) animals showed elongated and disorganized eyes, with the appearance of ectopic photoreceptor cells in 50% of the eyes analyzed (yellow arrows in the right image). The respective quantification of <i>opsin</i>+ and <i>tph</i>+ cells per eye is shown. Ectopic cells were not included in the analysis. <i>opsin</i>+ cells in control R15d, 49.25±4.37 (SD; n = 12 eyes); <i>β-cat1</i> (RNAi) R15d, 72.50±5.21 (SD; n = 6 eyes). <i>tph</i>+ cells in control R15d, 18.00±2.26 (SD; n = 12 eyes); <i>β-cat1</i> (RNAi) R15d, 20.33±1.86 (SD; n = 6 eyes). *p<0.05, ***p<0.001 (t test). <b>(C)</b> Double knockdown of <i>APC-1</i> (RNAi) and <i>β-cat4</i> (RNAi) in intact animals. Live images and FISH of <i>opsin</i> (red) and <i>tph</i> (green) after the indicated RNAi treatments with the respective quantification of <i>opsin</i>+ and <i>tph</i>+ cells per eye. <i>opsin</i>+ cells in <i>GFP</i> (RNAi), 48.17±4.80 (SD; n = 6 eyes); <i>APC-1</i>;<i>GFP</i> (RNAi), 38.13±3.76 (SD; n = 8 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi), 30.17±2.40 (SD; n = 6 eyes); <i>APC-1</i>;<i>β-cat4</i> (RNAi), 21.50±2.35 (SD; n = 6 eyes). <i>tph</i>+ cells in <i>GFP</i> (RNAi), 17.17±1.60 (SD; n = 6 eyes); <i>APC-1</i>;<i>GFP</i> (RNAi), 15.13±1.46 (SD; n = 8 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi),13.33±1.37 (SD; n = 6 eyes); <i>APC-1</i>;<i>β-cat4</i> (RNAi), 10.83±0.75 (SD; n = 6 eyes). *p<0.05, **p<0.01, ***p<0.001 (t test). <i>APC-1</i> (RNAi) and <i>β-cat4</i> (RNAi) caused smaller eyes than control. Notice that double RNAi of <i>APC-1</i> and <i>β-cat4</i> resulted in a more severe phenotype than each one alone. <b>(D)</b> Double knockdown of <i>β-cat1</i> (RNAi) and <i>β-cat4</i> (RNAi). FISH of <i>opsin</i> (red) and <i>tph</i> (green) after the indicated RNAi treatments in 9 days regenerating animals. The respective quantification of <i>opsin</i>+ and <i>tph</i>+ cells per eye is shown. <i>opsin</i>+ cells in <i>GFP</i> (RNAi), 39±1.7 (SD; n = 6 eyes); <i>β-cat1</i>;<i>GFP</i> (RNAi), 46.25±3.77 (SD; n = 8 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi), 27.67±4.03 (SD; n = 6 eyes); <i>β-cat1</i>;<i>β-cat4</i> (RNAi), 10±3.03 (SD; n = 6 eyes). <i>tph</i>+ cells in <i>GFP</i> (RNAi), 17±3.34 (SD; n = 6 eyes); <i>β-cat1</i>;<i>GFP</i> (RNAi), 21.12±1.96 (SD; n = 8 eyes); <i>β-cat4</i>;<i>GFP</i> (RNAi),13.33±1.37 (SD; n = 6 eyes); <i>β-cat1</i>;<i>β-cat4</i> (RNAi), 11±1.4 (SD; n = 6 eyes). *p<0.05, **p<0.01, ***p<0.001 (t test). <i>β-cat1</i> (RNAi) animals show large eyes with ectopic eye cells (yellow arrow); <i>β-cat4</i> (RNAi) animals show small eyes; and double <i>β-cat1</i>;<i>β-cat4</i> (RNAi) animals show smaller and more disorganized eyes than single <i>β-cat4</i> or <i>β-cat1</i> (RNAi) (white arrow indicates a row of delocalized photoreceptor and eye cells). Anterior is to the top. Scale bar = 20 μm (A), 50 μm (B, C,D).</p