Peroxiredoxin5 Controls Vertebrate Ciliogenesis by Modulating Mitochondrial Reactive Oxygen Species

Abstract

Aims:

Peroxiredoxin5 (Prdx5), a thioredoxin peroxidase, is an antioxidant enzyme that is widely studied for its antioxidant properties and protective roles in neurological and cardiovascular disorders. This study is aimed at investigating the functional significance of Prdx5 in mitochondria and at analyzing its roles in ciliogenesis during the process of vertebrate development.

Results:

We found that several Prdx genes were strongly expressed in multiciliated cells in developing Xenopus embryos, and their peroxidatic functions were crucial for normal cilia development. Depletion of Prdx5 increased levels of cellular reactive oxygen species (ROS), consequently leading to mitochondrial dysfunction and abnormal cilia formation. Proteomic and transcriptomic approaches revealed that excessive ROS accumulation on Prdx5 depletion subsequently reduced the expression level of pyruvate kinase (PK), a key metabolic enzyme in energy production. We further confirmed that the promotor activity of PK was significantly reduced on Prdx5 depletion and that the reduction in PK expression and its promoter activity led to ciliary defects observed in Prdx5-depleted cells.

Innovation:

Our data revealed the novel relationship between ROS and Prdx5 and the consequent effects of this interaction on vertebrate ciliogenesis. The normal process of ciliogenesis is interrupted by the Prdx5 depletion, resulting in excessive ROS levels and suggesting cilia as vulnerable targets of ROS.

Conclusion:

Prdx5 plays protective roles in mitochondria and is critical for normal cilia development by regulating the levels of ROS. The loss of Prdx5 is associated with excessive production of ROS, resulting in mitochondrial dysfunction and aberrant ciliogenesis.

Introduction

One of the major subjects in recent biological research concerns the regulation of excessive reactive oxygen species (ROS) in the body. ROS play important roles in many distinct biological processes, including mitochondrial function, the immune system, and regeneration (32). However, elevated ROS levels can result in serious human disorders, such as cancers, cardiovascular diseases, and neurodegenerative diseases (5, 13, 30). ROS are mostly produced in the mitochondria, and many antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, catalases, and peroxiredoxins (Prdxs), have evolved to protect cellular components from ROS-mediated cellular damage (32).

Prdxs are known to be widely expressed in human tissues. Currently, six Prdx genes have been identified in mammals, including humans. There are three different types of Prdxs, which are classified according to their catalytic active sites. Prdx6 is a one-cysteine Prdx that uses a single cysteine residue for catalytic activity. Prdx1–4 are two-cysteine Prdxs and use two cysteine residues for their peroxidatic and resolving reactions. Finally, Prdx5 is an atypical two-cysteine Prdx that uses two active cysteine residues in atypical locations. Prdx5 is known to protect cells from ROS by preferentially removing hydrogen peroxide (H₂O₂) and alkyl hydroperoxides (12, 16, 28). Other studies reported the localization of Prdx5 to different cellular organelles, that is, peroxisomes and mitochondria, and play a role in protecting them against neurological damage induced by excessive ROS (4, 17, 23, 38).

Innovation

Our study reveals the novel interaction between peroxiredoxin5 (Prdx5) and reactive oxygen species (ROS) that are involved in the process of ciliogenesis, thoroughly characterizes the ciliary defects induced by Prdx5 depletion, and reveals the relationship between ciliogenesis and ROS levels. Our data show that cilia are key and vulnerable cellular targets that can be damaged by excessive ROS. Further, we show conclusive evidence that elevated ROS levels affect cilia formation by reducing pyruvate kinase levels, which has not been previously demonstrated.

We believe that our study is the first intensive report on ROS-mediated ciliary defects and their underlying mechanisms and that it will likely be of significant interest for future prospects.

Cilia are highly conserved cellular structures that are widely distributed from unicellular organisms to complex multicellular organisms, including humans, and they carry out a variety of functions (27). In mammals, there are mainly two types of cilia, which are categorized based on their structure and function.

Most cells in resting stages possess a single cilium stemming from the mother centriole. These monocilia are often known as primary cilia and are mainly involved in signal reception or sensing in various tissues. Multicilia constitute another type of cilia that are responsible for motility. A single multiciliated cell may produce hundreds of cilia on its apical surface. Multicilia mainly exist in mucociliary epithelia, such as the airway epithelium and fallopian tubes, in which they generate a mucus fluid-flow over the epithelium, either to protect the tissues of the airway or to facilitate the migration of fertilized eggs through the fallopian tubes, respectively (7). In addition to their functional differences, primary cilia and multicilia also possess structural differences. For example, the axonemes of primary cilia have nine doublets of microtubules (9 + 0), whereas those of multicilia have an extra pair of central microtubules (9 + 2).

Recent studies have shown that ROS such as H₂O₂ function not only as agents that cause damage to cellular components, that is, DNA, proteins, and lipids, but also as intracellular messengers in multiple signaling cascades (28). Although it is considerably well understood how excessive ROS transduce a damage response to cellular organelles such as the endoplasmic reticulum (ER), peroxisome, and mitochondria, the consequences of excessive ROS levels in cilia require more detailed investigations.

In this study, we investigated the roles of Prdxs in ciliogenesis during the process of embryonic development. We observed that several Prdx genes were highly expressed in multiciliated cells and that Prdx5 depletion resulted in excessive ROS production. Consequently, excessive ROS levels disrupted both normal mitochondrial function and the process of ciliogenesis. In addition, silencing of Prdx5 resulted in considerable upregulation of phosphorylation of several signaling molecules such as AKT, ERK, JNK, eIF2α, and p38, revealing that the depletion of Prdx5 induces mitochondrial dysfunctions and ROS-mediated cellular responses. Finally, our results provided compelling evidences that a reduction in pyruvate kinase (PK) levels might be the direct cause of ciliary defects in Prdx5-depleted cells.

Results

Prdx5 is predominantly expressed in multiciliated cells

To investigate the functions of Prdx genes during vertebrate development, we examined the expression profiles of all six members of the Prdx family in developing Xenopus embryos. Prdx genes displayed dynamic expression patterns in developing Xenopus embryos, as revealed by whole-mount in situ hybridization (WISH; Supplementary Fig. S1. Prdx1 and Prdx3 were mainly expressed in the brain and developing kidney (Supplementary Fig. S1) (8), whereas Prdx2 was expressed in the ventral blood island and brain (Supplementary Fig. S1). Prdx4 displayed a low level of expression in the kidney and eyes (Supplementary Fig. S1), and Prdx5 was predominantly expressed in multiciliated cells (Fig. 1A), whereas Prdx6 was expressed in migrating myeloid cells (Supplementary Fig. S1).

FIG. 1.

Prdx5 is required for multicilia formation. (A) Prdx5 expression was determined by processing the developing Xenopus embryos at stage 33 for WISH analysis. The predominant Prdx5 expression is observed in the multiciliated cells in the embryonic epidermis of Xenopus. (B) Temporal expression pattern of Prdx5 was analyzed by reverse transcriptase-polymerase chain reaction. Prdx5 is a maternal gene, the expression of which increased from the late neurula stage. (C) Prdx5-Flag mRNA was co-injected with either Centrin-GFP or CLAMP-GFP mRNA into embryos, and Centrin-GFP, CLAMP-GFP, and Prdx5-Flag fusion proteins are visualized by confocal microscopy. Prdx5 does not co-localize with basal bodies but is enriched in the mitochondrial-rich zone in multiciliated cells. (D) hTERT-RPE1 cells were immunostained by using an anti-flag antibody to detect the expression of Prdx5-Flag. Co-staining of cells with Mito-Tracker and DAPI indicated that Prdx5-Flag (red) co-localized with Mito-Tracker (green) in hTERT-RPE1 cells. Nuclei are stained with DAPI and shown in blue. DAPI, 4′,6′-diamidino-2-phenylindole dihydrochloride; mRNA, messenger RNA; Prdx5, peroxiredoxin5; WISH, whole-mount in situ hybridization. Color images are available online.

The mucociliary epithelium functions as the first line of defense against external contaminants or pathogens. In the airway epithelium of mammals, mucociliary cells line the airway to protect the respiratory system (37). In Xenopus, the mucociliary epithelium exists in the embryonic epidermis to protect developing embryos.

The Xenopus mucociliary epidermis is an emerging model system for studying the physiology of the mucociliary epithelium (7, 34). Therefore, we exploited the Xenopus embryonic epidermis to study the biological functions of Prdxs in multiciliated cells. Among the Prdx genes, Prdx1 and Prdx5 were strongly expressed in multiciliated cells. Then, we conducted knockdown studies to investigate the significance of Prdx1 and Prdx5 in cilia formation. Among Prdx1 and Prdx5, the knockdown of Prdx1 did not significantly affect multicilia formation (data not shown); therefore, Prdx5 was selected for further studies to evaluate its roles in multicilia formation. Prdx5 expression increased sharply at embryonic stage 25, at which multicilia began to form (Fig. 1B). The subcellular localization of Prdx5 in multiciliated cells was investigated by expressing Flag-tagged Prdx5 (Prdx5-Flag). Prdx5 localized to the mitochondria-rich sub-apical region (i.e., beneath the basal bodies) of multiciliated cells (Fig. 1C). Moreover, when Prdx5-Flag was expressed in hTERT-RPE1 cells that were subsequently stained with Mito-Tracker, Prdx5 localized to the mitochondria (Fig. 1D), consistent with the role of Prdx5 in mitochondrial function (6, 17, 20).

Prdx5 depletion causes severe malformation of multicilia, and the Prdx5 catalytic cysteines are required for cilia formation

We further investigated Prdx5 function in multiciliated cells by inhibiting Prdx5 expression via an injection of antisense morpholino oligonucleotides (Prdx5-MO) into two-cell-stage Xenopus embryos. Prdx5-MO effectively inhibited the translation of wild type (WT) Prdx5 messenger RNA (mRNA), whereas mRNAs with a four-base pair mismatch (Prdx5-4MT) were not affected by Prdx5-MO, indicating the specificity of Prdx5-MO for Prdx5 transcripts (Fig. 2A).

FIG. 2.

Prdx5 knockdown prevents primary cilia formation. (A) The specificity and efficacy of Prdx5-MO were confirmed by co-injecting Prdx5-MO with WT Prdx5 mRNA or MO-resistant mRNA containing four-point mutations (Prxd5-4MT) into two-cell-stage Xenopus embryos. Prdx5-MO inhibits Prdx5 translation, whereas Prdx5-4MT expression is not affected by Prdx5-MO. (B) Prdx5-MO was injected into developing Xenopus embryos, and cilia were visualized by immunostaining (upper panel, red) or scanning EM (lower panel). Prdx5 knockdown disrupts multicilia formation in Xenopus embryonic epidermis, and the ciliary defects are effectively rescued by Prdx5-MO-resistant Prdx5 mRNA. (C) Clamp-GFP and Centrin-RFP mRNAs were co-injected with Prdx5-MO, and basal body maturation was analyzed by confocal microscopy. Prdx5 depletion impedes the docking and planar polarization of basal bodies in Xenopus multiciliated cells compared with those of control embryos. (D) Xenopus embryos are fixed at stage 26 and processed further to analyze the number and localization of basal bodies in control and Prdx5-depleted Xenopus multiciliated cells by TEM. Representative TEM images showing cytosolic basal bodies in Prdx5-depleted multiciliated cells failed to properly dock to the apical cell membrane as compared with control embryos. (E) The catalytic mutants of Prdx5 are generated by changing peroxidatic cysteine, resolving cysteine or both into serine to determine the significance of Prdx5 catalytic activity for ciliogenesis. (F) WT or mutant Prdx5 were ectopically expressed in Prdx5 morphant embryos. WT Prdx5 rescued the ciliary defects, whereas the catalytic mutants failed to rescue the phenotype. Cilia and cell membrane are shown in red and green, respectively. 4MT, four-base pair mismatch; Cp, peroxidatic cysteine; Cr, resolving cysteine; EM, electron microscopy; MO, morpholino oligonucleotides; TEM, transmission electron microscopy; WT, wild type. Color images are available online.

Next, we observed multicilia formation in Prdx5-MO-injected embryos (Prdx5 morphants) by immunofluorescent and electron microscopy (EM) imaging. Consistent with the results showing multiciliated cell-specific expression, MO-mediated depletion of Prdx5 in developing Xenopus embryos almost completely abolished multicilia formation in the embryonic mucociliary epidermis (Fig. 2B and Supplementary Fig. S5A). Moreover, the cilia-driven fluid-flow assay revealed that the velocity of the fluid-flow on the surface of the epidermis was significantly reduced in Prdx5 morphant embryos (Supplementary Fig. S2A, B; Supplementary Video S1A, B).

Ciliogenesis in multiciliated cells begins with the de novo formation of basal bodies, with hundreds of basal bodies docking to the apical surface of cells and initiating axoneme assembly (9, 25). Therefore, we analyzed the number and localization of basal bodies in multiciliated cells. Both confocal and EM imaging revealed that the numbers of basal bodies were similar between control and Prdx5 morphant embryos (data not shown); however, most of the basal bodies, which are marked by Centrin-GMP and CLAMP-RFP, in the cells of Prdx5 morphants failed to move apically for axoneme assembly (Fig. 2C, D).

Prdx5 catalyzes the reduction of H₂O₂, and two cysteine residues in the catalytic active site are critical for its reductase activity (16). To determine whether Prdx5 catalytic activity is necessary for ciliogenesis, we co-injected embryos with Prdx5-MO and Prdx5 catalytic mutants (in which the peroxidatic cysteine, resolving cysteine, or both cysteines were changed to serine; Fig. 2E). The embryos injected with Prdx5 mutants failed to rescue the ciliary defects (Fig. 2F) as compared with the embryos injected with WT Prdx5, which affectively reversed the ciliary defects. These data indicate that Prdx5 is critical for multicilia formation, presumably by affecting ciliogenesis after de novo formation of basal bodies. In addition, our data indicate that the catalytic activity of Prdx5 is necessary for cilia formation.

Prdx5 is necessary for primary cilia formation

We next examined the role of Prdx5 in primary cilia formation during the embryonic development. Xenopus embryos are known to generate primary cilia in the luminal surface of the neural tube. We observed that Prdx5 knockdown in Xenopus embryos disrupted primary cilia formation in the neural tube (Fig. 3A). In addition, Prdx5 depletion inhibited the convergent extension of neural tissue and resulted in a shortened body axis in neurula stage embryos (Fig. 3D, E), which was a previously reported consequence of defective cilia formation during embryonic development (24, 29).

FIG. 3.

Prdx5 is necessary for primary cilia formation. (A) Prdx5 depletion disrupted primary cilia formation in the neural tube in Xenopus embryos. Primary cilia in the neural tube (red arrows) are stained with the anti-acetylated tubulin antibody. (B) Prdx5 or control siRNA (10 nM each) was transfected into hTERT-RPE1 cells, and cilia were stained with an anti-acetylated tubulin antibody and DAPI. Prdx5 knockdown inhibits primary cilia formation in serum-starved hTERT-RPE1 cells. (C) Graphical representation of ciliated cells in control and Prdx5-transfected hTERT-RPE1 cells indicated that the percentage of ciliated cells in Prdx5 knockdown cells is significantly reduced as compared with the control cells. ***p < 0.001. (D) Neural tube closure was analyzed by WISH using Pax3 and Sox3 probes. Prdx5 depletion inhibited convergent extension, thereby disrupting neural tube closure in the Prdx5 morphant embryos. (E) The graphs showing the average neural fold widths and areas in control and Prdx5 morphant embryos. The relative width of neural fold was considerably reduced in Prdx5 morphants as compared with the control MO-injected embryos. Moreover, the graph showing the relative distance between the neural folds is significantly affected by Prdx5 knockdown. *p < 0.05; ***p < 0.001. siRNA, small interfering RNA. Color images are available online.

We further investigated the role of Prdx5 in primary cilia formation in hTERT-RPE1 cells after transfection with Prdx5 small interfering RNA (siRNA). Prdx5 knockdown in hTERT-RPE1 cells effectively decreased Prdx5 expression (Supplementary Fig. S3A), and Prdx5 depletion significantly reduced the number of ciliated cells (Fig. 3B, C). These data indicate that Prdx5 is necessary for primary cilia formation and that the function of Prdx5 in ciliogenesis is conserved among vertebrates.

Ciliary defects on Prdx5 depletion results from excessive ROS

Next, we sought to understand the molecular mechanisms underlying the ciliary defects observed after Prdx5 depletion. Prdx5 is known to eliminate mitochondrial ROS by scavenging peroxides, such as H₂O₂, organic hydroperoxides, and peroxynitrite, all of which can irreversibly harm cellular organelles and cells (3, 12). We speculated that an excess of ROS resulting from Prdx5 depletion may impair the ciliogenesis process. Indeed, Prdx5 knockdown significantly increased ROS levels in hTERT-RPE1 cells, and MitoQ, which is known to reduce ROS levels (15), effectively reduced the corresponding elevated ROS levels (Fig. 4A).

FIG. 4.

Ciliary defects on Prdx5 depletion result from excessive ROS. (A) The endogenous ROS level on Prdx5 depletion in hTERT-RPE1 cells was analyzed by flow cytometry. Prdx5 knockdown significantly increased ROS levels, and MitoQ reduced the elevated ROS levels induced by Prdx5 depletion in hTERT-RPE1 cells. (B) Xenopus embryos were fixed at stage 33 to analyze the expression of Trx2 in multiciliated cells, and strong expression of Trx2 was observed in multiciliated cells. Moreover, the Xenopus embryos were co-injected with Trx2 MO or Trx2 MO along with Prdx5 RNA. The depletion of Trx2 caused ciliary defects in Trx2 morphants, but these ciliary defects were rescued by the ectopic expression of Prdx5 in Trx2 morphants. (C) Prdx5-depleted hTERT-RPE1 cells were artificially quenched for ROS levels by treating them with MitoQ (100 nM). MitoQ treatment in Prdx5-depleted hTERT-RPE1 cells restored cilia formation. (D) The graphical representation of ciliated cells indicated that artificial quenching of ROS significantly restored the number of ciliated cells in MitoQ-treated Prdx5-depleted hTERT-RPE1 cells as compared with the untreated Prdx5-depleted hTERT-RPE1 cells. *p < 0.05; **p < 0.01. (E) hTERT-RPE1 cells were treated with GOX (1 mM) for the induction of ROS and then, the GOX-treated cells were stained with Mitosox Red. Cilia formation is significantly reduced in GOX-treated hTERT-RPE1 cells as compared with the control cells. (F) Graphical representation of ROS levels in GOX-treated hTERT-RPE1 cells. The percentage of Mitosox red fluorescence indicated the induction of ROS production in GOX-treated cells at specific time intervals. *p < 0.05. (G) Graph showing that percentage of ciliated cells in GOX-treated hTERT-RPE1 cells is remarkably reduced as compared with the control cells. ***p < 0.001. Data are presented as mean ± standard deviation. GOX, glucose oxidase; ROS, reactive oxygen species; Trx, thioredoxin. Color images are available online.

Since Prdxs function in Prdx/Trx (thioredoxin) systems to remove excessive H₂O₂ and need to be reduced by Trx for the next round of antioxidant reactions (2), we speculated that depletion of Trx would also cause ciliary defects and that these ciliary defects would be rescued by the overexpression of Prdx5. To test this speculation, we observed the expression of Trx2 in multiciliated cells and found that it was highly expressed in multiciliated cells. Moreover, depletion of Trx2 caused similar ciliary defects as Prdx5 depletion (Fig. 4B). We also observed that these defects were rescued by the overexpression of Prdx5, induced by injecting Prdx5 mRNA (Fig. 4B). Our findings suggest that Trx, in conjunction with Prdx5, has a significant role in cilia formation.

To confirm that the enhanced ROS levels were the major cause of ciliary dysfunction, we artificially quenched ROS by treating hTERT-RPE1 cells with MitoQ (Fig. 4A).We found that the percentage of ciliated cells was restored by MitoQ treatment of Prdx5-depleted hTERT-RPE1 cells (Fig. 4C, D). Further, artificial induction of excessive ROS levels via treatment with glucose oxidase (GOX) (1) inhibited primary cilia formation in hTERT-RPE1 cells (Fig. 4E–G). Taken together, our results indicate that excessive ROS are the major underlying causes of the ciliary defects observed after Prdx5 depletion. Thus, we suggest that Prdx5 regulates mitochondrial ROS levels and plays a significant role in cilia formation.

Prdx5 depletion causes mitochondrial dysfunction

We further investigated the underlying mechanism by which excessive ROS disrupt ciliogenesis in hTERT-RPE1 cells. Prdx5 mainly localizes to the mitochondria and protects them from oxidative damage (3). This prompted us to examine mitochondrial function after Prdx5 depletion. Using the membrane potential indicator JC-1, we observed that the mitochondrial membrane potential and adenosine triphosphate (ATP) production were dramatically reduced after Prdx5 knockdown in hTERT-RPE1 cells (Fig. 5A, B). Prdx5 knockdown also induced outer mitochondrial membrane blebbing (Fig. 5C, D) and reduced the number of mitochondria in Prdx5 morphant embryos (Fig. 5E). Further, the mitochondrial defects observed in Prdx5 morphants were recovered by a co-injection of Prdx5 mRNA with Prdx5-MO (Fig. 5C–E).

FIG. 5.

Prdx5 depletion causes mitochondrial dysfunction. (A) The membrane potential of the mitochondrial inner membrane was analyzed by JC-1 after Prdx5 knockdown. Prdx5 depletion strongly depolarized the mitochondrial membrane potential as compared with the control cells. (B) Luciferase-based ATP assay was performed to measure the endogenous ATP level in Prdx5-depleted cells and control cells. ATP levels were significantly lower in Prdx5-depleted cells as compared with control cells. **p < 0.01. (C) Mitochondrial morphology in Prdx5-depleted cells was analyzed by TEM. Mitochondrial morphology was abnormal in Prdx5-depleted cells. Yellow stars indicate normal mitochondria, whereas red arrows indicate abnormal mitochondria. (D) Graph showing the percentage of abnormal mitochondria in ciliated cells of Prdx5 morphants as compared with the ciliated cells of control embryos. Moreover, the percentage of abnormal mitochondria was significantly rescued in ciliated cells of Prdx5* injected embryos. (E) Graphical representation of number of mitochondria in the unit area indicated that Prdx5 depletion increased the number of abnormal mitochondria and reduced the total number of mitochondria. The number of abnormal mitochondria and total number of mitochondria were considerably rescued in Prdx5* injected embryos. *p < 0.05. ATP, adenosine triphosphate. Color images are available online.

The induction of mitochondrial dysfunction by excessive ROS is known to trigger several downstream signaling cascades, such as apoptosis, autophagy, and stress responses (14, 31). Therefore, we examined several downstream signaling pathways affected by mitochondrial dysfunction in Prdx5-depleted cells. Prdx5 depletion did not affect the activation of the cell death signaling, such as AMPK, caspase-3, or RIP1/3 (Fig. 6).

FIG. 6.

Prdx5 siRNA transfection did not significantly affect cell death pathways. (A, B) Prdx5 depletion did not affect the phosphorylation of AMPK or caspase-3 activation in hTERT-RPE1 cells. However, Prdx5 depletion significantly increased the phosphorylation of JNK, AKT, ERK, eIF2α, and p38 during cilia formation. (C) Graphs indicating that Prdx5 depletion did not induce necrotopsis pathways as the relative expression of RIP1 and MLKL were not affected in Prdx5-depleted cells. Full immunoblot shown in Supplementary Figure S5B. ⁺ p < 0.05; ⁺⁺ p < 0.01; **p < 0.001; n.s., not significant. FBS, fetal bovine serum.

However, Prdx5 siRNA transfection significantly upregulated the phosphorylation of several signaling molecules, such as AKT, ERK, JNK, eIF2α, and p38 (Fig. 6A, B, and Supplementary Fig. S5D). Among these changes, eIF2α phosphorylation was previously shown to globally reduce general translation as a mitochondrial stress response (40), and this is consistent with our finding that loss of Prdx5 induces significant mitochondrial dysfunction. In addition, AKT, ERK, JNK, and p38 have been shown to be activated by excessive ROS such as H₂O₂ (36, 39). Overall, these data indicate that Prdx5 depletion significantly disrupts mitochondrial function and induces ROS-mediated cellular responses.

Reduction in PK levels induced by excessive ROS results in ciliary defects

Mitochondrial dysfunction and the consequent elevation in ROS have been shown to alter the global metabolic status and transcriptome (19). We further investigated direct causes of cilia loss in Prdx5-depleted cells. To this end, we assessed the transcriptomic and proteomic profiles of Prdx5 morphant embryos (Fig. 7A). We focused on differentially expressed genes involved in mitochondrial ATP production and identified dozens of genes downregulated in multiciliated cells after Prdx5 knockdown (Supplementary Table S1). We further analyzed the expression patterns of candidate genes by performing WISH analysis.

FIG. 7.

Reduction in PK level induced by excessive ROS resulted in ciliary defects. (A) Changes in the transcriptome and proteome in Prdx5 morphant embryos were analyzed to identify the genes/proteins affected by Prdx5 depletion. Final candidates were examined by WISH to confirm their expression in multiciliated cells. (B) WISH analysis indicated that multiciliated cells of Xenopus embryos strongly expressed PK, and Prdx5 depletion decreased PK expression in multiciliated cells. In addition, Prdx5 expression induced by co-injecting Prdx5-MO-resistant Prdx5 mRNA (Prdx5*) into Prdx5 morphants rescued PK expression. (C) The activity of the PK promoter was measured by luciferase assay, and it was significantly reduced in Prdx5 morphant embryos. This reduced promoter activity is significantly rescued by Prdx5* injection into Prdx5 morphants. *p < 0.05; **p < 0.01. (D) Xenopus embryos were injected with PK MO and then immunostained with GFP and tubulin to observe the intensity of cilia in the epidermis of Xenopus multiciliated cells. The intensity of cilia is remarkably reduced in PK morphants as compared with the control. (E) Graph showing that PK knockdown disrupted ciliogenesis in Xenopus embryonic multiciliated cells as compared with the control cells. **p < 0.01. (F) hTERT-RPE1 cells were treated with PK siRNA and then stained with anti-acetylated tubulin and DAPI. PK depletion reduced primary cilia formation in hTERT-RPE1 cells. (G) Graphical representation of percentage of ciliated cells in PK-depleted hTERT-RPE1 cells as compared with the control cells. The number of ciliated cells was significantly decreased as a result of PK depletion. **p < 0.01. LC-MS, liquid chromatography-mass spectrometry; PK, pyruvate kinase. Color images are available online.

The muscle-specific PK gene, which is predominantly expressed in multiciliated cells, was among those downregulated in the morphants (Fig. 7B). PK is a critical enzyme that converts phosphoenolpyruvate to pyruvate, thereby generating ATP in the glycolytic pathway. WISH analysis further confirmed that PK expression, indeed, decreased sharply in the multiciliated cells of Prdx5 morphants (Fig. 7B). To confirm the reduction in PK transcription in Prdx5 morphants, we cloned the promoter region of PK and performed a promoter assay. Consistent with the WISH analysis, the activity of the PK promoter was significantly reduced in the Prdx5 morphants (Fig. 7C). The reduction in PK expression and its promotor activity was rescued by a co-injection of Prdx5 mRNA with Prdx5-MO (Fig. 7B, C). Further, the knockdown of PK in Xenopus embryos and hTERT-RPE1 cells severely disrupted multicilia (Fig. 7D, E) and primary cilia formation, respectively (Fig. 7F, G), indicating that PK is critical for ciliogenesis. We also confirmed that MitoQ treatment did not rescue defects in ciliogenesis in PK-depleted cells (Supplementary Fig. S4C, D), suggesting that PK acts downstream of ROS signaling in ciliogenesis.

A reduction in PK expression causes a reduction in pyruvate levels, and this not only affects ATP production through mitochondrial oxidative phosphorylation but may also affect the process of glycolysis by increasing NADH levels in the cytoplasm, which is necessary for the lactate formation. Thus, we speculated that impairments in glycolysis may cause additional energy crises in Prdx5-depleted cells. To rule out this possibility, we assessed the levels of NADH in Prdx5-depleted cells and found that NADH levels were not significantly different from those in controls (Supplementary Fig. S3C). These data indicate that glycolysis is not significantly affected by the reduction in PK and that the mitochondrial dysfunction and consequent increase in ROS is the primary cause of abnormal ciliogenesis, rather than impairment in glycolysis.

Discussion

Prdx5 is well known for its antioxidant effects, which are exerted by scavenging H₂O₂ and alkyl hydroperoxides in mammalian cells and Xenopus embryos (12). It plays significant protective roles in neurological disorders and in balancing the amount of ROS produced in cells, but its roles in vertebrate development have not yet been fully elucidated.

In this study, we systematically investigated the expression patterns of Prdx genes in developing Xenopus embryos (Supplementary Fig. S1) and found that multiciliated cells expressed multiple members of the Prdx family, that is, Prdx1 and Prdx5 (Supplementary Fig. S1). Cilia are important organelles that are required for a variety of developmental processes and are involved in various signaling pathways (22). Among the six Prdx genes, only knockdown of Prdx5 resulted in the failure of multicilia formation (Fig. 2B). Further research into the causes of the ciliary defects observed in Prdx5-depleted embryonic multicilia revealed that the basal bodies in multiciliated cells failed to properly dock to the apical surface to generate ciliary axonemes (Fig. 2C, D).

In addition to its role in multicilia, Prdx5 was also required for primary cilia formation. Prdx5 dysfunction in primary cilia formation is known to inhibit convergent extension and result in a shortened body axis (24, 29). Consistent with these previous reports, our results showed that knockdown of Prdx5 resulted in a shortened body axis in neurula stage embryos and blocked their convergent extension (Fig. 3D, E). Moreover, we showed that siRNA-mediated knockdown of Prdx5 significantly reduced the number of cilia in hTERT-RPE1 cells (Fig. 3B, C).

Prdx5 is an atypical two-cysteine Prdx protein, and both the peroxidatic and resolving cysteines are critical for its catalytic activities (16). In this study, we investigated the significance of both the peroxidatic and resolving cysteines in normal cilia formation by co-injecting Prdx5 catalytic mutants with Prdx5-MO. These Prdx5 mutants failed to rescue the ciliary defects induced by Prdx5 knockdown, indicating that the peroxidatic activity of Prdx5 is necessary for multicilia formation (Fig. 2E, F).

Prdx5 regulates the amount of ROS produced in cells and protects them from ROS-induced damage (26). The depletion of Prdx5 leads to increased levels of cellular ROS and ciliary defects. Our data showed that ciliary defects and enhanced ROS levels in Prdx5-depleted cells are directly related, that is, Prdx5 depletion leads to an excess of ROS, which impairs normal ciliogenesis. Removal of ROS by MitoQ treatment efficiently reversed the ciliary defects observed in Prdx5-depleted hTERT-RPE1 cells (Fig. 4C, D). Thus, in addition to the other known targets of ROS, such as the mitochondria, ER, and peroxisome, our results clearly suggest that the cilium is a vulnerable target of excessive ROS.

Prdx5 is widely distributed in the cytosol, mitochondria, nucleus, and peroxisomes, but the long form of Prdx5 is preferentially located in the mitochondria (16). Since the mitochondria are the vital source of intracellular ROS production, we focused on the antioxidant properties of Prdx5 in the mitochondria of multiciliated cells.

Our findings present compelling evidence that the major function of Prdx5 in multiciliated cells is to protect the mitochondrial function from the oxidative damage caused by ROS. The depletion of Prdx5 led to severe malformation of mitochondria and significantly disrupted the mitochondrial membrane potential and ATP production (Fig. 5). Consistent with its role in protecting the mitochondria, we confirmed that Prdx5 depletion further transduces a signal through p38 and eIF2α phosphorylation, which downregulates global translation as a mitochondrial stress response (Fig. 6A, B). Prdx5 depletion not only affected p38 and eIF2α phosphorylation but also significantly increased the phosphorylation of AKT, JNK, and ERK, which were previously shown to be activated by ROS, indicating that Prdx5 depletion causes mitochondrial dysfunction and elevates ROS (Fig. 6A, B).

Ciliogenesis is extremely important in vertebrate development, and abnormalities in ciliary development can lead to serious congenital disorders (11). Since our data showed that increased ROS production is a contributing factor in ciliary dysfunction in Prdx5-depleted cells, we analyzed the proteomic and transcriptomic changes induced by Prdx5 depletion for further insight into the direct causes of ciliary defects on Prdx5 depletion. Based on these analyses, it was clear that levels of the muscle-specific PK were significantly reduced on Prdx5 depletion, and this was further supported by WISH analysis showing no detectable expression in multiciliated cells (Fig. 7B). Indeed, PK expression was almost completely abolished in the multiciliated cells of Prdx5 morphant embryos. The activity of the PK promoter was significantly less responsive under Prdx5-depleted conditions (Fig. 7C). Moreover, PK depletion recapitulated the ciliary defects observed in Prdx5-depleted embryos.

The molecular mechanism by which PK affects cilia formation and function is currently not well understood. However, it has been shown that PK, along with other glycolytic enzymes, is present in the axoneme of the Chlamydomonas flagellum (21). An in-depth analysis is necessary to further elucidate the exact roles and mechanisms of metabolic enzymes involved in cilia formation.

In conclusion, we suggest that in addition to its protective role as an antioxidant in cells, Prdx5 is also involved in vertebrate development by significantly influencing the process of ciliogenesis. Moreover, the intact peroxidatic function of Prdx5 is critical for proper ciliary development.

Materials and Methods

In vitro fertilization

This study was performed in strict accordance with the guidelines of the Animal Care and Use Committee and in agreement with international laws and policies (National Institutes of Health Guide for the Care and Use of Laboratory Animals, publication no. 85-23, 1985). The Institutional Animal Care and Use Committee (IACUC) of the Ulsan National Institute of Science and Technology approved the experimental use of amphibians (approval no. UNISTIACUC-16-14). All members of our laboratory attended educational and training courses on the proper care and use of experimental animals. Adult Xenopus laevis obtained from the Korean Xenopus Resource Center for Research were housed at 18°C under conditions of 12 h light/12 h dark in containers recommended by the IACUC of the Ulsan National Institute of Science and Technology. Ovulation was induced in adult female amphibians by the subcutaneous injection of 1000 IU of human chorionic gonadotropin (Sigma-Aldrich, St. Louis, MO). The next day, the females were allowed to lay eggs in 60-mm plastic dishes. After washing three times in 0.1 × modified Barth's solution (MBS; 88 mM NaCl, 5 mM Hepes, 2.5 mM NaHCO₃, 1 mM KCl, 1 mM MgSO₄, and 0.7 mM CaCl₂, pH 7.8), the eggs were fertilized in the same solution. Thereafter, testes were obtained from adult male amphibians and minced in 3 to 4 mL of ice-cold 1 × MBS. After successful fertilization, the jelly coat was removed by gently swirling the embryos in a 2% l-cysteine solution. After removing unfertilized eggs and nonviable embryos, the healthy embryos were transferred to 0.5 × MBS containing 2% Ficoll 400 (GE Healthcare, Little Chalfont, UK) and cultured at 22 ± 0.5°C until stage 8.5.

Plasmids and reagents

A complementary DNA clone encoding full-length Prdx5 was obtained from ATCC (GenBank ID: NM_001092111.1). We designed a translation-blocking Prdx5-MO based on the sequence in the Xenbase database. We purchased the Prdx5-MO from Gene Tools (5′-ATGATACCGGGATACGAAGAGCCAT-3′). Flag-tagged Prdx5 fusion constructs (WT, mutant constructs; C47S, C151S, and C47S/C151S) were generated by polymerase chain reaction (PCR) and subcloned into the pCS2+ vector.

Embryo injections

Synthetic capped mRNAs were prepared by using the SP6 mMessage mMachine Kit (Ambion, Carlsbad, CA) and subsequently microinjected into embryos as previously described (18). To rescue the Prdx phenotype, a Prdx5-MO-resistant mRNA containing 4MT was synthesized. MOs (20–60 ng) and/or mRNAs were microinjected into blastomeres of two-cell-stage embryos.

Whole-mount in situ hybridization

To analyze gene expression in developing embryos in situ, WISH was performed as previously described (35). Briefly, embryos at the indicated stage were fixed in MEMFA buffer (4% paraformaldehyde in 0.1 M MOPS, 1 mM MgSO₄, and 2 mM EGTA, pH 7.4) for 2 h at room temperature. The antisense-RNA probes were labeled with digoxigenin-UTP by using the mMessage mMachine Kit, and the fixed embryos were incubated overnight with the antisense probes in the hybridization buffer at 55°C. The probes were detected with an alkaline phosphatase-labeled anti-digoxigenin antibody (1:1000; Roche, Basel, Switzerland) and BM purple dye.

Cilia-driven fluid-flow assay

Control and Prdx5 morphant embryos were anesthetized with benzocaine and placed in agarose-coated plastic dishes. Fluorescently labeled polystyrene beads with a diameter of 10 μm (Molecular Probes, Eugene, OR) were added on top of the embryos, and real-time images were recorded by time-lapse microscopy and cellSens Dimension Software (Olympus, Tokyo, Japan). The directionality and velocity of the beads were measured by merging 10 consecutive images.

Cell culture

hTERT-RPE1 cells were cultured in DMEM-F12 (Welgene, Seoul, Korea) containing 10% fetal bovine serum (Welgene) and 100 U/mL streptomycin/penicillin at 37°C in a humidified atmosphere of 5% carbon dioxide in air.

Western blot analysis

Whole embryos were homogenized in lysis buffer (137 mM NaCl, 20 mM Tris–HCl, 1% Nonidet-P40, and 10% glycerol, pH 8.0) containing 1 mM phenylmethylsulfonyl fluoride, 5 mM sodium orthovandate, and 1 × protease inhibitor cocktail (Sigma-Aldrich). Lysates were heated at 95°C in gel loading buffer for 5 min and electrophoresed on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel. Fusion proteins were detected with anti-flag-horseradish peroxidase (HRP)-conjugated (Sigma-Aldrich) and anti-HA-HRP conjugated (Roche) antibodies. Immunoreactive bands were detected by using the HyGLO Kit (Denville Scientific, South Plainfield, NJ).

Anti-RIP3 and anti-MLKL antibodies were purchased from Abcam (Cambridge, MA). Anti-RIP1 antibody was purchased from BD Transduction Laboratories (San Jose, CA). Anti-Prx5 antibody was purchased from Ab Frontier (Seoul, Korea). Anti-actin antibody was obtained from Sigma-Aldrich.

Immunofluorescent staining and confocal microscopy

The embryos at stage 26 were stained immunofluorescently according to previously described documented standards (10) by using a polyclonal anti-acetylated tubulin (1:400; Sigma-Aldrich) or a monoclonal anti-flag (1:400; Applied Biological Materials, Richmond, Canada) primary antibody, followed by incubation with a Cy3-conjugated anti-rabbit (1:400; Jackson ImmunoResearch Laboratories, West Grove, PA) or a fluorescein isothiocyanate-conjugated anti-mouse (1:400; Invitrogen, Carlsbad, CA) secondary antibody. hTERT-RPE1 cells were immunostained by using an anti-acetylated tubulin (1:500) or an anti-flag (1:500) antibody. Cells were co-stained with Mito-Tracker, a mitochondria-specific probe (1:1000; Invitrogen), and 4′,6′-diamidino-2-phenylindole dihydrochloride (1:5000; Invitrogen). Images were acquired with the Zeiss LSM7 PASCAL confocal microscope (Oberkochen, Germany) and analyzed with Image J and Adobe Photoshop Software.

Electron microscopy

To take EM imaging of embryos, the embryos were fixed at stage 26 in 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M NaH₂PO₄, pH 7.4 for 2 h at 4°C. After washing three times, the embryos were postfixed in 1% osmium tetroxide on ice for 2 h and then washed three times. After dehydration in a graded series of ethanol baths and infiltration in a 1:1 ratio of propylene oxide:epon, the embryos were embedded in Epon 812. Polymerization was carried out in resin at 70°C for 24 h. Ultrathin sections with a thickness of ∼70 nm were prepared with an MT-X ultramicrotome (RMC, Tucson, AZ) and collected on 100-mesh copper grids. After staining with 2% uranyl acetate for 7 min and lead citrate for 2 min, images were acquired with conventional transmission electron microscopy (TEM) (JEM-1400 Plus, at 120 kV) and Bio-HVEM (JEM-1000BEF, at 1000 kV) (Jeol, Tokyo, Japan).

Measurement of mitochondrial ROS levels

Mitochondrial ROS levels were determined by using Mitosox Red (Invitrogen). RPE-1 cells were treated with 1 mM GOX for the indicated periods, and then cells were stained with Mitosox Red for an additional 10 min. Then, cells were trypsinized and resuspended in phosphate buffered saline (PBS), and fluorescence was measured at specific time intervals with a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ).

Flow cytometry

hTERT-RPE1 cells were trypsinized, washed with PBS, and fixed in 70% ethanol for 30 min. To analyze the cell cycle by flow cytometry, cells were washed, resuspended, and stained with 10 μg/mL propidium iodide in PBS. The BD FACSVerse System (BD Biosciences, San Jose, CA) was used for flow cytometry. Cells were also stained with DCFH-DA or JC-1 in PBS at 37°C for 30 min in the dark. The stained cells were washed and then analyzed by flow cytometry.

Transcriptome and proteome profiling

For transcriptome profiling, the animal caps of WT and Prdx5 morphant embryos were explanted and cultured until Nieuwkoop and Faber stage 26, and total RNA was extracted by using TRIzol Reagent. The strand-specific library construction with poly-A enrichment was followed by the manufacturer's instruction (Illumina, San Diego, CA), and sequencing was carried out with Illumina HiSeq 2500 with 100PE configuration. The expression analysis was performed as previously described (33).

For proteome profiling, the animal caps of WT and Prdx5 morphant embryos were explanted and cultured until developmental stage 26 and they were then lysed in 100 mM triethylammonium bicarbonate, pH 8.0. The proteins were then reduced in 10 mM dithiothreitol at 60°C for 20 min and alkylated in 55 mM iodoacetamide at room temperature for 30 min, followed by trypsin digestion. The peptide fragments were analyzed by nano-ultra performance liquid chromatography (Waters, Milford, MA) and mass spectrometry using quadrupole-time-of-flight Premier (Waters). Peptides were separated by a 25 cm × 75 μm nanoACQUITY C18 column (Waters) using the LC system. Mobile phase A was composed of water with 0.1% FA (formic acid), and mobile B phase was composed of 0.1% FA in acetonitrile. The mass spectrometer was operated in the data-dependent mode, automatically switching between MS (mass spectrometry) and MS/MS acquisition modes. The method included a full sequential MS scan (m/z 150–1600, 0.6 s) and five MS/MS scans (m/z 100–1990, 0.6 s/scan) of the five most intense ions present in the full-scan mass spectrum. The spectra were analyzed with Mascot Software (version 2.2.1; Matrix Science, Boston, MA), and the identities of the peptides were determined by searching the Swiss-Prot database (version 51.6; 257,964 sequence entries). The search parameters were as follows: X. laevis for taxonomy, fragment ion mass tolerance of 0.5 Da, and parent ion tolerance of 0.1 Da. Two missed cleavages were allowed for the trypsin digestion. Carbamidomethylation of the cysteines and oxidation of methionines were considered as variable modifications. After searching the GO term “GO:0006754,” we selected proteins involved in the production of ATP in mitochondria. The proteins identified by this process are listed in Supplementary Table S1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD009323.

Statistical analysis

Data from WISH and reverse transcriptase-PCR were analyzed by using ImageJ software (National Institutes of Health^*). Results are presented as mean ± standard error (n = 5 with replicas for each sample). To determine statistical significance, results were analyzed by analysis of variance before Tukey post hoc test. A p-value of <0.05 was considered statistically significant.

Footnotes

Acknowledgments

This study was supported by grants from the National Research Foundation of Korea (NRF) (Grant No. NRF-2015R1A2A1A10053265) and the Ministry of Science, ICT, and Future Planning (MSIP) (Grant No. 2015R1A4A1042271), the Republic of Korea.

Author Disclosure Statement

All the authors declare no competing interests.

Abbreviations Used

References

al-Bekairi

, Nagi

, Shoeb

, and al-Sawaf

. Evidence for superoxide radical production by a simple flavoprotein: glucose oxidase. Biochem Mol Biol Int, 34: 233–238, 1994.

Aon

, Stanley

, Sivakumaran

, Kembro

, O'Rourke

, Paolocci

, and Cortassa

. Glutathione/thioredoxin systems modulate mitochondrial H₂O₂ emission: an experimental-computational study. J Gen Physiol, 139: 479–491, 2012.

Banmeyer

, Marchand

, Clippe

, and Knoops

. Human mitochondrial peroxiredoxin 5 protects from mitochondrial DNA damages induced by hydrogen peroxide. FEBS Lett, 579: 2327–2333, 2005.

Banmeyer

, Marchand

, Verhaeghe

, Vucic

, Rees

, and Knoops

. Overexpression of human peroxiredoxin 5 in subcellular compartments of Chinese hamster ovary cells: effects on cytotoxicity and DNA damage caused by peroxides. Free Radic Biol Med, 36: 65–77, 2004.

Barnham

, Masters

, and Bush

. Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov, 3: 205–214, 2004.

Billett

and Gould

. Fine structural changes in the differentiating epidermis of Xenopus laevis embryos. J Anat, 108: 465–480, 1971.

Brooks

and Wallingford

. Multiciliated cells. Curr Biol, 24: R973–R982, 2014.

Chae

, Lee

, Kim

, Jung Sim

, Ji

, Kim

, Ismail

, Park

, Kwon

, Kang

, Lee

, Bae

, Kim

, Min

, Kyu Kwon

, Park

, Han

, Kwon

, Park

, and Lee

. Peroxiredoxin1, a novel regulator of pronephros development, influences retinoic acid and Wnt signaling by controlling ROS levels. Sci Rep, 7: 8874, 2017.

Dawe

, Farr

, and Gull

. Centriole/basal body morphogenesis and migration during ciliogenesis in animal cells. J Cell Sci, 120: 7–15, 2007.

10.

Dollar

, Weber

, Mlodzik

, and Sokol

. Regulation of Lethal giant larvae by Dishevelled. Nature, 437: 1376–1380, 2005.

11.

Drummond

. Cilia functions in development. Curr Opin Cell Biol, 24: 24–30, 2012.

12.

Dubuisson

, Vander Stricht

, Clippe

, Etienne

, Nauser

, Kissner

, Koppenol

, Rees

, and Knoops

. Human peroxiredoxin 5 is a peroxynitrite reductase. FEBS Lett, 571: 161–165, 2004.

13.

Förstermann

. Oxidative stress in vascular disease: causes, defense mechanisms and potential therapies. Nat Clin Pract Cardiovasc Med, 5: 338–349, 2008.

14.

Holmström

and Finkel

. Cellular mechanisms and physiological consequences of redox-dependent signalling. Nat Rev Mol Cell Biol, 15: 411–421, 2014.

15.

James

, Sharpley

, Manas

, Frerman

, Hirst

, Smith

, and Murphy

. Interaction of the mitochondria-targeted antioxidant MitoQ with phospholipid bilayers and ubiquinone oxidoreductases. J Biol Chem, 282: 14708–14718, 2007.

16.

Knoops

, Goemaere

, Van der Eecken

, and Declercq

. Peroxiredoxin 5: structure, mechanism, and function of the mammalian atypical 2-Cys peroxiredoxin. Antioxid Redox Signal, 15: 817–829, 2011.

17.

Kropotov

, Usmanova

, Serikov

, Zhivotovsky

, and Tomilin

. Mitochondrial targeting of human peroxiredoxin V protein and regulation of PRDX5 gene expression by nuclear transcription factors controlling biogenesis of mitochondria. FEBS J, 274: 5804–5814, 2007.

18.

Lee

, Nishanian

, Mood

, Bong

, and Daar

. EphrinB1 controls cell-cell junctions through the Par polarity complex. Nat Cell Biol, 10: 979–986, 2008.

19.

Liemburg-Apers

, Willems

, Koopman

, and Grefte

. Interactions between mitochondrial reactive oxygen species and cellular glucose metabolism. Arch Toxicol, 89: 1209–1226, 2015.

20.

McDiarmid

and Altig

. Tadpoles: The Biology of Anuran Larvae. Chicago, IL: University of Chicago Press, 1999, pp. xiv, 444.

21.

Mitchell

, Pedersen

, Feely

, Rosenbaum

, and Mitchell

. ATP production in Chlamydomonas reinhardtii flagella by glycolytic enzymes. Mol Biol Cell, 16: 4509–4518, 2005.

22.

and Katsanis

. Cilia in vertebrate development and disease. Development, 139: 443–448, 2012.

23.

Park

, Choi

, Kim

, Chae

, Lee

, and Lee

. Peroxiredoxin 5 (Prx5) decreases LPS-induced microglial activation through regulation of Ca2+/calcineurin-Drp1-dependent mitochondrial fission. Free Radic Biol Med, 99: 392–404, 2016.

24.

Park

, Haigo

, and Wallingford

. Ciliogenesis defects in embryos lacking inturned or fuzzy function are associated with failure of planar cell polarity and Hedgehog signaling. Nat Genet, 38: 303–311, 2006.

25.

Park

, Mitchell

, Abitua

, Kintner

, and Wallingford

. Dishevelled controls apical docking and planar polarization of basal bodies in ciliated epithelial cells. Nat Genet, 40: 871–879, 2008.

26.

Peng

, Yang

, Guo

, Ng

, Liu

, Fung

, Tay

, Ge

, He

, Kung

, and Lin

. Catalase and peroxiredoxin 5 protect Xenopus embryos against alcohol-induced ocular anomalies. Invest Ophthalmol Vis Sci, 45: 23–29, 2004.

27.

Reiter

and Leroux

. Genes and molecular pathways underpinning ciliopathies. Nat Rev Mol Cell Biol, 18: 533–547, 2017.

28.

Rhee

, Woo

, Kil

, and Bae

. Peroxiredoxin functions as a peroxidase and a regulator and sensor of local peroxides. J Biol Chem, 287: 4403–4410, 2012.

29.

Ross

, May-Simera

, Eichers

, Kai

, Hill

, Jagger

, Leitch

, Chapple

, Munro

, Fisher

, Tan

, Phillips

, Leroux

, Henderson

, Murdoch

, Copp

, Eliot

, Lupski

, Kemp

, Dollfus

, Tada

, Katsanis

, Forge

, and Beales

. Disruption of Bardet–Biedl syndrome ciliary proteins perturbs planar cell polarity in vertebrates. Nat Genet, 37: 1135–1140, 2005.

30.

Sabharwal

and Schumacker

. Mitochondrial ROS in cancer: initiators, amplifiers or an Achilles' heel?. Nat Rev Cancer, 14: 709–721, 2014.

31.

Scherz-Shouval

and Elazar

. ROS, mitochondria and the regulation of autophagy. Trends Cell Biol, 17: 422–427, 2007.

32.

Sena

and Chandel

. Physiological roles of mitochondrial reactive oxygen species. Mol Cell, 48: 158–167, 2012.

33.

Session

, Uno

, Kwon

, Chapman

, Toyoda

, Takahashi

, Fukui

, Hikosaka

, Suzuki

, Kondo

, van Heeringen

, Quigley

, Heinz

, Ogino

, Ochi

, Hellsten

, Lyons

, Simakov

, Putnam

, Stites

, Kuroki

, Tanaka

, Michiue

, Watanabe

, Bogdanovic

, Lister

, Georgiou

, Paranjpe

, van Kruijsbergen

, Shu

, Carlson

, Kinoshita

, Ohta

, Mawaribuchi

, Jenkins

, Grimwood

, Schmutz

, Mitros

, Mozaffari

, Suzuki

, Haramoto

, Yamamoto

, Takagi

, Heald

, Miller

, Haudenschild

, Kitzman

, Nakayama

, Izutsu

, Robert

, Fortriede

, Burns

, Lotay

, Karimi

, Yasuoka

, Dichmann

, Flajnik

, Houston

, Shendure

, DuPasquier

, Vize

, Zorn

, Ito

, Marcotte

, Wallingford

, Ito

, Asashima

, Ueno

, Matsuda

, Veenstra

, Fujiyama

, Harland

, Taira

, and Rokhsar

. Genome evolution in the allotetraploid frog Xenopus laevis . Nature, 538: 336–343, 2016.

34.

Sim

, Kim

, Myung

, Kwon

, Lee

, and Park

. Xenopus: an alternative model system for identifying muco-active agents. PLoS One, 13: e0193310, 2018.

35.

Smith

and Levin

. Application of a locus-specific DNA hybridization probe in the analysis of the slow-feathering endogenous virus complex of chickens. Poult Sci, 70: 1957–1964, 1991.

36.

Son

, Kim

, Chung

, and Pae

. Reactive oxygen species in the activation of MAP kinases. Methods Enzymol, 528: 27–48, 2013.

37.

Stanke

. The contribution of the airway epithelial cell to host defense. Mediators Inflamm, 2015: 463016, 2015.

38.

Sun

, Kim

, Huang

, Kim

, Park

, Kim

, Yang

, Chung

, Lee

, Choi

, Min

, Park

, Kim

, Lee

, Chang

, Lee

, Yu

, and Lee

. Microglial peroxiredoxin V acts as an inducible anti-inflammatory antioxidant through cooperation with redox signaling cascades. J Neurochem, 114: 39–50, 2010.

39.

Ushio-Fukai

, Alexander

, Akers

, Yin

, Fujio

, Walsh

, and Griendling

. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem, 274: 22699–22704, 1999.

40.

Wek

, Jiang

, and Anthony

. Coping with stress: eIF2 kinases and translational control. Biochem Soc Trans, 34: 7–11, 2006.

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