Flt4 mRNA in adult rat central nervous system

Journal of Chemical Neuroanatomy 42 (2011) 56–64

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Distribution of vascular endothelial growth factor receptor-3/Flt4 mRNA in adult rat central nervous system Yun Hou, Yoo-Jin Shin, Esther Jiwon Han, Jeong-Sun Choi, Jang-Mi Park, Jung-Ho Cha, Jae-Youn Choi, Mun-Yong Lee * Department of Anatomy, College of Medicine, The Catholic University of Korea, 505 Banpo-dong, Socho-gu, 137-701, Seoul, South Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 25 February 2011 Received in revised form 21 May 2011 Accepted 7 June 2011 Available online 14 June 2011

Vascular endothelial growth factor receptor (VEGFR)-3/Flt4 binds VEGF-C and VEGF-D with high affinity. It has been suggested to be involved in neurogenesis and adult neuronal function. However, little is known about the localization of VEGFR-3 in the adult central nervous system (CNS). The present study presents, to our knowledge, the first detailed mapping of VEGFR-3 mRNA expression in adult rat brain and spinal cord by using in situ hybridization and reverse transcription-polymerase chain reaction analysis (RT-PCR). Varying VEGFR-3 expression intensity was detected in functionally diverse nuclei, with the highest levels in the mitral cells of the olfactory bulb, piriform cortex, anterodorsal thalamic nucleus, several nuclei of the hypothalamus, and the brainstem cranial nerve nuclei. VEGFR-3 mRNA was abundantly expressed in the ventral motor neurons of the spinal cord and in some circumventricular organs such as the median eminence and the area postrema. Moreover, the locus coeruleus and some of the nuclei of the reticular formation showed moderate-to-high hybridization signals. VEGFR-3 expression appeared to be localized mostly within neurons, but weak labeling was also found in some astrocytes. In particular, VEGFR-3 was highly expressed in ependymal cells of the ventral third ventricle and the median eminence, which were co-labeled with vimentin but not with glial fibrillary acidic protein, suggesting that these cells are tanycytes. RT-PCR analysis revealed similar levels of VEGFR-3 expression in all regions of the adult rat CNS. The specific but widespread distribution of VEGFR-3 mRNA in the adult rat CNS suggests that VEGFR-3 functions more broadly than expected, regulating adult neuronal function playing important roles in tanycyte function. ß 2011 Elsevier B.V. All rights reserved.

Keywords: VEGFR-3 Mapping Brain Spinal cord Tanycytes Hypothalamus In situ hybridization

1. Introduction The vascular endothelial growth factor (VEGF) family currently comprises 5 members in mammals: VEGF (also termed VEGF-A), VEGF-B, VEGF-C, VEGF-D, and the placental growth factor (PlGF) (reviewed in Roy et al., 2006; Yamazaki and Morita, 2006; Raab and Plate, 2007). These ligands bind in an overlapping pattern to 3 tyrosine kinase receptors: VEGF receptor-1 (VEGFR-1; fms-like tyrosine kinase-1; Flt-1), VEGFR-2 (fetal liver kinase receptor-1; Flk-1), and VEGFR-3 (Flt-4). VEGF-C and VEGF-D have been identified as the ligands for VEGFR-3. VEGFR-3 is expressed predominantly in the lymphatic endothelial cells and is regarded as an important regulator of lymphatic development and lymphangiogenesis (Ma¨kinen et al., 2001; Veikkola et al., 2001; Karkkainen et al., 2004). However, a newly discovered role for

* Corresponding author. Tel.: +82 2 2258 7261; fax: +82 2 536 3110. E-mail address: [email protected] (M.-Y. Lee). 0891-0618/$ – see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2011.06.001

VEGF-C as a trophic factor for neural progenitors in the developing brain (Le Bras et al., 2006) has created particular interest in the function of the VEGF-C/VEGFR-3 axis in the nervous system. We demonstrated that VEGFR-3 might be involved in neuronal precursor cell proliferation and neurogenesis in association with the radial glia in the developing forebrain and cerebellum (Choi et al., 2010; Hou et al., 2011). These studies also showed that the expression of VEGFR-3 mRNA is not confined solely to the fields of cellular proliferation, but it is constitutively expressed in almost all the cortical regions harboring mature neurons and postmitotic neurons in the mature cerebellum, suggesting a possible involvement of VEGFR-3 in adult neuronal function as well as in developmental processes. Furthermore, VEGFR-3 might be actively involved in regulating adult neurogenesis and glial reaction after focal and global forebrain ischemia (Shin et al., 2008, 2010a,b). Thus, these findings strongly suggest that VEGFR-3 expression might be highly active and widely distributed within the adult rat central nervous system (CNS). Although VEGFR-3 expression has already been described in some areas of the rat brain (Choi et al.,

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2010; Hou et al., 2011), a detailed study on the distribution of VEGFR-3 transcripts in the adult rat CNS has, to our knowledge, never been performed. To gain insights into VEGFR-3 function in the adult CNS by a molecular morphological approach, we aimed to establish the precise distribution of the VEGFR-3 mRNA in the rat brain and spinal cord using in situ nucleic acid hybridization, and to determine VEGFR-3 mRNA’s expression profile in the rat CNS using reverse transcription-polymerase chain reaction (RT-PCR). Together with in situ hybridization data for mouse from the Allen Brain Atlas project (Lein et al., 2007) this provides the comprehensive information of the expression pattern of VEGFR-3 in the adult rodent CNS. 2. Material and methods 2.1. Experimental animals and tissue processing All surgical interventions and presurgical and postsurgical animal care were provided in accordance with the Laboratory Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery provided by the IACUC (Institutional Animal Care and Use Committee) in the School of Medicine, The Catholic University of Korea. Ten male Sprague–Dawley rats (3–6 months old) were used in this study. Three rats were sacrificed for use in RT-PCR analysis and seven were sacrificed for use in in situ hybridization. For in situ hybridization histochemistry, animals were deeply anesthetized with 16.9% urethane (10 ml/kg) and killed by transcardial perfusion with a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.2). Brains and spinal cords were removed, post-fixed in the same fixative for 2 h at 4 8C, and equilibrated with 30% sucrose in 0.1 M PB. For Semi-quantitative RT-PCR analysis, animals were anesthetized with 16.9% urethane (10 ml/kg) and decapitated. The olfactory bulb, cerebral neocortex, hippocampus, thalamus/ hypothalamus, midbrain, pons, medulla oblongata, cerebellum, and lumbar spinal segments were dissected out and frozen immediately in liquid nitrogen. 2.2. Semi-quantitative RT-PCR Semi-quantitative RT-PCR analysis was carried out as described previously (Shin et al., 2008). In brief, total RNA was extracted from homogenates of nine areas from rat brains and spinal cords using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Firststrand cDNA was synthesized using Reverse Transcriptase M-MLV (Takara Korea Biomedical Inc., Korea) according to the manufacturer’s instructions. Equal amounts (1 ml) of the reverse transcription products were then PCR-amplified using Perfect Premix Version 2.1 (Ex Taq version; Takara Korea Biomedical Inc.). One picomole of primer, which was specific for rat VEGFR-3 (sense, 50 -ctgaggcagaatatcagtctggag-30 , antisense, 50 -agatgctcatacgtgtagttgtcc-30 ; GenBank accession no. NM_053652.1; nucleotides 1222–1763), was used in the amplification reaction. Amplification commenced with denaturation at 94 8C for 4 min followed by 25–30 cycles of 94 8C for 30 s, 58 8C for 30 s and 72 8C for 30 s. The final extension was made at 72 8C for 10 min. Ten microliters of each PCR reaction product were electrophoresed on 1.5% (w/v) agarose gels containing ethidium bromide (1 mg/ml). For semiquantitative measurements, we amplified the VEGFR-3 mRNAs with rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA and optimized the number of PCR cycles to maintain amplification within a linear range. RT-PCR products were quantified by photographic densitometry of the ethidium bromide-stained agarose gel, and VEGFR-3/GAPDH product ratios expressed as arbitrary units which represented the relative expression of this transcript in nine areas from rat brains and spinal cords. Three animals were used for PCR at each time point and the average value for the optical density was calculated from three independent RT-PCRs.

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rinses in TBS and equilibration in TBS/50 mM MgCl2 (pH 9.5) the enzyme reaction was performed using 4-nitroblue tetrazolium chloride (0.35 mg/ml) and 5-bromo4-chloro-3-indolyl phosphate (0.18 mg/ml) as substrates. Tissue sections were viewed with a microscope and photographed with a digital camera (Jenoptik, Germany). Images were acquired with the same intensity of light for the microscopy and the same parameters for the digital photography, and so only minor adjustments were made to establish uniform contrast for all figures. Images were converted to TIFF format and contrast levels adjusted by using Adobe Photoshop v. 7.0 (Adobe Systems, San Jose, CA, USA). After hybridization, as described above, some sections were incubated with biotin-conjugated mouse monoclonal anti-DIG antibody (Jackson ImmunoResearch, West Grove, PA, USA; dilution 1:200) overnight 4 8C. For tripleimmunofluorescence histochemistry, sections were incubated at 4 8C overnight with following antibodies; monoclonal mouse anti-vimentin (Chemicon International Inc., Temecula, CA, USA; dilution 1:200) and polyclonal rabbit anti-glial fibrillary acidic protein (GFAP; Chemicon; dilution 1:1500). Antibody staining was visualized with the following secondary antibodies; Cy3-conjugated streptavidin (Jackson ImmunoResearch; dilution 1:1500), Alexa488-conjugated anti-mouse antibody (Invitrogen; dilution 1:300) and Cy5-conjugated anti-rabbit antibody (Jackson ImmunoResearch; dilution 1:500). In controls, the primary antibody was omitted from the incubation solution. Counterstaining of cell nuclei was carried out by incubating the sections with DAPI (40 ,6-diamidino-20 -phenyindole; Roche; 0.5– 1 mg/ml) for 10 min. Slides were viewed with a confocal microscope (LSM 510 Meta; Carl Zeiss Co., Ltd., Germany) equipped with four lasers (Diode 405, Argon 488, HeNe 543, HeNe 633). Images were converted to TIFF format, and contrast levels were adjusted by using Adobe Photoshop v. 7.0.

3. Results 3.1. RT-PCR analysis The regional profile of VEGFR-3 mRNA was determined by semiquantitative RT-PCR on homogenates from the olfactory bulb, cerebral neocortex, hippocampus, thalamus/hypothalamus, midbrain, pons, medulla oblongata, cerebellum, and lumbar spinal segments. The intensity of the expected product size (542 bp) was similar for all regions tested (Fig. 1).

2.3. In situ hybridization histochemistry and triple labeling The sequences for VEGFR-3 from RT-PCR product corresponding to nucleotides 1222–1763 (see above) were cloned into the TA site of pGEM-T Easy vector (Promega Co., Madison, WI, USA), and sequenced. The antisense and sense riboprobes were labeled with digoxigenin (DIG) using in vitro transcription, as described in detail previously (Shin et al., 2008). Coronal cryostat sections were collected and washed twice with 2 standard saline citrate (SSC; 20 SSC: 3 M NaCl, 0.3 M sodium citrate, pH 7.0). Prehybridization was performed at 53 8C for 2 h in hybridization solution containing 50% formamide, 4 SSC, 5% dextran sulphate and 1 Denhardt’s solution. Sections were then incubated overnight at 53 8C with antisense or sense probes diluted in hybridization solution (150 ng/ml) and subsequently washed with 2 SSC at room temperature followed by successive washes at 62 8C in prewarmed 2 SSC, 2 SSC/50% formamide, 0.1 SSC/50% formamide and 0.1 SSC for 30 min each. After several rinses in Tris-buffered saline (TBS; 0.15 M NaCl, 0.1 M Tris, pH 7.5) and incubation with 10% normal sheep serum for 1 h the tissue was treated with alkaline phosphatase-conjugated sheep anti-DIG antibody (Roche, Germany; dilution 1:2000) overnight at 4 8C. Following several

Fig. 1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of VEGFR-3 gene expression in the adult rat central nervous system, including the olfactory bulb (lane 1), cerebral cortex (lane 2), hippocampus (lane 3), diencephalon (lane 4), midbrain (lane 5), pons (lane 6), medulla (lane 7), cerebellum (lane 8), and lumbar spinal cord (lane 9). RT-PCR analysis identified the VEGFR-3 gene as a 542bp product expressed at every level of the neuraxis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA served as an internal standard. VEGFR-3 expression has been normalized to the expression of GAPDH. Bars indicate the mean values derived from three independent RT-PCRs. Error bars indicate standard error of the mean.

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Y. Hou et al. / Journal of Chemical Neuroanatomy 42 (2011) 56–64 Table 1 (Continued )

3.2. In situ hybridization histochemistry

Anatomical region

In situ hybridization studies consistently and reproducibly labeled brain and spinal cord sections from 7 adult rats. Specificity was verified by the lack of signals when hybridization was carried out in the presence of an excess of a sense-stranded probe, even at a concentration 3 times that of the antisense probe (data not shown). Results of the in situ hybridization revealed widespread expression of VEGFR-3 mRNA in specific neuronal populations in diverse regions of the rat CNS, including the main olfactory bulb, cerebral cortex, hippocampus, several nuclei in the diencephalon and the brainstem, and the spinal cord (Table 1). On the basis of the cytological criteria, the signals appeared to be localized mainly within the cytoplasm of neurons, but weak signals were also observed in some glia-like small cells. Expression of VEGFR-3 is described in a rostral-to-caudal sequence. 3.2.1. Telencephalon In the main olfactory bulb, cells in the mitral cell and external plexiform layers were intensely labeled (Fig. 2A, B). In addition, neurons localized to the glomerular layer were moderately labeled and weaker labeling was observed in the internal granular layer. Hybridization in cortical neurons was relatively weak throughout the different subdivisions of the cerebral cortex, but could be clearly localized to layer V of the neocortex and, more prominently, in neurons of the piriform cortex (Fig. 2C, D, G). In the hippocampal formation, moderate signals were detected in the granule cell layer of the dentate gyrus, and in the pyramidal cell layer throughout the CA1–CA3 regions and subiculum (Fig. 2K), consistent with Shin et al.’s data (2008).

Table 1 Localization and relative labeling of VEGFR-3 mRNA in the rat central nervous system. Anatomical region Telencephalon Main olfactory bulb: Glomerular layer External plexiform layer Mitral cell layer Internal granular layer Septum Lateral septal nucleus Medial septal nucleus Striatum Olfactory tubercle Cerebral cortex Neocortex Piriform cortex Entorhinal cortex Hippocampal formation Fields CA1–3 of Ammon’s horn Dentate gyrus Subiculum Amygdaloid complex Medial amygdaloid nucleus Cortical amygdaloid nucleus Laterobasal nuclear complex Diencephalon Thalamus Anterodorsal thalamic nucleus Anteroventral thalamic nucleus Anteromedial thalamic nucleus Laterodorsal thalamic nucleus Paracentral thalamic nucleus Paratenial thalamic nucleus Paraventricular thalamic nucleus Reuniens thalamic nucleus Hypothalamus Arcuate hypothalamic nucleus

Relative labeling

++ +++ +++ + ++ + + +++ +/+ + +++ +/+ +

Ventromedial hypothalamic nucleus Paraventricular nucleus Supraoptic nucleus Periventricular hypothalamic nucleus Suprachiasmatic nucleus Retrochiasmatic nucleus Anteroventral periventricular nucleus Medial preoptic nucleus Medial preoptic area Mammillary complex Tuberomammillary nucleus Medial habenular nucleus Mesencephalon Periaqueductal gray Oculomotor nucleus Red nucleus Substantia nigra Compact part Reticular part Interpeduncular nucleus Superior colliculus Inferior colliculus Median raphe nucleus Dorsal raphe nucleus

Relative labeling + + + + + + + + + + + +

+ ++ ++ ++ + + + + + +

++ +++ +++ + + ++ +/+ + +/+ + ++ ++

Metencephalon and myelencephalon Locus ceruleus Pontine nuclei Ventral tegmental nucleus Reticulotegmental nucleus Pontine reticular nucleus Ventral nucleus lateral lemniscus Mesencephalic trigeminal nucleus Trigeminal motor nucleus Principal sensory nucleus Spinal trigeminal nucleus Superior olivary complex Facial motor nucleus Vestibular nuclei Cochlear nuclei Hypoglossal nucleus Vagal motor nucleus External cuneate nucleus Lateral reticular nucleus Nucleus of the solitary tract Posterodorsal tegmental nucleus Gigantocellular reticular nuclei

+ + + + + + + + + + + + + + + + + + + + +

Circumventricular organ Median eminence Area postrema Choroid plexus

++ +++ ++

Spinal cord Dorsal horn Ventral horn

++ +++

++ ++ + +

+ + + + + + + + + + +

+ + + + +

+ + +

Level of VEGFR-3 expression: weak (+); moderate (++); strong (+++).

++ ++ ++ ++ ++ +

+ + + + + + + +

++ + + +

+ +

+++

The basal nuclei were weakly labeled, particularly in the striatum and medial septal nuclei (Fig. 2C, D). However, moderate signals were observed in the lateral septal nucleus (Fig. 2C), and some nuclei of the amygdaloid complex, such as the medial and cortical amygdaloid nuclei (data not shown). 3.2.2. Diencephalon In the thalamus, only some nuclei—the anterior thalamic complex, including the anterodorsal, anteroventral, and anteromedial thalamic nuclei, and the laterodorsal thalamic and paraventricular thalamic nuclei—were intensely labeled, with the most intense signals in the anterodorsal thalamic nucleus (Fig. 2D, E, I, K). In addition, the paratenial thalamic and reuniens thalamic nuclei were weakly labeled (data not shown). Other

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Fig. 2. Distribution of VEGFR-3 mRNA in coronal sections through the main olfactory bulb to the diencephalon of adult rat brain. (A, B) The main olfactory bulb showing intensely labeled cells in the mitral cell layer (Ml) and external plexiform layer (EPl). In addition, weak labeling was observed in the internal granular layer (IGr), and moderate labeling in cells of the glomerular layer (Gl). (B) Higher-magnification view of the boxed area in A. (C) Coronal section of the forebrain at the level of the anterior commissure (ac) showing a moderate signal in the lateral septal nucleus (LS) and in the medial preoptic area (MPA) and a very prominent signal in the piriform cortex (arrowheads). CPu, the caudate putamen; LV, the lateral ventricle. (D) Coronal section of the diencephalon through the optic chiasm (ox). The asterisk indicates the suprachiasmatic nucleus showing a moderate signal; st, the stria terminalis. (E–F) Higher-magnification view of the boxed areas in D showing a strong signal in the anterodorsal thalamic nucleus (AD in E) and supraoptic nucleus (F), and a moderate signal in the anteroventral thalamic nucleus (AV in E). D3V, the dorsal third ventricle; sm, the stria medullaris. (G) Highermagnification view in D showing a moderate signal in layer V of the cerebral cortex. (H) Coronal section through the hypothalamus showing a strong signal in the paraventricular nucleus (Pa). 3V, the third ventricle. (I) Coronal section through the thalamus showing intensely labeled neurons in the anterodorsal thalamic nucleus (AD); neurons in the anteroventral (AV) and laterodorsal (LD) thalamic nuclei are less strongly labeled. (K–J) Coronal section at the level of the median eminence (ME). Areas expressing VEGFR-3 at this level include the hippocampus; medial habenular (MHb); and laterodorsal thalamic (LD), retrochiasmatic (RCh), and arcuate hypothalamic nuclei (Arc), with highest levels in the latter. (J) Higher-magnification view of the boxed areas in K. Note that the ependymal cells of the median eminence were also labeled. (L) Coronal section through the posterior commissure (pc). Labeling is seen in the tuberomammillary (TM) and arcuate nuclei. (M) Higher-magnification view of the boxed area in L showing that the ependymal cells of the subcommissural organ (SCO) are devoid of signals, whereas the intense signals are seen in the adjacent ependymal cells of the third ventricle. (N) Higher-magnification view of the boxed area in L showing the intense signals in the arcuate nucleus. Scale bars = 500 mm for C, D, K, L; 200 mm for A, B, E, H; 100 mm for F, G, I, J, M, N.

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thalamic nuclei or epithalamic nuclei, except the medial habenular nucleus, showed no prominent cellular labeling (Fig. 2K). In the hypothalamus, intense hybridization signals were seen in the arcuate, paraventricular, supraoptic, and periventricular hypothalamic nuclei (Fig. 2D, F, H, J–L, N). A moderate density of VEGFR-3 mRNA was also observed in some nuclei of the preoptic area and in the suprachiasmatic, retrochiasmatic, and tuberomammillary nuclei (Fig. 2C, D, K, L). In addition, intense expression of VEGFR-3 in the ependymal layer of the ventral third ventricle and the median eminence implies that it was likely to be expressed in tanycytes (Fig. 2J, K). This assumption was confirmed by triple labeling with VEGFR-3, vimentin, a tanycyte marker (Chauvet et al., 1998), and GFAP. In the floor and the ventral part of the third ventricle, VEGFR-3-expressing cells, the cell bodies of which were located in the ependymal layer, exhibited vimentin-positive, but GFAP-negative long processes toward the periventricular parenchyma (Fig. 3A–H). 3.2.3. Brainstem In the midbrain, the most intense signals were observed in the oculomotor and red nuclei (Fig. 4A). Other nuclei of the midbrain, such as the interpeduncular, median raphe, and dorsal raphe nuclei showed moderate hybridization signals (Fig. 4A, B, D). In addition, moderate signal levels were associated with cells located in the central gray, lateral to the aqueduct (Fig. 4A, B). Weak labeling was detected in the substantia nigra and the superior and inferior colliculi (Fig. 4A–C). In the pons, intense-to-moderate hybridization signals were exhibited by the locus coeruleus, superior olivary complex, pontine nuclei, and mesencephalic and motor trigeminal nuclei (Fig. 4B, E– I). Furthermore, the nuclei of the pontine reticular formation, including the ventral tegmental, pontine reticular, and reticulotegmental nuclei, showed moderate hybridization signals (Fig. 4B, D, E). An intense hybridization signal was observed in the Purkinje cell layer of the cerebellar cortex (Fig. 4G), as observed previously (Hou et al., 2011).

In the medulla oblongata, many cranial nerve nuclei such as the facial, vagal motor, hypoglossal, spinal trigeminal, vestibular, and cochlear nuclei displayed moderate-to-intense hybridization signals (Fig. 5A–F). In addition, low-to-moderate hybridization signals were observed in the nuclei of the reticular formation including the gigantocellular reticular, intermediate reticular, and lateral reticular nuclei (Fig. 5A, B, E). 3.2.4. Circumventricular organ Within circumventricular organs tested, the area postrema showed the most intense hybridization signals (Fig. 5F). The median eminence (Fig. 2J, K) and some epithelial cells of the choroid plexus in the third ventricle (data not shown) showed moderate hybridization signals. However, no prominent cellular labeling was detected in the subfornical organ and neurohypophysis (data not shown). In addition, no signal was observed within the ependymal cells of the subcommissural organ, although the adjacent ependymal cells of the third ventricle showed intense hybridization signals (Fig. 2L, M). 3.2.5. Spinal cord Along the entire length of spinal cord, intense-to-moderate hybridization signals were observed in the dorsal and ventral horns, with the highest intensity signals in the motor neurons of the ventral horn (Fig. 5G, H). 4. Discussion Here, we present a detailed transcriptional map of VEGFR-3 of the adult rat brain and spinal cord. Our results together with data from Allen Brain Atlas (Lein et al., 2007) display that expression of VEGFR-3 mRNA is widely distributed throughout the rodent CNS, from the olfactory bulbs to the spinal cord. Moreover, RT-PCR analysis detected the VEGFR-3 542 bp PCR product at every level of the neuraxis. However, VEGFR-3 mRNA was not homogeneously distributed, and hybridization signals of varying intensity were

Fig. 3. Characterization of VEGFR-3-expressing cells in the hypothalamus. (A–D) Triple labeling for VEGFR-3, vimentin, and GFAP showing that VEGFR-3 expression is intense in ependymal cells at the level of the arcuate nucleus (Arc), typically corresponding to tanycytes. 3V, the third ventricle. (E–H) Higher-magnification views of the boxed areas in A–D, respectively. Note that VEGFR-3 expression is intense in ependymal cells whose vimentin-positive, but GFAP-negative long processes extended toward the periventricular parenchyma. Scale bars = 100 mm for A–D; 50 mm for E–H.

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Fig. 4. Distribution of VEGFR-3 mRNA in coronal sections through the upper brainstem. (A) Coronal section at the level of the superior colliculus showing the moderate-tointense signals in the oculomotor (3), red (R), and interpeduncular nuclei (IP). Aq, the cerebral aqueduct. (C) Higher-magnification view of the boxed area in A with weakly labeled neurons in the compact (SNC) and reticular (SNR) parts of the substantia nigra; cp, the cerebral peduncle. (B, D) Coronal section at the inferior colliculus showing the moderate-to-intense signals in the pontine (Pn), median raphe (MnR), ventral tegmental (VTg), and reticulotegmental nuclei (RtTg); ml, the medial lemniscus; PnO, the oral pontine reticular nucleus; VLL, the ventral nucleus of the lateral lemniscus. (D) Higher-magnification view of the boxed area in B. (E, G) Two different levels of the pons. (E) Pons with labeled neurons in the mesencephalic trigeminal (Me5; upper boxed area in E) and trigeminal motor nuclei (lower boxed area in E), and at higher magnification in F and H, respectively. 4V, the fourth ventricle; PnC, the caudal pontine reticular nucleus. (G) Pons with labeled neurons in the locus coeruleus (boxed area in G), posterodorsal tegmental nucleus (PDTg), superior olivary complex (SOC), principal sensory trigeminal nucleus (Pr5), and ventral cochlear nucleus (VCA). Note the intense signal in cells located in the Purkinje cell layer (arrowheads) of the cerebellum; 8n, the vestibulocochlear nerve; s5, sensory root of the trigeminal nerve. (I) Higher-magnification of the boxed area in G showing the intensely labeled neurons in the locus coeruleus. Scale bars = 500 mm for A, B, E, G; 100 mm for C, D, F, H; 50 mm for I.

localized to specific CNS regions (Table 1). The morphological features of labeled cells in the gray matter and the absence of labeled cells within the fiber tracts indicate that neurons mainly, if not only, express the signals. However, astrocytes, especially those located in the periventricular zone of the third ventricle, some choroid plexus epithelial cells, and ependymal cells hybridized to the VEFGR-3 probe. In particular, VEGFR-3 is more intensely expressed in ependymal cells of the third ventricle and the median eminence, but not in those bordering the lateral ventricle and the central canal of the spinal cord. The most numerous cell population expressing VEGFR-3 was located in the brainstem, where the most intense signals were associated with the cranial nerve nuclei, the pontine nuclei, and the locus coeruleus. In particular, most cranial nerve motor nuclei including the oculomotor, trigeminal motor, facial motor, vagal motor, and hypoglossal nuclei displayed the most intense signals. In addition, the red nucleus, ventral motor neurons of the spinal cord, and Purkinje cells of the cerebellum exhibited intense labeling, suggesting the association of VEGFR-3 expression with the motor system. However, noticeable expression was also present in nuclei of the sensory system, such as the cochlear and vestibular nuclei, and in the reticular formation. Although neurons expressing VEGFR-3 represent a variety of phenotypes and are involved in many distinct physiological functions, it is likely that the characterization of specific neurons showing intense

VEGFR-3 signals for will help better understand its physiological role. Prominent hybridization signals were also observed in several hypothalamic nuclei, including the arcuate, paraventricular, and supraoptic nuclei; the ependymal layer of the third ventricle; and the median eminence. In particular, ependymal expression was most prominent in the ventral half portion of the third ventricle and in the median eminence corresponding to the expected location of tanycytes (Bjelke and Fuxe, 1993; Chauvet et al., 1998; Mathew, 2008), and could be tentatively attributed to tanycytes, which are vimentin positive and GFAP negative (Chauvet et al., 1995, 1998). Tanycytes play roles in absorption and transportation processes of neuroendocrine hypothalamic neurons and in sensing cerebrospinal fluid glucose concentration (Bjelke and Fuxe, 1993; Garcı´a et al., 2003; Yamamura et al., 2004). Thus, intense signals for VEGFR-3 in neurosecretory hypothalamic neurons and in tanycytes suggest its involvement in the neurosecretory function of the hypothalamus. Interestingly, tanycytes retain the morphological features of embryonic radial glial cells, and further, have been suggested to possess neural cell progenitor properties (Xu et al., 2005). We showed that radial glia in embryonic forebrain, Mu¨ller glial cells and Bergmann glia expressed VEGFR-3 (Choi et al., 2010; Hou et al., 2011). Furthermore, VEGFR-3 is upregulated in SVZ astrocytes, type B cells in the SVZ of stroke-lesioned rats, which share some

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Fig. 5. Distribution of VEGFR-3 mRNA at different rostrocaudal levels of the medulla (A–F) and the lumbar spinal cord (G–H). (A, B) Cranial medulla with heavily labeled neurons in the lateral (LVe), medial (MVe), spinal vestibular (SpVe), facial (7), and dorsal cochlear nuclei (DC). In addition, a moderate signal was observed in prepositus hypoglossal (PrH), spinal trigeminal (Sp5), and gigantocellular reticular nuclei (Gi), 4V, the fourth ventricle; g7, the genu facial nerve; icp, inferior cerebellar peduncle; IRt, the intermediate reticular nucleus; py, the pyramidal tract. (C, D) Higher-magnification views of the boxed areas in A. (E, F) Caudal medulla with labeled neurons in the vagal motor (10), solitary tract (Sol), and hypoglossal nuclei (12). Note the intense hybridization signal in the area postrema (AP); cc, the central canal; LRt, the lateral reticular nucleus. (G) Spinal cord with labeled neurons in the dorsal and ventral horns; DF, dorsal funiculus; LF, lateral funiculus; VF, ventral funiculus. (H) Higher-magnification view of the boxed area in G. Note intense signals in motor neurons of the ventral horn. Scale bars = 500 mm for A, B, E–G; 200 mm for C, D, H.

characteristics with radial glia-like cells (Shin et al., 2010a). An important aspect of our study is that the VEGFR-3 expression in the persistent radial glia of the mature brain comprising tanycytes, Mu¨ller cells, and Bergmann glia, as well as in the radial glia in the developing brain indicates its utility as a marker of radial glia in the developing and adult brain. Prominent expression was also observed in some circumventricular organs, including the median eminence, the area postrema, and some choroid plexus epithelial cells. The association of VEGFR3 expression with the circumventricular organs, however, is not a general feature; there was no significant expression in other organs, including the subfornical and subcommissural organs and the neurohypophysis. The functional significance of VEGFR-3 within these circumventricular organs is not known. However, it is

interesting to note that the circumventricular organs, including the median eminence, the area postrema, and some choroid plexus ependymal cells constitute a novel source of neural stem cells (Itokazu et al., 2006; Pecchi et al., 2007; Bennett et al., 2009). Thus, it is possible that VEGFR-3 is expressed in these unique neurogenic zones, which are thought to retain the capacity of generating new neurons and glia in the adult brain. This suggestion is supported by our previous findings (Choi et al., 2010) that VEGFR-3 is expressed in the subventricular zone of the lateral ventricle and the rostral migratory stream of the mature forebrain, where neural stem cells reside (Doetsch et al., 1999; Ihrie and Alvarez-Buylla, 2008; Mendoza-Torreblanca et al., 2008). In contrast to the more widespread distribution pattern in the hypothalamus, expression of VEGFR-3 is discrete in the thalamus,

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where only a few nuclei were labeled. In particular, the anterior thalamic complex, including the anterodorsal, anteroventral, and anteromedial nuclei, and the laterodorsal nucleus, which have great similarities in afferent and efferent connectivity (for review, see Groenewegen and Witter, 2004) showed prominent hybridization signals with the highest expression levels in the anterodorsal thalamic nuclei. The midline thalamic nuclei also showed weak-to-moderate signals, but prominent expression was not detected in the sensory or motor thalamic nuclei. Thus, it was apparent that VEGFR-3 mRNA was mostly localized in the associated thalamic nuclei rather than in the principal thalamic relay nuclei. Although our results and data from Allen Brain Atlas (Lein et al., 2007) showed that the general distribution pattern of VEGFR-3 mRNA in the CNS was remarkably similar in rat and mouse, species differences concerning the expression in the cerebral cortex and the basal ganglia do exist. In the rat forebrain, prominent hybridization signals for this receptor were observed in layer V neurons of the cerebral cortex and pyramidal neurons of the hippocampus, while the basal ganglia including the striatum and the globus pallidus contained far less intensely labeled cells. However, data from Allen Brain Atlas (Lein et al., 2007) showed that hybridization signals with similar intensity were evenly distributed in the cerebral cortex and the basal ganglia of the mouse forebrain. There seems to be no simple explanation for these species differences, because the distribution of VEGFR-3 mRNA in two species was in excellent agreement in other brain areas studied. For the final identification of VEGFR-3 expressing cell type in the different brain areas, it will be necessary to perform double-labeling study. However, in most areas, it was apparent that VEGFR-3 mRNA was localized in principal projection neurons, e.g., mitral and tufted cells of the olfactory bulb, layer V neurons of the cerebral cortex, pyramidal neurons of the hippocampus, motor neurons of the brainstem and the spinal cord, and Purkinje cells and deep cerebellar nuclei (Hou et al., 2011) of the cerebellum. Thus, VEGFR-3 mRNA is expressed in neuronal populations involved in a large range of functions, but it remains to be elucidated whether the high expression level of VEGFR-3 in specific nuclei reflects the distinct physiological function. Le Bras et al. (2006) showed that VEGF-C and VEGFR-3 are expressed in neural progenitor cells of Xenopus laevis and mouse embryos, suggesting that VEGF-C–VEGFR-3 signaling provides a direct trophic support to neural progenitor cells in the embryonic brain. In addition, the expression profiles of VEGF-C and VEGFR-3 shared overlapping expression patterns in the control and ischemic hippocampus (Shin et al., 2008). Thus, our current data suggest the widespread expression pattern of VEGF-C and VEGF– D, ligands of VEGFR-3, and their close relationship with its receptor in the adult rat CNS. Therefore, future investigations are needed to elucidate the possible autocrine or paracrine mechanisms of the VEGF-C/D and VEGFR-3 ligand receptor system in the adult CNS. In conclusion, our results show that VEGFR-3 is widely distributed throughout the rat CNS, and that a corresponding diversity exists with respect to the physiological function, i.e., it is expressed in functionally diverse areas including the motorrelated areas, sensory system, limbic system, and the reticular formation. The specific but widespread distribution of VEGFR-3 in the rat CNS suggests that VEGFR-3 might have broader functions in the adult CNS than previously thought. More than just the involvement in developmental processes, it could be associated with a particular role in different neuronal populations. In addition, prominent VEGFR-3 expression in the hypothalamus, the circumventricular organs, and tanycytes might be associated with specific but hitherto unknown physiological functions.

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