Expression of TMEFF1 MRNA in the Mouse Central Nervous System

Molecular Brain Research 86 (2001) 48–55www.elsevier.com/ locate /bres

Research report

Expression of TMEFF1 mRNA in the mouse central nervous system:precise examination and comparative studies of TMEFF1 and

TMEFF2a a a a aNaohide Kanemoto , Masato Horie , Kuniko Omori , Naoki Nishino , Mari Kondo ,

b aKoichi Noguchi , Akira TanigamiaOtsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10, Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan

bDepartment of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1, Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan

Accepted 10 October 2000

Abstract

TMEFF1 and TMEFF2 are putative transmembrane proteins comprised of one epidermal growth factor (EGF)-like domain and twofollistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated thatrecombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA inthe mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression ofTMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, theyexhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fifth layer of the cerebralneocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 ishighly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex andcranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulateddifferently and that they play region-specific roles in the central nervous system. 2001 Elsevier Science B.V. All rights reserved.

Theme: Development and regeneration

Topic: Neurotrophic factors: expression and regulation

Keywords: TMEFF1; TMEFF2; Survival factor; Mouse; In situ hybridization

1. Introduction Previously, we cloned a paralogous gene, TMEFF2 (alsocalled tomoregulin [15]), and investigated precise expres-

TMEFF1 (‘Transmembrane protein with EGF-like and sion sites and function in mammalian organisms. Northern-two follistatin-like domains 1’) was first identified in blot analysis showed that TMEFF2 is predominantlyXenopus laevis as X7365, a novel transmembrane protein expressed in the both human and mouse brain. In situcontaining a signal peptide, two follistatin modules, an hybridization analysis of mouse tissue also revealed ex-EGF-like domain and a short cytoplasmic region [5]. The pression throughout the brain, specifically in various typesfunction of TMEFF1 is largely unknown, although its of neurons and glial cells. Furthermore, we demonstratedexpression patterns have been characterized in Xenopus that a recombinant protein containing the putative extracel-laevis, and a human ortholog has been cloned and mapped lular domain of TMEFF2 promoted survival of neurons into human chromosome 9q31 [6]. primary culture [10]. The similarity between TMEFF1 and

TMEFF2 suggests that the two proteins share similarfunctions. To better understand TMEFF functions, we have*Corresponding author. Tel.: 181-88-665-2888; fax: 181-88-637-investigated the precise localization of TMEFF1 mRNA in1035.

E-mail address: [email protected] (A. Tanigami). the mouse central nervous system.

0169-328X/01/$ – see front matter 2001 Elsevier Science B.V. All rights reserved.PI I : S0169-328X( 00 )00257-6

N. Kanemoto et al. / Molecular Brain Research 86 (2001) 48 –55 49

2. Materials and methods 2.4. In situ hybridization

2.1. Probe preparation Slide-mounted sections were fixed in 4% paraformal-dehyde in phosphate-buffered saline (PBS) for 20 min (for

Mouse TMEFF1 cDNA sequences are registered as paraffin sections, this step was omitted, and the paraffinoverlapped ESTs in GenBank (accession numbers was removed instead), followed by two washes in PBS.AA023493 and AA020026). Mouse TMEFF1 cDNA was Slides were then immersed in 0.2 N HCl solution for 2 minamplified from mouse brain Marathon-ReadyE cDNA and washed with PBS. Sections were subjected to diges-(Clontech) by PCR using the primer sequences 59-AGAG- tion in proteinase K (10 mg/ml for frozen sections, or 150GCAAGAGCAACTGCTC-39 (P1) and 59-TAGGAAC- mg/ml for paraffin sections) in 0.1 M Tris–HCl pH 7.5, 5TCCCGTCGGAAGC-39 (P2). These P1 and P2 primers mM EDTA for 5 min in case of the lung, for 15 min (brainwere designed based on the AA023493 and AA020026 and testis) or 30 min (heart) at room temperature, followedsequences, respectively. The expected product size is 458 by two PBS washes. Slides were postfixed for 20 min inbp, corresponding to a segment (nucleotides 94 to 551) of the fixative solution described above, then washed witha recently registered partial cDNA sequence (GenBank PBS. Next, slides were acetylated with 0.25% aceticaccession number AJ400622). For Northern-blot analysis, anhydrate in 0.1 M triethanolamine for 10 min, washed

32the amplified cDNA was labeled with a-[ P]dCTP by with PBS and dehydrated in serial ascended ethanol (70,random-primed labeling. For in situ hybridization, the 80, 95 and 100%). Sections were then defatted with

amplified cDNA was subcloned into the pGEM -T vector chloroform for 10 min, immersed in 100% ethanol, and(Promega). Nucleotide sequences of the subcloned cDNA subjected to hybridization. For hybridization, DIG-labeledwere determined using an ABI377 autosequencer (Perkin probes were diluted to 1 mg/ml with hybridization bufferElmer). This plasmid was linearized with NotI (for sense (0.3 M NaCl, 20 mM Tris–HCl pH 8.0, 50% formamide,probe) or NcoI (for antisense probe). Digoxigenin (DIG)- 13Denhart’s solution, 0.2% N-lauroylsarcosine, 0.5 mg/labeled sense and antisense probes were generated with T7 ml yeast tRNA and 0.2 mg/ml salmon testes DNA) andand SP6 polymerases, respectively, using the DIG RNA pre-heated at 808C for 5 min; the hybridization mixtureLabeling Kit (Roche Molecular Biochemicals). was applied to each slide and incubated overnight at 558C.

After hybridization, slides were washed with 43SSC at608C, followed by washing in high-stringency wash solu-2.2. Northern-blot analysistion (50% formamide, 23SSC) at 608C. Next, slides wererinsed with RNase buffer (0.5 M NaCl, 10 mM Tris–HClGene expression was evaluated using a mouse multiplepH 8.0 and 0.5 M EDTA) three times for 10 min each attissue northern (MTN) blot (Clontech). After prehybridiza-

32 378C, incubated with 2 mg/ml RNase in RNase buffer fortion, the membrane was hybridized with a-[ P] dCTP-30 min at 378C, then rinsed twice in RNase buffer for 10labeled mouse TMEFF1 and b-actin cDNAs according tomin. Sections were washed again in high-stringency washthe manufacturer’s procedure. Washed membranes werebuffer at 608C for 30 min, and color detection of theautoradiographed for 18 h at 2808C.hybridized probe was performed using the DIG NucleicAcid Detection Kit (Roche Molecular Biochemicals) ac-

2.3. Tissue preparationcording to the manufacturer’s instructions. Anatomicallocalizations were verified using the atlas of Franklin and

2.3.1. Brains Paxinos [7].Two-month-old (129/SvEv3C57BL/6j)F male mice1

were anesthetized with pentobarbital, and fresh brains wereremoved quickly and immediately frozen on powdered dryice. Tissue sections (14–20 mm thick) were cut using a 3. Resultscryostat onto glass slides coated with 3-aminopropyltri-ethoxysilane. 3.1. Northern-blot analysis

2.3.2. Testes, lungs, and hearts Northern-blot analysis of mouse tissues showed that,Testes, lungs, and hearts were removed from mice by like TMEFF2, TMEFF1 mRNA (2.9 kb, major band; 2.2

perfusion with 4% paraformaldehyde. Fixed testes tissue kb, minor band) is expressed predominantly in the brainwas embedded in paraffin, and lung and heart tissues were [10]. High levels of TMEFF1 (2.9 kb, major band; 1.7 kb,

frozen on dry ice and embedded in Tissue-Tek O.C.T. minor band) expression were also detected in the testis,Compound (Sakura Finetechnical). Paraffin sections and and moderate expression was observed in the lung andfrozen sections were cut to 5 mm- and 10 mm-thickness, heart (Fig. 1). However, the predominant expression in therespectively, and mounted onto glass slides coated with brain implicates involvement of TMEFF1 in the central3-aminopropyltriethoxysilane. nervous system.

50 N. Kanemoto et al. / Molecular Brain Research 86 (2001) 48 –55

3.2. In situ hybridization

3.2.1. Gross examination of TMEFF1 expression sitesDistribution of TMEFF1 mRNA was examined grossly

in sagittal brain sections. DIG-labeled antisense probe gaveprominent signals in the forebrain olfactory region, cere-bral cortex and hippocampus (Fig. 2A), while a senseprobe gave no signal (Fig. 2B).

3.2.2. Precise examination of TMEFF1 expression sitesPrecise expression sites of TMEFF1 were investigated

using serial coronal brain sections and intermittently cutsagittal brain sections; sites where expression was detectedare summarized in Table 1. In the olfactory bulb, strongsignals were observed in cells of the mitral cell layer andthe external plexiform layer (Fig. 3A). As the positive cellsin the external plexiform layer are large in size and locatedin the outer part of the layer, they are thought to beperiglomerular cells. The anterior olfactory nucleus, teniatecta, pirform cortex (Fig. 3B), basolateral nucleus andbasomedial nucleus of the amygdaloid nuclear complex(Fig. 3C), and entorhinal cortex (Fig. 3D) also expressTMEFF1. Expression of TMEFF1 was detected through-out the cerebral neocortex; in particular, the pyramidalcells of fifth layer of the cingulate cortex, motor cortex(Fig. 3E), somatosensory cortex and posterior parietalassociation area gave significant signals. In the hippocam-

Fig. 1. Expression of the TMEFF1 gene as determined by Northern blot pus, expression of TMEFF1 appeared stronger in the CA332analysis. The mouse MTN blot (Clontech) was hybridized with P- and CA1 pyramidal layer and subiculum than in the CA2

labeled TMEFF1 and b-actin probes. pyramidal layer and dentate gyrus (Fig. 3F). Moderate

Fig. 2. Localization of TMEFF1 mRNA in the mouse brain and demonstration of probe specificity. Sagittal brain sections from 2-month-old(129/SvEv3C57BL/6j)F mice were probed with a DIG-labeled mouse TMEFF1 antisense riboprobe (A) or sense riboprobe (B). Hi, hippocampus; Cx,1

cerebral cortex; Ob, olfactory bulb. Bar51 mm.


Table 1aLocalization of TMEFF1 and TMEFF2 mRNA in the mouse central nervous system

b bLocus TMEFF1 Remarkable portions TMEFF2 Remarkable portions

Olfactory bulb 111 Mi, Peri 111 Mi, GrOAnterior olfactory nu. 11 11

Olfactory tubercle 1 1

Tenia tecta 111 111

Medial septam nu. 1 11

Piriform cortex 111 111

Amygdaloid nu. complex 111 BLP, BMP 111 Co, BLP, BMPEntorhinal cortex 11 111

Cingulate cortex 111 11

Motor cortex 111 V 11

Somatosensory cortex 111 V 11

Posterior parietal association area 111 V 11

Retrosplenial agranular cortex 11 111 IIICorpus callosum 1 111

Fimbria hippocampus 1 111

Cingulum 1 111

External capsule 1 111

Internal capsule 1 111

Dorsal hippocampal commissure 1 111

Hippocampus 111 CA1, CA3 111 CA2, CA3, DGSubiculum 111 1

Medial habenular nu. 2 111

Paraventricular hypothal. nu. 11 1

Reuniens thalamic nu. 2 1

Ventromedial hypothalamic nu. 11 2

Premammillary nu., ventral 2 11

Substantia nigra, compact 11 11

Oculomotor nu. (III) 2 111

Trochlear nu. (IV) 2 111

Median raphe nu. 1 11

Dorsal raphe nu., inferior part 1 2

Motor trigeminal nu. (V) 11 111

Locus coeruleus 111 2

Facial nu. (VII) 2 111

Ambiguus nu. 2 11

Dosal motor nu. vagus nu. (X) 1 111

Cerebellar cortex 1 111 Pij, GrLateral (dentate) cerebellar nu. 111 2

Hypoglossal nu. (XII) 1 111

Pars anterior of the hypophysis 11 2

Optic nerve 1 11

Spinal cord 2 11

a Terminology was based on Franklin and Paxinos [7]. Relative signal intensities for each gene are graded as follows: 111 high; 11 moderate; 1 low; 2

background.b This description was added to denote the presence of remarkably strong signals in the more limited portions of each locus. Mi, mitral cell; Peri,periglomerular cell; GrO, granule layer of the olfactory bulb; BLP, posterior part of the basolateral amygdaloid nucleus; BMP, posterior part of thebasomedial amygdaloid nucleus; Co, cortical amygdaloid nucleus; V and III, layer fifth and third of cerebral cortex; CA1-3, CA1-3 field; DG, dentate gyrus;Pij, Purkinje cell; Gr, granular layer.

expression was detected in the paraventricula nucleus (Fig. Leydig’s cells, which synthesize testosterone. In the lung,3G) and ventromedial hypothalamic nucleus (Fig. 3H) of expression of TMEFF1 was detected in all types ofthe hypothalamus, the anterior lobe of the hypophysis (Fig. bronchial epithelial cells and in type II pneumocytes.3I), the compacta part of the substantia nigra, and the TMEFF1 probe gave signal throughout the heart, includingmotor trigeminal nucleus. A prominent signal was also not only areas containing cardiac myocytes but also heartseen in the locus coeruleus (Fig. 3J). In the cerebellum, valves (data not shown).strong expression was observed in the dentate nucleus(Fig. 3K). 3.3. Comparative studies of TMEFF1 and TMEFF2

In addition to the central nervous system, we investi-gated sites of TMEFF1 expression in the testis, lung, and To compare expression sites of TMEFF1 and TMEFF2,heart. In the testis, strong signals were observed in data quoted from our previous study of TMEFF2 [10] are


Fig. 3. Localization of TMEFF1 mRNA in the mouse brain and hypophysis. Brain and pituitary sections from 2-month-old male mice were hybridizedwith DIG-labeled TMEFF1 antisense probe. (A) coronal section of the olfactory bulb. EPl, external plexiform layer; Mi, mitral cell layer; Gl, glomerularlayer; GrO, grannule layer. (B) coronal section of the forebrain olfactory region. DTT, dorsal tenia tecta; VTT, ventral tenia tecta; AO, anterior olfactorynucleus; Pir, piriform cortex. (C) coronal section of the amygdaloid nuclear complex. BLP, posterior part of the basolateral amygdaloid nucleus; BMP,posterior part of the basomedial amygdaloid nucleus; LV, lateral ventricle. (D) coronal section of the entorhinal cortex. LEnt, lateral entorhinal cortex; ec,external capsule. (E) coronal section of the cerebral neocortex. M, motor cortex; RSA, retrosplenial agranular cortex; PPtA, posterior parietal associationarea. (F) sagital section of the hippocampus. CA1-3, CA1-3 field; DG, dentate gyrus; S, subiculum. (G) coronal section of the anterior hypothalamus. Pa,paraventricular nucleus. (H) coronal section of the posterior hypothalamus. VMH, ventromedial hypothalamic nucleus. (I) coronal section of thehypophysis. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe. (J) coronal section of the locus coeruleus. LC, locus coeruleus. (K) coronal sectionof the cerebellar deep nucleus. Lat, lateral (dentate) cerebellar nucleus. Bar5100 mm.


Fig. 3. (continued)

also presented in Table 1. In the olfactory pathway, both of the cortex, whereas strong signals for TMEFF2 weregenes are strongly expressed, while periglomerular cells of limited to the third layer of the retrosplenial agranularthe olfactory bulb express TMEFF1 specifically, and the cortex. In the hippocampus, stronger expression ofcortical nucleus of the amygdala and entorhinal cortex TMEFF1 was found in the subiculum and the fields ofexpress TMEFF2 predominantly. The pyramidal cells of CA3 and CA1. In contrast, TMEFF2 is more highlythe fifth layer of the fore part of the cerebral cortex gave expressed in the fields of CA2 and CA3, and the dentatemore remarkable signals for TMEFF1 than the other layers gyrus. Specific or predominant signals of TMEFF1 were


also noted in the locus coeruleus, the dentate nucleus of the ground or low levels, in the motor trigeminal nucleus,cerebellum, the paraventricula nucleus and the ventrome- facial nucleus, ambiguus nucleus, dorsal motor nucleus ofdial hypothalamic nucleus of the hypothalamus, and the the vagus, and the hypoglossal nucleus, all of which areanterior lobe of the hypophysis. In contrast, stronger involved in feeding processes, e.g. mastication and swal-expression of TMEFF2 was observed in the medial lowing [13]. We speculate that the TMEFF2 deficiency inhabenular, cranial nerve nuclei (III, IV, V, VII, X and XII), homozygous mice may impair the ability to feed, resultingambiguus nucleus, Purkinje-cell and granular layers of the in severe under-nutrition.cerebellar cortex, premammillary nucleus, median raphe TMEFF1 is strongly expressed in the locus coeruleus,nucleus and spinal cord. In nerve fiber tracts, e.g. corpus which is a major center for the noradrenergic neurons [13],callosum, fimbria hippocampus, cingulum, external cap- and in the wide area of cerebral neocortex (e.g. motorsule, internal capsule, dosal hippocampus commissure and cortex, somatosensory cortex, posterior pariental associa-optic nerve, signals for both genes were detected. This tion area, etc.), in particular, the pyramidal cells of fifthindicates that both TMEFF1 and TMEFF2 genes are layer. No TMEFF2 signal is detected in the locusexpressed in glial as well as neuronal cells. In particular, coeruleus, and strong signals in the cerebral neocortex areprominent expression in these populations appears to be a limited to the third layer of the retrosplenial agranularcharacteristic of the TMEFF2 gene. cortex. Given the expression patterns of TMEFF1 and

TMEFF2, our previous observation that the recombinantTMEFF2 protein failed to support survival of cortical

4. Discussion neurons is intriguing; it is possible that TMEFF1 exerts atrophic effect on the majority of cortical neurons. TMEFF1

TMEFF1 and TMEFF2 are novel members of the EGF- is also expressed in the paraventricula nucleus of thelike protein family; both proteins contain two follistatin- hypothalamus and the anterior lobe of the hypophysis. Aslike domains and a single EGF-like domain, and are indicated by experiments by Eib and colleagues instructurally unique within this family. Various neurotrophic Xenopus laevis, TMEFF1 may function in the neuroen-growth factors and neural proteoglycans, including docrine system [5].neuregulins [3,4,9,11,17], agrin [1,2,8,14], neurocan [12] Northern-blot analysis reveals the presence of large andand brevican [16] have EGF-like domains. Notably, agrin, small transcripts in the brain, possibly the result ofwhich plays a key role in the aggregation of acetylcholine alternative splicing or different polyA signal usage. Sincereceptors during synaptogenesis in the neuromuscular our in situ hybridization probe is unable to discriminatejunction, also contains follistatin-like domains that share between the two transcripts, further study is needed toconsiderable similarity with those of the TMEFFs [5,10]. clarify whether the transcripts differentially localize.These structural similarities suggest that the TMEFF In this study, we have demonstrated that TMEFF1 andproteins also possess neurotrophic acitvities. We recently TMEFF2 exhibit different patterns of expression. Thesedemonstrated that a purified recombinant TMEFF2 poly- results indicate that expression of TMEFF1 and TMEFF2peptide containing the putative extracellular domain can is differentially regulated, and that both genes play region-function as a survival factor for hippocampal and mesence- specific roles in the central nervous system.phalic neurons, but not for cortical neurons [10]. Theseresults indicate that TMEFF2 is a novel trophic factor forsome types of neurons. Furthermore, the activation of

AcknowledgementserbB4 tyrosine kinase receptor by TMEFF2 has beenshown [15]. The high amino acid sequence homology

We thank Drs E. Kondo and H. Tsujino for help with theshared by TMEFF1 and TMEFF2 implies that TMEFF1technical instruction and T. Iwanaga and M. Okano formay possess similar trophic activity, possibly for differenttechnical assistance.types of neurons. Therefore, our comparison of TMEFF1

and TMEFF2 mRNA expression provides clues to thespecific roles of TMEFF1 and TMEFF2 in the centralnervous system. References

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Expression of TMEFF1 MRNA in the Mouse Central Nervous System