Abstract
Primary cilia are microtubule-based protuberances that project from the eukaryotic cell body to sense the extracellular environment. Ciliogenesis is closely correlated to the cell cycle and defects of cilia are related to human systemic diseases such as primary ciliary dyskinesia. However, the role of ciliogenesis in cortical development remains unclear. Here, we demonstrate that Cenpj, a protein that is required for centriole biogenesis, plays a role in regulating cilium disassembly in vivo. Depletion of Cenpj in neural progenitor cells results in long cilia and abnormal cilia disassembly. Radial glial cells Cenpj depletion exhibit uncompleted cell division, reduced cell proliferation, and increased cell apoptosis in the developing mouse cerebrum cortex, leading to microcephaly. In addition, Cenpj depletion causes long and thin primary cilia and motile cilia in adult neural stem cells and reduced cell proliferation in the subventricular zone. Furthermore, we show that Cenpj regulates cilia disassembly and neurogenesis through Kif2a, a plus-end-directed motor protein. These data collected from mice of both sexes provide insights into how ciliogenesis plays roles in cortical development and how primary microcephaly is induced by Cenpj mutations in humans.
SIGNIFICANCE STATEMENT Autosomal recessive primary microcephaly is a neurodevelopmental disorder with the major symptoms of reduction of circumference of the head, brain volume, and cortex thickness with normal brain architecture in birth. We used conditional Cenpj deletion mice and found that neural progenitor cells (NPCs) exhibited long primary cilia and abnormal cilium appendages. The defective cilium disassembly caused by Cenpj depletion might correlate to reduced cell proliferation, uncompleted cell division, cell apoptosis, and microcephaly in mice. Cenpj also regulates the cilium structure of adult neural stem cells and adult neurogenesis in mice. Additionally, our results illustrate that Cenpj regulates cilia disassembly and neurogenesis through Kif2a, indicating that primary cilia dynamics play a crucial role in NPC mitosis and adult neurogenesis.
Keywords: CenpJ, cerebral cortex, neurogenesis, primary cilia, radial glial cell, subventricular zone
Introduction
Centrosomes are the organelles at the poles of the spindle that can persist into interphase as microtubule organizing centers. The core is a ninefold symmetrical centriole, and their ability to nucleate cytoplasmic microtubules (MTs) is a property of the surrounding pericentriolar material (PCM). The centriole has a dual life, existing not only as the core of the centrosome, but also as the basal body (BB) for primary cilia assembly. As a result, the structure and function of the centriole, the centrosome, and the cilia have an impact on many aspects of development and physiology. The primary cilium, a continuation and extension of microtubule doublets from the BB structure, is localized to the apical region of a radial glial (RG) cell. In the G0 quiescent phase, the RG cell processes a primary cilium out of the ventricular end foot surface. Upon entry into the cell cycle, the primary cilium disassembles quickly to release the centrioles to form a bipolar spindle (Kobayashi and Dynlacht, 2011; Nigg and Stearns, 2011). Cilium assembly and disassembly are highly correlated to the cell cycle and centrosome (Pugacheva et al., 2007; Qin et al., 2007; Robert et al., 2007). The dynamics of primary cilia, unique in their ability to function as sensors and conveyors of critical signals in a complex environment, may have a supervisory role in the neurogenesis of RG cells.
Mutations of Cenpj or CPAP (a centrosomal-P4.1-associated protein) cause microcephaly, dwarfism, low birthweight, and intellectual disability (Al-Dosari et al., 2010), which are thought to arise from a decline in neural progenitor cells (NPCs) during development (Insolera et al., 2014). Depletion of Cenpj causes severe defects in centriole duplication and increases the population of monopolar, asymmetric bipolar, and multipolar spindles in mitotic cells (Cho et al., 2006; Kohlmaier et al., 2009; Schmidt et al., 2009; Tang et al., 2009; Kitagawa et al., 2011). In addition, Cenpj acts as a scaffold for the cilium disassembly complex, which includes Nde1, Aurora A, and OFD1, which is recruited to the ciliary base for timely cilium disassembly (Gabriel et al., 2016). Mouse embryos with complete elimination of Cenpj present prolonged mitosis and a widespread, p53-dependent cell death phenotype resulting in a reduction in RG cells and other types of NPCs (McIntyre et al., 2012; Bazzi and Anderson, 2014; Insolera et al., 2014; Garcez et al., 2015). A recent study has shown that the cells of cerebral organoids derived from Cenpj-mutant microcephaly patients present long cilia (Gabriel et al., 2016). Contrary to observations in humans, flies with Dsas-4 (the ortholog of Cenpj) mutations show no cilia or flagella (Basto et al., 2006) and deletion of Cenpj by shRNA treatment blocked cilia formation and caused cilia shortening in CAD (Cath. a-differentiated) cells (Wu and Tang, 2012). These observations indicate that Cenpj plays a role in NPC proliferation and cilia formation. However, how the balance between centrosome and cilia functions regulates cortical development is largely unknown.
By conditionally deleting Cenpj in mouse NPCs, we found longer primary cilia and abnormal cilium appendages in RG cells. The defective cilium disassembly upon Cenpj depletion led to reduced cell proliferation, uncompleted cell division, cell apoptosis, and microcephaly in mice. Cenpj depletion also caused long motile cilia with defective structures in ependymal cells and a reduction in adult neural stem cells in mice. Additionally, we found that Kif2a, a member of the kinesin-13 motor proteins, contributes to Cenpj-regulated cilia disassembly and neurogenesis. These findings indicate that primary cilia dynamics are essential for mitosis of RG cells and are required for constructing a normal cerebral cortex.
Materials and Methods
Animals.
Adult CenpjLacZ mice (Cenpjtm1a (EUCOMM)Wtsi) were obtained from the Welcome Trust Sanger Institute Mouse Genetics Projects. Cenpjfl/fl mice were generated by crossing CenpjLacZ mice with Rosa26Flp mice. Cenpjfl/+ Emx1-Cre mice were produced by crossing Cenpjfl/fl mice with Emx1-Cre mice. Conditional knock-out (CenpjCKO) mice were produced by crossing Cenpjfl/+ Emx1-Cre mice with Cenpjfl/+ Emx1-Cre mice. CenpjCKOCiliaGFP mice were obtained by crossing Cenpjfl/+ Emx1-Cre mice with CiliaGFP mice (R26Sstr3GFP, a gift from Dr. Bradley Yoder at the University of Alabama at Birmingham). Animal housing and experimental procedures in this study were in compliance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Biophysics, Chinese Academy of Sciences. All mice had ad libitum access to food and water and were housed in the institutional animal care facility (specific pathogen free) with a 12 h light/dark schedule. Both males and females were used for all experiments.
Cell culture.
ARPE19 cells were grown in medium containing DMEM, 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies). 293T cells were grown under standard conditions in DMEM supplemented with 10% FBS. For serum starvation experiments, cells were grown in medium without serum for the described time period and stimulated with 10% FBS-containing medium. Cells were maintained at 37°C with 5% CO2.
Western blotting.
The tissue and culture cells were lysed in RIPA (added protease inhibitor). Protein samples were separated by SDS-PAGE and transferred to nitrocellulose membranes. After the nitrocellulose membranes were blocked with 5% milk for 1 h, the nitrocellulose member was incubated with the primary antibody at 4°C overnight and then incubated with horseradish peroxidase-conjugated secondary antibody for 45 min at room temperature. Enhanced chemiluminescence reagent (Thermo Scientific) was used as a Western blotting substrate to visualizing the protein bands. The antibodies used as follows: GAPDH (1:5000, KC-5G5; KangChen), Cenpj (1:200, 11517-1-AP; Proteintech), and Kif2a (1:5000, PAB12407; Abnova).
Immunostaining.
Brain slices were fixed in 4% paraformaldehyde in PBS, pH 7.4, and culture cells were fixed in ice-cold methanol, permeabilized with 0.1% Triton X-100 in PBS for 15 min, and blocked with 10% donkey serum in PBS for 2 h at room temperature (RT). Antibody incubations were performed overnight at 4°C for brain slices or 1 h at RT for cell cultures, followed by three washes in PBS. Fluorescent-conjugated secondary antibody incubation for 2 h at RT. Images were acquired using a confocal laser scanning microscope (FV1000MPE-BX61W1; Olympus) and were analyzed using Fluo View (Olympus) with Imaris (Bitplane) and Photoshop (Adobe Systems) software. Primary antibodies used were as follows: Zo-1 (1:300, 339100; Invitrogen), γ-Tubulin (1:5000, T3559; Sigma-Aldrich), γ-Tubulin (1:5000, T5326; Sigma-Aldrich), Acetylated Tubulin (1:30000, T7451; Sigma-Aldrich), Cenpj (1:50, 11517-1-AP; Proteintech), Pericentrin (1:300, 611814; BD Biosciences), GFP (1:500, GFP-1020; Aves Laboratories), Cleaved Caspase 3 (1:300, 9664; Cell Signaling Technology), Tbr2 (1:300, ab23345; Abcam), Satb2 (1:300, ab34735; Abcam), Ctip2 (1:300, ab18465; Abcam), Ki67 (1:300, ab9260; Millipore), BrdU (1:300, ab6326; Abcam), Pax6 (1:300, PRB-278P; Covance), Sox2 (1:300, sc-17320; Santa Cruz Biotechnology), EdU (Click-iT EdU Alexa Fluor 594 Imaging Kit, C10339; Thermo Scientific), GFAP (1:500, G9269,Sigma-Aldrich), and β-Catenin (1:300, 610153; BD Biosciences). Secondary antibodies used were as follows: donkey anti-mouse, anti-rabbit, anti-chicken, anti-rat or anti-goat Alexa Fluor-546-, Alexa Fluor-488-, and Alexa Fluor-647-conjugated antibodies (1:500; Invitrogen). DNA was stained with 49,6-diamidino-2-phenylindole (1:10000, D1306; Invitrogen).
Plasmids and in utero electroporation.
Cenpj and Kif2a shRNA sequences were cloned into pLL3.7 vector (Addgene, catalog #11795) as described previously (Wang et al., 2009). Kif2a sequences were cloned into pEGFP-C1 vector and ptdTomato-C1 vector. Cenpj sequences were cloned into ptdTomato-C1 vector. Electroporation was performed as described previously (Wang et al., 2009). Briefly, timed pregnant CD-1 mice (E13.5) were deeply anesthetized with isofluorane and the uterine horns were exposed through a midline incision. One microliter of plasmid DNA (1–2 μg/μl) mixed with Fast Green (Sigma-Aldrich) was manually microinjected into the brain lateral ventricle through the uterus using a bevelled and calibrated glass micropipette (Drummond Scientific), followed by 5 50 ms pulses of 50 mV with a 1 s interval delivered across the uterus with 2 9 mm electrode paddles positioned on either side of the head (BTX, ECM830).
Cortical slice culture and time-lapse imaging.
Approximately 12 h after in utero electroporation, embryos were removed and the brain was extracted into ice-cold artificial CSF (ACSF) containing the following (in mm): 125 NaCl, 5 KCl, 1.25 NaH2PO4, 1 MgSO4 2 CaCl2, 25 NaHCO3, and 20 glucose; pH 7.4, 310 mOsm l-1. Brains were embedded in 3.5% low-melting-point agarose in ACSF and sectioned at 300 μm using a vibratome (Leica microsystems). Brain slices that contained GFP-expressing cells were transferred on to a slice culture insert (Millicell) in a glass-bottom Petri dish (MatTek) with culture medium containing (by volume): 66% BME, 25% Hank's, 5% FBS, 1% N-2, 1% penicillin/streptomycin/glutamine (Invitrogen) and 66% d-(+)-glucose (Sigma-Aldrich). Cultures were maintained in a humidified incubator at 37°C with constant 5% CO2 supply. Two hours later, Petri dishes with slice cultures were transferred to an inverted microscope FV1000 (Olympus). Time-lapse images of dividing radial glia cells were acquired every 10 min for ∼5–8 h. Images were analyzed using Fluo View (Olympus) and Photoshop (Adobe Systems).
Real-time PCR assay.
Total RNA was extracted from cells by using SV Total RNA Isolation System (Promega). Reverse transcription was performed with the Prime Script II first Strand cDNA Synthesis Kit (Takara) and real-time PCR was performed by using SYBR Premix Ex Taq (Tli RNaseH Plus; Takara). The real-time PCR contained 5 μl of qPCR Mix, 0.4 μl of forward primer, 0.4 μl of reverse primer, 1 μl of cDNA template, and 3.2 μl of ddH2O and in the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 15 s. The data were normalized to that of the internal control. Primer sequences are described in the supplemental information.
RNA-sequencing (RNA-Seq) and library construction.
For RNA-Seq analysis, a timed pregnant mouse at E15.5 were anesthetized. Embryos were removed separately and genotyping was performed to identify the Cenpjf/+ and CenpjCKO pups. Cortical slices of Cenpjf/+ and CenpjCKO embryos were prepared as described above. The ventricular zone (VZ)/subventricular zone (SVZ) regions were dissected separately and RNA was isolated using RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. After the samples were tested, the eukaryotic mRNA was enriched by magnetic beads with Oligo (dT). Subsequently, using fragmentation buffer to break the mRNA into short fragments, t mRNA was used as a template to synthesize a strand of cDNA with six random bases and then the buffer, dNTPs, and DNA polymerase I were used to synthesize the double-stranded cDNA, followed by AMPure XP beads of purified double-stranded cDNA. The purified double-stranded cDNA was subjected to terminal repair, a tail was added, and the sequencing adapter was ligated. The size of the fragment was then selected with AMPure XP beads and, finally, PCR enrichment was performed to obtain the final cDNA library. RNA-Seq was performed by the Novogene Bioinformatics Institute, Beijing.
Scanning electron microscopy.
Mice were anesthetized and perfused with 0.1 m PBS buffer pH 7.4 containing 2.5% glutaraldehyde (Electron Microscopy Sciences) and 2% formaldehyde (Sigma-Aldrich). Brains were then removed and fixed overnight in the same solution at 4°C and cut into 500-μm-thick vibratome sections in PBS, washed three times with PBS, and then dehydrated through an ethanol gradient (30%, 60%, 90%, and 100%). Samples were dried without introducing surface tension artifacts by using critical point drier. After drying, samples were then introduced into the chamber of the sputter coater and coated with a very thin film of gold before SEM examination. 3D reconstruction was analyzed by Amira and Imaris.
Transmission electron microscopy.
Mouse embryos were perfused with 2% glutaraldehyde and 2% paraformaldehyde in PBS and then fixed in perfusion solution overnight at 4°C. The samples were washed 3 times with PBS then secondarily fixed in 2% osmium tetroxide (Sigma-Aldrich) in cacodylate buffer for 2 h at room temperature. The samples were washed three times with ddH2O, stained with uranyl acetate then dehydrated through an ethanol gradient (30%, 60%, 90%, and 100%) followed by propylene oxide. Subsequently, samples were embedded in low-viscosity resin (TAAB Laboratories). Sections were cut on a microtome (Leica, EM UC6+FC6) and then viewed on a transmission electron microscope (Tecnai Spirit, 120 kV).
3D model reconstruction.
We took serial images of the brain tissue at 80 nm intervals with a focused ion beam scanning electron microscope (FIB-SEM). We manually drew the morphology of the cilium of the ependymal cells in each image and then created a surface using these consecutive pictures by Imaris software. A surface represents a cilium. All cilia were drawn in this manner. Multicilia 3D morphology of ependymal cells was reconstructed.
Quantification of polarity defects.
We used the algorithm previously published for the characterization of PCP (Planar Cell Polarity) defects in the same system (Boutin et al., 2014). For the analysis of translation polarity defects in ependymal multiciliated cells, we imported images into MATLAB and saved regions of interest. Then we traced the cell borders and basal body based on Zo-1 and γ-tubulin staining respectively. Centers of mass were calculated for BBs and cellular outline within every cell. Angles of BB orientation vectors (BBOVs) were measured and CSD across all BBOVs within every field of view was assessed. Each experimental condition was characterized by mean and CSDs across all fields of view. CSD values were calculated for BBOV angles. The angles measured within one image plane were normalized by subtracting from their values the average angle of a corresponding field of view, this difference was added to 90° and plotted on the summarized graph.
Statistical analysis.
All data were represented as the mean ± SD. Comparisons between two groups were performed using t tests. The quantification of polarity defects used an unpaired Mann–Whitney test. The quantification graphs were analyzed by using GraphPad Prism software. Sample size and p-values are described in the figure legends. Differences were considered significant at p < 0.05.
Results
Cenpj deletion in mice causes abnormal cilia and microcephaly
To define the function of Cenpj in ciliogenesis in vivo and the cellular basis of centrosome-associated microcephaly, CenpjCKO mice were developed by crossing Cenpjfl/fl mice with Emx1-Cre mice, which selectively express Cre recombinase in dorsal telencephalic NPCs (Fig. 1A). Western blot analysis of the mouse cortex lysate at E15.5 confirmed that the expression of Cenpj was depleted in the CenpjCKO cortex (Fig. 1B,C). Brain sections were immunostained for Cenpj and pericentrin, a pericentriolar material protein, and no Cenpj signals were detected in centrosomes on the VZ (Fig. 1D). With Cenpj deletion in NPCs of the dorsal telencephalon, the brain hemisphere circumference of CenpjCKO embryos was greatly decreased compared with that of the control Cenpj+/+ embryos at E15.5 (Fig. 1E,F). Nissl staining of brain slices at E15.5 revealed significantly reduced thickness of the cortex with a normal layered structure upon Cenpj depletion (Fig. 1G,H). We also checked the cortical development of the Cenpj+/+ and CenpjCKO cortex at P7. Although body size was not influenced by Cenpj depletion, a reduced brain size, smaller hemisphere circumference and thinner cortex were observed in the CenpjCKO mice (Fig. 1I–K). The numbers of early-born Ctip2-expressing deep layer neurons and late-born Statb2-expressing superficial layer neurons were both reduced in the CenpjCKO cortex (Fig. 1L,M). Consistently, we observed a decrease in Foxp2-expressing layer VI neurons and Cux1-expressing layer II/III neurons following Cenpj depletion but with normal lamination (Fig. 1N–P). The phenotypes were consistent with the phenotypes of microcephaly patients caused by Cenpj mutations (Leal et al., 2003; Gul et al., 2006; Al-Dosari et al., 2010); therefore, we used the mouse model to investigate Cenpj-related microcephaly pathogenic mechanisms.
To detect the cilia in vivo, a Cre recombinase-inducible CiliaGFP mouse (O'Connor et al., 2013) was used. CenpjCKOCiliaGFP RG cells had longer cilia than Cenpj+/+CiliaGFP RG cells (Fig. 2A,B). As observed by scanning electron microscope, RG cells of the CenpjCKO mice consistently contained long cilia with a median length of 1.93 ± 0.29 μm compared with RG cells of Cenpj+/+ mice, which contained cilia with a median length of 1.11 ± 0.15 μm at E15.5 (Fig. 2C,D). At the end of cortical development, monociliated progenitor cells in the VZ differentiate into multiciliated ependymal cells (Spassky et al., 2005). Therefore, an FIB-SEM was used to capture serial images at 80 nm intervals. Multicilia 3D morphology of ependymal cells was reconstructed (Fig. 2E, Movies 1, 2, 3, 4). The reconstructed images showed that the motile cilia length (11.4 ± 1.6 μm) of ependymal cells was increased in CenpjCKO mice compared with that in Cenpj+/+ mice (7.2 ± 2.5 μm) (Fig. 2E,F). In addition, CenpjCKO cilia were disarrayed on the cell surface compared with organized Cenpj+/+ cilia (Fig. 2E). The diameters of the CenpjCKO cilia were smaller than those of the Cenpj+/+ cilia (Fig. 2G). Furthermore, on the adult brain ventricle surface of CenpjCKO mice, the motile cilia were curled and tangled with bulges and curly tips when observed by scanning electron microscope (Fig. 2H, arrow).
Cilia emerge from a tubulin-based structure called the BB (Yang et al., 2005; Mohan et al., 2013). Ultrastructural analysis revealed that the majority of primary cilium BBs of CenpjCKO RG cells lacked one or more appendages, including distal appendages (arrowhead) and subdistal appendages (arrow), at E15.5 (Fig. 2I,J). Similarly, the ultrastructure of the motile cilia appendages in adult ependymal cells exhibited abnormal organization upon Cenpj depletion. Unlike the wild-type mice, which had distal and subdistal appendages, the appendages of CenpjCKO mice were hair-like with some parts gathered into a bulging shape. We found ∼97.9% of RG cells with abnormally disorganized appendages, including distal appendages (arrowhead) and subdistal appendages (arrow), and striated rootlets (asterisks) in the SVZ of adult CenpjCKO mice (Fig. 2K). The aberrant arrangement of the microtubule doublets was also observed in CenpjCKO motile cilia by transmission electron microscopy (Fig. 2L,M). Together, these results suggest that Cenpj plays a role in regulating cilia structural elements, including length, width, microtubule structure, and appendage organization, in embryonic and adult NPCs.
Cenpj controls NPC division by regulating cilia disassembly
It has been demonstrated that ciliary dynamics, including assembly and disassembly, can affect the timing of the cell cycle (Kim and Tsiokas, 2011; Basten and Giles, 2013). Previous research has suggested that primary cilia disassemble when cells enter mitosis to release the centrosome to assemble the spindle body (Wang et al., 2014). Consistent with previous observations (Paridaen et al., 2013), we found ∼8.2% RGCs with disassembled cilia by immunostaining of γ-tubulin in the RG cells of Cenpj+/+CiliaGFP mice, but this number increased dramatically to 33.8% of CenpjCKOCiliaGFP RG cells at E15.5 (Fig. 3A,B). Consistently, en face views of the VZ showed an increased percentage of prophase and metaphase RG cells and a decreased percentage of anaphase and telophase RG cells on the VZ surface upon Cenpj depletion (Fig. 3C,D). However, Cenpj depletion has no obvious effect on cell–cell adhesion of RG cells as shown by N-cadherin immunofluorescent staining (Fig. 3C). To further observe dividing cells, immunostaining of phosphorylation of histone H3 was used to label mitotic RG cells. We observed that more dividing cells were in prophase and metaphase in the CenpjCKO VZ compared with the Cenpj+/+ VZ (Fig. 3E,F), suggesting that the disassembly of cilia blocked the cell cycle of RG cells.
Next, we evaluated whether Cenpj depletion delays the mitosis of RG cells. We first reduced Cenpj expression by short-hairpin RNAs (shCenpj-a, shCenpj-b and shCenpj-c; Fig. 3G,H). Cenpj shRNA constructs were electroporated into the mouse cortex via in utero electroporation (IUE) at E13.5 and the brains were examined 3 d later (E16.5). In contrast to shControl-GFP-electroporated samples, most of the shCenpj-GFP-transfected cells were found in the intermediate zone, suggesting that Cenpj depletion impairs neurogenesis, possibly including migration (Fig. 3I,J). This neurogenesis defect was rescued by exogenous expression of Cenpj-tdTomato plasmid (Fig. 3K–M). High-efficiency shCenpj-a was selected to be electroporated at E13.5 and cell divisions of RG cells were recorded by time-lapse microscopy. Compared with normal interkinetic nuclear migration and mitosis, RG cells stayed in prophase/metaphase for an abnormally long time following Cenpj depletion (Fig. 3N, Movies 5, 6). To investigate whether Cenpj regulates cilium disassembly to affect the cell cycle, shCenpj-a-transfected ARPE19 cells were starved for cilia induction first and then cultured with serum stimulation for 24 h. The ciliated cells were counted 0 and 24 h after serum stimulation. Twenty-four hours after serum stimulation, the deletion of Cenpj led to cilia disassembly delay (Fig. 3O,P) and a significant reduction in the proportion of mitotic cells (Fig. 3Q). Together, our results indicate that abnormal cilia disassembly caused by Cenpj depletion influences RG cell divisions.
Because RG cells are mother cells of other types of NPCs involved in cortical development, we next investigated how abnormal cilia caused by Cenpj depletion exert an influence on NPC behavior. NPCs of Cenpj+/+ and CenpjCKO mice at E14.5 were sequentially pulse-chased by using EdU and BrdU, two thymidine analogs, spaced 22 h apart (Fig. 4A). A reduced number of cells with EdU incorporation in CenpjCKO cortices indicated that fewer cells entered S phase at E14.5 (Fig. 4B). Additionally, 66.37 ± 3.13% Cenpj+/+ NPCs and 41.32 ± 3.54% CenpjCKO NPCs in the VZ/SVZ underwent further rounds of cell division in the next 22 h (Fig. 4C), suggesting that cortical NPCs with Cenpj depletion showed less proliferating potential for consecutive divisions during cortical development. In addition, signals from the apoptosis marker Caspase 3 were observed in the CenpjCKO cortex at E15.5 (Fig. 4D,E). We next examined the cortical development and neurogenesis capacities of NPCs upon Cenpj depletion. Compared with the control group, decreased expression of the NPC markers Sox2 and Pax6 was observed in the CenpjCKO cortex compared with the Cenpj+/+ cortex at E15.5 (Fig. 4F,G,I). In addition, the population of intermediate progenitor cells identified by Tbr2 was also reduced upon Cenpj depletion (Fig. 4F,H). Together, these results suggest that depletion of Cenpj reduces NPC proliferation capacities and progenitor number, resulting in cortical thickness reduction during brain development.
Cenpj knock-out reduces adult neural stem cells
The CenpjCKO adult mutants also showed a strong microcephalic phenotype in which the telencephalic area estimated by circumference measurement of CenpjCKO mice was reduced by 38.0% in 2-month-old mice (Fig. 5A,B). Nissl staining of Cenpj+/+ and CenpjCKO brain slices showed remarkable reductions in the thickness of the cerebral cortex in adult mice upon Cenpj depletion (Fig. 5C,D). Within the VZ/SVZ of the adult mammalian brain, adult neural stem cells (aNSCs) remain mitotically active and continuously generate olfactory bulb interneurons (Codega et al., 2014). Glial fibrillary acidic protein (GFAP)-positive B1 cells are considered to be aNSCs (Doetsch et al., 1999; García et al., 2004; Mirzadeh et al., 2008). To examine the aNSCs in the VZ/SVZ, we visualized the aNSCs and niche astrocytes in the VZ/SVZ by immunostaining for GFAP. GFAP+ cells were highly enriched on the surface of the lateral ventricle in Cenpj+/+ brains, but only a few GFAP+ cells were observed in CenpjCKO adult brains (Fig. 5E). Consistent with this observation, dramatically reduced Ki67+ cells were also observed in the VZ/SVZ of the CenpjCKO brains (Fig. 5E). Furthermore, there were fewer SOX2+ type C cells in the VZ/SVZ of the CenpjCKO brains (Fig. 5F,G). The VZ/SVZ in the adult mouse brain is a highly organized microenvironment composed of aNSCs and multiciliated ependymal cells, which are responsible for the niche cytoarchitecture and the regulation of the self-renewal and differentiation potential of aNSCs (Lim et al., 2000; Kuo et al., 2006; Ramírez-Castillejo et al., 2006; Gajera et al., 2010; Paez-Gonzalez et al., 2011). En face and side views of the ventricle were labeled by immunofluorescence staining for β-catenin, which is a marker of ependymal cell membranes, and GFAP, which is a marker of B1 cells, in the Cenpj+/+CiliaGFP and CenpjCKOCiliaGFP brains at postnatal day 40 (P40) (Fig. 5H,I). GFAP+ B1 cells were reduced in CenpjCKO brains and the orientation of the motile cilia on the ependymal cells was disarrayed in CenpjCKO brains (Fig. 5H,I). We also found that the lengths of the primary cilia on the B1 cells in CenpjCKO brains were increased compared with those in Cenpj+/+ brains (Fig. 5H,I). Cilia BBs migrate toward the anterior side of a cell, creating so-called translational polarity, which is critical for the coordinated movement of cilia (Mirzadeh et al., 2010; Wallingford, 2010). We further addressed the BB patches by confocal microscopy using the BB marker γ-tubulin and the ependymal cell membrane marker Zo-1 to reveal the structure of the Cenpj+/+ and CenpjCKO VZ/SVZ. BB patches are essential for the directional beating of motile cilia. BB patches of CenpjCKO adult mice often migrated in a different direction from the neighboring cells, whereas the majority of Cenpj+/+ BB patches were in the same direction (Fig. 5J,K). Moreover, aNSCs continuously generate olfactory bulb interneurons (Codega et al., 2014). Neuron-specific class III β-tubulin (Tuj1) was expressed in immature postmitotic neurons. As expected, Tuj1+ cells were reduced in the olfactory bulb of CenpjCKO adult mice compared with those in the olfactory bulb of Cenpj+/+ adult mice, especially in the granule cell layer. Moreover, decreased Dcx+ cells were also observed in the granule cell layer (Fig. 5L). Cenpj depletion not only affects aNSC proliferation and adult neurogenesis, but also influences the primary cilia of B1 cells and motile cilia of the ependymal cells, showing long cilia with disorganized arrangements.
Cenpj regulates cilia disassembly and cortical development through Kif2a
To understand the molecular mechanism underlying how Cenpj depletion leads to cilium abnormality and microcephaly, we performed RNA-Seq to analyze the transcriptome changes in Cenpj-depleted cortices at E15.5. An RNA-Seq correlation test showed the high similarity of the expression patterns between Cenpj+/+ and CenpjCKO samples (Fig. 6A): 11,569 genes were expressed in both CenpjCKO and Cenpj+/+ samples and 1513 genes and 370 genes were expressed in only Cenpj+/+ or CenpjCKO samples, respectively (Fig. 6B). By comparing gene expression levels in Cenpj+/+ and CenpjCKO samples, we found 3517 differentially expressed genes (1643 upregulated and 1874 downregulated) (Fig. 6C, log2 (fold change) > 1, q-value < 0.005). Gene ontology term analysis of the downregulated genes in E15.5 CenpjCKO samples showed a significant enrichment of terms related to cell cycle, cell migration, neurogenesis, neuronal differentiation, and axonogenesis, whereas the upregulated genes were enriched in cell death and cilium assembly (Fig. 6D,E). KEGG signaling pathway analysis revealed that the differentially expressed genes are predicted to be involved in Wnt, MAPK, Hippo, AMPK, and Notch, as well as other signaling pathways (Fig. 6F). With Cenpj depletion, we found that a number of genes involved in cilium disassembly were downregulated in the developing mouse cortex at E15.5, including Kif2a, Kif24, Plk1, Nek2, and Ccp110 (Fig. 6G), whereas some cilium assembly genes, such as Rab8a and Mks1 (Fig. 6H), were upregulated, which is consistent with the RNA-Seq data.
Cilium assembly and disassembly are tightly controlled by Wnt signaling pathways (Liang et al., 2016; Sánchez and Dynlacht, 2016). Given that Kif2a is a component of the Wnt pathway and plays a role in MT depolymerizing and disassembly of primary cilia (Trinczek et al., 2004; Miyamoto et al., 2015) and that Kif2a transcription was reduced upon Cenpj depletion, we hypothesized that Cenpj may regulate cilia disassembly through the PLK1-Kif2a signaling pathway. To test this hypothesis, we transfected GFP-Kif2a into ARPE19 cells and immunostained with γ-tubulin and anti-Cenpj antibodies. Kif2a was colocalized with Cenpj and γ-tubulin in centrosomes (Fig. 7A). This colocalization of Kif2a and Cenpj also existed when the mother centrioles served as the BBs for the primary cilia (Fig. 7B). To determine whether Cenpj regulates cilium disassembly via Kif2a, we first evaluated whether knock-down of Kif2a alone by shRNA disrupted neurogenesis in vivo. Given that the shRNAs were highly efficient in knocking down Kif2a (Fig. 7C,D), these shRNA constructs were electroporated into the mouse cortex via IUE at E13.5 and the brains were examined 3 d later (E16.5). Similar to Cenpj knock-down, Kif2a depletion via two different shRNA plasmids also impaired neurogenesis (Fig. 7E,F). The neurogenesis defects were rescued by expression of the Kif2a-td plasmid (Fig. 7G–I).
To determine whether Kif2a is a downstream effector of Cenpj, thus regulating cilium disassembly, we cotransfected serum-starved ARPE19 cells with control shRNA shKif2a, shCenpj, a combination of shCenpj and GFP-Kif2a, or GFP-Kif2a alone and then cultured the cells with serum stimulation for 24 h. To evaluate the effect on cilia disassembly, we counted the ciliated cells 0 and 24 h after serum stimulation. We found that deletion of either Cenpj or Kif2a led to a delay in cilia disassembly (Fig. 8A,B). Additionally, the cilia disassembly caused by Cenpj depletion was rescued by expressing Kif2a, whereas overexpression of Kif2a alone had no effect on cilia disassembly (Fig. 8A,B). We next investigated whether Kif2a functions as a downstream effector of Cenpj in regulating cortical neurogenesis. We observed that more neurons migrated to the cortical plate after Kif2a expression in the shCenpj cortex (Fig. 8C,D). Moreover, Sox2+ cells among GFP+ Cenpj-knock-down cells were greatly reduced compared with control samples, consistent with the finding that Cenpj depletion reduced the NPC pool in the developing mouse cortex. This Sox2+ NPC reduction was rescued by expressing Kif2a (Fig. 8C,E). Together, our results indicate that Kif2a may be a downstream effector facilitating Cenpj to regulate cilium disassembly and is responsible for NPC generation and cortical development.
Discussion
In this study, we have illustrated the role of Cenpj in regulating cilia disassembly in mouse NPCs. We found that depletion of Cenpj caused long primary cilia of RG cells at the embryonic stage and long curl motile cilia on ependymal cells at the adult stage. We also demonstrated that Kif2a contributes to Cenpj-controlled primary cilia disassembly, which may be responsible for NPC cycle delay and neurogenesis (Fig. 9).
Microcephaly mutations happen most often in centrosomal proteins. Mutations in Nde1, Cdk5rap2, Magoh, and Cenpj induce microcephaly through a combination of loss of progenitor self-renewal, premature differentiation, and progenitor apoptosis (Pawlisz et al., 2008; Lizarraga et al., 2010; Silver et al., 2010; McIntyre et al., 2012; Houlihan and Feng, 2014). The apoptosis induced by Nde1 and Cenpj mutations is p53 dependent (Bazzi and Anderson, 2014; Houlihan and Feng, 2014). Overexpressing the centriole duplication protein PLK4 with a microcephaly phenotype is caused by aneuploidy-linked cell death (Marthiens et al., 2013). Aspm mutants exhibit mild microcephaly resulting from cell death associated with genomic instability and DNA damage (Fujimori et al., 2014; Williams et al., 2015), whereas Wdr62 mutant mice have a modestly reduced brain size resulting from premature cell-cycle exit, which causes defects in the mitotic progression of embryonic neural progenitors (Bogoyevitch et al., 2012; Chen et al., 2014). Centrosomes are templates for ciliogenesis, so it is conceivable that altered ciliogenesis could underlie the pathophysiology of microcephaly to some extent. Recent studies have shown that the proper timing of cilium disassembly mediated by Nde1 and TcTex1 is critically regulated during the cell cycle (Kim et al., 2011; Li et al., 2011; Maskey et al., 2015) and that mutations in Nde1 cause microcephaly. There have been some studies identifying the role of centrosome proteins in cilium in regulating neural stem cells during cortical development (Breunig et al., 2008; Han et al., 2008; Willaredt et al., 2008; Wilson et al., 2012; Hu et al., 2014). Our results added more information, supporting the idea that ciliogenesis controlled by Cenpj is required for regulation of NPC cycles, in addition to its function in cell apoptosis in microcephaly disorders (Gabriel et al., 2016).
Cenpj is a conserved centrosomal protein and is crucial for centrosome biogenesis and many other important processes in addition to the cilia-related function. Cenpj regulates centriole duplication (Gönczy, 2012; Fırat-Karalar and Stearns, 2014; Hirono, 2014) and elongation (Schmidt et al., 2009). CenpjCKO mice show neurogenesis defects and microcephaly due to a loss of centrioles, but spindles can still be formed (Insolera et al., 2014). Cenpj also plays an essential role as a scaffold for cytoplasmic PCM complexes and contributes to the recruitment of PCM to centrioles (Zheng et al., 2014). Many studies have shown that Cenpj depletion causes defects in the size of centrosomes at the spindle poles (Kirkham et al., 2003; Cho et al., 2006; Dzhindzhev et al., 2010; Gopalakrishnan et al., 2011; Zheng et al., 2014; Conduit et al., 2015). Therefore, Cenpj mutations or deletions lead to increase of the cells with monopolar, abnormal bipolar, and multipolar spindles, resulting in mitosis arrest and apoptosis (Cho et al., 2006; Kitagawa et al., 2011). In addition, cells with abnormal asymmetric bipolar spindles exhibit spindle orientation defects leading to mitotic cleavage randomization (Kitagawa et al., 2011). The disassembly of cilia affects the natural structure of centrosome and possibly impairs microtubule nucleation capacity, which could also result in spindle disorganization and mispositioning. Therefore, our results suggest that neurogenesis failure could be a correlative outcome of the defects in centrosome biogenesis and ciliary disassembly in mice.
From the RNA-Seq data, we note that genes in the Wnt signaling pathway, which is important for regulating gene transcription and maintaining microtubule stabilization, were downregulated upon Cenpj depletion, suggesting that related abnormal cilium morphology and dynamics might be regulated via Wnt signals. Indeed, a number of Wnt signaling components have been observed in the axoneme and at the BBs and the noncanonical Wnt signaling pathway has been linked to ciliary assembly (Wallingford and Mitchell, 2011; Lienkamp et al., 2012) and disassembly (Lee et al., 2012). Recent studies, however, have revealed that a mitotic kinase Polo-like kinase 1 (Plk1) plays a pivotal role in primary cilia disassembly (Lee et al., 2012; Seeger-Nukpezah et al., 2012; Wang et al., 2013). In response to the ligand Wnt5a, Plk1 forms a complex with Dvl2. The Dvl2-Plk1 complex is required to stabilize and inhibit HEF1 degradation, leading to Aurora A activation, which phosphorylates and activates HDAC6 to increase axonemal microtubule instability, leading to ciliary disassembly (Lee et al., 2012). PLK1 also phosphorylates histone deacetylase 6 (HDAC6) to promote tubulin deacetylation and to destabilize the axonemal MTs of primary cilia (Wang et al., 2013). Moreover, PLK1 promotes primary cilia disassembly through the KIF2A pathway via its MT-depolymerizing activity in a growth-signal-dependent manner (Miyamoto et al., 2015). Our data support the model that Cenpj-regulated cilia disassembly is due to PLK1-KIF2A pathway dysfunction. Kif2a belongs to the kinesin-13 gene family. Unlike conventional kinesin motor proteins involved in intracellular transport, kinesin-13 proteins do not walk along MTs, but have the unique activity of ATP-dependent MT depolymerization (Walczak et al., 2013). Cenpj may recruit Kif2a at the subdistal appendages, which depolymerize centrosomal (cytoplasmic) MT to arrest transport of materials such as tubulin into cilia (Bhogaraju et al., 2013). Further studies are required to clarify how the MT-depolymerizing activity of Kif2a is transmitted to primary cilia disassembly.
In most mammals, the VZ/SVZ continues to produce neurons and glial cells throughout adulthood. Our results indicate a defect in the postnatal stem cell niche of the CenpjCKO adult mice. The VZ/SVZ adult neurogenic niche is established at postnatal stages from a subpopulation of embryonic RG cells. RG cells constitute a heterogeneous population of cells that give rise to both type-B1 aNSCs and multiciliated ependymal cells in addition to neurons and glial cells (Guérout et al., 2014). Depletion of Cenpj in RG cells results in abnormal ciliary structures, such as disorganized BB structures, long cilia, and misarranged microtubule structures. Similarly, aNSCs and ependymal cells in the SVZ of the CenpjCKO adult mice also show long cilia and disordered appendages. Depletion of Cenpj not only results in a decrease in embryonic neurogenesis but also influences adult neurogenesis. Ependymal cells and CSF are integral components of the adult VZ/SVZ niche (Lehtinen et al., 2011) and disruption of motile cilia in ependymal cells is likely to indirectly affect VZ/SVZ progenitors. Because Cenpj depletion also affects the structure of motile cilia in ependymal cells and therefore CSF flow, new approaches to selectively ablate cilia in VZ/SVZ B1 progenitors, but not ependymal cells, are required to understand the role of primary cilia in these periventricular aNSCs.
The primary cilia at the embryonic stage and the adult motile cilia showed similar phenotypes following Cenpj depletion and motile cilia are terminally differentiated, indicating that there is still an intrinsic pathway controlled by Cenpj interrupted in all cilia independent of the cell cycle. Cenpj is required for centrosome duplication and possesses the ability to destabilize, not only cytoplasmic microtubules, but also cilium disassembly (Hung et al., 2004; Tang et al., 2009). A lack of the Cenpj CC5 domain causes long cilia (Gabriel et al., 2016), whereas the N-terminal tubulin-binding domain is responsible for centriole and cilium elongation (Wu and Tang, 2012). We observed that, in addition to cilia length regulation, Cenpj plays roles in controlling the cilium axoneme and BB structure. The bulging and curly tip phenotype seen following Cenpj depletion may be the result of aberrant arrangement of the microtubule doublets. Individuals with microcephaly have characteristic facies, skeletal dysplasia, and abnormal dentition and have an increased risk of cerebrovascular disease; insulin resistance, severe neonatal cholestasis, and histological dysplasia of the kidneys have also been found in microcephaly patients (Berger et al., 1998; Bober and Jackson, 2017). We speculate that the cilia abnormalities in these organs could be one of the causes of these defects. Our mouse model with Cenpj deletion provides an ideal model in which to test this possibility in preclinical studies.
Footnotes
This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant XDA16020601, XDB32010100),the National Basic Research Program of China (Grants 2017YFA0103303 and 2017YFA0102601), the National Natural Science Foundation of China (Grant 31371100, 31771140, 31671072), and Beijing Brain Initiation (Z181100001518004). We thank Bradley Yoder (University of Alabama at Birmingham) for kindly sharing and transferring the mouse strain to us; Tian Xue (University of Science and Technology of China) for sharing the ARPE19 cell line with us; Jianguo Zhang, Lei Sun, and Can Peng (Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences) for help in making EM samples and taking EM images; and Arnold Kriegstein (University of California at San Francisco) and his laboratory members for discussions and reagent support.
The authors declare no competing financial interests.
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