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IFT46 plays crucial roles in craniofacial and cilia development
Biochemical and Biophysical Research Communications xxx (2016) 1e7
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IFT46 plays crucial roles in craniofacial and cilia development Inji Park a, 1, Hyun-Kyung Lee a, 1, Chowon Kim a, Tayaba Ismail a, Yoo-Kyung Kim a, Jeen-Woo Park a, Oh-Shin Kwon a, Beom Sik Kang a, Dong-Seok Lee a, Tae-Joo Park b, Mae-Ja Park c, Sun-Cheol Choi d, **, Hyun-Shik Lee a, * a
ABRC, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, South Korea School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, South Korea c Department of Anatomy, College of Medicine, Kyungpook National University, Daegu, 41944, South Korea d Department of Biomedical Sciences, University of Ulsan, College of Medicine, Seoul, 05505, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2016 Accepted 15 June 2016 Available online xxx
The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development. © 2016 Elsevier Inc. All rights reserved.
Keywords: IFT46 Ciliogenesis Neurogenesis Craniofacial development
1. Introduction Cilia are microtubule-based organelles that project from the cell surface in a wide range of organisms [1,2]. Cilia are divided into two subtypes based on structural features. Motile cilia, having a 9 þ 2 structure, are important for rolling eggs in the fallopian tubes and eliminating dirts from the trachea [1]. Primary cilia, on the other hand, having a 9 þ 0 structure (no central pair), are important for transmitting various signals to the cytoplasm of cells [2e4]. Recently, the involvement of cilia in embryogenesis has gained attention because abnormal cilia formation was found to cause various human genetic diseases known as ciliopathies, including blindness, obesity, situs inversus, polycystic kidney disease, and neural tube closure defects [5e8]. Ciliopathies involve abnormal
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S.-C. Choi), [email protected] (H.-S. Lee). 1 These authors contributed equally to this work.
proteins or the loss of proteins that are normally localized in the cilia or have important functions in ciliogenesis. Previous studies indicated that disruption of Kif7, which is connected to the centrosome, causes fetal hydrolethalus, acrocallosal syndromes, and Bardet-Biedl syndrome (BBS) [9]. Further, loss of DNAH5 and DNAH11, which are associated with the axonemal dynein arm and radical spoke protein, induce primary ciliary dyskinesia [10,11]. Ciliogenesis begins with attachment of the distal end of mother centrioles to a vesicle, mediated by the transition fiber [12]. Fusion of plasma and ciliary membranes leads to bud exposure and then elongation to a stable length [13,14]. During this process, various protein components required for cilia assembly are not synthesized in the cilium, but rather, transported by IFT systems [13]. IFT is a highly conserved system regulating bi-directional movement of IFT particles, including ciliary components, along the axoneme in the cilium [13,15]. The interactions between IFT components have been characterized in the green algae Chlamydomonas reinhardtii [16]. This system maintains ciliary structure and functions by interacting with kinesin and dynein molecular motors and transporting ciliary cargo [17e19]. Based on the motor
http://dx.doi.org/10.1016/j.bbrc.2016.06.083 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
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proteins involved, IFT systems form two different complexes (A and B). Complexes A and B are composed of at least 6 and 13 subunits respectively. Complex B is typically involved in retrograde trafficking, whereas complex A is in anterograde trafficking [20]. Diverse components having functions in the IFT system have been identified; however, their effects on developmental processes are not completely understood. IFT46 is a core protein in the retrograde trafficking machinery and functions by forming a trimetric complex with other IFT complex B core proteins including IFT52, IFT70, and IFT88 [21]. Cterminus deletion construct of IFT46 showed unstable complex B structure, whereas N-terminus deletion construct showed a loss of the outer dynein arms in the cilium of C. reinhardtii [21]. Recent reports suggest that IFT46 knockdown induces the shortening of cilia in various ciliated tissues such as Kupffer’s vesicle, pronephric ducts, ears, and spinal cord of zebrafish [22]. In present study, we characterized IFT46 using Xenopus embryos and the hTERT-immortalized retinal pigment epithelial cell line (RPE)-1 to determine the functions of IFT46 during vertebrate embryogenesis. We observed strong expression of IFT46 in the head, spinal cord, and multiciliated cells. Moreover, knockdown of IFT46 resulted in a loss of cilia in various tissues. IFT46 morphants exhibited a shortened axis, neural tube closure defects, and abnormal craniofacial development. The present work exhibits that IFT46 plays an important role in neural and craniofacial development as well as ciliogenesis. 2. Materials and methods 2.1. mRNA synthesis and Xenopus embryo microinjection cDNA of IFT46 and IFT46 (5MT), not recognized by IFT46 MO, were subcloned into pCS107 using a Flag-tagged primer having BamH1 and EcoR1 restriction sites. IFT46 morpholino (MO) consisted of 24 nucleotides: 50 -GATGTGTTTCTGCCTCTTCCATCC-30 (Gene Tools, Philomath, OR, USA). Capped mRNAs were synthesized using the mMessage mMachine kit (Ambion, Austin, TX, USA). The pCS107/IFT46-Flag and pCS107/IFT46 (5MT)-Flag constructs were linearized with Apa1. mRNAs and MOs were co-injected into the both blastomeres of 2cell staged embryos. 2.2. Western blot analysis Protein lysates were prepared by homogenizing embryos in lysis buffer (137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1% Nonidet-P40, 10% glycerol) supplemented with 1 mM phenylmehtylsulfonyl fluoride, 5 mM sodium orthovanadate, and 1 protease inhibitor mixture. Lysates were heated at 95 C in loading buffer for 5 min and electrophoresed by 12% SDS-PAGE; western blots were probed with monoclonal anti-Flag (1:1000, Applied Biological Materials, Richmond, British Columbia, Canada) and goat anti-mouse horseradish peroxidase-conjugated antibodies (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immune-reactive bands were detected using an ECL kit (Hyclone, Logan, UT, USA).
2.4. Whole mount in situ hybridization Embryos were fixed in MEMFA (4% paraformaldehyde, 0.1 M MOPS (pH 7.4), 1 mM MgSO4, 2 mM EGTA) overnight at 4 C and then dehydrated before storage in 100% methanol at 20 C. To prepare the anti-sense digoxigenin (Dig)-labeling probe, the IFT46, SOX3, and PAX3 DNA templates were linearized using ApaI, HindIII, and NotI enzymes, respectively. Probes were generated using mMessage mMachine kit and were detected using alkaline phosphatase-labeled anti-Dig antibody (1:1000, Roche, Basel, Switzerland) and BM purple dye. Whole mount in situ hybridization was performed with appropriate staged embryos using the RNA probes of IFT46, SOX3, and PAX3.
2.5. siRNA transfection in hTERT-RPE1 cells We used hTERT-immortalized retinal pigment epithelial cell-1 (RPE-1 cells) to evaluate primary cilia. hTERT-RPE1 cells were cultured at 37 C, 5% CO2 in DMEM F-12 (Welgene, Seoul, Korea) containing 100 U/mL streptomycin/penicillin. Three types of siRNAs were transfected into hTERT-RPE1 cells using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA) for 24 h. Serum starvation was conducted to induce primary cilia for 48 h.
2.6. Immunofluorescence and confocal microscopy Both blastomeres of 2-cell staged embryos were injected and fixed at stage 32 in MEMFA (4% paraformaldehyde, 0.1 M MOPS (pH 7.4), 1 mM MgSO4, 2 mM EGTA) overnight at 4 C. Embryos were incubated in blocking buffer (PBS containing 1% bovine serum albumin and 5% heat-inactivated lamb serum). The primary antibody used for protein detection was monoclonal anti-acetylated tubulin (1:500, Sigma, St. Louis, MO, USA), and samples were visualized with Alexa 568 anti-mouse IgG (1:2000, Invitrogen, Carlsbad, CA, USA) and Alexa 488 anti-rabbit IgG (1:2000, Invitrogen) or Alexa 488 anti-mouse IgG (1:2000, Invitrogen) as secondary antibodies on a Zeiss LSM7 PASCAL confocal microscope (Carl Zeiss AG, Jena, Germany). Next, 100% methanol was used to fix hTERT-RPE1 cells in a LabTEK chamber (Nunc, Rochester, NY, USA), followed by immunofluorescence using the primary antibodies monoclonal anti-acetylated tubulin (1:500, Sigma) and monoclonal anti-Flag antibodies (1:500, Applied Biological Materials), which were visualized using Alex 568 anti-mouse (1:2000, Invitrogen). Three-dimensional projections, image processing and analysis were performed using a Zeiss LSM7 confocal microscope, Image J (NIH, Bethesda, MD, USA).
2.7. Alcian blue staining We harvested the stage 50 Xenopus embryos and fixed overnight at 4 C. Embryos were stained with 0.05% Alcian blue (Sigma) for 2 h, and bleached. Embryos were made clear using a mixture of 2:1 benzyl benzoate: benzyl alcohol.
2.3. Reverse transcription-polymerase chain reaction (RT-PCR)
2.8. Statistical analysis
cDNAs were prepared by reverse transcription using a Primescript 1st-strand cDNA synthesis kit (Takara, Shiga, Japan) from RNAs extracted from Xenopus embryos from stages 0e40 using a standard protocol. The PCR products were separated on a 1% agarose gel and images were captured using Wise Capture I-1000 (Daihan Scientific, Wonju, Kangwon-do, South Korea).
Data from RT-PCR and immunofluorescence staining were analyzed using ImageJ software. The results are presented as the mean ± standard error of the mean (n ¼ 5 with replicas for each sample). To determine the levels of significance, the results were analyzed based on standard deviation followed by ANOVA with Tukey post-hoc test.
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Fig. 1. Spatio-temporal expression patterns of IFT46 during embryogenesis. (A) Xenopus embryos were harvested at various stages and RT-PCR was performed using standard methods. The numbers indicate embryonic stages and ornithine decarboxylase (ODC) was a loading control. IFT46 is expressed as a maternal gene. (B) Xenopus embryos were harvested at stage 32 and processed via whole mount in situ hybridization using Dig-labeled antisense probes of IFT46. IFT46 was expressed in the central nervous system and multiciliated cells. The right figure is higher magnification view. (C) Both blastomeres of 2-cell stage embryos were injected with IFT46 mRNA containing a Flag tag and harvested at stage 27. Embryos were immunostained with acetylated tubulin-monoclonal antibody (green) and Flag-polyclonal antibodies (red). IFT46 was localized along the axoneme of motile cilia of epithelial cells. White arrows indicate IFT46 proteins. Right figure is higher magnification view (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2. Loss of IFT46 prevents motile cilia formation in Xenopus epithelial cells. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and mutant mRNA, IFT46 (5MT), not recognized by MOs. Embryos were harvested at stage 32 and immunofluorescence staining of motile cilia with acetylated tubulin-monoclonal antibody (red) was carried out. (B) Relative quantification of green fluorescence per motile cilia of control MO- or IFT46 MO-injected embryos with IFT46 (5MT) mRNA. IFT46 MO and IFT46 (5MT) mRNA coinjected embryos showed partially rescued ciliogenesis. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MOinjected. (C) Western blot analysis of exogenous IFT46-Flag mRNA expression (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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Fig. 3. Loss of IFT46 hinders primary cilia formation. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and fixed embryos at stage 32. Primary cilia in the neural tube lumen were detected by immunofluorescence staining using acetylated tubulin-antibody (gray) and confocal microscopy. (B) Treatment of IFT46 siRNA for 24 h and serum starvation was performed to induce primary cilia for 48 h. We performed immunofluorescence staining of the nucleus and primary cilia using DAPI (blue) and acetylated tubulin-monoclonal antibody (red) respectively. Compared with the control, IFT46 siRNA-treated cells show short or no primary cilia. Data are shown as mean ± SD. ***p < 0.001 compared with control siRNA-treated. (C) RT-PCR was performed to evaluate the knockdown of IFT46. b-actin was used as a loading control (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3. Results 3.1. IFT46 are expressed in the retina, central nervous system, and multiciliated cells during Xenopus embryogenesis To investigate mRNA expression patterns of IFT46, we conducted RT-PCR and whole mount in situ hybridization. The spatioexpression pattern of IFT46 revealed maternal nature of IFT46 gene (Fig. 1A). In addition, enhanced expression of IFT46 was detected during the blastula stage (st. 8) and neurula stage (st. 16) (Fig. 1A). Moreover, the spatial expression pattern following whole mount in situ hybridization showed strong expression of IFT46 in animal hemisphere at early stages (st. 8, data not shown) and central nervous system, including the head, retina, and ciliated cell in the epidermis at later stages (st. 32) (Fig. 1B). Double immunostaining against acetylated tubulin and Flag-tag (exogenous IFT46) confirmed the IFT46 expression in multiciliated cells of epidermis. Interestingly, IFT46 was localized along the motile ciliary axoneme including microtubule tracks as well as the ciliary tip (Fig. 1C). These results indicate that IFT46 is involved in neurulation and ciliogenesis during Xenopus development. 3.2. Loss of IFT46 causes ventralized phenotype To examine the functions of IFT46 in early vertebrate
development, we performed loss of functions experiments using MO to block the translation of IFT46 mRNA through competitively interacting with mRNA. Immunoblotting analysis showed that IFT46 MO inhibited the translation of exogenous IFT46 (Fig. 2C). Embryos injected with IFT46 MO displayed ventralized phenotype including shortened trunks along the anterior-posterior axis compared with control embryos (Supplementary Fig. S1). Our findings clearly exhibit that loss of IFT46 disrupts anterior-posterior axis elongation as well as head development. 3.3. IFT46 is crucial for primary and motile cilia formation in vivo and in vitro The involvement of IFT46 in ciliogenesis was confirmed by immunostaining the cilia of IFT46 morphants using antibodies against acetylated tubulin. Immunostaining showed significant reduction in number and length after IFT46 knockdown (Fig. 2A). The fluorescence intensity in the cilia of IFT46 knockdown embryos was 5fold lower than in control embryos (Fig. 2B). We verified the specificity of ciliogenesis defects due to IFT46 knockdown by performing a rescue experiment. We microinjected IFT46 MO with IFT46 mutant mRNA (IFT46 5MT), not recognized by MO, but translated to functional IFT46 proteins. Exogenous IFT46 proteins partially rescued the number and length of motile cilia up to 70% (Fig. 2A and B). Exogenous IFT46 expression was confirmed by
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
Fig. 4. Loss of IFT46 induces severe reduction in neural development and cartilage formation. (A) 10 ng of IFT46 MO was injected into the both blastomeres of 2-cell staged embryos with 2 ng of dominant-negative BMP-4 receptor (DN-BR) mRNA. Animal caps were dissected from injected embryos at stage 8.5 and collected at stage 18. The animal cap was processed for RT-PCR using standard methods. ODC was used as a loading control. No-RT indicates the control in the absence of reverse transcriptase. Quantification of the relative expression levels of BF-1, XAG, N-CAM, AP2a, and ZIC3 normalized to ODC expression. (B) Both blastomeres of 2-cell staged embryos were injected with 5 ng of IFT46 MO. Tadpoles were fixed at stage 50 and subjected to Alcian blue staining for cartilage formation. IFT46 morphants showed severe defects on cartilage formation (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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immunoblotting (Fig. 2C). These results indicate that loss of IFT46 prevented motile cilia formation in Xenopus epithelial cells. Next, we investigated the effect of IFT46 on primary cilia formation and motile cilia elongation by examining primary cilia morphology in the neural tube lumen after embryos were sectioned. The primary cilia in IFT46 morphants showed similar defects as motile cilia (Fig. 3A). Moreover, by depleting IFT46 in RPE1 cells using siRNA, the effects of loss of IFT46 were confirmed on primary cilia formation. Primary cilia were induced by serum starvation for 48 h and examined by using acetylated tubulin antibodies specifically expressed in cilia. The number of ciliated cells was greatly decreased in IFT46 depletion cells (Fig. 3B). The efficiency of IFT46-specific siRNA was confirmed by RT-PCR (Fig. 3C). Hence, IFT46 is essential for motile and primary cilia formation. 3.4. IFT46 is important for neural development According to ventralized phenotype of the IFT46 morphants, we examined the effects of IFT46 on neural tube closure defects. Whole mount in situ hybridization with the neural tube marker PAX3, being governed by Wnt/planar cell polarity (PCP), sonic hedgehog (shh) signaling pathways and neural plate marker SOX3, being regulated by Wnt/PCP signaling pathway showed that IFT46 morphants displayed neural tube closure defects and wider neural plates (Supplementary Fig. S2). Our results show that loss of IFT46 inhibits convergence-extension movement during gastrulation and perhaps by influencing Wnt/PCP and sonic hedgehog signaling pathways. Next, to investigate the role of IFT46 in neurogenesis, we introduced dominant-negative BMP-4 receptor (DN-BR) to block BMP-4 signaling with IFT46 MO. Loss of IFT46 did not influence DNBR-induced pan-neural marker gene expression, N-CAM. However, loss of IFT46 decreased the level of DN-BR-induced BF1, XAG (an anterior neural marker), and AP2a, ZIC3 (a neural crest cell marker) expression (Fig. 4A). Moreover, we observed knockdown of IFT46 specificity by rescue experiments. These results clearly indicate that IFT46 affects neurogenesis by influencing neural gene expression. 3.5. Loss of IFT46 induces severe reduction of craniofacial cartilage formation Mostly craniofacial cartilage is induced by neurogenesis, including neural crest cells [23]. To investigate the role of IFT46 in craniofacial development, we examined cartilage morphology by alcian blue staining using antibodies against glycosaminoglycan, present in cartilage. As shown in Fig. 4B, IFT46 knockdown caused severe reduction of cartilage formation including the branchial stream, hyoid stream, and non-neural crest. Taken together, these results suggest that IFT46 regulates craniofacial development and neurogenesis. 4. Discussion Ciliary epithelial cells are essential for the proper functioning of various organs, and the loss of ciliary function induces ciliopathies including neural tube closure defects and abnormal craniofacial development [24]. IFT46 was first characterized in C. reinhardtii as a retrograde trafficking system protein interacting with ciliary components including IFT88, IFT70, and IFT52 and links to the motor protein kinesin [25]. Knockdown of IFT46 causes unstable complex B structure, loss of the outer dynein arm in the cilia [26], and shortened cilia in numerous ciliated tissues including Kupffer’s vesicle, pronephric ducts, ears, and spinal cord [22]; however, effects of
IFT46 on embryogenesis need further investigation. Some signaling pathways including Wnt/PCP and Shh are needed for proper nervous system development, particularly for neural tube closure [27]. Primary cilia act as platforms on which signaling proteins are localized [27]. Previous studies using mice showed that disruption of IFT resulted in improper neural tube patterning [27]. IFT88 and IFT172 mutants led to neural tube mispatterning through the shortening of cilia and low Shh signaling via the loss of GliA and GliR [27]. In addition, disruption of the PCP effectors Inturned and Fuzzy can cause ciliary loss and neural tube closure defects [28]. Disheveled mutant mice also displayed neural tube closure defects similar to those in IFT or Shh signaling mutant mice [29]. Ciliogenesis and signaling pathways such as PCP and Shh influence neural development. However, the involvement of IFT46 in signaling transduction for neurogenesis and craniofacial formation is unknown. Most craniofacial cartilage is induced by neurogenesis, including neural crest cells. Cranial neural crest stem cells that differentiate into chondrocytes containing primary cilia consist of craniofacial cartilage [23,30]. Recent reports suggested that primary cilia are important for the maintenance of craniofacial cartilage [31]. Conditional deletion of Kif3a resulted in the loss of primary cilia, excessive Hedgehog signaling, and aberrant neural crest cell proliferation in a Gli-dependent manner as well as unusual craniofacial formation [31]. In addition, IFT88 transgenic mice showed upregulation of Hedgehog signaling and thick cartilage [31]. In current study, we investigated that IFT46 is essential for neurogenesis including neural tube closure (Fig. 4 and Supplementary Fig. S2) and knockdown of IFT46 resulted in a loss of primary cilia regulating neural crest cell gene expression and induced unusual craniofacial development (Fig. 4). AP2a and ZIC3, which are associated with neural crest cells, are required for craniofacial and skeletal morphogenesis [32,33]. It is likely that loss of IFT46 was responsible for the reduction in craniofacial cartilage formation via neural crest cell expression of proteins such as AP2a and ZIC3. We also demonstrated that IFT46 is required for motile and primary cilia elongation by using Xenopus embryos and hTERTimmortalized RPE-1. In conclusion, our results suggest that IFT46 regulates ciliogenesis, neurogenesis and craniofacial development by modulating specific signaling pathways such as Wnt/PCP and shh. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) funded by the Korea government [MSIP] (Grant No. 2015R1A4A1042271 and NRF-2015R1A2A1A10053265), Republic of Korea. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.083. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.06.083. References [1] S. Roy, The motile cilium in development and disease: emerging new insights, Bioessays 31 (2009) 694e699. [2] S.C. Goetz, K.V. Anderson, The primary cilium: a signalling centre during vertebrate development, Nat. Rev. Genet. 11 (2010) 331e344. [3] N.F. Berbari, A.K. O’Connor, C.J. Haycraft, B.K. Yoder, The primary cilium as a
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
IFT46 plays crucial roles in craniofacial and cilia development Inji Park a, 1, Hyun-Kyung Lee a, 1, Chowon Kim a, Tayaba Ismail a, Yoo-Kyung Kim a, Jeen-Woo Park a, Oh-Shin Kwon a, Beom Sik Kang a, Dong-Seok Lee a, Tae-Joo Park b, Mae-Ja Park c, Sun-Cheol Choi d, **, Hyun-Shik Lee a, * a
ABRC, CMRI, School of Life Sciences, BK21 Plus KNU Creative BioResearch Group, Kyungpook National University, Daegu, 41566, South Korea School of Life Sciences, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 44919, South Korea c Department of Anatomy, College of Medicine, Kyungpook National University, Daegu, 41944, South Korea d Department of Biomedical Sciences, University of Ulsan, College of Medicine, Seoul, 05505, South Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 May 2016 Accepted 15 June 2016 Available online xxx
The intraflagellar transport (IFT) system is essential for bidirectional movement of ciliary components from the basal body to the tip beneath the ciliary sheath and is conserved for cilia and flagella formation in most vertebrates. IFT complex A is involved in anterograde trafficking, whereas complex B is involved in retrograde trafficking. IFT46 is well known as a crucial component of IFT complex B, however, its developmental functions are poorly understood. In this study, we investigated the novel functions of IFT46 during vertebrate development, especially, ciliogenesis and neurogenesis, because IFT46 is strongly expressed in both multiciliated cells of epithelial and neural tissues. Knockdown of IFT46 using morpholino microinjections caused shortening of the body axis as well as the formation of fewer and shorter cilia. Furthermore, loss of IFT46 down-regulated the expression of the neural plate and neural tube markers, thus may influence Wnt/planar cell polarity and the sonic hedgehog signaling pathway during neurogenesis. In addition, loss of IFT46 caused craniofacial defects by interfering with cartilage formation. In conclusion, our results depict that IFT46 plays important roles in cilia as well as in neural and craniofacial development. © 2016 Elsevier Inc. All rights reserved.
Keywords: IFT46 Ciliogenesis Neurogenesis Craniofacial development
1. Introduction Cilia are microtubule-based organelles that project from the cell surface in a wide range of organisms [1,2]. Cilia are divided into two subtypes based on structural features. Motile cilia, having a 9 þ 2 structure, are important for rolling eggs in the fallopian tubes and eliminating dirts from the trachea [1]. Primary cilia, on the other hand, having a 9 þ 0 structure (no central pair), are important for transmitting various signals to the cytoplasm of cells [2e4]. Recently, the involvement of cilia in embryogenesis has gained attention because abnormal cilia formation was found to cause various human genetic diseases known as ciliopathies, including blindness, obesity, situs inversus, polycystic kidney disease, and neural tube closure defects [5e8]. Ciliopathies involve abnormal
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (S.-C. Choi), [email protected] (H.-S. Lee). 1 These authors contributed equally to this work.
proteins or the loss of proteins that are normally localized in the cilia or have important functions in ciliogenesis. Previous studies indicated that disruption of Kif7, which is connected to the centrosome, causes fetal hydrolethalus, acrocallosal syndromes, and Bardet-Biedl syndrome (BBS) [9]. Further, loss of DNAH5 and DNAH11, which are associated with the axonemal dynein arm and radical spoke protein, induce primary ciliary dyskinesia [10,11]. Ciliogenesis begins with attachment of the distal end of mother centrioles to a vesicle, mediated by the transition fiber [12]. Fusion of plasma and ciliary membranes leads to bud exposure and then elongation to a stable length [13,14]. During this process, various protein components required for cilia assembly are not synthesized in the cilium, but rather, transported by IFT systems [13]. IFT is a highly conserved system regulating bi-directional movement of IFT particles, including ciliary components, along the axoneme in the cilium [13,15]. The interactions between IFT components have been characterized in the green algae Chlamydomonas reinhardtii [16]. This system maintains ciliary structure and functions by interacting with kinesin and dynein molecular motors and transporting ciliary cargo [17e19]. Based on the motor
http://dx.doi.org/10.1016/j.bbrc.2016.06.083 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
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proteins involved, IFT systems form two different complexes (A and B). Complexes A and B are composed of at least 6 and 13 subunits respectively. Complex B is typically involved in retrograde trafficking, whereas complex A is in anterograde trafficking [20]. Diverse components having functions in the IFT system have been identified; however, their effects on developmental processes are not completely understood. IFT46 is a core protein in the retrograde trafficking machinery and functions by forming a trimetric complex with other IFT complex B core proteins including IFT52, IFT70, and IFT88 [21]. Cterminus deletion construct of IFT46 showed unstable complex B structure, whereas N-terminus deletion construct showed a loss of the outer dynein arms in the cilium of C. reinhardtii [21]. Recent reports suggest that IFT46 knockdown induces the shortening of cilia in various ciliated tissues such as Kupffer’s vesicle, pronephric ducts, ears, and spinal cord of zebrafish [22]. In present study, we characterized IFT46 using Xenopus embryos and the hTERT-immortalized retinal pigment epithelial cell line (RPE)-1 to determine the functions of IFT46 during vertebrate embryogenesis. We observed strong expression of IFT46 in the head, spinal cord, and multiciliated cells. Moreover, knockdown of IFT46 resulted in a loss of cilia in various tissues. IFT46 morphants exhibited a shortened axis, neural tube closure defects, and abnormal craniofacial development. The present work exhibits that IFT46 plays an important role in neural and craniofacial development as well as ciliogenesis. 2. Materials and methods 2.1. mRNA synthesis and Xenopus embryo microinjection cDNA of IFT46 and IFT46 (5MT), not recognized by IFT46 MO, were subcloned into pCS107 using a Flag-tagged primer having BamH1 and EcoR1 restriction sites. IFT46 morpholino (MO) consisted of 24 nucleotides: 50 -GATGTGTTTCTGCCTCTTCCATCC-30 (Gene Tools, Philomath, OR, USA). Capped mRNAs were synthesized using the mMessage mMachine kit (Ambion, Austin, TX, USA). The pCS107/IFT46-Flag and pCS107/IFT46 (5MT)-Flag constructs were linearized with Apa1. mRNAs and MOs were co-injected into the both blastomeres of 2cell staged embryos. 2.2. Western blot analysis Protein lysates were prepared by homogenizing embryos in lysis buffer (137 mM NaCl, 20 mM Tris-HCl (pH 8.0), 1% Nonidet-P40, 10% glycerol) supplemented with 1 mM phenylmehtylsulfonyl fluoride, 5 mM sodium orthovanadate, and 1 protease inhibitor mixture. Lysates were heated at 95 C in loading buffer for 5 min and electrophoresed by 12% SDS-PAGE; western blots were probed with monoclonal anti-Flag (1:1000, Applied Biological Materials, Richmond, British Columbia, Canada) and goat anti-mouse horseradish peroxidase-conjugated antibodies (1:10,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The immune-reactive bands were detected using an ECL kit (Hyclone, Logan, UT, USA).
2.4. Whole mount in situ hybridization Embryos were fixed in MEMFA (4% paraformaldehyde, 0.1 M MOPS (pH 7.4), 1 mM MgSO4, 2 mM EGTA) overnight at 4 C and then dehydrated before storage in 100% methanol at 20 C. To prepare the anti-sense digoxigenin (Dig)-labeling probe, the IFT46, SOX3, and PAX3 DNA templates were linearized using ApaI, HindIII, and NotI enzymes, respectively. Probes were generated using mMessage mMachine kit and were detected using alkaline phosphatase-labeled anti-Dig antibody (1:1000, Roche, Basel, Switzerland) and BM purple dye. Whole mount in situ hybridization was performed with appropriate staged embryos using the RNA probes of IFT46, SOX3, and PAX3.
2.5. siRNA transfection in hTERT-RPE1 cells We used hTERT-immortalized retinal pigment epithelial cell-1 (RPE-1 cells) to evaluate primary cilia. hTERT-RPE1 cells were cultured at 37 C, 5% CO2 in DMEM F-12 (Welgene, Seoul, Korea) containing 100 U/mL streptomycin/penicillin. Three types of siRNAs were transfected into hTERT-RPE1 cells using Lipofectamine RNAiMAX (Life Technologies, Carlsbad, CA, USA) for 24 h. Serum starvation was conducted to induce primary cilia for 48 h.
2.6. Immunofluorescence and confocal microscopy Both blastomeres of 2-cell staged embryos were injected and fixed at stage 32 in MEMFA (4% paraformaldehyde, 0.1 M MOPS (pH 7.4), 1 mM MgSO4, 2 mM EGTA) overnight at 4 C. Embryos were incubated in blocking buffer (PBS containing 1% bovine serum albumin and 5% heat-inactivated lamb serum). The primary antibody used for protein detection was monoclonal anti-acetylated tubulin (1:500, Sigma, St. Louis, MO, USA), and samples were visualized with Alexa 568 anti-mouse IgG (1:2000, Invitrogen, Carlsbad, CA, USA) and Alexa 488 anti-rabbit IgG (1:2000, Invitrogen) or Alexa 488 anti-mouse IgG (1:2000, Invitrogen) as secondary antibodies on a Zeiss LSM7 PASCAL confocal microscope (Carl Zeiss AG, Jena, Germany). Next, 100% methanol was used to fix hTERT-RPE1 cells in a LabTEK chamber (Nunc, Rochester, NY, USA), followed by immunofluorescence using the primary antibodies monoclonal anti-acetylated tubulin (1:500, Sigma) and monoclonal anti-Flag antibodies (1:500, Applied Biological Materials), which were visualized using Alex 568 anti-mouse (1:2000, Invitrogen). Three-dimensional projections, image processing and analysis were performed using a Zeiss LSM7 confocal microscope, Image J (NIH, Bethesda, MD, USA).
2.7. Alcian blue staining We harvested the stage 50 Xenopus embryos and fixed overnight at 4 C. Embryos were stained with 0.05% Alcian blue (Sigma) for 2 h, and bleached. Embryos were made clear using a mixture of 2:1 benzyl benzoate: benzyl alcohol.
2.3. Reverse transcription-polymerase chain reaction (RT-PCR)
2.8. Statistical analysis
cDNAs were prepared by reverse transcription using a Primescript 1st-strand cDNA synthesis kit (Takara, Shiga, Japan) from RNAs extracted from Xenopus embryos from stages 0e40 using a standard protocol. The PCR products were separated on a 1% agarose gel and images were captured using Wise Capture I-1000 (Daihan Scientific, Wonju, Kangwon-do, South Korea).
Data from RT-PCR and immunofluorescence staining were analyzed using ImageJ software. The results are presented as the mean ± standard error of the mean (n ¼ 5 with replicas for each sample). To determine the levels of significance, the results were analyzed based on standard deviation followed by ANOVA with Tukey post-hoc test.
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Fig. 1. Spatio-temporal expression patterns of IFT46 during embryogenesis. (A) Xenopus embryos were harvested at various stages and RT-PCR was performed using standard methods. The numbers indicate embryonic stages and ornithine decarboxylase (ODC) was a loading control. IFT46 is expressed as a maternal gene. (B) Xenopus embryos were harvested at stage 32 and processed via whole mount in situ hybridization using Dig-labeled antisense probes of IFT46. IFT46 was expressed in the central nervous system and multiciliated cells. The right figure is higher magnification view. (C) Both blastomeres of 2-cell stage embryos were injected with IFT46 mRNA containing a Flag tag and harvested at stage 27. Embryos were immunostained with acetylated tubulin-monoclonal antibody (green) and Flag-polyclonal antibodies (red). IFT46 was localized along the axoneme of motile cilia of epithelial cells. White arrows indicate IFT46 proteins. Right figure is higher magnification view (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Fig. 2. Loss of IFT46 prevents motile cilia formation in Xenopus epithelial cells. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and mutant mRNA, IFT46 (5MT), not recognized by MOs. Embryos were harvested at stage 32 and immunofluorescence staining of motile cilia with acetylated tubulin-monoclonal antibody (red) was carried out. (B) Relative quantification of green fluorescence per motile cilia of control MO- or IFT46 MO-injected embryos with IFT46 (5MT) mRNA. IFT46 MO and IFT46 (5MT) mRNA coinjected embryos showed partially rescued ciliogenesis. Data are shown as mean ± SD, and the number of embryos per sample is 25. ***p < 0.001 compared with control MOinjected. (C) Western blot analysis of exogenous IFT46-Flag mRNA expression (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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Fig. 3. Loss of IFT46 hinders primary cilia formation. (A) Both blastomeres of 2-cell staged embryos were injected with IFT46 MO and fixed embryos at stage 32. Primary cilia in the neural tube lumen were detected by immunofluorescence staining using acetylated tubulin-antibody (gray) and confocal microscopy. (B) Treatment of IFT46 siRNA for 24 h and serum starvation was performed to induce primary cilia for 48 h. We performed immunofluorescence staining of the nucleus and primary cilia using DAPI (blue) and acetylated tubulin-monoclonal antibody (red) respectively. Compared with the control, IFT46 siRNA-treated cells show short or no primary cilia. Data are shown as mean ± SD. ***p < 0.001 compared with control siRNA-treated. (C) RT-PCR was performed to evaluate the knockdown of IFT46. b-actin was used as a loading control (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
3. Results 3.1. IFT46 are expressed in the retina, central nervous system, and multiciliated cells during Xenopus embryogenesis To investigate mRNA expression patterns of IFT46, we conducted RT-PCR and whole mount in situ hybridization. The spatioexpression pattern of IFT46 revealed maternal nature of IFT46 gene (Fig. 1A). In addition, enhanced expression of IFT46 was detected during the blastula stage (st. 8) and neurula stage (st. 16) (Fig. 1A). Moreover, the spatial expression pattern following whole mount in situ hybridization showed strong expression of IFT46 in animal hemisphere at early stages (st. 8, data not shown) and central nervous system, including the head, retina, and ciliated cell in the epidermis at later stages (st. 32) (Fig. 1B). Double immunostaining against acetylated tubulin and Flag-tag (exogenous IFT46) confirmed the IFT46 expression in multiciliated cells of epidermis. Interestingly, IFT46 was localized along the motile ciliary axoneme including microtubule tracks as well as the ciliary tip (Fig. 1C). These results indicate that IFT46 is involved in neurulation and ciliogenesis during Xenopus development. 3.2. Loss of IFT46 causes ventralized phenotype To examine the functions of IFT46 in early vertebrate
development, we performed loss of functions experiments using MO to block the translation of IFT46 mRNA through competitively interacting with mRNA. Immunoblotting analysis showed that IFT46 MO inhibited the translation of exogenous IFT46 (Fig. 2C). Embryos injected with IFT46 MO displayed ventralized phenotype including shortened trunks along the anterior-posterior axis compared with control embryos (Supplementary Fig. S1). Our findings clearly exhibit that loss of IFT46 disrupts anterior-posterior axis elongation as well as head development. 3.3. IFT46 is crucial for primary and motile cilia formation in vivo and in vitro The involvement of IFT46 in ciliogenesis was confirmed by immunostaining the cilia of IFT46 morphants using antibodies against acetylated tubulin. Immunostaining showed significant reduction in number and length after IFT46 knockdown (Fig. 2A). The fluorescence intensity in the cilia of IFT46 knockdown embryos was 5fold lower than in control embryos (Fig. 2B). We verified the specificity of ciliogenesis defects due to IFT46 knockdown by performing a rescue experiment. We microinjected IFT46 MO with IFT46 mutant mRNA (IFT46 5MT), not recognized by MO, but translated to functional IFT46 proteins. Exogenous IFT46 proteins partially rescued the number and length of motile cilia up to 70% (Fig. 2A and B). Exogenous IFT46 expression was confirmed by
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
Fig. 4. Loss of IFT46 induces severe reduction in neural development and cartilage formation. (A) 10 ng of IFT46 MO was injected into the both blastomeres of 2-cell staged embryos with 2 ng of dominant-negative BMP-4 receptor (DN-BR) mRNA. Animal caps were dissected from injected embryos at stage 8.5 and collected at stage 18. The animal cap was processed for RT-PCR using standard methods. ODC was used as a loading control. No-RT indicates the control in the absence of reverse transcriptase. Quantification of the relative expression levels of BF-1, XAG, N-CAM, AP2a, and ZIC3 normalized to ODC expression. (B) Both blastomeres of 2-cell staged embryos were injected with 5 ng of IFT46 MO. Tadpoles were fixed at stage 50 and subjected to Alcian blue staining for cartilage formation. IFT46 morphants showed severe defects on cartilage formation (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
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immunoblotting (Fig. 2C). These results indicate that loss of IFT46 prevented motile cilia formation in Xenopus epithelial cells. Next, we investigated the effect of IFT46 on primary cilia formation and motile cilia elongation by examining primary cilia morphology in the neural tube lumen after embryos were sectioned. The primary cilia in IFT46 morphants showed similar defects as motile cilia (Fig. 3A). Moreover, by depleting IFT46 in RPE1 cells using siRNA, the effects of loss of IFT46 were confirmed on primary cilia formation. Primary cilia were induced by serum starvation for 48 h and examined by using acetylated tubulin antibodies specifically expressed in cilia. The number of ciliated cells was greatly decreased in IFT46 depletion cells (Fig. 3B). The efficiency of IFT46-specific siRNA was confirmed by RT-PCR (Fig. 3C). Hence, IFT46 is essential for motile and primary cilia formation. 3.4. IFT46 is important for neural development According to ventralized phenotype of the IFT46 morphants, we examined the effects of IFT46 on neural tube closure defects. Whole mount in situ hybridization with the neural tube marker PAX3, being governed by Wnt/planar cell polarity (PCP), sonic hedgehog (shh) signaling pathways and neural plate marker SOX3, being regulated by Wnt/PCP signaling pathway showed that IFT46 morphants displayed neural tube closure defects and wider neural plates (Supplementary Fig. S2). Our results show that loss of IFT46 inhibits convergence-extension movement during gastrulation and perhaps by influencing Wnt/PCP and sonic hedgehog signaling pathways. Next, to investigate the role of IFT46 in neurogenesis, we introduced dominant-negative BMP-4 receptor (DN-BR) to block BMP-4 signaling with IFT46 MO. Loss of IFT46 did not influence DNBR-induced pan-neural marker gene expression, N-CAM. However, loss of IFT46 decreased the level of DN-BR-induced BF1, XAG (an anterior neural marker), and AP2a, ZIC3 (a neural crest cell marker) expression (Fig. 4A). Moreover, we observed knockdown of IFT46 specificity by rescue experiments. These results clearly indicate that IFT46 affects neurogenesis by influencing neural gene expression. 3.5. Loss of IFT46 induces severe reduction of craniofacial cartilage formation Mostly craniofacial cartilage is induced by neurogenesis, including neural crest cells [23]. To investigate the role of IFT46 in craniofacial development, we examined cartilage morphology by alcian blue staining using antibodies against glycosaminoglycan, present in cartilage. As shown in Fig. 4B, IFT46 knockdown caused severe reduction of cartilage formation including the branchial stream, hyoid stream, and non-neural crest. Taken together, these results suggest that IFT46 regulates craniofacial development and neurogenesis. 4. Discussion Ciliary epithelial cells are essential for the proper functioning of various organs, and the loss of ciliary function induces ciliopathies including neural tube closure defects and abnormal craniofacial development [24]. IFT46 was first characterized in C. reinhardtii as a retrograde trafficking system protein interacting with ciliary components including IFT88, IFT70, and IFT52 and links to the motor protein kinesin [25]. Knockdown of IFT46 causes unstable complex B structure, loss of the outer dynein arm in the cilia [26], and shortened cilia in numerous ciliated tissues including Kupffer’s vesicle, pronephric ducts, ears, and spinal cord [22]; however, effects of
IFT46 on embryogenesis need further investigation. Some signaling pathways including Wnt/PCP and Shh are needed for proper nervous system development, particularly for neural tube closure [27]. Primary cilia act as platforms on which signaling proteins are localized [27]. Previous studies using mice showed that disruption of IFT resulted in improper neural tube patterning [27]. IFT88 and IFT172 mutants led to neural tube mispatterning through the shortening of cilia and low Shh signaling via the loss of GliA and GliR [27]. In addition, disruption of the PCP effectors Inturned and Fuzzy can cause ciliary loss and neural tube closure defects [28]. Disheveled mutant mice also displayed neural tube closure defects similar to those in IFT or Shh signaling mutant mice [29]. Ciliogenesis and signaling pathways such as PCP and Shh influence neural development. However, the involvement of IFT46 in signaling transduction for neurogenesis and craniofacial formation is unknown. Most craniofacial cartilage is induced by neurogenesis, including neural crest cells. Cranial neural crest stem cells that differentiate into chondrocytes containing primary cilia consist of craniofacial cartilage [23,30]. Recent reports suggested that primary cilia are important for the maintenance of craniofacial cartilage [31]. Conditional deletion of Kif3a resulted in the loss of primary cilia, excessive Hedgehog signaling, and aberrant neural crest cell proliferation in a Gli-dependent manner as well as unusual craniofacial formation [31]. In addition, IFT88 transgenic mice showed upregulation of Hedgehog signaling and thick cartilage [31]. In current study, we investigated that IFT46 is essential for neurogenesis including neural tube closure (Fig. 4 and Supplementary Fig. S2) and knockdown of IFT46 resulted in a loss of primary cilia regulating neural crest cell gene expression and induced unusual craniofacial development (Fig. 4). AP2a and ZIC3, which are associated with neural crest cells, are required for craniofacial and skeletal morphogenesis [32,33]. It is likely that loss of IFT46 was responsible for the reduction in craniofacial cartilage formation via neural crest cell expression of proteins such as AP2a and ZIC3. We also demonstrated that IFT46 is required for motile and primary cilia elongation by using Xenopus embryos and hTERTimmortalized RPE-1. In conclusion, our results suggest that IFT46 regulates ciliogenesis, neurogenesis and craniofacial development by modulating specific signaling pathways such as Wnt/PCP and shh. Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) funded by the Korea government [MSIP] (Grant No. 2015R1A4A1042271 and NRF-2015R1A2A1A10053265), Republic of Korea. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.083. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.06.083. References [1] S. Roy, The motile cilium in development and disease: emerging new insights, Bioessays 31 (2009) 694e699. [2] S.C. Goetz, K.V. Anderson, The primary cilium: a signalling centre during vertebrate development, Nat. Rev. Genet. 11 (2010) 331e344. [3] N.F. Berbari, A.K. O’Connor, C.J. Haycraft, B.K. Yoder, The primary cilium as a
Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083
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Please cite this article in press as: I. Park, et al., IFT46 plays crucial roles in craniofacial and cilia development, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.06.083