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Discipline
Biological
Keywords
Hippo Pathway
Planarians
Cell Proliferation
Cell Dedifferentiation
Cell Fate
Observation Type
Standalone
Nature
Standard Data
Submitted
Mar 20th, 2019
Published
Jun 7th, 2019
  • Abstract

    In the last years, the Hippo signaling has emerged as an essential pathway to control tissue and organ homeostasis, and its deregulation leads to tumoral transformation in all animals studied. However, the underlying cellular and molecular mechanism of this transformation are still not clear. The core of the Hippo pathway includes a kinase cassette composed by the kinases Hippo, Salvador, Mats, and Warts that in turn phosphorylates the nuclear effector of the pathway, Yorkie, targeting it to proteasomal degradation. Recently, the functional study of hippo, in planarians, flatworms that continuously change their size and renew their tissues according to nutrients, allowed demonstrating that the underlying mechanism of cellular transformation was not an increase in cell proliferation but inducing cell cycle arrest and promoting cell dedifferentiation. However, the functional study of yorkie did not allow to relate it with the Hippo function. Here, we show that inhibition of salvador and warts phenocopies the defects observed in hippo RNAi planarians. Since Warts is responsible for phosphorylating yorkie, this data suggests that not only the kinase cassette of the Hippo pathway is functionally conserved but also the nuclear effector.

  • Figure
  • Introduction

    The Hippo signaling plays a central role in the control of essential cellular mechanisms required for proper cell turnover in all organisms, as stem-cell maintenance, cell differentiation, cell fate decisions, and cell survival. Although the downregulation of Hippo signaling is clearly related with the appearance of overgrowths and tumorigenesis, the underlying mechanism is still poorly understood. The core of the Hippo pathway is a kinase cassette, composed by the kinases Hippo, Salvador, Mats, and Warts. Warts, in turn, phosphorylates the nuclear effector of the pathway, Yorkie, targeting it to proteasomal degradation. Thus, only when the Hippo kinases are not active Yorkie is not phosphorylated and can enter the nucleus to regulate transcription. There are multiple upstream signals that regulate the pathway, most of them related to cell-cell and cell-matrix contact. Recently, the role of hippo during cell renewal has been functionally studied in planarians, flatworms well known for their abilities to regenerate any missing organ, even the head, in a few days. Planarians do not only regenerate but continuously renew their tissues since they grow and shrink in size according to nutrients throughout their lives. These features are due to the presence of a unique population of adult stem cells, together with the continuous activation of signaling cues that coordinate cell proliferation, cell death, and cell fate decisions, allowing planarians to maintain proportioned and functional organs during homeostasis and regeneration. This continuous active regulation of the stem cell and postmitotic cell compartments makes the planarian an ideal in vivo model to understand the mechanisms underlying homeostatic cell renewal. It was recently shown that inhibition of hippo in planarians promoted the appearance of undifferentiated regions and overgrowths. Cellular studies demonstrated that the underlying mechanism of overgrowth formation was not an increase in cell proliferation but in arresting the cell cycle, inhibiting apoptosis and promoting cell dedifferentiation. Due to the complex phenotype obtained, the functional study of yorkie did not allow to relate it with the Hippo function. Here, we show that inhibition of the core kinase elements salvador and warts promotes the appearance of undifferentiated regions and overgrowths, phenocopying the hippo RNAi phenotype. Since Warts is responsible to phosphorylate yorkie, this data suggests that not only the kinase cassette of the Hippo pathway is functionally conserved but also the nuclear effector.

  • Objective

    The specific objective is to check whether inhibition of the core kinase elements of Hippo pathway salvador and warts promotes the appearance of overgrowths in planarians, thus phenocopying the hippo RNAi phenotype. The general objective is to demonstrate that, despite the functional study of yorkie did not allow to demonstrate its role as an effector of the pathway in planarians, the main elements, including Warts, which directly regulates Yorkie nuclearization, are indeed conserved.

  • Results & Discussion

    The two genes of the Hippo pathway core kinase Smed-salvador and Smed-warts (for simplicity, salvador and warts) were identified in the Schmidtea mediterranea transcriptomes database (PlanMine) (Suppl. Info. 1). An alignment of the planarian sequences with the homologs of different model species demonstrates the sequence conservation (Suppl. Info. 2). In situ hybridization (ISH) showed that both genes are ubiquitously expressed since they do not show any tissue-specific pattern (Fig. 1A). An in silico search in a single cell database of planarians SCS also corroborates that they are present in several planarian cell types, as also found for hippo and yorkie genes (Suppl. Fig. 1).

    To inhibit the function of salvador and warts during homeostatic cell renewal in planarians, we injected dsRNA of both genes into planarians during 3 weeks in the ventral pre-pharyngeal region (see Methods and Fig. 1B). Planarians were starved during the experiments; thus they were shrinking in size. After the inhibition of salvador or warts, animals presented unpigmented regions, mainly around the body margin, which became bigger or evolved into unpigmented overgrowths (Fig. 1C and C’). Taking into account that, the region where were performed injections is not the same where we observe phenotype, and that control animals were also injected and did not present any phenotype, we can conclude that the injections are not related with the appearance of the overgrowths/unpigmented regions. The analysis of the expression pattern of genes associated with cell differentiation (arrestin and b-catenin2) revealed that salvador and warts (RNAi) animals are not able to properly maintain differentiated structures as the visual and the digestive system (labeled with arrestin (VC1) and b-catenin2, respectively (Fig. 1D). The eyes of these animals are smaller, and the optic chiasm is thinner and interrupted in the case of warts (RNAi). The digestive system is also thinner and interrupted in warts (RNAi) animals (Fig. 1D). The loss of differentiated structures in these animals could be due to the inability of cells to maintain the differentiated state, as previously shown for hippo RNAi animals.

    It has been shown that, despite not showing an increase in the number of cells, neither an increase in cell proliferation, hippo RNAi animals show an increase of cells in M phase. To test if this was also the case for salvador and warts (RNAi) animals, we analyzed the expression of pH3 with a specific antibody. The results show that the number of pH3+ cells was increased after three weeks of salvador and warts inhibition (Fig. 1E).

  • Conclusions

    The observation that salvador and warts (RNAi) animals show the same phenotype as that of hippo RNAi animals suggests that the kinase cassette of the Hippo pathway appears conserved in planarians. In salvador and warts (RNAi) animals the mitotic arrest and the inability of cells to maintain a stable differentiated fate could underlay the appearance of the overgrowths, as observed in hippo RNAi animals. Since Warts is the kinase dedicated to Yorkie phosphorylation, the results also suggest that Yorkie could be the effector of the pathway in planarians and that its activation leads to the observed phenotype.

  • Limitations

    To demonstrate that the Hippo kinase core elements regulate Yorkie phosphorylation, specific experiments would be required, for instances to test the levels of phosphorylated Yorkie in warts and Salvador RNAi planarians with respect to controls.

  • Alt. Explanations

    It would be possible that the kinase cassette of the Hippo pathway is conserved, and even that Yorkie also acts downstream of the pathway in planarians. However, it could also be that some of the defects observed in planarians depend on the activity of the kinases but not of Yorkie, since the Hippo kinase cassette has been demonstrated to exert several Yorkie independent function.

  • Conjectures

    It would be necessary to characterize more in deep the cellular origin of the overgrowths, and if their appearance depends on the activation of Yorkie.

  • Methods

    Planarian culture

    Asexual planarians from a clonal strain of S. mediterranea BCN-10 were maintained at 20°C in PAM water (planarian artificial medium) as described. Animals were fed with veal liver and starved for at least 1 week before starting the experiments.

    RNA interference analysis

    dsRNA was synthesized by in vitro transcription (Roche) and microinjection performed as previously described, following the standard protocol of 3×32 nl injection of dsRNA for 3 consecutive days. To achieve the inhibition of salvador and warts, we performed this protocol during 3 consecutive weeks in starved planarians.

    Whole-mount in situ hybridization

    Sense and anti-sense RNA probes were synthesized in vitro using Sp6 or T7 polymerase (Roche) and DIG-modified (Perkin Elmer) nucleotides. RNA probes were purified and precipitated with ethanol and 7.5 M ammonium acetate. For in situ hybridization, animals were fixed and processed as previously described. After probe development, animals were mounted with 70% glycerol in PBS.

    Whole-mount immunostaining

    Immunostaining was performed as described. The following antibodies were used: rabbit anti-phospho-histone-H3-Ser10 (anti-H3P) (1:500; Cell Signaling Technology) and rabbit anti-β-catenin2 (anti-Bcat2) (; 1:2000). To avoid technical variance and obtain a reliable quantification of H3P+cells, at least 2 independent experiments were performed.

    Imaging

    Immunostained samples were imaged using a MZ16F stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik). Images were processed using Fiji software. Quantifications were performed by hand using the “multi-point selection” tool of Fiji. Data were analyzed by Student t-test.

  • Funding statement

    Universitat de Barcelona (APIF fellowship). Received by NdS. FPI grant number BES-2015-071578. Received by EP-C. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Ministerio de educación y ciencia (grant number BFU2017-83755-P and BFU2014-56055-P). Received by ES and TA. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. AGAUR (Generalitat de Catalunya) (grant number 2009SGR1018).

  • Acknowledgements

    We acknowledge all the members of ES Lab.

  • Ethics statement

    Not Applicable.

  • References
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    Matters14.5/20

    Functional characterization of salvador and warts in planarians

    Affiliation listing not available.
    Abstractlink

    In the last years, the Hippo signaling has emerged as an essential pathway to control tissue and organ homeostasis, and its deregulation leads to tumoral transformation in all animals studied. However, the underlying cellular and molecular mechanism of this transformation are still not clear. The core of the Hippo pathway includes a kinase cassette composed by the kinases Hippo, Salvador, Mats, and Warts that in turn phosphorylates the nuclear effector of the pathway, Yorkie, targeting it to proteasomal degradation. Recently, the functional study of hippo, in planarians, flatworms that continuously change their size and renew their tissues according to nutrients, allowed demonstrating that the underlying mechanism of cellular transformation was not an increase in cell proliferation but inducing cell cycle arrest and promoting cell dedifferentiation. However, the functional study of yorkie did not allow to relate it with the Hippo function. Here, we show that inhibition of salvador and warts phenocopies the defects observed in hippo RNAi planarians. Since Warts is responsible for phosphorylating yorkie, this data suggests that not only the kinase cassette of the Hippo pathway is functionally conserved but also the nuclear effector.

    Figurelink

    Figure 1. Functional characterization of salvador and warts in planarians.

    (A) In situ hybridization for salvador and warts with sense and anti-sense probe. Anti-sense probe reveals a ubiquitous expression pattern.

    (B) Cartoon illustrating the experimental design used for salvador and warts RNAi during planarian homeostasis. Animals were starved for 1 week before the experiment and were then injected on 3 consecutive days each week for 3 weeks. Starvation was maintained throughout.

    (C) Stereomicroscopic image of live salvador (RNAi) and warts (RNAi) planarians showing the overgrowths and unpigmented regions in marginal regions of the body (n = 10). White arrows indicate overgrowths and white discontinuous boxes indicate unpigmented regions. (C’) Magnifications of unpigmented areas and overgrowths.

    (D) Analysis of unpigmented regions. Top panel: the visual system stained with anti-arrestin (VC-1). Bottom panel: digestive system labeled with the anti-Bcat2 antibody. White arrows indicate defects in the visual and digestive system.

    (E) Quantification of mitotic cells (H3P+) in salvador and warts (RNAi) animals (n ≥ 10). Error bars represent standard deviation. Data were analyzed by Student t-test. **p < 0.01. Scale bars: (A) and (D) 1 mm; (E) top panel 100 µm; (E) bottom panel 250 µm.

    Introductionlink

    The Hippo signaling plays a central role in the control of essential cellular mechanisms required for proper cell turnover in all organisms, as stem-cell maintenance, cell differentiation, cell fate decisions, and cell survival[1][2][3][4][5][6]. Although the downregulation of Hippo signaling is clearly related with the appearance of overgrowths and tumorigenesis, the underlying mechanism is still poorly understood[7][8][9][10]. The core of the Hippo pathway is a kinase cassette, composed by the kinases Hippo, Salvador, Mats, and Warts[11]. Warts, in turn, phosphorylates the nuclear effector of the pathway, Yorkie, targeting it to proteasomal degradation. Thus, only when the Hippo kinases are not active Yorkie is not phosphorylated and can enter the nucleus to regulate transcription[11]. There are multiple upstream signals that regulate the pathway, most of them related to cell-cell and cell-matrix contact[12]. Recently, the role of hippo during cell renewal has been functionally studied in planarians, flatworms well known for their abilities to regenerate any missing organ, even the head, in a few days[13]. Planarians do not only regenerate but continuously renew their tissues since they grow and shrink in size according to nutrients throughout their lives. These features are due to the presence of a unique population of adult stem cells, together with the continuous activation of signaling cues that coordinate cell proliferation, cell death, and cell fate decisions, allowing planarians to maintain proportioned and functional organs during homeostasis and regeneration[14][15][16]. This continuous active regulation of the stem cell and postmitotic cell compartments makes the planarian an ideal in vivo model to understand the mechanisms underlying homeostatic cell renewal[17][18]. It was recently shown that inhibition of hippo in planarians promoted the appearance of undifferentiated regions and overgrowths[19]. Cellular studies demonstrated that the underlying mechanism of overgrowth formation was not an increase in cell proliferation but in arresting the cell cycle, inhibiting apoptosis and promoting cell dedifferentiation[19]. Due to the complex phenotype obtained, the functional study of yorkie did not allow to relate it with the Hippo function[19][20]. Here, we show that inhibition of the core kinase elements salvador and warts promotes the appearance of undifferentiated regions and overgrowths, phenocopying the hippo RNAi phenotype. Since Warts is responsible to phosphorylate yorkie, this data suggests that not only the kinase cassette of the Hippo pathway is functionally conserved but also the nuclear effector.

    Objectivelink

    The specific objective is to check whether inhibition of the core kinase elements of Hippo pathway salvador and warts promotes the appearance of overgrowths in planarians, thus phenocopying the hippo RNAi phenotype. The general objective is to demonstrate that, despite the functional study of yorkie did not allow to demonstrate its role as an effector of the pathway in planarians, the main elements, including Warts, which directly regulates Yorkie nuclearization, are indeed conserved.

    Results & Discussionlink

    The two genes of the Hippo pathway core kinase Smed-salvador and Smed-warts (for simplicity, salvador and warts) were identified in the Schmidtea mediterranea transcriptomes database (PlanMine)[21] (Suppl. Info. 1). An alignment of the planarian sequences with the homologs of different model species demonstrates the sequence conservation (Suppl. Info. 2). In situ hybridization (ISH) showed that both genes are ubiquitously expressed since they do not show any tissue-specific pattern (Fig. 1A). An in silico search in a single cell database of planarians SCS[22] also corroborates that they are present in several planarian cell types, as also found for hippo and yorkie genes (Suppl. Fig. 1).

    To inhibit the function of salvador and warts during homeostatic cell renewal in planarians, we injected dsRNA of both genes into planarians during 3 weeks in the ventral pre-pharyngeal region (see Methods and Fig. 1B). Planarians were starved during the experiments; thus they were shrinking in size. After the inhibition of salvador or warts, animals presented unpigmented regions, mainly around the body margin, which became bigger or evolved into unpigmented overgrowths (Fig. 1C and C’). Taking into account that, the region where were performed injections is not the same where we observe phenotype, and that control animals were also injected and did not present any phenotype, we can conclude that the injections are not related with the appearance of the overgrowths/unpigmented regions. The analysis of the expression pattern of genes associated with cell differentiation (arrestin and b-catenin2) revealed that salvador and warts (RNAi) animals are not able to properly maintain differentiated structures as the visual and the digestive system (labeled with arrestin (VC1) and b-catenin2, respectively (Fig. 1D). The eyes of these animals are smaller, and the optic chiasm is thinner and interrupted in the case of warts (RNAi). The digestive system is also thinner and interrupted in warts (RNAi) animals (Fig. 1D). The loss of differentiated structures in these animals could be due to the inability of cells to maintain the differentiated state, as previously shown for hippo RNAi animals[19].

    It has been shown that, despite not showing an increase in the number of cells, neither an increase in cell proliferation, hippo RNAi animals show an increase of cells in M phase. To test if this was also the case for salvador and warts (RNAi) animals, we analyzed the expression of pH3 with a specific antibody. The results show that the number of pH3+ cells was increased after three weeks of salvador and warts inhibition (Fig. 1E).

    Conclusionslink

    The observation that salvador and warts (RNAi) animals show the same phenotype as that of hippo RNAi animals suggests that the kinase cassette of the Hippo pathway appears conserved in planarians. In salvador and warts (RNAi) animals the mitotic arrest and the inability of cells to maintain a stable differentiated fate could underlay the appearance of the overgrowths, as observed in hippo RNAi animals[19]. Since Warts is the kinase dedicated to Yorkie phosphorylation[11], the results also suggest that Yorkie could be the effector of the pathway in planarians and that its activation leads to the observed phenotype.

    Limitationslink

    To demonstrate that the Hippo kinase core elements regulate Yorkie phosphorylation, specific experiments would be required, for instances to test the levels of phosphorylated Yorkie in warts and Salvador RNAi planarians with respect to controls.

    Alternative Explanationslink

    It would be possible that the kinase cassette of the Hippo pathway is conserved, and even that Yorkie also acts downstream of the pathway in planarians. However, it could also be that some of the defects observed in planarians depend on the activity of the kinases but not of Yorkie, since the Hippo kinase cassette has been demonstrated to exert several Yorkie independent function[23].

    Conjectureslink

    It would be necessary to characterize more in deep the cellular origin of the overgrowths, and if their appearance depends on the activation of Yorkie.

    Methodslink

    Planarian culture

    Asexual planarians from a clonal strain of S. mediterranea BCN-10 were maintained at 20°C in PAM water (planarian artificial medium) as described[24]. Animals were fed with veal liver and starved for at least 1 week before starting the experiments.

    RNA interference analysis

    dsRNA was synthesized by in vitro transcription (Roche) and microinjection performed as previously described[25], following the standard protocol of 3×32 nl injection of dsRNA for 3 consecutive days. To achieve the inhibition of salvador and warts, we performed this protocol during 3 consecutive weeks in starved planarians.

    Whole-mount in situ hybridization

    Sense and anti-sense RNA probes were synthesized in vitro using Sp6 or T7 polymerase (Roche) and DIG-modified (Perkin Elmer) nucleotides. RNA probes were purified and precipitated with ethanol and 7.5 M ammonium acetate. For in situ hybridization, animals were fixed and processed as previously described[26]. After probe development, animals were mounted with 70% glycerol in PBS.

    Whole-mount immunostaining

    Immunostaining was performed as described[27]. The following antibodies were used: rabbit anti-phospho-histone-H3-Ser10 (anti-H3P) (1:500; Cell Signaling Technology) and rabbit anti-β-catenin2 (anti-Bcat2) ([28]; 1:2000). To avoid technical variance and obtain a reliable quantification of H3P+cells, at least 2 independent experiments were performed.

    Imaging

    Immunostained samples were imaged using a MZ16F stereomicroscope (Leica) equipped with a ProgRes C3 camera (Jenoptik). Images were processed using Fiji software. Quantifications were performed by hand using the “multi-point selection” tool of Fiji. Data were analyzed by Student t-test.

    Funding Statementlink

    Universitat de Barcelona (APIF fellowship). Received by NdS. FPI grant number BES-2015-071578. Received by EP-C. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. Ministerio de educación y ciencia (grant number BFU2017-83755-P and BFU2014-56055-P). Received by ES and TA. The funder had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. AGAUR (Generalitat de Catalunya) (grant number 2009SGR1018).

    Acknowledgementslink

    We acknowledge all the members of ES Lab.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not Applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

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