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- The Company of Biologists
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- © 2021 The Company of Biologists. All rights reserved.
- ISSN
- 0950-1991
- eISSN
- 0950-1991
- DOI
- 10.1242/dev.041129
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Abstract
RESEARCH ARTICLE 4021 Development 136, 4021-4031 (2009) doi:10.1242/dev.041129 FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain 1 1 1 1 1 2 Benoit V. Jacquet , Raul Salinas-Mondragon , Huixuan Liang , Blair Therit , Justin D. Buie , Michael Dykstra , 3 4 5 1, Kenneth Campbell , Lawrence E. Ostrowski , Steven L. Brody and H. Troy Ghashghaei * Neuronal specification occurs at the periventricular surface of the embryonic central nervous system. During early postnatal periods, radial glial cells in various ventricular zones of the brain differentiate into ependymal cells and astrocytes. However, mechanisms that drive this time- and cell-specific differentiation remain largely unknown. Here, we show that expression of the forkhead transcription factor FoxJ1 in mice is required for differentiation into ependymal cells and a small subset of FoxJ1 astrocytes in the lateral ventricles, where these cells form a postnatal neural stem cell niche. Moreover, we show that a subset of FoxJ1 cells harvested from the stem cell niche can self-renew and possess neurogenic potential. Using a transcriptome comparison of FoxJ1- null and wild-type microdissected tissue, we identified candidate genes regulated by FoxJ1 during early postnatal development. The list includes a significant number of microtubule-associated proteins, some of which form a protein complex that could regulate the transport of basal bodies to the ventricular surface of differentiating ependymal cells during FoxJ1-dependent ciliogenesis. Our results suggest that time- and cell-specific expression of FoxJ1 in the brain acts on an array of target genes to regulate the differentiation of ependymal cells and a small subset of astrocytes in the adult stem cell niche. KEY WORDS: Subventricular zone, Adult stem cell niche, Ependymal cells, Astrocytes, FoxJ1, Mouse INTRODUCTION lining of the ventricles (Mirzadeh et al., 2008), resembling The developing neuroepithelium consists of multipotent stem cells rosette/pinwheel structures, and are organized in the developing with radial glial features (Campbell and Gotz, 2002; Noctor et al., epithelia of multiple species and organ systems (Zallen, 2007). 2002) that give rise to various neuronal and glial cell types in the Ependymal cells are known to influence the SCN in several ways; for embryonic central nervous system (Rakic, 1972; Pinto and Gotz, example, by regulating neurogenesis through secretion of noggin 2007). Around the time of birth, radial glial cells are thought to (Lim et al., 2000). Furthermore, subsets of cells in this epithelial layer differentiate into astrocytes and ependymal cells that line the are thought to function as quiescent neural stem cells (Doetsch et al., cerebral ventricles in the mature brain (Schmechel and Rakic, 1979; 1997; Johansson et al., 1999; Spassky et al., 2005; Coskun et al., Merkle et al., 2004; Spassky et al., 2005); however, the molecular 2008) and to maintain a second layer of transit amplifying mechanisms responsible for this differentiation are largely unknown. progenitors (TAPs) that proliferate rapidly (Doetsch et al., 1999a; The differentiation into the astrocytes and ependymal cells that Doetsch et al., 1999b). TAPs give rise to a third layer comprising separate the subventricular zone (SVZ) from the fluid-filled space migrating neuroblasts that travel through the rostral migratory stream in the lateral ventricles is of particular interest as these cells help (RMS) to the OB (Lois and Alvarez-Buylla, 1993; Lois and Alvarez- form an adult stem cell niche (SCN). The emergence and Buylla, 1994; Ghashghaei et al., 2007a). Upon arrival at the OB, the maintenance of the adult SCN in the SVZ is thought to support neuroblasts differentiate into interneurons during early postnatal and olfactory bulb (OB) neurogenesis throughout adulthood (Alvarez- adult periods (Lois and Alvarez-Buylla, 1993; Wichterle et al., 1999). Buylla and Lim, 2004). Identification of the factors required for postnatal establishment and Maintenance of adult neurogenesis is thought to depend on cellular maintenance of cellular integrity within the adult SCN is essential for composition within the SCN, which is compartmentalized into three deciphering functional differences between adult and embryonic functionally distinct layers. The first is an epithelial layer consisting regulation of neurogenesis and is crucial if the adult SCN is to be of ependymal cells and a subset of astrocytes, both of which are in utilized in cell-based therapies. contact with the cerebrospinal fluid circulating in the lateral ventricles A crucial aspect of ependymal cell differentiation is ciliogenesis. (Doetsch et al., 1997). The basal processes of these unique astrocytes Cilia are evolutionarily conserved structures that are classified as form clusters surrounded by a few ependymal cells on the cellular motile or primary (Mitchell, 2004). Motile cilia depend on molecular motors and a central microtubule pair for their motility, North Carolina State University, College of Veterinary Medicine, Department of whereas primary cilia are specialized as environmental sensors and Molecular Biomedical Sciences, Raleigh, NC 27606, USA. North Carolina State lack the motility apparatus. Motile cilia in primitive flagellated University, College of Veterinary Medicine, Department of Population Health and organisms and mammalian sperm flagella exist as a single axonemal Pathobiology, Raleigh, NC 27606, USA. Division of Developmental Biology, Department of Pediatrics, Children’s Hospital Medical Center, University of Cincinnati structure. Additionally, a population of cells containing a single College of Medicine, Cincinnati, OH 45229, USA. Cystic Fibrosis Research Center, motile cilium is present within the embryonic node during somite University of North Carolina, Chapel Hill, NC 27599, USA. Pulmonary and Critical development that is responsible for establishing left-right Care Medicine, Department of Internal Medicine, Washington University School of Medicine, St Louis, MO 63110, USA. asymmetry (Nonaka et al., 1998). However, in epithelial cells of the airway, the oviduct, the choroid plexus and the ependymal cells, cilia *Author for correspondence ([email protected]) are expressed as multiple axonemal structures. Recently, the forkhead transcription factor FoxJ1 has been shown to be necessary Accepted 18 September 2009 DEVELOPMENT 4022 RESEARCH ARTICLE Development 136 (23) and sufficient for the generation of motile cilia throughout the body both vectors for tracking transduction. For cell transplantation studies, –/–EGFP +/+EGFP FoxJ1 or FoxJ1 cells were injected into the lateral ventricles in Xenopus and zebrafish (Yu et al., 2008; Stubbs et al., 2008). of wild-type and FoxJ1-null animals using a stereotaxic apparatus. Cell However, the role of FoxJ1 in the differentiation of ependymal cells preparations were obtained using fluorescence-activated cell sorting (Dako and cellular organization in the SCN has remained unknown. Here Cytomation MoFlo; NCSU Flow Cytometry and Cell Sorting Laboratory), we report the expression and function of FoxJ1 in the differentiation followed by resuspension in artificial cerebrospinal fluid (Ghashghaei et al., of the postnatal and adult SCN. 2007b) supplemented with 10 ng of epidermal growth factor (Egf) and 6 + fibroblast growth factor (Fgf2) to a final concentration of 1 10 EGFP MATERIALS AND METHODS cells/l. Animals For both transplantation and lentiviral injections, newborn pups (P0) were Animals were used under Institutional Animal Care and Use Committee anesthetized by means of hypothermia as described previously (Ghashghaei regulations and approval at North Carolina State University, and were et al., 2007b). For shFoxJ1-lenti and control-lenti vector injections, 1 l of housed at Laboratory Animal Research facilities at the College of Veterinary each vector (10 infectious units/ml) was injected into the anterior lateral EGFP –/– (Ostrowski et al., 2003), FoxJ1 (Brody et al., 2000) Medicine. FoxJ1 ventricles of P0 pups using stereotaxic surgery (n3 per construct). For cell –/–EGFP and FoxJ1 mice and their littermate controls were sacrificed at transplantation studies, 1 l of resuspended cells was injected into each multiple developmental stages. For embryonic studies, the day of vaginal 6 + hemisphere (10 EGFP cells/l). Wild-type pups were injected plug was considered embryonic day 0 (E0). For fixed analysis of tissue, intraventricularly with control-lenti vector (n3), shFoxJ1-lenti vector (n3) animals were transcardially perfused with 4% paraformaldehyde in 0.1 M –/–EGFP –/– +/+EGFP or FoxJ1 cells (n3). FoxJ1 pups were injected with FoxJ1 phosphate buffered saline (PBS), pH 7.4. Following perfusion, brains were cells (n3). Following surgery, injected pups were placed under a heating removed and postfixed for a minimum of 24 hours. lamp to revive rapidly. The entire surgical procedure lasted less than 15 For microarray experiments, mice at the multiple developmental time minutes for each pup. Pups were placed back with the dam 15-20 minutes points were sex-genotyped by PCR amplification of the sex-determining after recovery and sacrificed at P21. region on the Y chromosome (Sry; forward primer, 5-TGGGAC - TGGTGACAATTGTC-3; reverse primer, 5-GAGTACAGGTG TGC - FoxJ1-independent hydrocephalic models AGCTCT-3). RNA from male mice was extracted and processed for P0 wild-type B6/C57 mice were used for kaolin induction of hydrocephalus. microarray analyses. Kaolin (Sigma, K1512-500G) was dissolved to a 25% working concentration in artificial cerebrospinal fluid as described previously Tissue processing and immunohistochemistry (Ghashghaei et al., 2006). Pups were placed on ice for 1 minute to induce For immunohistochemistry, brains were sectioned at 50 m on a vibratome anesthesia by means of hypothermia and 3 l of kaolin solution was injected (Leica VT 1000 S). E10 embryos and whole heads of postnatal day 0 (P0) into their cisterna magna over a period of less than 3 minutes. Injected pups newborn mice were fixed overnight, cryopreserved in 30% sucrose in PBS were placed under a heating lamp until breathing stabilized over a 20-minute (containing 0.05% sodium azide), frozen in TissueTek at –80°C overnight recovery period. Recovered pups were returned to their mother and those and sectioned at 15-20 m on a cryostat (Leica). Brain sections were blocked that developed hydrocephalus were sacrificed at P6 and P21 for analysis of in 10% goat serum or 10% donkey serum (for experiments involving goat differentiation in the SCN. primary antibodies) with 1% Triton X-100 (Sigma, S26-36-23) in PBS, –/– Dnaic1 mice (Ostrowski et al., 2009) were used as a genetic model for followed by incubation with primary antibodies at 4°C overnight. assessing the effects of hydrocephalus on SCN development and postnatal Appropriate goat or donkey secondary antibodies conjugated to Alexa 488, neurogenesis in the OB. The developmental time course of hydrocephalus Cy3 and Alexa 647 were used for visualization (all diluted 1:1000, 1-hour –/– –/– in Dnaic1 mice was similar to that in FoxJ1 mice, starting between P1 incubation at room temperature). Primary antibodies used included: rabbit –/– and P6 with a progressive increase in ventricular volume by P21. Dnaic1 anti-Dlx2 (D. Eisenstat, University of Manitoba, Canada; 1:1000), mouse and wild-type littermates were perfused at P6 and P21 as described (n3 per anti-FoxJ1 (1:200), mouse anti-Gfap (Milipore; 1:1000), rabbit anti-Gfap genotype for each age group). The brains were sectioned and assessed for (Dakocytomation; 1:1000), chicken anti-GFP (Abcam; 1:2000), rabbit anti- SCN cell types as described above. Gsh2 (Gsx2 – Mouse Genome Informatics; K. Campbell; 1:4000), mouse anti-Lhx6 (Novus Biologicals; 1:200), rabbit anti-S100 (Sigma; 1:1000), Neurosphere cultures +/+EGFP rabbit anti-Blbp (Milipore; 1:500), mouse anti-RC1 (Cbx8 – Mouse Genome Brains from P0 and P21 FoxJ1 mice were rapidly collected, followed Informatics; Developmental Studies Hybridoma Bank University of Iowa; by microdissection and dissociation of the SVZ and RMS. Cells were sorted 1:250), mouse anti-BrdU (BD Bioscience; 13:1000), rabbit anti-Pax6 using a Dako Cytomation MoFlo and immediately cultured in Neurobasal (Milipore; 1:500), mouse anti-Ncam (Abcam; 1:500), rat anti-CD31 (BD medium supplemented with growth factors as described previously (Jacquet Pharmigen; 1:500), rat anti-CD133 (eBioscience; 1:1000), mouse anti-nestin et al., 2009). The neurospheres obtained were passaged every 5 days by (Chemicon; 1:1000), rabbit anti--catenin (Invitrogen; 1:500) and rabbit dissociation into individual cells followed by culturing in Neurobasal anti--tubulin (Sigma; 1:1000). Some sections were counterstained with + medium. After every passage, the percentage of EGFP neurospheres was Alexa-647-conjugated Nissl stain (Invitrogen; 1:1000) for cytoarchitectonic calculated by counting the number of neurospheres in each well (n6 for assessment of labeled tissue. each passage). Neurospheres were differentiated on laminin- and poly- For in situ hybridization, a probe corresponding to nucleotides 784-1368 L-lysine-coated chambered glass slides in Neurobasal medium without of GenBank sequence NM_008240 (FoxJ1) was amplified by PCR from growth factors for 10 days. Differentiated cultures were fixed with 4% FoxJ1 cDNA (ATCC, Manassas, VA, USA). Digoxigenin (DIG)-labeled paraformaldehyde and immunostained as in tissue processing. antisense and sense riboprobes were generated using the DIG RNA Labeling Data analysis Kit (Roche Diagnostics). Cryosections of adult brains were hybridized with Tissue and cell culture analyses were performed using a confocal riboprobes at 55°C for 30 hours. After washing and blocking, sections were incubated with anti-DIG antibody conjugated with alkaline phosphatase microscope (Nikon Eclipse C1) and data were quantified using standard (1:1000) overnight at 4°C. DIG labeling was visualized using Nitro Blue stereological estimation methods as described previously (Ghashghaei et al., Tetrazolium/5-bromo-4-chloro-3-indolyl phosphate development. 2006). Significance was determined using Student’s t-tests and all values were expressed as mean ± s.e.m. Intraventricular lentiviral and cell injections Effective FoxJ1-specific shRNA sequences were identified (see Fig. S7 in Laser capture microscopy +/+EGFP the supplementary material). The shFoxJ1-lenti and control-lenti vectors Brains obtained from P1, P7 and P14 FoxJ1 male mice were collected were generated by transfecting viral constructs into human293 cells as in a 10% RNA preservation solution (50% ammonium sulfate, 10 mM described previously (Olsen, 1998; O’Rourke et al., 2005; Jacquet et al., EDTA, 25 mM sodium citrate in RNase-free water, pH5.2) in PBS, 2009). EGFP reporter was expressed under the chicken -actin promoter in embedded in RNase-free OCT medium and frozen at –80°C overnight. DEVELOPMENT Development of the adult neural stem cell niche RESEARCH ARTICLE 4023 Brains were then sectioned on a cryostat at 10 m and collected on anti-mouse) conjugated to horseradish peroxidase (Millipore; 1:1000) at room MembraneSlides (Zeiss; NF 1.0 PEN; 1 mm, nuclease and human nucleic temperature for 1 hour. All membranes were developed for visualizing labeled acid free). The slides were UV-irradiated 30 minutes prior to use. Slides with bands with an ECL Kit (Pierce, 32106). +/+EGFP sagittal FoxJ1 sections were rapidly placed under a Zeiss Axiovert 200 microscope coupled to a PALM microlaser system with epifluorescence RESULTS capability. A 355 NANAO Laser was used for microdissection of The forkhead transcription factor FoxJ1 is +/+EGFP FoxJ1 domains in the forebrain (laser spot size, 7.5 m; pulse power, expressed in the adult SCN 100 mW; pulse duration, 0.8-1.5 milliseconds). Excised tissue was captured Examination of adult mice expressing enhanced GFP regulated by into Eppendorf tubes containing 10 l of TRIzol (Invitrogen). The RNA was EGFP the FoxJ1 promoter (FoxJ1 ) (Ostrowski et al., 2003) indicated extracted by tissue homogenization in TRIzol, followed by RNA cleanup high levels of FoxJ1 expression in the ventricular epithelium and (Qiagen, Valencia, CA, USA). the hippocampal dentate gyrus (Fig. 1A; see Fig. S1 in the Microarray experiments supplementary material). In situ hybridization and EGFP Total RNA extracted from laser-captured FoxJ1 domains was used for immunohistochemistry confirmed the validity of transgene microarray experiments. Expression profiling using Affymetrix GeneChip expression in the SVZ (Fig. 1B,C). High-magnification marker Mouse Gene 1.0 ST arrays was performed at the University of North analysis revealed that most ependymal cells positive for the calcium Carolina Neuroscience Center, Functional Genomics Core Facility, binding protein S100 express FoxJ1 in the SVZ (Fig. 1D). Subsets including cRNA synthesis and Chip hybridization. Data obtained from EGFP+ EGFP– of FoxJ1 cells in contact with the ventricles co-expressed the FoxJ1 and FoxJ1 domains from wild-type and FoxJ1-null brains surface protein CD133 (Prominin 1 – Mouse Genome Informatics), were compared at the three developmental time points, and those with an average fold-change of 1.5 or more (s.e.m. across three ages 0.1) were which has recently been shown to label ependymal cells in the adult selected for subsequent analysis. Hierarchical clustering was performed on SVZ (Fig. 1E) (Coskun et al., 2008). In addition, an intriguing subset fold-changes calculated from normalized expression values on the log scale of astrocytes in the SVZ, positive for the glial fibrillary acidic using MeV Multi Experimental Viewer software (version 4.1). A selection protein (Gfap), expressed FoxJ1 (Fig. 1F,G). By contrast, FoxJ1 was of genes encoding cytoskeleton-associated proteins with a statistically largely absent in TAPs positive for the epidermal growth factor consistent fold-change across three postnatal developmental time points was receptor (Egfr; Fig. 1H). Most FoxJ1 astrocytes had an elongated confirmed by quantitative real-time PCR (qRT-PCR) as described below. process that contacted CD31 (Pecam 1 – Mouse Genome Expression of the identified genes in the ependymal layer was confirmed in Informatics) blood vessels within the SVZ (Fig. 1I) (Tavazoie et al., sections obtained from the Allen Brain Atlas at http://www.brain-map.org 2008). (see Fig. S9 in the supplementary material). The data discussed in this In the RMS, migrating neuroblasts expressing the polysialylated publication have been deposited in NCBI’s Gene Expression Omnibus (Edgar et al., 2002) and are accessible through GEO Series accession neuronal cell adhesion molecule (PSA-NCAM), Gfap astrocytes + EGFP number GSE18678. and S100 glia (not shown) lacked FoxJ1 expression (Fig. 1J- EGFP K). However, most FoxJ1 cells in the RMS were positive for Quantitative real-time PCR the immature radial glia markers brain lipid-binding protein (Blbp) Perinatal FoxJ1 expression levels and the expression levels of selected genes and nestin (Fig. 1L). A few cells also expressed low levels of the encoding cytoskeleton-associated proteins were determined by qRT-PCR on paired homeobox domain transcription factor Pax6 (Fig. 1L). Thus, independent samples (n3 per age). PCR reactions were performed in EGFP FoxJ1 cells may constitute a distinct progenitor population in duplicate using an iQ Cycler detection system (BioRad). The fold-change the RMS. between paired samples was calculated using normalized data. cDNA was EGFP + synthesized from RNA extracts according to the Taqman RT Kit protocol In the OB, a large proportion of FoxJ1 cells were Gfap EGFP (ABI, Foster City, CA, USA). Primers were designed using Primer3 astrocytes, but neurons were devoid of FoxJ1 expression (Fig. software (http://frodo.wi.mit.edu/primer3/input.htm). Primer sequences are 1M,N). Taken together, this cellular characterization indicates that provided in Table S2 in the supplementary material. Gene expression data FoxJ1 is primarily expressed by ependymal cells and a small subset –C were calculated using the 2 method (Livak and Schmittgen, 2001). EGFP of astrocytes in the SVZ. We also found that FoxJ1 cells in the + + + RMS constitute a potential population of Blbp nestin Pax6 Reverse transcriptase (RT)-PCR EGFP progenitors, whereas most FoxJ1 cells in the OB are a subset of Total RNA was isolated using TRIzol extracts from whole brains, and the astrocytes. Protoscript II RT-PCR Kit (New England Biolabs) was used for cDNA preparation. PCR amplification was performed using gene-specific primers: 5-GAGCTGGAACCACTCAAAGC-3 and 5-GGAACATGGGTGG - Ependymal cells and FoxJ1 astrocytes appear in ATGAAAC-3 for FoxJ1. Twenty-five cycles of amplification were the SVZ during two distinct postnatal time-points performed by denaturing at 94°C for 30 seconds, annealing at 51°C for 30 Based on cell- and region-specific localization of FoxJ1 in the brain, seconds and elongation at 72°C for 2 minutes. EGFP EGFP we next mapped FoxJ1 expression prior to birth. FoxJ1 expression in the rostral forebrain included the choroid plexus, as Co-immunoprecipitation and western blotting well as the radial glial cells on and near the ventricular surface of the Animals were euthanized by isofluorane overdose and brains from wild-type –/– or FoxJ1 mice at various ages were rapidly collected and lysed for protein lateral ganglionic eminence (LGE) and the olfactory ventricles (Fig. extraction. For detection of FoxJ1 protein levels, extracted proteins from E20 2A; see Fig. S2 in the supplementary material). Perinatal mapping and P0 mice were transferred to a membrane and incubated with rabbit anti- and quantification revealed that FoxJ1 was significantly upregulated FoxJ1 antibody (Affinity Bioreagents; 1:200) at 4°C overnight. For at birth (P0), compared with just before birth (E20; Fig. 2A-E). At immunoprecipitation, lysates were incubated with primary antibodies against EGFP P1, the density of FoxJ1 cells increased along the walls of the -tubulin (Sigma) at 4°C overnight. Immunoprecipitated proteins were then ventricles throughout the brain, where the new cells were cuboidal purified using Protein G-agarose beads according to the manufacturer’s EGFP+ in morphology rather than elongated, similar to FoxJ1 radial instructions (Invitrogen). Samples were run on a reducing SDS-PAGE gel glial cells in the LGE (Fig. 2F,F). By P6, many of the expanding followed by semi-dry transfer to a nitrocellulose membrane. The membrane FoxJ1 cells began to express S100 and clearly resembled was then blocked (5% skimmed milk powder), exposed to primary antibodies ependymal cells (Fig. 2G,H), matching the timing of ependymal cell against Dnaic (Milipore), Dnahc (Santa Cruz) and Kif6 (AbCam). Membranes were incubated with the appropriate secondary antibodies (goat anti-rabbit or maturation reported previously (Spassky et al., 2005). Notably, the DEVELOPMENT 4024 RESEARCH ARTICLE Development 136 (23) astrocytes in the SVZ at this stage, but found a subset of radial glia that appear to persist in the SVZ and RMS during early postnatal development. To time the emergence of FoxJ1 astrocytes found in the adult SCN (Fig. 1F,G,I), we labeled for S100 as a marker for ependymal cells, and used a monoclonal antibody against Gfap (mGfap) for SVZ astrocytes. The majority of FoxJ1 cells prior to P19 were nascent + – + – S100 mGfap ependymal cells and Blbp mGfap radial glial-like cells (Fig. 2K). Interestingly, between P19 and P21, a subset of FoxJ1 cells intercalated between ependymal cells began to express mGfap + – + (Fig. 2K). These FoxJ1 S100 mGfap cells became more abundant between 6 weeks (1.6±0.5% of all astrocytes in the SVZ) and 6 months (2.1±0.6% of all astrocytes in the SVZ) postnatal (n3 per age group; Fig. 2L). Thus, after the significant upregulation of FoxJ1 in the forebrain after birth, FoxJ1 ependymal cells differentiate during the first postnatal week. This is followed by a second wave of differentiation during the end of the third postnatal week, when a small subset of FoxJ1 SVZ astrocytes begin to differentiate and their density gradually increases for the first 6 months of life. FoxJ1 is required for postnatal differentiation of ependymal cells and FoxJ1 astrocytes in the SVZ Changes in temporal and spatial expression of FoxJ1 in the brain inspired the hypothesis that the two postnatal waves of differentiation in FoxJ1 cells are required for SCN maturation during the LGE-to-SVZ transition. To test this hypothesis, we used –/– FoxJ1 knockout mice (FoxJ1 ), which exhibit random asymmetry defects associated with the absence of motile cilia throughout –/– development (Brody et al., 2000). FoxJ1 mice that do not exhibit Fig. 1. FoxJ1 expression in the adult brain. (A)Nissl (red) stained aberrant left/right asymmetry survive into early adulthood, but the EGFP sagittal section of an 8-week-old adult FoxJ1 mouse brain reveals surface of their cerebral ventricles is devoid of motile cilia and they EGFP expression in the hippocampus (Hipp), cerebellum (CBL), a subset develop hydrocephalus (Fig. 3A-D). Importantly, growth defects of diencephalic nuclei (DIEN), SVZ, RMS and OB. (B,C)In situ were quantified in multiple brain regions at P21, and those in the hybridization (B, blue) and immunostaining using a monoclonal FoxJ1 –/– FoxJ1 OB were most significant (66±12% reduction in volume; antibody (FoxJ1-mAb; C, red) confirmed transgene expression in the SVZ, RMS and OB. (D-N)High-magnification confocal micrographs of Fig. 3A-B; see Fig. S3 in the supplementary material). EGFP –/– FoxJ1 -expressing cell types in the SVZ (D-I), RMS (J-L) and OB (M,N). Retarded growth in the FoxJ1 mouse OB, together with the EGFP A small subset of cells positive with a monoclonal FoxJ1 antibody and restricted expression of FoxJ1 in the LGE and olfactory EGFP for FoxJ1 , co-labeled with a polyclonal Gfap antibody (G, pGfap, ventricles, suggested a possible alteration of specification events in + – blue; arrow, a triple-labeled FoxJ1 astrocyte; arrowhead, a FoxJ1 these structures, whereby OB neurogenesis occurs during embryonic astrocyte). Many FoxJ1 astrocytes in the SVZ have an elongated and perinatal periods (Hinds, 1968a; Hinds, 1968b; Bayer, 1983; process (I, arrowheads) that contacts blood vessels positive for CD31 Gong and Shipley, 1995). Surprisingly, we only found subtle EGFP (I, asterisk, blue). In the RMS, many FoxJ1 cells express the changes in the patterning, density and proliferation of known immature/radial glial marker brain lipid binding protein (Blbp; progenitor domains in the LGE and in the olfactory ventricular zone L, arrowheads). A few also express the progenitor-specific intermediate prior to birth (see Fig. S4 in the supplementary material), and filament nestin and the paired homeobox domain transcription factor –/– Pax6 (L, arrowheads). Scale bars: 100m in A,B; 10m in C-N. embryos and newborn FoxJ1 animals appeared grossly normal in size and in brain organization. Additionally, primary cilia were expressed, in a similar manner to in wild-type animals, on the apical surface of radial glia lining the ventricular zone of the embryonic + –/– emergence of S100 ependymal cells was delayed in regions with FoxJ1 LGE, suggesting that FoxJ1 is not required for genesis of EGFP embryonic FoxJ1 expression (e.g. the ventricular zone of the primary cilia (see Fig. S4 in the supplementary material). These LGE). Accordingly, ependymal cells appeared to differentiate earlier findings, together with the marked upregulation of FoxJ1 at birth, (between P1 and P6) in non-striatal regions of the ventricular zone, prompted us to hypothesize that FoxJ1 expression could have a such as the ventricular zone of the septum and the dorsal ventricular temporally focused role in postnatal establishment of the SCN. To surface underneath the corpus callosum. examine this possibility, we first asked whether constituents of the + + –/– Intriguingly, a subset of FoxJ1 Blbp radial glia found in the postnatal SCN were appropriately specified in the P6 FoxJ1 SVZ LGE (see Fig. S2 in the supplementary material) persisted in the using S100 labeling. Indeed, we found that S100 cuboidal cells + –/– SVZ at P6, and were anatomically distinct from maturing FoxJ1 failed to emerge in the ventricular wall of P6 FoxJ1 mice (see Fig. + + + and S100 cells. FoxJ1 Blbp cells were predominantly situated S5 in the supplementary material), suggesting that FoxJ1 is required in the TAP layer (Fig. 2G,I) and RMS (Fig. 2J), and only a few were for ependymal cell differentiation. intercalated among ependymal cells. Therefore, by P6, an initial To determine whether a mature adult SCN ever develops in + –/– wave of differentiation in FoxJ1 cells was specific to the maturation FoxJ1 mice, cellular integrity of astrocytes and ependymal cells of ependymal cells. However, we could not detect any FoxJ1 was examined at P21, at which time the adult-stage SCN is DEVELOPMENT Development of the adult neural stem cell niche RESEARCH ARTICLE 4025 Fig. 2. Emergence of FoxJ1 cells in the EGFP postnatal SCN. (A-E)FoxJ1 expression in the E20 mouse embryo is present in nestin cells with radial glial morphology (A, inset) on the walls of the lateral ganglionic eminence (LGE), the olfactory ventricle (RMS) and the OB. In the SVZ, transforming EGFP+ + FoxJ1 nestin radial glia (B, inset) begin to lose their uniform polarized processes. Upregulation of FoxJ1 at birth was quantified by real-time PCR (C), reverse transcriptase (RT)-PCR (D) and western blotting (E). (F,F) At P1, FoxJ1 radial glia persist deep in the SVZ and RMS (F, arrowheads), while ependymal cells start to appear in the SVZ adjacent to the ventricular lumen (F, cuboidal cells, red arrows). (G-J)At P6, the ependymal layer expands EGFP+ + (G) and FoxJ1 S100 cells become pronounced (H). Dashed lines demarcate the SVZ in G. Ependymal cells do not express the radial glial marker Blbp (H, inset). A distinct population of EGFP+ + FoxJ1 Blbp cells persist in the deep regions of the SVZ (boxed area labeled radial glia in G is magnified in I) and in the RMS (J). (K)Low- magnification micrographs of sagittal sections from EGFP P14, P19 and P21 FoxJ1 brains stained for mGfap (red) and S100 (blue). High-magnification panels are confocal micrographs of a flat embedded ependymal layer prepared from the corresponding sagittal sections. Arrival of the newly discovered FoxJ1 astrocytes (outlined by the dotted line) occurs between P19 and P21. (L)The density of distinct types of FoxJ1 cells at multiple postnatal time points. The data in L are stereological estimates obtained from triple-labeled tissues (cell density 4 3 number of cells 10 /mm , n3 per age group). established. Transmission and scanning electron microscopy SVZ, and they were significantly smaller than wild-type cells (Fig. –/–EGFP clearly demonstrated the absence of motile cilia on the surface of 4A). As suspected, nearly all FoxJ1 cells expressed Blbp, –/– the FoxJ1 SVZ, as well as the disrupted organization of the suggesting that they had failed to mature (Fig. 4B). Additionally, the –/– + ependymal layer in these mice (Fig. 3C-D). Despite the absence of FoxJ1 SVZ included a significant increase in density of mGfap –/– motile cilia, primary cilia were present in the FoxJ1 SVZ (see cells resembling reactive astrocytes, with densely distributed + –/–EGFP Fig. S6 in the supplementary material). As suspected, S100 processes adjacent to FoxJ1 cells (Fig. 4B, asterisks). – –/–EGFP mGfap ependymal cells were almost absent (Fig. 3E,E) and However, a significantly smaller percentage of FoxJ1 cells in –/– + +/+EGFP CD133 expression was substantially decreased in the FoxJ1 SVZ the SVZ were Gfap compared with those in FoxJ1 mice (Fig. 3F,F). These findings demonstrated that in the absence of (9±0.3% vs 19±0.8%, respectively; Fig. 4B). Thus, FoxJ1 is, in part, FoxJ1 expression, ependymal cells fail to mature at any required for differentiation of FoxJ1 astrocytes that emerge by P21 developmental stage. In their place, we found a significant number in the wild-type SVZ. of residual immature Blbp cells (Fig. 3G,G), suggesting that Next, to examine the rosette/pinwheel architecture of ependymal –/– subsets of FoxJ1 radial glia may be arrested in an undifferentiated and astrocytic cells on the surface of the lateral ventricles, we state. obtained wholemount preparations of the ependymal layer and immunostained for Gfap together with -catenin and -tubulin (Fig. FoxJ1 is cell-autonomously required for 4C). -catenin, an important constituent of the Wnt signaling differentiation of the SCN pathway (Schlessinger et al., 2009), is expressed on the lateral and Based on the perturbed cell-specific phenotypes, we set out to apical surfaces of wild-type ependymal cells. -tubulin is a clearly assess the autonomous and non-autonomous effects of FoxJ1 component of a microtubule-organizing complex at the base of deletion on the cellular constituents of the adult SCN. To accomplish individual cilia and is crucial for their growth and maintenance EGFP –/– this, we crossed our FoxJ1 reporter mice into the FoxJ1 (Oakley, 1992) and clearly decorates the apical surface of –/–EGFP background to obtain FoxJ1 animals. The overall density of multiciliated ependymal cells (Mirzadeh et al., 2008). The –/–EGFP +/+EGFP FoxJ1 cells was higher than in FoxJ1 controls in the rosette/pinwheel organization of ependymal cells and astrocytes was DEVELOPMENT 4026 RESEARCH ARTICLE Development 136 (23) –/– Fig. 3. FoxJ1 mice fail to establish the SCN. (A,A) Photomicrographs of P21 brains harvested –/– from wild-type (A) and FoxJ1 (A) littermates. Arrows point to the OB and arrowheads to the cerebellum. (B,B) Sagittal sections of P21 brains –/– harvested from wild-type and FoxJ1 littermates. Arrows point to the RMS, boxed areas demarcate regions shown in high-magnification confocal images in E-G. (C,C) Transmission electron micrographs obtained from the ventricular zone of P21 wild-type –/– (C) and FoxJ1 (C) littermates. Colored cells were classified based on description by a previous study (Doetsch et al., 1997). The red arrowhead points to –/– unusual aggregates within FoxJ1 undifferentiated ependymal cells. (D,D) Scanning electron micrographs obtained from the ventricular surface of –/– the SVZ show a complete lack of cilia in FoxJ1 mice but their clear presence in wild-type animals. + + (E-F) S100 and CD133 ependymal cells in wild- –/– type mice and their absence in FoxJ1 SVZs. (G,G) Residual Blbp radial glia are in the position of –/– the ependymal cells in the FoxJ1 SVZ. –/– absent on the surface of FoxJ1 ventricles, concomitant with a Finally, to confirm whether FoxJ1 functions cell-autonomously, disruption of junctional -catenin expression (Fig. 4C). To and whether the wild-type or knockout environments influenced the determine whether or not disrupted junctional -catenin expression observed phenotypes, we performed transplantation studies in –/– +/+EGFP –/–EGFP was due to the loss of junctional complexes in the FoxJ1 SVZ, we newborn mice. P0 FoxJ1 and FoxJ1 cells were cross- conducted transmission electron microscopy (TEM) analyses. transplanted into P0 wild-type and knockout mice without EGFP Junctional complexes along the walls of mutant cells were readily reporter expression, and host mice were allowed to survive to P21 detectable, although their orientation and positioning were severely (Fig. 5A). We found that wild-type EGFP cells transplanted into –/– disrupted (see Fig. S6 in the supplementary material). Thus, FoxJ1 ventricles incorporated into the host SVZ and differentiated –/– –/– disrupted -catenin expression in the FoxJ1 SVZ is not due to a into multiciliated ependymal cells despite their FoxJ1 –/–EGFP complete loss of junctional complexes within mutant cells along the environment (Fig. 5B,C). FoxJ1 cells injected into wild-type ventricles. ventricles also incorporated into the wild-type ependymal layer, but –/–EGFP FoxJ1 cells included unusually large aggregates of - failed to mature into multiciliated ependymal cells (S100; arrows) tubulin within their cytoplasm (Fig. 4C, arrowheads), suggesting or astrocytes (mGfap; asterisks) and maintained a Blbp identity that, in the absence of FoxJ1 expression, -tubulin fails to be (n3; Fig. 5B,D). Concomitantly, CD133 cilia were present in more +/+EGFP distributed and docked at the apical surface of differentiating than 80±16% of FoxJ1 cells transplanted into knockout host ependymal cells. TEM analysis of the P21 SCN revealed the brains (n3), whereas there was no apparent CD133 expression by –/– –/–EGFP presence of multiple basal bodies within FoxJ1 cells lining the FoxJ1 cells transplanted into wild-type host brains (Fig. ventricles (Fig. 4D). This suggests that the aggregates of - 5B,D). As in other experiments, aggregates of -tubulin were –/–EGFP tubulin-immunoreactive particles are likely to correspond to basal consistently found in the transplanted FoxJ1 cells, whereas –/– – bodies within the cytoplasm of FoxJ1 cells. To determine adjacent EGFP wild-type ependymal cells included speckles of - whether the aggregation of basal bodies was developmentally tubulin on their apical surface (Fig. 5C, arrowheads). In summary, related to their replication during early postnatal ciliogenesis, we results from knockout, shRNA and cross-transplantation conducted TEM analysis of the P6 SVZ. We found that structures experiments conclusively show that FoxJ1 is required for the containing basal bodies, referred to as deuterosomes (Spassky et differentiation of ependymal cells and a subset of FoxJ1 astrocytes al., 2005), were visible within differentiating wild-type in the ventricular zone of the brain in a cell-autonomous manner. –/– ependymal cells, but could not be found in the FoxJ1 cells –/– lining the ventricles (Fig. 4D). Instead, aggregates of unorganized FoxJ1 phenotypes are independent of –/– basal bodies were found within the cytoplasm of P6 FoxJ1 hydrocephalus –/– cells, suggesting that the formation of deuterosomes, and possibly A possible cause of defects in the P21 FoxJ1 ventricular zone might the transport of basal bodies, was disrupted in the absence of be due to the presence of hydrocephalus. We explored this issue by FoxJ1 expression. examining ependymal cell and astrocytic integrity in two independent To rule out potential bystander effects from whole-body knockout models of hydrocephalus, one genetic and the other obstructive. First, of FoxJ1, we cloned and tested a short hairpin RNA (shRNA) genetic deletion of a major component of the axonemal dyneins in sequence for acute knockdown of FoxJ1 expression (see Fig. S7 in the motile cilia [axonemal intermediate chain 1 (Dnaic1)] resulted in supplementary material). We found identical phenotypes to those in altered cilia motility and subsequent non-obstructive hydrocephalus –/–EGFP –/– FoxJ1 cells, suggesting a cell-autonomous role for FoxJ1 in the (Dnaic1 ; see Fig. S8 in the supplementary material). The initiation differentiation of the SCN (see Fig. S7 in the supplementary material). and progression of hydrocephalus in these mice is similar to that in DEVELOPMENT Development of the adult neural stem cell niche RESEARCH ARTICLE 4027 –/– Fig. 4. Disrupted cellular differentiation in the FoxJ1 SVZ. (A)Confocal photomicrographs from the surface of the ventricles in the SVZs of +/+EGFP –/–EGFP –/–EGFP FoxJ1 and FoxJ1 mice. FoxJ cells were traced in the SVZ (red dotted lines). Cell density was estimated using stereological cell 4 3 counting (number of cells 10 /mm ). Cell diameter was defined as the longest distance between two ends of each cell and is presented inm (33 +/+EGFP –/–EGFP cells in three individuals per genotype; mean ± s.e.m.; asterisk, P<0.01, Student’s t-test). (B)FoxJ1 and FoxJ1 mice were assessed for –/–EGFP + expression of Blbp and mGfap on the surface of the SVZ. Reactive astrogliosis in the FoxJ1 SVZ did not overlap with the EGFP domain –/– containing FoxJ1 cells, but was deep within the adjacent TAP domain and striatal tissue (asterisks). mGfap panel to the right shows overlap of +/+EGFP –/–EGFP + FoxJ1 and FoxJ1 cells in the P21 SVZ with mGfap astrocytes at high-magnification (arrow). The bar chart illustrates the percentage of +/+EGFP –/–EGFP –/–EGFP FoxJ1 and FoxJ1 cells positive for Blbp and mGfap (3 mice per genotype; n50 cells per mouse). (C)FoxJ1 ventricles completely lack the rosette/pinwheel organization and -catenin and -tubulin expression is massively disrupted. Large aggregates of -tubulin are found within –/– +/+ –/– FoxJ1 cells (arrowheads). (D)TEM images of basal body (red arrowheads) organization in the FoxJ1 and FoxJ1 SVZ. At P6, deuterosomes found in the wild-type SVZ cannot be detected in FoxJ1-null mice (red arrowheads). By P21, basal bodies have been successfully transported to, and –/– docked at, the apical surface of wild-type ependymal cells, whereas in FoxJ1 cells only aggregates of basal bodies can be found in an unorganized manner. Scale bar: 1m. –/– EGFP + – FoxJ1 mice. In another model, injection of kaolin (Marlin et al., SVZs of P0 and P21 FoxJ1 mice. EGFP and EGFP cells were 1978) into the cerebra magna of P0 wild-type mice resulted in FACS sorted and cultured in the presence of growth factors for –/– significant hydrocephalus by P6. The SCNs of Dnaic1 and kaolin- several days (Fig. 6A,B). Both populations gave rise to injected mice were analyzed at P6 and P21. In contrast to an absence neurospheres, which are clones of neural stem cells (Reynolds and + –/– – of S100 ependymal cells in FoxJ1 mice, this population was Weiss, 1992). Interestingly, cells within EGFP neurospheres began EGFP clearly present and aligned along the ventricular surface of both to express FoxJ1 after 1 week (Fig. 6B, red arrows), suggesting hydrocephalus models (see Fig. S8 in the supplementary material). either transformation or differentiation into FoxJ1 cells from the However, both models displayed varying degrees of reactive EGFP pool of neurosphere progenitors during the in vitro culture astrogliosis at P21, which was also present on the ventricular surface period. The number and size of neurospheres were similar between –/– + – of FoxJ1 mice and has been reported in other mice with EGFP and EGFP groups, and both groups could be passaged at EGFP hydrocephalus (Kuo et al., 2006). Thus, the absence of ependymal least ten times, suggesting that FoxJ1 cells possess self-renewal cells and disrupted differentiation in a subset of astrocytes in capacity (Fig. 6C). Both groups of neurospheres were then plated –/– the FoxJ1 SVZ appears to be independent of increased onto laminin and poly-L-lysine for 10 days and their progeny intraventricular pressure due to hydrocephalus. differentiated into astrocytes, oligodendrocytes and neurons (Fig. 6D). The preferential shift toward gliogenesis was noted in both EGFP + – Developmental potential of FoxJ1 cells in the EGFP - and EGFP -derived neurospheres, but many neurons were SCN also found within cultures. This finding suggests that a + EGFP Our discovery of a small subset of FoxJ1 astrocytes in the SCN subpopulation of FoxJ1 cells possesses self-renewal and raised the question of whether they possess properties of neural stem neurogenic potential in vitro, and these cells may function as stem cells. To address this, cells were dissociated from microdissected cells in the adult SCN. DEVELOPMENT 4028 RESEARCH ARTICLE Development 136 (23) Fig. 5. FoxJ1 is cell-autonomously required for ependymal and astrocytic cell differentiation. (A)Cross-transplantation of –/–EGFP +/+EGFP – FoxJ1 knockout (KO) and FoxJ1 wild-type cells into EGFP –/– wild-type and FoxJ1 KO hosts. (B)Wild-type cells differentiate into + + ependymal cells (arrows, S100 ; CD133 ) and a subset of astrocytes (asterisks, Gfap ) despite their KO host environment. KO cells remain immature (Blbp ), despite their wild-type host environment. (C)Aggregates of -tubulin are present in KO cells or within the EGFP KO host SVZ (arrowheads). (D)The percentage of EGFP cells expressing different markers in the SCN (n3 mice per genotype, 50 EGFP cells per mouse). Data are mean ± s.e.m.; asterisks, P<0.01, Student’s t-test. EGFP Fig. 6. Differentiation potential of Fox cells in the postnatal EGFP SCN. (A,B)P21 SCN-derived FoxJ1 cells were FACS-sorted (A) and Identification of target genes influenced by FoxJ1 cultured to form neurospheres (B). FoxJ1 promoter activity was expression maintained in floating neurospheres derived from EGFP fractions and The role of FoxJ1 in differentiation of the postnatal SCN naturally became visible in some EGFP -derived neurospheres (arrows) after 7 leads to the question of how FoxJ1 carries out its developmental EGFP+ EGFP– days of growth in vitro. (C)FoxJ1 and FoxJ1 neurospheres functions. To identify candidate genes in the brain regulated by generated similar numbers of neurospheres at P21. The percentage of +/+EGFP FoxJ1, we compared the expression of genes in FoxJ1 and EGFP+ neurospheres that included FoxJ1 cells gradually increased in –/–EGFP – EGFP FoxJ1 domains of the forebrain during the span of adult SCN EGFP -derived cells, suggesting that at least a subset of FoxJ1 cells development (P1, P7, P14) using laser capture micro-dissection (Fig. from the SCN can self-renew. The numbers of neurospheres are per 10 2 EGFP 7A) and microarray transcriptome analysis. This analysis revealed mm culture wells. (D)FoxJ1 -derived neurospheres gave rise to + + + neurons (TuJ1 ), astrocytes (mGfap ) and oligodendrocytes (NG2 ) after that 198 genes were altered more than 1.5-fold in a consistent 10 days of differentiation on laminin and poly-L-lysine. A subset of manner at P1, P7 and P14. We used a stringent statistical criterion to EGFP FoxJ1 cells persisted in differentiated cultures (green cells). The only include genes with an s.e.m. of less than or equal to 0.1 of the 5 2 numbers of cells in the bar chart are per 10 m . average fold-change of their expression across the three ages (<10% error). Of the identified genes, 197 were consistently downregulated –/–EGFP in FoxJ1 domains, whereas only one gene that met the the 24 genes encode microtubule-associated proteins, including six statistical criterion above was consistently upregulated at P1, P7 and distinct isoforms of dynein axonemal heavy chain, and three dynein P14. intermediate chain-like proteins (Fig. 7B). Additionally, genes for We next asked which of the identified genes had known cellular or three members of the kinesin family of motor proteins (Kif6/9/27) –/– molecular functions and were expressed in the ependymal zone of the were significantly and consistently downregulated in the FoxJ1 adult brain using the Allen Brain Atlas. This analysis narrowed the list SCN. The list also included a radial spokehead-like gene (Rshl3), a to 55 genes with known functions and ependymal zone expression, all gene encoding the microtubule-associated protein Tekt4, and another –/–EGFP of which were consistently downregulated in the FoxJ1 brain for tubulin-tyrosine ligase-like family member 6 (Ttll6). To test the (see Table S1 in the supplementary material). Remarkably, of the 55 validity of microarray data, changes in expression of genes encoding known downregulated genes, 24 encoded cytoskeleton-associated microtubule-associated proteins were confirmed by qRT-PCR on three –/– molecular motors, and sperm/flagellar-associated proteins (43% of independent samples from FoxJ1 and wild-type brains at P1, P7 and identified genes encoded proteins with known functions). Sixteen of P14 (Fig. 7B). DEVELOPMENT Development of the adult neural stem cell niche RESEARCH ARTICLE 4029 EGFP Fig. 7. Identification of candidate genes regulated by FoxJ1 during postnatal maturation of the SCN. (A)FoxJ1 domains in the rostral –/– forebrain were laser-capture microdissected and processed for microarray analysis. (B)Comparison of wild-type and FoxJ1 array data resulted in –/–EGFP the identification of 16 genes encoding microtubule-associated proteins that are significantly downregulated in the FoxJ1 domains. (C)Western blotting of immunoprecipitated -tubulin complexes from P6 mice revealed a potential interaction with Dnahc, Dnaic and Kif6 proteins –/– in wild-type mice, and the absence of these complexes in the FoxJ1 brain. The potential role of candidate genes in the subset of FoxJ1-expressing astrocytes that appear late during the generation of motile cilia in ependymal cells differentiation of the SCN, and that their cellular organization in the The functional classification of the identified genes pointed to a postnatal SVZ unequivocally relies on transcriptional activity by master regulatory role for FoxJ1 in driving the expression of the FoxJ1. motility apparatus within cilia in the mouse brain, as has been shown FoxJ1 belongs to a recently annotated class of transcription in Xenopus and zebrafish (Yu et al., 2008; Stubbs et al., 2008). To factors with at least 30 family members that share a common shed light on whether or not the significant downregulation of forkhead (Fox) DNA-binding domain (Lehmann et al., 2003; Katoh dynein and kinesin motor proteins was related to aggregation of - and Katoh, 2004). FoxJ1 plays a direct role in the development of –/– tubulin in the cytoplasm of FoxJ1 ependymal cells, we analyzed motile cilia (Brody et al., 2000; Yu et al., 2008; Stubbs et al., 2008), their expression and biochemical interactions in the brain. First, we and motile cilia are important elements of ependymal cell found that most of the identified axonemal dynein and kinesin differentiation and function in the central nervous system (Spassky proteins had a restricted expression in the ependymal layer of the et al., 2005; Sawamoto et al., 2006). Our analyses demonstrate that adult SVZ (see Fig. S9 in the supplementary material). FoxJ1-dependent differentiation in ependymal cells includes the Next, to determine the potential interaction of identified proteins expression of a significant number of cytoskeletal proteins with -tubulin, total proteins from P6 wild-type and FoxJ1-null associated with motile cilia, and indicate their potential role in forebrains were extracted and -tubulin was immunoprecipitated. regulating the transport of -tubulin-containing basal bodies to the Western blotting was then performed on immunoprecipitated apical surface of differentiating ependymal cells. Although the focus proteins to determine whether Dnahc, Dnaic and Kif6 were forming of this study was on FoxJ1-dependent expression of microtubule- a complex with -tubulin. All three proteins were detected in associated proteins and their potential role in the transport of basal immunoprecipitated wild-type extracts, indicating that they formed bodies, the list of identified genes included additional functional a complex with -tubulin at the peak of ependymal cell families (see Table S1 in the supplementary material). This finding differentiation (Fig. 7C). By contrast, levels of all three proteins suggests a potentially broader function for FoxJ1-dependent –/– were extremely low or absent in FoxJ1 extracts and undetectable differentiation in the SCN than in just the genesis of motile cilia. The –/– in -tubulin-immunoprecipitated material from FoxJ1 brains (Fig. function of most of the identified genes in SCN differentiation 7C). These findings indicate that physical interactions among - remains to be elucidated. tubulin, axonemal dyneins and specific isoforms of kinesin motor proteins may be responsible for the transport and distribution of basal bodies to the ventricular surface of ependymal cells prior to the genesis of motile cilia (Fig. 8). DISCUSSION The ependymal component of the postnatal stem cell niche is thought to be crucial for neurogenesis in the OB, but its precise role in the regulation of neurogenesis remains little studied. We found that ependymal cells within distinct anatomical regions of the ventricular zone appear to differentiate at distinct postnatal time- points within the walls of the ventricles. Ependymal cells in the striatal wall, which are components of a neural stem cell niche, differentiate later during postnatal periods than other ependymal Fig. 8. Working model of the cell-autonomous function of FoxJ1 cells in the medial or dorsal walls of the lateral ventricles. This for genesis of motile cilia in ependymal cells. A physical interaction finding indicates the potential existence of regionally distinct among -tubulin, dynein and kinesin motor proteins may regulate the ependymal subtypes within the walls of the ventricles; however, we transport of -tubulin rings (basal bodies) on the apical (ventricular) found that in the absence of FoxJ1 expression, all ependymal cells surface of ependymal cells. In the absence of FoxJ1, expression of these within the lateral ventricles fail to differentiate during postnatal motor proteins is severely depleted, resulting in aggregation of –/– periods, regardless of their position. We also discovered a small -tubulin rings in FoxJ1 radial glia. DEVELOPMENT 4030 RESEARCH ARTICLE Development 136 (23) Cell-autonomous function of FoxJ1 in the genesis (Meletis et al., 2008; Carlen et al., 2009). It will be intriguing to of motile cilia in ependymal cells determine whether the shared expression of FoxJ1 by ependymal We demonstrated that, in the absence of FoxJ1, radial glia in the cells and a small subset of astrocytes is the basis of plasticity ventricular zone of the late embryonic brain fail to differentiate into inherent to the adult SCN. ependymal cells. The unique molecular feature of FoxJ1-null cells Although the cellular compartments of the adult SCN are fairly includes intracellular accumulation of -tubulin protein, which is well characterized, the identity of adult neural stem cells has consistent with the previous finding of undocked -tubulin- remained largely elusive. Our in vitro findings reveal that subsets –/– EGFP containing basal bodies in multiciliated FoxJ1 lung epithelial cells of FoxJ1 cells harvested from the P21 SVZ generate (Brody et al., 2000; You et al., 2004). These findings suggest that neurospheres, can self-renew and have the potential to give rise to generation of multiple basal bodies in multiciliated epithelial and astrocytes, oligodendrocytes and neurons, thus functionally ependymal cells is independent of FoxJ1 activity. FoxJ1-dependent resembling adult neural stem cells. Together with previous studies docking of basal bodies has been linked to Ezrin-associated demonstrating that ependymal cells are not capable of generating signaling (Gomperts et al., 2004) and Rho-mediated enrichment of neurospheres (Doetsch et al., 1999a; Laywell et al., 2000; Capela actin (Pan et al., 2007) at the apical surface of airway epithelial cells. and Temple, 2002), our in vitro results suggest that the small subset + + However, mechanisms of basal body transport prior to docking are of FoxJ1 astrocytes in the SVZ and FoxJ1 progenitor-like cells in still largely unknown. It is currently thought that this transport is the RMS are likely to be the source of the stem cell properties mediated by the fusion of basal bodies to vesicles (Vladar and detected in our neurosphere experiments. Thus, FoxJ1 radial-glia- Axelrod, 2008), and no distinction has been made between transport like cells prior to P21, and FoxJ1 astrocytes after P21, may directly of basal bodies for generation of motile versus primary cilia. Our contribute to progenitor populations in the LGE and SVZ –/– finding that primary cilia are normal in the FoxJ1 brain is highly perinatally, thus functioning as a subset of adult stem cells in vivo. suggestive of divergent mechanisms of basal body transport for the In further support of this possibility, we found a subset of FoxJ1 genesis of motile and primary cilia. astrocytes in the hippocampal dentate gyrus, where they may also –/– The accumulation of -tubulin in FoxJ1 cells further suggests function as a subset of adult neural stem cells. Although a recent that replicated basal bodies may be distributed to the apical surface study attempted to lineage-trace FoxJ1 cells using viral vectors of ependymal cells utilizing proteins involved in intracellular (Carlen et al., 2009), genetic lineage tracing will be required to transport, the expression of which depends on FoxJ1 activity. Hints conclusively determine whether any of the FoxJ1 cell types toward potential mechanisms for this transport came from our identified in our study participate in early postnatal and adult –/– transcriptome comparison of FoxJ1 and wild-type brains. Of the neurogenesis. candidate genes downregulated in FoxJ1-null brains, a significant In summary, this study demonstrates the cellular requirement for number are microtubule-associated proteins. In particular, three FoxJ1 in the differentiation of the ependymal layer within the isoforms of kinesin motor proteins (Kif6/9/27) can be predicted to central nervous system. The timing of this unique differentiation be responsible for transport of -tubulin and axonemal proteins to process is not uniform across all ventricular zones of the brain. The the apical (ventricular) surface of differentiating ependymal cells. In ependymal differentiation along the striatal walls of the ventricles, support of this possibility, we showed that Kif6 is found in a where neurogenesis persists throughout life, is delayed compared complex that includes -tubulin, Dnahc and Dnaic. Finally, the with other ventricular zones. Curiously, the FoxJ1 promoter confined expression of the majority of the identified genes to the appears to be preferentially active at low levels during ependymal layer of the SVZ is highly suggestive of a direct role for embryogenesis in the LGE and olfactory ventricular zone, whereas FoxJ1 in the expression of the identified genes and their potential it is inactive in other neurogenic niches of the embryonic brain. The function in the generation of motile cilia. Thus, we propose that cluster of FoxJ1-dependent genes identified in this study appears to Kif6, and potentially Kif9 and Kif27, motor proteins could be be highly focused on the regulation of microtubule-based responsible for the transport of basal bodies to the surface of intracellular transport. Our findings suggest that the expression of differentiating ependymal cells (Fig. 7D). It will be intriguing to the identified genes might be related to the transport of replicated determine whether or not any vesicular proteins are part of this basal bodies to the apical surface of the differentiating ependymal complex. The sequence of signaling and molecular events driven by cells during the genesis of motile cilia. The role of FoxJ1-dependent FoxJ1-dependent transcriptional activity during ciliogenesis remains differentiation of the SCN in postnatal neurogenesis remains to be to be elucidated. determined in vivo. + Acknowledgements FoxJ1 astrocytes in the SCN We thank M. Iyengar, A. Sheikh and J. Burroughs for technical assistance. M. We also discovered a small subset of FoxJ1 astrocytes that Vernon from the Functional Genomic Core Facility at University of North emerge in the postnatal SCN around P21 in mice and gradually Carolina conducted the microarray hybridizations and helped organize the data. D. Eisenstat (U. Manitoba) kindly provided the Dlx2 antibody. We thank J. increase in density by 6 months of age in the wild-type SVZ. The Weimer, S. Magness, A. Barnes and J. Horowitz for comments and discussions emergence of these astrocytes between P19 and P21 is dependent during various stages of this work. This work was entirely supported by startup on FoxJ1 expression as their density is severely depleted in the funds to H.T.G. from the College of Veterinary Medicine and Department of –/– FoxJ1 SVZ. Recent studies have described the expansion of a Molecular Biomedical Sciences at NCSU. unique set of astrocytes in the aged ependymal layer (Luo et al., Supplementary material 2006), a phenomenon later shown to be involved in the repair of Supplementary material for this article is available at ependymal cells in elderly mice (Luo et al., 2008). It is possible http://dev.biologists.org/cgi/content/full/136/23/4021/DC1 that the small population of resident FoxJ1 astrocytes might References participate in the repair of the aged ependymal layer. In support Alvarez-Buylla, A. and Lim, D. A. (2004). For the long run: maintaining germinal of this, recent studies have revealed that FoxJ1 promoter-active niches in the adult brain. Neuron 41, 683-686. cells in the spinal canal and SVZ participate in neurogenesis and Bayer, S. A. (1983). 3H-thymidine-radiographic studies of neurogenesis in the rat gliogenesis in the injured spinal cord and in response to stroke olfactory bulb. Exp. Brain Res. 50, 329-340. DEVELOPMENT Development of the adult neural stem cell niche RESEARCH ARTICLE 4031 Brody, S. L., Yan, X. H., Wuerffel, M. K., Song, S. K. and Shapiro, S. D. (2000). Luo, J., Shook, B. A., Daniels, S. B. and Conover, J. C. (2008). Subventricular Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice. Am. J. zone-mediated ependyma repair in the adult mammalian brain. J. Neurosci. 28, Respir. Cell Mol. Biol. 23, 45-51. 3804-3813. Campbell, K. and Gotz, M. (2002). Radial glia: multi-purpose cells for vertebrate Marlin, A. E., Wald, A., Hochwald, G. M. and Malhan, C. (1978). Kaolin- brain development. Trends Neurosci. 25, 235-238. induced hydrocephalus impairs CSF secretion by the choroid plexus. Neurology Capela, A. and Temple, S. (2002). LeX/ssea-1 is expressed by adult mouse CNS 28, 945-949. stem cells, identifying them as nonependymal. Neuron 35, 865-875. Meletis, K., Barnabe-Heider, F., Carlen, M., Evergren, E., Tomilin, N., Carlen, M., Meletis, K., Goritz, C., Darsalia, V., Evergren, E., Tanigaki, K., Shupliakov, O. and Frisen, J. (2008). Spinal cord injury reveals multilineage Amendola, M., Barnabe-Heider, F., Yeung, M. S., Naldini, L. et al. (2009). differentiation of ependymal cells. PLoS Biol. 6, e182. Forebrain ependymal cells are Notch-dependent and generate neuroblasts and Merkle, F. T., Tramontin, A. D., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. astrocytes after stroke. Nat. Neurosci. 12, 259-267. (2004). Radial glia give rise to adult neural stem cells in the subventricular zone. Coskun, V., Wu, H., Blanchi, B., Tsao, S., Kim, K., Zhao, J., Biancotti, J. C., Proc. Natl. Acad. Sci. USA 101, 17528-17532. Hutnick, L., Krueger, R. C., Jr, Fan, G. et al. (2008). CD133+ neural stem cells Mirzadeh, Z., Merkle, F. T., Soriano-Navarro, M., Garcia-Verdugo, J. M. and in the ependyma of mammalian postnatal forebrain. Proc. Natl. Acad. Sci. USA Alvarez-Buylla, A. (2008). Neural stem cells confer unique pinwheel 105, 1026-1031. architecture to the ventricular surface in neurogenic regions of the adult brain. Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1997). Cellular Cell Stem Cell 3, 265-278. composition and three-dimensional organization of the subventricular germinal Mitchell, D. R. (2004). Speculations on the evolution of 9+2 organelles and the zone in the adult mammalian brain. J. Neurosci. 17, 5046-5061. role of central pair microtubules. Biol. Cell 96, 691-696. Doetsch, F., Caille, I., Lim, D. A., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. Noctor, S. C., Flint, A. C., Weissman, T. A., Wong, W. S., Clinton, B. K. and (1999a). Subventricular zone astrocytes are neural stem cells in the adult Kriegstein, A. R. (2002). Dividing precursor cells of the embryonic cortical mammalian brain. Cell 97, 703-716. ventricular zone have morphological and molecular characteristics of radial glia. Doetsch, F., Garcia-Verdugo, J. M. and Alvarez-Buylla, A. (1999b). J. Neurosci. 22, 3161-3173. Regeneration of a germinal layer in the adult mammalian brain. Proc. Natl. Nonaka, S., Tanaka, Y., Okada, Y., Takeda, S., Harada, A., Kanai, Y., Kido, M. Acad. Sci. USA 96, 11619-11624. and Hirokawa, N. (1998). Randomization of left-right asymmetry due to loss of Edgar, R., Dornrachev, M. and Lash, A. E. (2002). Gene Expression Omnibus: nodal cilia generating leftward flow of extraembryonic fluid in mice lacking NCBI gene expression and hybridization array data repository. Nucleic Acids Res. KIF3B motor protein. Cell 95, 829-837. 30, 207-210. O’Rourke, J. P., Olsen, J. C. and Bunnell, B. A. (2005). Optimization of equine Ghashghaei, H. T., Weber, J., Pevny, L., Schmid, R., Schwab, M. H., Lloyd, K. infectious anemia derived vectors for hematopoietic cell lineage gene transfer. C., Eisenstat, D. D., Lai, C. and Anton, E. S. (2006). The role of neuregulin- Gene Ther. 12, 22-29. ErbB4 interactions on the proliferation and organization of cells in the Oakley, B. R. (1992). Gamma-tubulin: the microtubule organizer? Trends Cell Biol. subventricular zone. Proc. Natl. Acad. Sci. USA 103, 1930-1935. 2, 1-5. Ghashghaei, H. T., Lai, C. and Anton, E. S. (2007a). Neuronal migration in the Olsen, J. C. (1998). Gene transfer vectors derived from equine infectious anemia adult brain: are we there yet? Nat. Rev. Neurosci. 8, 141-151. virus. Gene Ther. 5, 1481-1487. Ghashghaei, H. T., Weimer, J. M., Schmid, R. S., Yokota, Y., McCarthy, K. D., Ostrowski, L. E., Hutchins, J. R., Zakel, K. and O’Neal, W. K. (2003). Targeting Popko, B. and Anton, E. S. (2007b). Reinduction of ErbB2 in astrocytes expression of a transgene to the airway surface epithelium using a ciliated cell- promotes radial glial progenitor identity in adult cerebral cortex. Genes Dev. 21, specific promoter. Mol. Ther. 8, 637-645. 3258-3271. Ostrowski, L. E., Yin, W., Rogers, T. D., Busalacchi, K. B., Chua, M., O’Neal, Gomperts, B. N., Gong-Cooper, X. and Hackett, B. P. (2004). Foxj1 regulates W. K. and Grubb, B. R. (2009). Conditional deletion of Dnaic1 in a murine basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells. J. model of primary ciliary dyskinesia causes chronic rhinosinusitis. Am. J. Respir. Cell Sci. 117, 1329-1337. Cell Mol. Biol. (in press). Gong, Q. and Shipley, M. T. (1995). Evidence that pioneer olfactory axons Pan, J., You, Y., Huang, T. and Brody, S. L. (2007). RhoA-mediated apical actin regulate telencephalon cell cycle kinetics to induce the formation of the enrichment is required for ciliogenesis and promoted by Foxj1. J. Cell Sci. 120, olfactory bulb. Neuron 14, 91-101. 1868-1876. Hinds, J. W. (1968a). Autoradiographic study of histogenesis in the mouse Pinto, L. and Gotz, M. (2007). Radial glial cell heterogeneity-the source of diverse olfactory bulb. I. Time of origin of neurons and neuroglia. J. Comp. Neurol. 134, progeny in the CNS. Prog. Neurobiol. 83, 2-23. 287-304. Rakic, P. (1972). Mode of cell migration to the superficial layers of fetal monkey Hinds, J. W. (1968b). Autoradiographic study of histogenesis in the mouse olfactory neocortex. J. Comp. Neurol. 145, 61-83. bulb. II. Cell proliferation and migration. J. Comp. Neurol. 134, 305-322. Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from Jacquet, B. V., Patel, M., Iyengar, M., Liang, H., Therit, B., Salinas- isolated cells of the adult mammalian central nervous system. Science 255, Mondragon, R., Lai, C., Olsen, J. C., Anton, E. S. and Ghashghaei, H. T. 1707-1710. (2009). Analysis of neuronal proliferation, migration and differentiation in the Sawamoto, K., Wichterle, H., Gonzalez-Perez, O., Cholfin, J. A., Yamada, M., postnatal brain using equine infectious anemia virus-based lentiviral vectors. Spassky, N., Murcia, N. S., Garcia-Verdugo, J. M., Marin, O., Rubenstein, J. Gene Ther. 16, 1021-1033. L. et al. (2006). New neurons follow the flow of cerebrospinal fluid in the adult Johansson, C. B., Momma, S., Clarke, D. L., Risling, M., Lendahl, U. and brain. Science 311, 629-632. Frisen, J. (1999). Identification of a neural stem cell in the adult mammalian Schlessinger, K., Hall, A. and Tolwinski, N. (2009). Wnt signaling pathways central nervous system. Cell 96, 25-34. meet Rho GTPases. Genes Dev. 23, 265-277. Katoh, M. and Katoh, M. (2004). Human FOX gene family. Int. J. Oncol. 25, Schmechel, D. E. and Rakic, P. (1979). A Golgi study of radial glial cells in 1495-1500. developing monkey telencephalon: morphogenesis and transformation into Kuo, C. T., Mirzadeh, Z., Soriano-Navarro, M., Rasin, M., Wang, D., Shen, J., astrocytes. Anat. Embryol. (Berl) 156, 115-152. Sestan, N., Garcia-Verdugo, J., Alvarez-Buylla, A., Jan, L. Y. et al. (2006). Spassky, N., Merkle, F. T., Flames, N., Tramontin, A. D., Garcia-Verdugo, J. M. Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in and Alvarez-Buylla, A. (2005). Adult ependymal cells are postmitotic and are the subventricular neurogenic niche 2. Cell 127, 1253-1264. derived from radial glial cells during embryogenesis. J. Neurosci. 25, 10-18. Laywell, E. D., Rakic, P., Kukekov, V. G., Holland, E. C. and Steindler, D. A. Stubbs, J. L., Oishi, I., Izpisua Belmonte, J. C. and Kintner, C. (2008). The (2000). Identification of a multipotent astrocytic stem cell in the immature and forkhead protein Foxj1 specifies node-like cilia in Xenopus and zebrafish adult mouse brain. Proc. Natl. Acad. Sci. USA 97, 13883-13888. embryos. Nat. Genet. 40, 1454-1460. Lehmann, O. J., Sowden, J. C., Carlsson, P., Jordan, T. and Bhattacharya, S. S. Tavazoie, M., Van, d., V, Silva-Vargas, V., Louissaint, M., Colonna, L., Zaidi, (2003). Fox’s in development and disease. Trends Genet. 19, 339-344. B., Garcia-Verdugo, J. M. and Doetsch, F. (2008). A specialized vascular niche Lim, D. A., Tramontin, A. D., Trevejo, J. M., Herrera, D. G., Garcia-Verdugo, J. for adult neural stem cells. Cell Stem Cell 3, 279-288. M. and Alvarez-Buylla, A. (2000). Noggin antagonizes BMP signaling to create Vladar, E. K. and Axelrod, J. D. (2008). Dishevelled links basal body docking and a niche for adult neurogenesis. Neuron 28, 713-726. orientation in ciliated epithelial cells. Trends Cell Biol. 18, 517-520. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression Wichterle, H., Garcia-Verdugo, J. M., Herrera, D. G. and Alvarez-Buylla, A. data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. (1999). Young neurons from medial ganglionic eminence disperse in adult and Methods 25, 402-408. embryonic brain. Nat. Neurosci. 2, 461-466. Lois, C. and Alvarez-Buylla, A. (1993). Proliferating subventricular zone cells in You, Y., Huang, T., Richer, E. J., Schmidt, J. E., Zabner, J., Borok, Z. and Brody, the adult mammalian forebrain can differentiate into neurons and glia. Proc. S. L. (2004). Role of f-box factor foxj1 in differentiation of ciliated airway Natl. Acad. Sci. USA 90, 2074-2077. epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol. 286, L650-L657. Lois, C. and Alvarez-Buylla, A. (1994). Long-distance neuronal migration in the Yu, X., Ng, C. P., Habacher, H. and Roy, S. (2008). Foxj1 transcription factors are adult mammalian brain. Science 264, 1145-1148. master regulators of the motile ciliogenic program. Nat. Genet. 40, 1445-1453. Luo, J., Daniels, S. B., Lennington, J. B., Notti, R. Q. and Conover, J. C. (2006). Zallen, J. A. (2007). Planar polarity and tissue morphogenesis. Cell 129, 1051- The aging neurogenic subventricular zone. Aging Cell 5, 139-152. 1063. DEVELOPMENT
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Development – The Company of Biologists
Published: Dec 1, 2009