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Molecular cloning of a cDNA encoding mouse A15, a member of the transmembrane 4 superfamily, and its preferential expression in brain neurons
Neuroscience Research 35 (1999) 281 – 290 www.elsevier.com/locate/neures
Molecular cloning of a cDNA encoding mouse A15, a member of the transmembrane 4 superfamily, and its preferential expression in brain neurons Yoshitaka Hosokawa a,1, Eiko Ueyama1 b, Yoshihiro Morikawa b, Yumiko Maeda a, Masao Seto a,*, Emiko Senba b a
Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, 1 -1 Kanakoden, Chikusa-ku, Nagoya 464 -8681, Japan b Department of Anatomy and Neurobiology, Wakayama Medical College, 811 -1 Kimiidera, Wakayama 641 -0012, Japan Received 29 June 1999; accepted 14 September 1999
Abstract A15, a member of the transmembrane 4 superfamily (TM4SF), was isolated by differential screening of the cDNAs that are preferentially expressed on immature T cells. As a first step in the study of the biological function of the A15 molecule, we isolated cDNAs encoding the entire coding region of mouse A15. Nucleotide sequence analysis of the cDNAs revealed that mouse A15 shares 97% amino acid sequence identity with its human counterpart. The mouse A15 protein product has not yet been characterized, but is predicted to be 244 amino acids with four hydrophobic domains. Northern blot analysis of the RNA samples from various mouse tissues disclosed that the A15 transcripts are expressed most strongly in the brains, and are detectable in the colon, muscle, heart, kidney, and spleen. In situ hybridization of the mouse brain with ribo-probe established that the A15 transcripts are expressed primarily in neurons of the frontal cortex, olfactory bulb, dentate gyrus, caudoputamen, and CA3 region of the hypothalamus as well as in Purkinje cells in the cerebellar cortex, which strongly suggests that A15 may have a special function in the fundamental neuronal functioning of the higher nervous system. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: TM4SF; Tetraspanin; CCG-B7; Ion channel; In situ hybridization; A15
1. Introduction The transmembrane 4 superfamily (TM4SF), also known as the tetraspanin, consists of a group of structurally related proteins that are characterized by four transmembrane domains (Wright and Tomlinson, 1994; Maeker et al., 1997). Membership of this family has now grown to more than 20 known genes (Hotta et al., 1988; Classon et al., 1989; Amiot, 1990; Angelisova et al., 1990; Oren et al., 1990; Azorsa et al., 1991; Boucheix et al., 1991; Imai et al., 1992; Emi et * Corresponding author. Tel.: + 81-52-7626111 (ext. 8840); fax: +81-52-7635233. E-mail address: [email protected] (M. Seto) 1 Yoshitaka Hosokawa and Eiko Ueyama contributed equally to this work.
al., 1993; Szala et al., 1993; Jankowski et al. 1994; Dong et al., 1995; Takagi et al., 1995; Hasegawa et al., 1996), some of which are also found in organisms as primitive as schsitosomes and nematodes. The existence of so many different TM4SF members in organisms as evolutionarily diverse as humans and schistosomes strongly implies essential functions for these genes in cellular functioning. Many of them are expressed on leukocytes and it is likely that they mediate signal transduction events that play a critical role in the regulation of cell proliferation, motility, and differentiation. Furthermore, two of the members, CD63/ME491 and KAl-1, have been shown to be involved, respectively, in tumor progression and metastasis (Hotta et al., 1988; Dong et al., 1995). A15, a member of TM4SF, was isolated by differential screening of the cDNAs that are preferentially
0168-0102/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 9 3 - 0
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expressed on immature T cells as compared with the peripheral blood leukocytes (PBLs) (Emi et al., 1993). Thus, it is conceivable that the A15 protein product may have a special part to play in T cell development. Subsequently, the CCG-B7 cDNA clone was isolated from a human frontal cortex cDNA library (Li et al., 1993). The library was screened with triplet repeat probes that contained ten repeats of either CCG or CTG. The CCG-B7 clone has seven CCG repeats in its 5% upstream end of the initiation codon. A homology search disclosed that the predicted CCG-B7 coding region is identical to that of A15 protein. Thus, the A15/CCG-B7 gene appears to contain triplet nucleotide repeats that are often associated with several neuropsychiatric diseases (Warren, 1996), such as Huntington’s chorea, myotonic dystrophy, and fragile X syndrome, where patients’ genes have been shown to contain amplified triplet nucleotide sequences. It is unclear whether the A15 gene is a candidate gene responsible for such neuropsychiatric diseases. A15/CCG-B7 cDNA was also isolated by means of a different approach using a monoclonal antibody and was named TALLA-1 (Takagi et al., 1995). As a first step toward elucidating the biological function of the A15 protein, we isolated cDNAs for mouse A15 and demonstrated that mouse A15 transcripts are expressed preferentially in brain neurons. The distribution of the transcripts in the brain was determined by using in situ hybridization (ISH).
2. Materials and methods
2.1. PCR cloning of mouse A15 cDNA Male mice (C57BL/B6) were obtained from SLC Japan (Shizuoka, Japan) and kept in the laboratory animal center. Total RNA was isolated from mouse spleen via homogenization in guanidium thiocyanate and centrifugation through cesium chloride. Firststrand cDNA was generated by using 5 mg of the total
RNA samples from mouse spleen, 200 units of superscript II reverse transcriptase (Stratagene, La Jolla, CA) and 200 ng of random primer in a total volume of 40 ml. Following first strand cDNA synthesis, 35 cycles of PCR were performed in a total volume of 50 ml containing 2 ml of first-strand cDNA, 1 mM of each primer, 100 mM dNTP, 1.5 mM MgCl2, 10 mM Tris–Cl (pH 8.3), 50 mM KCl, and 1 unit Taq DNA polymerase (Takara, Kyoto, Japan). The PCR parameters were: 94°C, 45 s; 55°C, 90 s; and 72°C, 90 s for 35 cycles. The primer sequences corresponded to the human A15 sequences and were as follows: Primer 1: 5%-ATGGAGACCAAACCTGTGAT-3% Primer 2: 5%-TTACACCATCTCATACTGAT-3% The locations of primers 1 and 2 are shown in Fig. 1. The RT-PCR products (clone mA15-1) were purified from a low-melting-point agarose gel and subcloned into the plasmid vector. To obtain cDNAs that correspond to the 5% and 3% ends of mouse A15 transcript, PCR was performed to screen the mouse spleen cDNA library. The mouse spleen random-primed cDNA library was constructed in the phage vector lgt 10 as described (Seto et al., 1992). Oligonucleotide primers were then designed to correspond to the sequences near the 5% and 3% end of clone mA15-1 (primers 3 and 4 in Fig. 1) and the sequences flanking the cloning site in lgt 10 (primers R and L in Fig. 1). Phage lysates (5 ml) of the cDNA library were directly subjected to the PCR reaction. The PCR parameters were: 94°C, 45 s; 55°C, 90 s; and 72°C, 90 s for 35 cycles. The primer sequences used were as follows. Primer 3: 5%-CCAGAAGACGAAGGAGTAGATGATGAGGAG-3% Primer 4: 5%-TTCATGGAGACTAACATGGG-3% Primer F: 5%-AGCAAGTTCAGCCTGGTTAA-3% Primer R: 5%-CTTATGAGTATTTCTTCCAGGGTA-3% The locations of the primers are also shown in Fig. 1. The PCR products (clones mA15-2 and mA15-3) were purified from a low-melting-point agarose gel and subcloned into the plasmid vector.
Fig. 1. Mouse A15 cDNA. Three clones for mouse A15 cDNA are indicaded by thick lines. The open box shows the open reading frame. The PCR primers are represented by arrows.
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Fig. 2. Nucleotide sequence of mouse A15 cDNA and its predicted amino acid sequence. The nucleotides are numbered in the 5% to 3% direction, and the amino acid sequence is numbered sequentially from the first methionine. The amino acid sequence of human A15 is shown beneath the mouse sequence for comparison. Four putative transmembrane domains are underlined. The dashes represent identical amino acids and the terminal codon is indicated by an asterisk. GenBank accession number; AF052492.
2.2. Northern blot analysis Total RNAs (5 mg per lane) from various tissues of C57BL/B6 mice were separated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde and then transferred to a Hybond N nylon membrane. The hybridization probe was a mouse A15 coding region probe (nucleotides 6 – 737 in Fig. 2). The probe was labeled with [a32P]dCTP by using a randomprimed labeling kit (Nippon Gene, Tokyo, Japan). The hybridization was carried out at 42°C overnight
in a solution containing 45% formamide, 5×SSPE, 1 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml salmon sperm DNA. The membrane was finally washed for 30 min at 65°C with 0.5× SSC (1×SSC; 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS.
2.3. DNA sequencing DNA sequencing was performed with the aid of an ABI PRISM dye terminator cycle sequencing kit
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(Perkin Elmer, Foster, CA) on a 372 automated DNA sequencer (Applied Biosystems, Foster City, CA).
2.4. Animals and tissue preparation for ISH Male mice (C57BL/B6) were housed in plastic cages with soft sawdust bedding under a 12 h light/dark cycle (lights on at 08:00 h) and free access to food and water. Adult animals weighing about 20 g (7 – 8 weeks old, n =3) were used. They were decapitated under deep anesthesia with diethylether, and the brains were excised and immediately frozen with powdered dry ice. The other group of animals (n = 4) were perfused under deep sodium pentobarbital anesthesia (75 mg/kg i.p.) with 0.85% saline (100 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 400 ml), after which the brains, trigeminal ganglia and several peripheral tissues and organs were excised. These samples were post-fixed in the same fixative overnight, stored in 30% (w/v) sucrose in 0.1 M phosphate buffer for 2 days at 4°C, and then frozen with powdered dry ice. Serial 6 mm sections were prepared in a cryostat at − 20°C and mounted onto glass slides coated with 3-amino propyltriethoxysilane. These sections were stored at −80°C until they were processed.
2.5. ISH A fragment (732 bp) of mouse A15 cDNA was amplified by PCR and subcloned into the EcoRV site of Bluescript II SK(+ ) plasmid (Stratagene) and digested with Sal I (antisense probe) or Not I (sense probe). This was followed by in vitro transcription using T3 RNA polymerase (antisense probe) or T7 RNA polymerase (sense probe). To inactivate RNase and to promote the permeability of the tissues, the sections were incubated with 1 mg/ml of proteinase K in 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 10 min at 37°C. After rinsing with 0.1 M phosphate buffer, the samples were incubated with 0.2 N HCl for 10 min and then with 0.25% acetic anhydrate in 0.1 M triethanolamine for 10 min to prevent nonspecific binding of the probes. The sections were rinsed again with 0.1 M phosphate buffer and dehydrated through a graded ethanol series (70–100%). Hybridization using the riboprobe for A15 mRNA was performed overnight at 55°C in a buffer (1×TE; 50% formamide; 0.6 M NaCl; 1× Denhardt’s solution; 0.25% SDS; 500 mg/ml yeast tRNA; 10% dextran sulfate; 0.1 M dithiothreitol) containing 0.5–1.0× 106 cpm of labeled probes per slide. After hybridization, the slides were immersed in 5× SSC (pH 7.2) containing 10 mM DTT for 10 min at 50°C to remove glass covers and the sections were rinsed in 2 × SSC containing 50% formamide and 10 mM DTT for 30 min at 65°C. The sections were then incubated with RNase A (Sigma Chemicals, St Louis,
MO) in Tris–EDTA buffer containing 0.5M NaCl for 30 min at 37°C. After rinsing with a graded SSC series (2×SSC–0.1× SSC), the samples were dehydrated with 70% ethanol and air-dried. For autoradiography, the mounted sections were coated with Kodak NTB3 emulsion (diluted 1:2 with distilled water at 40°C) and exposed for 1–3 weeks in a tightly sealed dark box at 4°C. After development in a Kodak D19 developer and fixation in 24% sodium thiosulfate, the sections were counterstained with 1% Neutral Red solution for morphological identification. Sections were observed and photographed under dark-field lateral illumination (XF-WFL, Nikon, Tokyo, Japan).
3. Results
3.1. cDNA cloning of mouse A15 In an effort to isolate mouse A15 cDNA, we first performed reverse transcription-polymerase chain reaction (RT-PCR) of the total RNA samples from the mouse spleen. The oligonucleotide primers used corresponded to the human A15 sequence primers 1 and 2 as shown in Fig. 1. Mouse A15 cDNA fragments (732 bp) were amplified and subcloned into the plasmid vector. Sequence analysis of the clone (mA15-1 in Fig. 1) revealed that they show 97% amino acid sequence identity with the corresponding region of the human A15 cDNA. Thus, the cDNA fragment was found to encode a middle portion of mouse A15 cDNA. To isolate cDNAs that correspond to the 5% and 3% further ends of mouse A15 transcript, PCR was again performed to screen the mouse spleen cDNA library constructed in lgt 10 phage vector. Oligonucleotide primers were designed to correspond to the sequences near the 5% and 3% end of clone mA15-1 (primers 3 and 4 in Fig. 1) and to the sequences flanking the cloning site in lgt 10 (primers R and F in Fig. 1). To identify the appropriate PCR products corresponding to the 5% and 3% end of A15 cDNA, the end-labeled oligonucleotides (primers 1 and 2 in Fig. 1) were used as internal probes for Southern blot analysis (data not shown). The hybridized PCR products were then subcloned into the plasmid vector. Sequencing analysis of these clones (mA15-2 and mA15-3 in Fig. 1) revealed that they indeed encode the 5% and 3% end of the mouse A15 coding region, demonstrating that we were able to isolate cDNAs covering the entire coding region of mouse A15. Nucleotide sequence analysis of the cDNAs revealed that overall, mouse A15 shares 97% amino acid sequence identity with its human counterpart. Nucleotide sequence of the mouse A15 cDNA and its predicted amino acid sequence are shown in Fig. 2. The mouse A15 protein product has not yet been characterized, but
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is predicted to be 244 amino acids with four putative transmembrane domains. However, the PCR cloning did not allow us to isolate the 5%-untranslated end of the full-length mouse A15 cDNA.
3.2. Northern blot analysis To clarify the biological role of A15, it was of interest to examine whether A15 transcripts are expressed in tissues other than immature T cells. Northern blot analysis of the RNA samples from various mouse tissues revealed that A15 transcripts are expressed most strongly in the brain, and are detectable in the colon, muscle, heart, kidney and spleen (Fig. 3). This finding is intriguing in that the human CCG-B7/ A15 cDNA was found to contain, in its 5%-untranslated region, triplet nucleotide repeats that are often associated with several neuropsychiatric diseases.
3.3. ISH To examine the detailed localization of expression of the A15 transcript in mouse brain, we performed ISH with mouse A15 ribo-probe. The A15 signal intensity in each brain ares is summarized in Table 1. Signals for A15 mRNA were found to be concentrated in neurons and its distribution was rather ubiquitous. However, a
Fig. 3. Northern blot analysis of the various mouse tissues with A15 cDNA. The total RNAs (5 mg) were electrophoresed on a 1% agarose/formaldehyde gel and blotted on a nylon membrane. The membrane was hybridized with 32P-labeled mouse A15 cDNA as a probe. The ethidium bromide-stained gel used in the northern blot analysis is shown at the bottom, and tissue sources are also indicated in the figure.
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wide variety of signal densities were also observed, i.e. neurons in discrete brain regions expressed higher levels of A15 mRNA signals as described below (Section 3.3.1–Section 3.3.7). It is possible that these variations may partly reflect the difference of cell densities. Almost no signals were identified in fiber tracts. Sections hybridized with sense probes showed no accumulation of signals (Fig. 4D, F), indicating the specificity of the probe and the validity of hybridization method.
3.3.1. Cortex and olfactory areas A high signal density for A15 mRNA was diffusely distributed in layers II–VI of the cerebral cortex throughout the forebrain (Fig. 4). The highest signals were observed in layer II. In cortical areas, densities were especially high in the cingulate cortex (Cg), piriform cortex (Pir), dorsal endopiriform nucleus (DEn) and tenia tecta (TT) (Fig. 4). In the olfactory bulb, a high signal density was observed in the mitral cell layer (not shown). Moderate signals were distributed in the granule cells in the internal granular layer and in the perigromerular cells in the glomerular layer. 3.3.2. Basal ganglia and limbic system Moderate signals were spread evenly throughout the caudate-putamen (CPu) and accumbens nucleus (Acb) (Fig. 4A, B). Signals in the globus pallidus were very low in comparison to those of the CPu (Fig. 5A). Moderate signals could also be seen in the bed nucleus of the stria terminalis (BST) and in the lateral septal nucleus (LS) (Fig. 4B, Fig. 5A). Labeled neurons were scattered in the entopeducular nucleus (EP) (Fig. 5C). The hippocampus showed the highest signal density in the pyramidal cell layer of the CA3 region and in the granule cells of the dentate gyrus (DG) (Fig. 4E, Fig. 5C, Fig. 6A). However, the signals in the CA1–CA2 regions of the hippocampal formation were relatively low compared to that of CA3 region and dentate gyrus. The subiculum (S) showed low signal densities (Fig. 6A) and the amygdala high signal densitites (Fig. 4C,Fig. 4E, Fig. 5C). The strongest signals were observed in the central (ACe) and cortical (ACo) amygdaloid nuclei. 3.3.3. Thalamus ISH signals in the thalamus were relatively weak (Fig. 4C, Fig. 4E, Fig. 5B, Fig. 5C). Low levels were found in the midline thalamic nuclei, such as paraventricular nucleus (PV), central medial nucleus (CM), reuniens nucleus (Re), rhomboideus nucleus (Rh) of thalamus (Fig. 5B, C). Labeled cells were serially distributed from the zona incerta (ZI) to reticular thalamic nucleus (Rt). A low signal level was observed in the ventral lateral geniculate nucleus (VLG), while the level in the dorsal lateral geniculate nucleus was lower (Fig. 4E). Low signal levels could also be seen in the rest of the thalamic nulcei.
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Table 1 Distribution of A15 mRNAs in the mouse brain Brain area Cerebral cortex Neocortex (Cx) Tenia tecta (TT) Cingulate cortex (Cg) Olfactory system Olfactory bulb Glomerular layer Mitral cell layer Internal granular layer Olfactory tubercle (Tu) Dorsal endopiriform nucleus (DEn) Piriform cortex (Pir) Basal ganglia and septum Caudoputamen (Cpu) Globus pallidus (GP) Accumbens nucleus (Acb) Bed nucleus of stria terminalis (BST) Entopeduncular nucleus (EP) Lateral septal nucleus (LS) Hippocampal formation CA1 CA2 CA3 Dentate gyrus (DG) Subiculum (S) Amygdaloid complex Central amygdaloid nucleus (ACe) Cortical amygdaloid nucleus (ACo) Basolateral amygdaloid nucleus Medial amygdaloid nucleus Thalamus Paraventricular nucleus (PV) Central medial nucleus (CM) Rhomboid nucleus (Rh) Reunience nucleus (Re) Medial and lateral habenula (MHb, LHb) Reticular nucleus (Rt) Ventrolateral nucleus (VL) Parafacicular nucleus Ventral Lateral geniculate nucleus (VLG) Medial geniculate nucleus (MG) Zona incerta (ZI) Hypothalamus Anterior hypothalamic nucleus Paraventricular hypothalamic nucleus (PVH) Ventromedial hypothalamic nucleus (VMH) Lateral hypothalamic nucleus (LH) Arcuate nucleus (Arc) Premammillary nucleus (PM) Medial and lateral mammillary nuclei (MM, LM) Brainstem Central grey (CG) Superior colliculus (SC) Inferior colliculus (IC) Interpeduncular nucleus (IPN) Substantia nigra (SN) Red nucleus (RN)
Signal intensitya
+++ +++ +++
++ +++ ++ ++ +++ ++++ ++ + ++ ++ + ++ +++ +++ ++++ ++++ + +++ +++ +++ +++ + + + + + + +/− + + + ++ ++ + ++ + + ++ +
+ + ++ + +/− +/−
Pontine nucleus (Pn) Parabrachial nucleus (PB) Dorsal tegmental nucleus (DT) Principal sensory trigeminal nucleus (Pr5) Ventral cochlear nucleus (VCN) Medial vestibular nucleus Spinal trigeminal nucleus Nucleus of the solitary tract Lateral reticular nucleus Motor cranial nerve nuclei
++ ++ ++ + + + + + + +
Cerebellar cortex Purkinje cell layer (PCL) Granular layer (GL) Molecular layer
+++ ++ −
Trigeminal ganglion
+++
a Grading of signal intensity; ++++: very high, +++: high, ++: moderate, +: low, +/−: very low.
3.3.4. Hypothalamus Overall, the signal density in the hypothalamus was moderate (Fig. 4C, Fig. 4E, Fig. 5C, Fig. 5D). Labeled neurons were concentrated in the medially located nuclei, such as the anterior hypothalamic nucleus, paraventricular nucleus (PVH) (Fig. 5B), ventromedial nucleus (VMH) (Fig. 5C), arcuate nucleus (Arc), premammillary nucleus (PM) and medial mammillary nucleus (MM) (Fig. 5D). Moderately labeled neurons were scattered in the lateral part of the hypothalamus. 3.3.5. Brainstem Midbrain — structures in the midbrain expressed a relatively low signal density (Fig. 6A, B). Few signals could be identified in the central grey (CG), dorsal raphe nucleus (DR), interpeduncular nucleus (IP) and medial geniculate nucleus (MG). The superior colliculus (SC) displayed low level signals, but the inferior colliculus (IC) showed more (Fig. 6C). Other areas of the midbrain, such as the substantia nigra (SN) and red nucleus (RN) were very low in signal levels. Pons — areas displaying moderate signal concentrations included the pontine nuclei (Pn) (Fig. 6A, B) and nuclei on the floor of the IVth ventricle including dorsal tegmental nuclei (DT) and locus coeruleus (LC). The pontine nuclei showed one of the highest signal densities in the brainstem. Other areas with moderate to low concentrations of signal density included the parabrachial nucleus (PB), principal sensory trigeminal nucleus (Pr5) and ventral cochleal nucleus (VCN) (Fig. 6C). Moderately labeled neurons were scattered in the raphe magnus and pontine reticular nuclei. Motoneurons in the trigeminal motor nucleus and facial nucleus also exhibited moderate signal density. Medulla — Low signal concentrations were identified throughout the nucleus tractus solitarii, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, nucleus ambiguus and lateral reticular nucleus (not shown).
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3.3.6. Cerebellum High signal concentrations were identified in the Purkinje cell layer (PCL) and moderate signal levels in
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the granular layer (GL) (Fig. 6D), while only signals of control level could be identified in the molecular layer and white matter of the cerebellum.
Fig. 4. Dark-field photomicrographs showing signals for A15 mRNA in forebrain areas. Coronal sections are arranged rostrocaudally. C and D and E and F are serial sections hybridized with antisense (C, E) and sense (D, F) probes for A15 mRNA. Scale bar; 1 mm. Abbreviations in Figures: ac, anterior comissure; Acb, accumbens nucleus (n.); ACe, central amygdaloid n.; ACo, cortical amygdaloid n.; Arc, arcuate n.; BST, bed nucleus of stria terminalis; Cer, cerebellum; CG, central grey; Cg, cingulate cortex; CM, central medial thalamic n.; CPu, caudoputamen; Cx, neocortex; DEn, dorsal endopiriform n.; DR, dorsal raphe n.; DT, dorsal tegmental n.; EP, entopeduncular n.; GL, granular layer; GP, globus pallidus; ic, internal capsule; IC, inferior colliculus; IP, interpeduncular n.; LC, locus coeruleus; LG, lateral geniculate n.; LH, lateral hypothalamic n.; LM, lateral mammillary n.; LS, lateral septal n.; LSD, dorsal lateral septal n.; MG, medial geniculate n.; MHb, medial habenular n.; MM, medial mammillary n.; MMn, median medial mammillary n.; mt, mammillothalamic tract; ot, optic tract; PB, parabrachial n.; Pir, piriform cortex; PCL, Purkinje cell layer; PM, premammillary n.; Pn, pontine n.; Pr5, principal sensory trigeminal n.; PV; paraventricular nucleus; PVH, paraventricular hypothalamic n.; Re, reuniens thalamic n.; Rh, rhomboid thalamic n.; RN, red n.; Rt, reticular thalamic n.; S, subiculum; SC, superior colliculus; sm, stria medullaris thalami; SN, substantia nigra; Tu, olfactory tubercle; TT, tenia tecta; VCN, ventral cochlear n.; VL, ventrolateral thalamic n.; VMH, ventral medial hypothalamic n.; ZI, zona incerta.
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Fig. 5. Dark-field photomicrographs showing precise localization of signals for A15 mRNA in the basal ganglia (A), thalamus (B, C) and hypothalamus (C, D). Scale bars; 500 mm (A–C), 250 mm (D).
3.3.7. Trigeminal ganglion All of the neurons in the trigeminal ganglion expressed high levels of A15 mRNA signals (Fig. 6E).
4. Discussion TM4SF consists of a group of structurally related membrane proteins that have in common four conserved membrane spanning regions, certain cystein residues, and short sequence motifs (Wright and Tomlinson, 1994; Maeker et al., 1997). Many of them have a flair for promiscuous associations with other molecules, including integrins and other TM4SFs. The biological function of the members of this family are largely unknown, but it is likely that they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We previously isolated a cDNA for human A15, a member of the TM4SF by using differential screening of the cDNAs that are preferentially expressed in immature T cells as compared with the PBLs (Emi et al.,
1993). In the present study, we isolated cDNAs encoding mouse A15 and demonstrated that the transcript is expressed preferentially in the brain neurons. The mouse A15 predicted from the cDNA sequence consists of 244 amino acid residues and it was found to share 97% amino acid sequence identity with its human counterpart. This extremely high conservation between mouse and human A15 supports the possibility that A15 may play a role in fundamental biological processes such as cell proliferation, motility, and differentiation. Although TM4SF shows no significant homology to any known proteins, there are certain features of the family that are characteristic of ligand-gated ion channels, including the acetylcholine receptor family. The transmembrane domains of the TM4SF are relatively rich in polar residues and have highly conserved Glu or Gln residues buried deep in the third and fourth transmembrane domains. Thus, it may be possible that A15, a member of TM4SF, functions as an ion channel in brain neurons. Our earlier northern blot analysis showed that the human A15 transcripts are expressed in HPB-ALL, Jurkat, and MOLT3 cell lines, but not in HUT102 or
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PBL cells, which are thought to be mature T cells. This pattern suggests that A15 transcripts are expressed in the immature stage of T cell development (Emi et al.,
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1993). We also examined whether A15 transcripts are expressed in tissues other than immature T cells. Northern blot analysis of the RNA samples from various
Fig. 6. Dark-field photomicrographs showing signals for A15 mRNA in the brainstem (A, B, C), cerebellum (D) and trigeminal ganglion (E). D and F are serial sections hybridized with antisense (D) and sense (F) probes for A15 mRNA. Scale bars; 1 mm (A – C, E), 500 mm (D, F).
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mouse tissues revealed that mouse A15 transcripts are expressed most strongly in the brain. This finding led us to examine in detail the distribution of A15 expression in the brain by means of ISH. Our ISH analysis indicated that A15 mRNA appears to be expressed in all the neurons in the brain, and that uneven and characteristic distribution pattern was also observed among these neurons. Especially noteworthy was the extremely high level of expression in the cortex and hippocampus. Neurons in the caudoputamen, nucleus accumbens, bed nucleus of stria terminalis, amygdala and hypothalamus were also found to express moderate to high levels of ISH signals. Such distribution patterns strongly suggest that A15 may have a fundamental neuronal function especially of higher nervous system. Chromosomal location of the human A15 gene was previously assigned to Xq11 (Virtaneva et al., 1994), and it has been noted that this region harbors genes associated with X-linked mental retardations (Frezal and Schinzel, 1991). Given the fact that the A15 transcripts are expressed primarily in the brain neurons, it will be of particular interest to examine whether the A15 gene is responsible for such neuropsychiatric diseases. In summary, the present study describes the cDNA cloning of mouse A15, a member of the TM4SF and demonstrates that the transcripts are expressed preferentially in the brain neurons as well as in immature T cells. Much more needs to be learned about the biological functions of A15, however. In order to further clarify the function of A15, it is essential to identify molecules which associate with A15 on the cell membrane. Gene targeting with mouse ES cells should prove to be another important means to identify and characterize the biological function of A15 in the nervous system.
Acknowledgements This work was supported in part by a Grant-in-aid for the Second Term Comprehensive Ten Year Strategy for Cancer Control from the Ministry of Health and Welfare, a Grant-in-aid for Science on Primary Areas (Cancer Research), from the Ministry of Education, Science and Culture, Japan, a Grant-in-aid from the Japanese Foundation for Multidisciplinary Treatment of Cancer, and the Bristol – Myers Squibb Unrestricted Biomedical Research Grants.
References Amiot, M., 1990. Identification and analysis of cDNA clones encoding CD53 — a pan-leukocyte antigen related to membrane transport proteins. J. Immunol. 145, 4322–4325. .
Angelisova, P., Vlcek, C., Stefanova, I., Lipoldova, M., Horejsi, V., 1990. The human leukocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens. Immunogenetics 32, 281 – 285. Azorsa, D.O., Hyman, J.A., Hildreth, J.K., 1991. CD63/Pltgp40: a platelet activation antigen identical to the stage-specific melanoma associated antigen ME491. Blood 178, 280 – 284. Boucheix, C., Benoit, P., Franchet, P., et al., 1991. Molecular cloning of the CD9 antigen. J. Biol. Chem. 266, 117 – 122. Classon, B.J., Williams, A.F., Willis, A.C., Seed, B., Stamenkovic, I., 1989. The primary structure of the human leukocyte antigen CD37, a species homologue of the rat MRC OX-44 antigen. J. Exp. Med. 169, 1497 – 1502. Dong, J-T., Lamb, P.W., Rinker-Schaeffer, C.W., et al., 1995. KAl1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 268, 884 – 886. Emi, N., Kitaori, K., Seto, M., Ueda, R., Saito, H., Takahashi, T., 1993. Isolation of a novel cDNA clone showing marked similarity to ME491/CD63 superfamily. Immunogenetics 37, 193 –198. Frezal, J., Schinzel, A., 1991. Report of the committee on clinical disorders, chromosome aberrations and uniparental dismony. Cytogenet. Cell. Genet. 58, 986 – 1052. Hasegawa, H., Utsunomiya, Y., Kishimoto, K., Yanagisawa, K., Fujita, S., 1996. SFA-1, a novel cellular gene induced by human T-cell leukemia virus type 1, is a member of the transmembrane 4 superfamily. J. Virol. 70, 3258 – 3263. Hotta, H., Ross, A.H., Huebner, K., et al., 1988. Molecualr cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res. 48, 2955 – 2962. Imai, T., Fukudome, K., Takagi, S., et al., 1992. C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63. J. Immunol. 151, 6470 – 6481. Jankowski, S.A., Mitchell, D.S., Snith, S.H., Trent, J.M., Meltzer, P.S., 1994. SAS, a gene amplified in human sarcomas, encodes a new member of the transmembrane 4 superfamily of the proteins. Oncogene 9, 1205 – 1211. Li, S.-H., Mclnnis, M.G., Margolis, R.L., Antonarakis, S.E., Ross, C.A., 1993. Novel tripet repeat containing genes in human brain — cloning, expression, and length polymorphisms. Genomics 16, 572 – 579. Maeker, H.T., Todd, S.C., Levy, S., 1997. The tetraspanin superfamily: molecular facilitators. FASEB J. 11, 428 – 442. Oren, R., Takahashi, S., Doss, C., Levy, R., Levy, S., 1990. TAPA-1, the target of an antiproliferative antibody, defines a new family of transmembrane proteins. Mol. Cell. Biol. 10, 4007 – 4015. Seto, M., Yamamoto, K., Iida, S., et al., 1992. Gene rearrangement and overexpression of PRAD1 in lymphoid malignancy with t(11;14)(q13;q32) translocation. Oncogene 7, 1401 – 1406. Szala, S., Kasai, Y., Steplewski, Z., Rodeck, U., Koprowski, H., Williams, A.F., 1993. Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens. Proc. Natl. Acad. Sci. USA 87, 6833– 6837. Takagi, S., Fujikawa, K., Imai, T., et al., 1995. Identification of a highly specific surface marker of T-cell acute lymphoblastic leukemia and neuroblastoma as a new member of the transmembrane 4 superfamily. Int. J. Cancer 61, 706 – 715. Virtaneva, K.I., Emi, N., Marken, J., et al., 1994. Chromosomal localization of three human genes coding for A15, L6, and S5.7(TAPA1): all members of the transmembrane 4 superfamily of proteins. Immunogenetics 39, 329 – 334. Warren, S.T., 1996. The expanding world of trinucleotide repeats. Science 271, 1374 – 1375. Wright, M.D., Tomlinson, M.G., 1994. The ins and outs of the transmembrane 4 superfamily. Immunol. Today 15, 588–594.
Molecular cloning of a cDNA encoding mouse A15, a member of the transmembrane 4 superfamily, and its preferential expression in brain neurons Yoshitaka Hosokawa a,1, Eiko Ueyama1 b, Yoshihiro Morikawa b, Yumiko Maeda a, Masao Seto a,*, Emiko Senba b a
Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, 1 -1 Kanakoden, Chikusa-ku, Nagoya 464 -8681, Japan b Department of Anatomy and Neurobiology, Wakayama Medical College, 811 -1 Kimiidera, Wakayama 641 -0012, Japan Received 29 June 1999; accepted 14 September 1999
Abstract A15, a member of the transmembrane 4 superfamily (TM4SF), was isolated by differential screening of the cDNAs that are preferentially expressed on immature T cells. As a first step in the study of the biological function of the A15 molecule, we isolated cDNAs encoding the entire coding region of mouse A15. Nucleotide sequence analysis of the cDNAs revealed that mouse A15 shares 97% amino acid sequence identity with its human counterpart. The mouse A15 protein product has not yet been characterized, but is predicted to be 244 amino acids with four hydrophobic domains. Northern blot analysis of the RNA samples from various mouse tissues disclosed that the A15 transcripts are expressed most strongly in the brains, and are detectable in the colon, muscle, heart, kidney, and spleen. In situ hybridization of the mouse brain with ribo-probe established that the A15 transcripts are expressed primarily in neurons of the frontal cortex, olfactory bulb, dentate gyrus, caudoputamen, and CA3 region of the hypothalamus as well as in Purkinje cells in the cerebellar cortex, which strongly suggests that A15 may have a special function in the fundamental neuronal functioning of the higher nervous system. © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: TM4SF; Tetraspanin; CCG-B7; Ion channel; In situ hybridization; A15
1. Introduction The transmembrane 4 superfamily (TM4SF), also known as the tetraspanin, consists of a group of structurally related proteins that are characterized by four transmembrane domains (Wright and Tomlinson, 1994; Maeker et al., 1997). Membership of this family has now grown to more than 20 known genes (Hotta et al., 1988; Classon et al., 1989; Amiot, 1990; Angelisova et al., 1990; Oren et al., 1990; Azorsa et al., 1991; Boucheix et al., 1991; Imai et al., 1992; Emi et * Corresponding author. Tel.: + 81-52-7626111 (ext. 8840); fax: +81-52-7635233. E-mail address: [email protected] (M. Seto) 1 Yoshitaka Hosokawa and Eiko Ueyama contributed equally to this work.
al., 1993; Szala et al., 1993; Jankowski et al. 1994; Dong et al., 1995; Takagi et al., 1995; Hasegawa et al., 1996), some of which are also found in organisms as primitive as schsitosomes and nematodes. The existence of so many different TM4SF members in organisms as evolutionarily diverse as humans and schistosomes strongly implies essential functions for these genes in cellular functioning. Many of them are expressed on leukocytes and it is likely that they mediate signal transduction events that play a critical role in the regulation of cell proliferation, motility, and differentiation. Furthermore, two of the members, CD63/ME491 and KAl-1, have been shown to be involved, respectively, in tumor progression and metastasis (Hotta et al., 1988; Dong et al., 1995). A15, a member of TM4SF, was isolated by differential screening of the cDNAs that are preferentially
0168-0102/99/$ - see front matter © 1999 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 0 1 0 2 ( 9 9 ) 0 0 0 9 3 - 0
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expressed on immature T cells as compared with the peripheral blood leukocytes (PBLs) (Emi et al., 1993). Thus, it is conceivable that the A15 protein product may have a special part to play in T cell development. Subsequently, the CCG-B7 cDNA clone was isolated from a human frontal cortex cDNA library (Li et al., 1993). The library was screened with triplet repeat probes that contained ten repeats of either CCG or CTG. The CCG-B7 clone has seven CCG repeats in its 5% upstream end of the initiation codon. A homology search disclosed that the predicted CCG-B7 coding region is identical to that of A15 protein. Thus, the A15/CCG-B7 gene appears to contain triplet nucleotide repeats that are often associated with several neuropsychiatric diseases (Warren, 1996), such as Huntington’s chorea, myotonic dystrophy, and fragile X syndrome, where patients’ genes have been shown to contain amplified triplet nucleotide sequences. It is unclear whether the A15 gene is a candidate gene responsible for such neuropsychiatric diseases. A15/CCG-B7 cDNA was also isolated by means of a different approach using a monoclonal antibody and was named TALLA-1 (Takagi et al., 1995). As a first step toward elucidating the biological function of the A15 protein, we isolated cDNAs for mouse A15 and demonstrated that mouse A15 transcripts are expressed preferentially in brain neurons. The distribution of the transcripts in the brain was determined by using in situ hybridization (ISH).
2. Materials and methods
2.1. PCR cloning of mouse A15 cDNA Male mice (C57BL/B6) were obtained from SLC Japan (Shizuoka, Japan) and kept in the laboratory animal center. Total RNA was isolated from mouse spleen via homogenization in guanidium thiocyanate and centrifugation through cesium chloride. Firststrand cDNA was generated by using 5 mg of the total
RNA samples from mouse spleen, 200 units of superscript II reverse transcriptase (Stratagene, La Jolla, CA) and 200 ng of random primer in a total volume of 40 ml. Following first strand cDNA synthesis, 35 cycles of PCR were performed in a total volume of 50 ml containing 2 ml of first-strand cDNA, 1 mM of each primer, 100 mM dNTP, 1.5 mM MgCl2, 10 mM Tris–Cl (pH 8.3), 50 mM KCl, and 1 unit Taq DNA polymerase (Takara, Kyoto, Japan). The PCR parameters were: 94°C, 45 s; 55°C, 90 s; and 72°C, 90 s for 35 cycles. The primer sequences corresponded to the human A15 sequences and were as follows: Primer 1: 5%-ATGGAGACCAAACCTGTGAT-3% Primer 2: 5%-TTACACCATCTCATACTGAT-3% The locations of primers 1 and 2 are shown in Fig. 1. The RT-PCR products (clone mA15-1) were purified from a low-melting-point agarose gel and subcloned into the plasmid vector. To obtain cDNAs that correspond to the 5% and 3% ends of mouse A15 transcript, PCR was performed to screen the mouse spleen cDNA library. The mouse spleen random-primed cDNA library was constructed in the phage vector lgt 10 as described (Seto et al., 1992). Oligonucleotide primers were then designed to correspond to the sequences near the 5% and 3% end of clone mA15-1 (primers 3 and 4 in Fig. 1) and the sequences flanking the cloning site in lgt 10 (primers R and L in Fig. 1). Phage lysates (5 ml) of the cDNA library were directly subjected to the PCR reaction. The PCR parameters were: 94°C, 45 s; 55°C, 90 s; and 72°C, 90 s for 35 cycles. The primer sequences used were as follows. Primer 3: 5%-CCAGAAGACGAAGGAGTAGATGATGAGGAG-3% Primer 4: 5%-TTCATGGAGACTAACATGGG-3% Primer F: 5%-AGCAAGTTCAGCCTGGTTAA-3% Primer R: 5%-CTTATGAGTATTTCTTCCAGGGTA-3% The locations of the primers are also shown in Fig. 1. The PCR products (clones mA15-2 and mA15-3) were purified from a low-melting-point agarose gel and subcloned into the plasmid vector.
Fig. 1. Mouse A15 cDNA. Three clones for mouse A15 cDNA are indicaded by thick lines. The open box shows the open reading frame. The PCR primers are represented by arrows.
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Fig. 2. Nucleotide sequence of mouse A15 cDNA and its predicted amino acid sequence. The nucleotides are numbered in the 5% to 3% direction, and the amino acid sequence is numbered sequentially from the first methionine. The amino acid sequence of human A15 is shown beneath the mouse sequence for comparison. Four putative transmembrane domains are underlined. The dashes represent identical amino acids and the terminal codon is indicated by an asterisk. GenBank accession number; AF052492.
2.2. Northern blot analysis Total RNAs (5 mg per lane) from various tissues of C57BL/B6 mice were separated by electrophoresis on a 1% agarose gel containing 2.2 M formaldehyde and then transferred to a Hybond N nylon membrane. The hybridization probe was a mouse A15 coding region probe (nucleotides 6 – 737 in Fig. 2). The probe was labeled with [a32P]dCTP by using a randomprimed labeling kit (Nippon Gene, Tokyo, Japan). The hybridization was carried out at 42°C overnight
in a solution containing 45% formamide, 5×SSPE, 1 × Denhardt’s solution, 0.5% SDS, and 100 mg/ml salmon sperm DNA. The membrane was finally washed for 30 min at 65°C with 0.5× SSC (1×SSC; 0.15 M sodium chloride and 0.015 M sodium citrate) and 0.1% SDS.
2.3. DNA sequencing DNA sequencing was performed with the aid of an ABI PRISM dye terminator cycle sequencing kit
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(Perkin Elmer, Foster, CA) on a 372 automated DNA sequencer (Applied Biosystems, Foster City, CA).
2.4. Animals and tissue preparation for ISH Male mice (C57BL/B6) were housed in plastic cages with soft sawdust bedding under a 12 h light/dark cycle (lights on at 08:00 h) and free access to food and water. Adult animals weighing about 20 g (7 – 8 weeks old, n =3) were used. They were decapitated under deep anesthesia with diethylether, and the brains were excised and immediately frozen with powdered dry ice. The other group of animals (n = 4) were perfused under deep sodium pentobarbital anesthesia (75 mg/kg i.p.) with 0.85% saline (100 ml) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4, 400 ml), after which the brains, trigeminal ganglia and several peripheral tissues and organs were excised. These samples were post-fixed in the same fixative overnight, stored in 30% (w/v) sucrose in 0.1 M phosphate buffer for 2 days at 4°C, and then frozen with powdered dry ice. Serial 6 mm sections were prepared in a cryostat at − 20°C and mounted onto glass slides coated with 3-amino propyltriethoxysilane. These sections were stored at −80°C until they were processed.
2.5. ISH A fragment (732 bp) of mouse A15 cDNA was amplified by PCR and subcloned into the EcoRV site of Bluescript II SK(+ ) plasmid (Stratagene) and digested with Sal I (antisense probe) or Not I (sense probe). This was followed by in vitro transcription using T3 RNA polymerase (antisense probe) or T7 RNA polymerase (sense probe). To inactivate RNase and to promote the permeability of the tissues, the sections were incubated with 1 mg/ml of proteinase K in 10 mM Tris–HCl (pH 8.0) and 1 mM EDTA for 10 min at 37°C. After rinsing with 0.1 M phosphate buffer, the samples were incubated with 0.2 N HCl for 10 min and then with 0.25% acetic anhydrate in 0.1 M triethanolamine for 10 min to prevent nonspecific binding of the probes. The sections were rinsed again with 0.1 M phosphate buffer and dehydrated through a graded ethanol series (70–100%). Hybridization using the riboprobe for A15 mRNA was performed overnight at 55°C in a buffer (1×TE; 50% formamide; 0.6 M NaCl; 1× Denhardt’s solution; 0.25% SDS; 500 mg/ml yeast tRNA; 10% dextran sulfate; 0.1 M dithiothreitol) containing 0.5–1.0× 106 cpm of labeled probes per slide. After hybridization, the slides were immersed in 5× SSC (pH 7.2) containing 10 mM DTT for 10 min at 50°C to remove glass covers and the sections were rinsed in 2 × SSC containing 50% formamide and 10 mM DTT for 30 min at 65°C. The sections were then incubated with RNase A (Sigma Chemicals, St Louis,
MO) in Tris–EDTA buffer containing 0.5M NaCl for 30 min at 37°C. After rinsing with a graded SSC series (2×SSC–0.1× SSC), the samples were dehydrated with 70% ethanol and air-dried. For autoradiography, the mounted sections were coated with Kodak NTB3 emulsion (diluted 1:2 with distilled water at 40°C) and exposed for 1–3 weeks in a tightly sealed dark box at 4°C. After development in a Kodak D19 developer and fixation in 24% sodium thiosulfate, the sections were counterstained with 1% Neutral Red solution for morphological identification. Sections were observed and photographed under dark-field lateral illumination (XF-WFL, Nikon, Tokyo, Japan).
3. Results
3.1. cDNA cloning of mouse A15 In an effort to isolate mouse A15 cDNA, we first performed reverse transcription-polymerase chain reaction (RT-PCR) of the total RNA samples from the mouse spleen. The oligonucleotide primers used corresponded to the human A15 sequence primers 1 and 2 as shown in Fig. 1. Mouse A15 cDNA fragments (732 bp) were amplified and subcloned into the plasmid vector. Sequence analysis of the clone (mA15-1 in Fig. 1) revealed that they show 97% amino acid sequence identity with the corresponding region of the human A15 cDNA. Thus, the cDNA fragment was found to encode a middle portion of mouse A15 cDNA. To isolate cDNAs that correspond to the 5% and 3% further ends of mouse A15 transcript, PCR was again performed to screen the mouse spleen cDNA library constructed in lgt 10 phage vector. Oligonucleotide primers were designed to correspond to the sequences near the 5% and 3% end of clone mA15-1 (primers 3 and 4 in Fig. 1) and to the sequences flanking the cloning site in lgt 10 (primers R and F in Fig. 1). To identify the appropriate PCR products corresponding to the 5% and 3% end of A15 cDNA, the end-labeled oligonucleotides (primers 1 and 2 in Fig. 1) were used as internal probes for Southern blot analysis (data not shown). The hybridized PCR products were then subcloned into the plasmid vector. Sequencing analysis of these clones (mA15-2 and mA15-3 in Fig. 1) revealed that they indeed encode the 5% and 3% end of the mouse A15 coding region, demonstrating that we were able to isolate cDNAs covering the entire coding region of mouse A15. Nucleotide sequence analysis of the cDNAs revealed that overall, mouse A15 shares 97% amino acid sequence identity with its human counterpart. Nucleotide sequence of the mouse A15 cDNA and its predicted amino acid sequence are shown in Fig. 2. The mouse A15 protein product has not yet been characterized, but
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is predicted to be 244 amino acids with four putative transmembrane domains. However, the PCR cloning did not allow us to isolate the 5%-untranslated end of the full-length mouse A15 cDNA.
3.2. Northern blot analysis To clarify the biological role of A15, it was of interest to examine whether A15 transcripts are expressed in tissues other than immature T cells. Northern blot analysis of the RNA samples from various mouse tissues revealed that A15 transcripts are expressed most strongly in the brain, and are detectable in the colon, muscle, heart, kidney and spleen (Fig. 3). This finding is intriguing in that the human CCG-B7/ A15 cDNA was found to contain, in its 5%-untranslated region, triplet nucleotide repeats that are often associated with several neuropsychiatric diseases.
3.3. ISH To examine the detailed localization of expression of the A15 transcript in mouse brain, we performed ISH with mouse A15 ribo-probe. The A15 signal intensity in each brain ares is summarized in Table 1. Signals for A15 mRNA were found to be concentrated in neurons and its distribution was rather ubiquitous. However, a
Fig. 3. Northern blot analysis of the various mouse tissues with A15 cDNA. The total RNAs (5 mg) were electrophoresed on a 1% agarose/formaldehyde gel and blotted on a nylon membrane. The membrane was hybridized with 32P-labeled mouse A15 cDNA as a probe. The ethidium bromide-stained gel used in the northern blot analysis is shown at the bottom, and tissue sources are also indicated in the figure.
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wide variety of signal densities were also observed, i.e. neurons in discrete brain regions expressed higher levels of A15 mRNA signals as described below (Section 3.3.1–Section 3.3.7). It is possible that these variations may partly reflect the difference of cell densities. Almost no signals were identified in fiber tracts. Sections hybridized with sense probes showed no accumulation of signals (Fig. 4D, F), indicating the specificity of the probe and the validity of hybridization method.
3.3.1. Cortex and olfactory areas A high signal density for A15 mRNA was diffusely distributed in layers II–VI of the cerebral cortex throughout the forebrain (Fig. 4). The highest signals were observed in layer II. In cortical areas, densities were especially high in the cingulate cortex (Cg), piriform cortex (Pir), dorsal endopiriform nucleus (DEn) and tenia tecta (TT) (Fig. 4). In the olfactory bulb, a high signal density was observed in the mitral cell layer (not shown). Moderate signals were distributed in the granule cells in the internal granular layer and in the perigromerular cells in the glomerular layer. 3.3.2. Basal ganglia and limbic system Moderate signals were spread evenly throughout the caudate-putamen (CPu) and accumbens nucleus (Acb) (Fig. 4A, B). Signals in the globus pallidus were very low in comparison to those of the CPu (Fig. 5A). Moderate signals could also be seen in the bed nucleus of the stria terminalis (BST) and in the lateral septal nucleus (LS) (Fig. 4B, Fig. 5A). Labeled neurons were scattered in the entopeducular nucleus (EP) (Fig. 5C). The hippocampus showed the highest signal density in the pyramidal cell layer of the CA3 region and in the granule cells of the dentate gyrus (DG) (Fig. 4E, Fig. 5C, Fig. 6A). However, the signals in the CA1–CA2 regions of the hippocampal formation were relatively low compared to that of CA3 region and dentate gyrus. The subiculum (S) showed low signal densities (Fig. 6A) and the amygdala high signal densitites (Fig. 4C,Fig. 4E, Fig. 5C). The strongest signals were observed in the central (ACe) and cortical (ACo) amygdaloid nuclei. 3.3.3. Thalamus ISH signals in the thalamus were relatively weak (Fig. 4C, Fig. 4E, Fig. 5B, Fig. 5C). Low levels were found in the midline thalamic nuclei, such as paraventricular nucleus (PV), central medial nucleus (CM), reuniens nucleus (Re), rhomboideus nucleus (Rh) of thalamus (Fig. 5B, C). Labeled cells were serially distributed from the zona incerta (ZI) to reticular thalamic nucleus (Rt). A low signal level was observed in the ventral lateral geniculate nucleus (VLG), while the level in the dorsal lateral geniculate nucleus was lower (Fig. 4E). Low signal levels could also be seen in the rest of the thalamic nulcei.
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Table 1 Distribution of A15 mRNAs in the mouse brain Brain area Cerebral cortex Neocortex (Cx) Tenia tecta (TT) Cingulate cortex (Cg) Olfactory system Olfactory bulb Glomerular layer Mitral cell layer Internal granular layer Olfactory tubercle (Tu) Dorsal endopiriform nucleus (DEn) Piriform cortex (Pir) Basal ganglia and septum Caudoputamen (Cpu) Globus pallidus (GP) Accumbens nucleus (Acb) Bed nucleus of stria terminalis (BST) Entopeduncular nucleus (EP) Lateral septal nucleus (LS) Hippocampal formation CA1 CA2 CA3 Dentate gyrus (DG) Subiculum (S) Amygdaloid complex Central amygdaloid nucleus (ACe) Cortical amygdaloid nucleus (ACo) Basolateral amygdaloid nucleus Medial amygdaloid nucleus Thalamus Paraventricular nucleus (PV) Central medial nucleus (CM) Rhomboid nucleus (Rh) Reunience nucleus (Re) Medial and lateral habenula (MHb, LHb) Reticular nucleus (Rt) Ventrolateral nucleus (VL) Parafacicular nucleus Ventral Lateral geniculate nucleus (VLG) Medial geniculate nucleus (MG) Zona incerta (ZI) Hypothalamus Anterior hypothalamic nucleus Paraventricular hypothalamic nucleus (PVH) Ventromedial hypothalamic nucleus (VMH) Lateral hypothalamic nucleus (LH) Arcuate nucleus (Arc) Premammillary nucleus (PM) Medial and lateral mammillary nuclei (MM, LM) Brainstem Central grey (CG) Superior colliculus (SC) Inferior colliculus (IC) Interpeduncular nucleus (IPN) Substantia nigra (SN) Red nucleus (RN)
Signal intensitya
+++ +++ +++
++ +++ ++ ++ +++ ++++ ++ + ++ ++ + ++ +++ +++ ++++ ++++ + +++ +++ +++ +++ + + + + + + +/− + + + ++ ++ + ++ + + ++ +
+ + ++ + +/− +/−
Pontine nucleus (Pn) Parabrachial nucleus (PB) Dorsal tegmental nucleus (DT) Principal sensory trigeminal nucleus (Pr5) Ventral cochlear nucleus (VCN) Medial vestibular nucleus Spinal trigeminal nucleus Nucleus of the solitary tract Lateral reticular nucleus Motor cranial nerve nuclei
++ ++ ++ + + + + + + +
Cerebellar cortex Purkinje cell layer (PCL) Granular layer (GL) Molecular layer
+++ ++ −
Trigeminal ganglion
+++
a Grading of signal intensity; ++++: very high, +++: high, ++: moderate, +: low, +/−: very low.
3.3.4. Hypothalamus Overall, the signal density in the hypothalamus was moderate (Fig. 4C, Fig. 4E, Fig. 5C, Fig. 5D). Labeled neurons were concentrated in the medially located nuclei, such as the anterior hypothalamic nucleus, paraventricular nucleus (PVH) (Fig. 5B), ventromedial nucleus (VMH) (Fig. 5C), arcuate nucleus (Arc), premammillary nucleus (PM) and medial mammillary nucleus (MM) (Fig. 5D). Moderately labeled neurons were scattered in the lateral part of the hypothalamus. 3.3.5. Brainstem Midbrain — structures in the midbrain expressed a relatively low signal density (Fig. 6A, B). Few signals could be identified in the central grey (CG), dorsal raphe nucleus (DR), interpeduncular nucleus (IP) and medial geniculate nucleus (MG). The superior colliculus (SC) displayed low level signals, but the inferior colliculus (IC) showed more (Fig. 6C). Other areas of the midbrain, such as the substantia nigra (SN) and red nucleus (RN) were very low in signal levels. Pons — areas displaying moderate signal concentrations included the pontine nuclei (Pn) (Fig. 6A, B) and nuclei on the floor of the IVth ventricle including dorsal tegmental nuclei (DT) and locus coeruleus (LC). The pontine nuclei showed one of the highest signal densities in the brainstem. Other areas with moderate to low concentrations of signal density included the parabrachial nucleus (PB), principal sensory trigeminal nucleus (Pr5) and ventral cochleal nucleus (VCN) (Fig. 6C). Moderately labeled neurons were scattered in the raphe magnus and pontine reticular nuclei. Motoneurons in the trigeminal motor nucleus and facial nucleus also exhibited moderate signal density. Medulla — Low signal concentrations were identified throughout the nucleus tractus solitarii, dorsal motor nucleus of the vagus nerve, hypoglossal nucleus, nucleus ambiguus and lateral reticular nucleus (not shown).
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3.3.6. Cerebellum High signal concentrations were identified in the Purkinje cell layer (PCL) and moderate signal levels in
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the granular layer (GL) (Fig. 6D), while only signals of control level could be identified in the molecular layer and white matter of the cerebellum.
Fig. 4. Dark-field photomicrographs showing signals for A15 mRNA in forebrain areas. Coronal sections are arranged rostrocaudally. C and D and E and F are serial sections hybridized with antisense (C, E) and sense (D, F) probes for A15 mRNA. Scale bar; 1 mm. Abbreviations in Figures: ac, anterior comissure; Acb, accumbens nucleus (n.); ACe, central amygdaloid n.; ACo, cortical amygdaloid n.; Arc, arcuate n.; BST, bed nucleus of stria terminalis; Cer, cerebellum; CG, central grey; Cg, cingulate cortex; CM, central medial thalamic n.; CPu, caudoputamen; Cx, neocortex; DEn, dorsal endopiriform n.; DR, dorsal raphe n.; DT, dorsal tegmental n.; EP, entopeduncular n.; GL, granular layer; GP, globus pallidus; ic, internal capsule; IC, inferior colliculus; IP, interpeduncular n.; LC, locus coeruleus; LG, lateral geniculate n.; LH, lateral hypothalamic n.; LM, lateral mammillary n.; LS, lateral septal n.; LSD, dorsal lateral septal n.; MG, medial geniculate n.; MHb, medial habenular n.; MM, medial mammillary n.; MMn, median medial mammillary n.; mt, mammillothalamic tract; ot, optic tract; PB, parabrachial n.; Pir, piriform cortex; PCL, Purkinje cell layer; PM, premammillary n.; Pn, pontine n.; Pr5, principal sensory trigeminal n.; PV; paraventricular nucleus; PVH, paraventricular hypothalamic n.; Re, reuniens thalamic n.; Rh, rhomboid thalamic n.; RN, red n.; Rt, reticular thalamic n.; S, subiculum; SC, superior colliculus; sm, stria medullaris thalami; SN, substantia nigra; Tu, olfactory tubercle; TT, tenia tecta; VCN, ventral cochlear n.; VL, ventrolateral thalamic n.; VMH, ventral medial hypothalamic n.; ZI, zona incerta.
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Fig. 5. Dark-field photomicrographs showing precise localization of signals for A15 mRNA in the basal ganglia (A), thalamus (B, C) and hypothalamus (C, D). Scale bars; 500 mm (A–C), 250 mm (D).
3.3.7. Trigeminal ganglion All of the neurons in the trigeminal ganglion expressed high levels of A15 mRNA signals (Fig. 6E).
4. Discussion TM4SF consists of a group of structurally related membrane proteins that have in common four conserved membrane spanning regions, certain cystein residues, and short sequence motifs (Wright and Tomlinson, 1994; Maeker et al., 1997). Many of them have a flair for promiscuous associations with other molecules, including integrins and other TM4SFs. The biological function of the members of this family are largely unknown, but it is likely that they are involved in diverse processes such as cell activation and proliferation, adhesion and motility, differentiation, and cancer. We previously isolated a cDNA for human A15, a member of the TM4SF by using differential screening of the cDNAs that are preferentially expressed in immature T cells as compared with the PBLs (Emi et al.,
1993). In the present study, we isolated cDNAs encoding mouse A15 and demonstrated that the transcript is expressed preferentially in the brain neurons. The mouse A15 predicted from the cDNA sequence consists of 244 amino acid residues and it was found to share 97% amino acid sequence identity with its human counterpart. This extremely high conservation between mouse and human A15 supports the possibility that A15 may play a role in fundamental biological processes such as cell proliferation, motility, and differentiation. Although TM4SF shows no significant homology to any known proteins, there are certain features of the family that are characteristic of ligand-gated ion channels, including the acetylcholine receptor family. The transmembrane domains of the TM4SF are relatively rich in polar residues and have highly conserved Glu or Gln residues buried deep in the third and fourth transmembrane domains. Thus, it may be possible that A15, a member of TM4SF, functions as an ion channel in brain neurons. Our earlier northern blot analysis showed that the human A15 transcripts are expressed in HPB-ALL, Jurkat, and MOLT3 cell lines, but not in HUT102 or
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PBL cells, which are thought to be mature T cells. This pattern suggests that A15 transcripts are expressed in the immature stage of T cell development (Emi et al.,
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1993). We also examined whether A15 transcripts are expressed in tissues other than immature T cells. Northern blot analysis of the RNA samples from various
Fig. 6. Dark-field photomicrographs showing signals for A15 mRNA in the brainstem (A, B, C), cerebellum (D) and trigeminal ganglion (E). D and F are serial sections hybridized with antisense (D) and sense (F) probes for A15 mRNA. Scale bars; 1 mm (A – C, E), 500 mm (D, F).
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mouse tissues revealed that mouse A15 transcripts are expressed most strongly in the brain. This finding led us to examine in detail the distribution of A15 expression in the brain by means of ISH. Our ISH analysis indicated that A15 mRNA appears to be expressed in all the neurons in the brain, and that uneven and characteristic distribution pattern was also observed among these neurons. Especially noteworthy was the extremely high level of expression in the cortex and hippocampus. Neurons in the caudoputamen, nucleus accumbens, bed nucleus of stria terminalis, amygdala and hypothalamus were also found to express moderate to high levels of ISH signals. Such distribution patterns strongly suggest that A15 may have a fundamental neuronal function especially of higher nervous system. Chromosomal location of the human A15 gene was previously assigned to Xq11 (Virtaneva et al., 1994), and it has been noted that this region harbors genes associated with X-linked mental retardations (Frezal and Schinzel, 1991). Given the fact that the A15 transcripts are expressed primarily in the brain neurons, it will be of particular interest to examine whether the A15 gene is responsible for such neuropsychiatric diseases. In summary, the present study describes the cDNA cloning of mouse A15, a member of the TM4SF and demonstrates that the transcripts are expressed preferentially in the brain neurons as well as in immature T cells. Much more needs to be learned about the biological functions of A15, however. In order to further clarify the function of A15, it is essential to identify molecules which associate with A15 on the cell membrane. Gene targeting with mouse ES cells should prove to be another important means to identify and characterize the biological function of A15 in the nervous system.
Acknowledgements This work was supported in part by a Grant-in-aid for the Second Term Comprehensive Ten Year Strategy for Cancer Control from the Ministry of Health and Welfare, a Grant-in-aid for Science on Primary Areas (Cancer Research), from the Ministry of Education, Science and Culture, Japan, a Grant-in-aid from the Japanese Foundation for Multidisciplinary Treatment of Cancer, and the Bristol – Myers Squibb Unrestricted Biomedical Research Grants.
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