Molecular Brain Research 86 (2001) 4855

www.elsevier.com/ locate/ bres

Research report
Expression of TMEFF1 mRNA in the mouse central nervous system:
precise examination and comparative studies of TMEFF1 and
TMEFF2
a a a a a
Naohide Kanemoto , Masato Horie , Kuniko Omori , Naoki Nishino , Mari Kondo ,
b a
Koichi Noguchi , Akira Tanigami
a
Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10, Kagasuno, Kawauchi-cho, Tokushima 771-0192, Japan
b
Department 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 two
follistatin-like domains. Both TMEFF1 and TMEFF2 are predominantly expressed in the brain. We previously demonstrated that
recombinant TMEFF2 protein can promote survival of neurons in primary culture and determined expression sites of TMEFF2 mRNA in
the mouse central nervous system. To extend our understanding of TMEFF protein functions, we compared precise sites of expression of
TMEFF1 and TMEFF2 mRNA using in situ hybridization analysis. Although both TMEFF genes are widely expressed in the brain, they
exhibit different patterns of expression. TMEFF1 showed comparatively higher signals in the pyramidal cells of fth layer of the cerebral
neocortex, CA3, CA1 and subiculum regions of the hippocampus, locus coeruleus, and dentate cerebellar nucleus. In contrast, TMEFF2 is
highly expressed in the medial habenular, CA2, CA3 and dentate gyrus region of the hippocampus, corpus callosum, cerebellar cortex and
cranial nerve nuclei (III, IV, VII, X, XII). The results presented here indicate that expression of TMEFF1 and TMEFF2 are regulated
differently and that they play region-specic 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 (also
called 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 rst identied in blot analysis showed that TMEFF2 is predominantly
Xenopus laevis as X7365, a novel transmembrane protein expressed in the both human and mouse brain. In situ
containing 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, specically in various types
function of TMEFF1 is largely unknown, although its of neurons and glial cells. Furthermore, we demonstrated
expression 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 in
to human chromosome 9q31 [6]. primary culture [10]. The similarity between TMEFF1 and
TMEFF2 suggests that the two proteins share similar
functions. 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 in
1035.
E-mail address: atanigam@otsuka.gr.jp (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) 4855 49
2. Materials and methods 2.4. In situ hybridization
2.1. Probe preparation Slide-mounted sections were xed in 4% paraformal-
dehyde in phosphate-buffered saline (PBS) for 20 min (for
Mouse TMEFF1 cDNA sequences are registered as parafn sections, this step was omitted, and the parafn
overlapped 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 min
amplied 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 150
GCAAGAGCAACTGCTC-39 (P1) and 59-TAGGAAC- mg/ ml for parafn sections) in 0.1 M TrisHCl pH 7.5, 5
TCCCGTCGGAAGC-39 (P2). These P1 and P2 primers mM EDTA for 5 min in case of the lung, for 15 min (brain
were designed based on the AA023493 and AA020026 and testis) or 30 min (heart) at room temperature, followed
sequences, respectively. The expected product size is 458 by two PBS washes. Slides were postxed for 20 min in
bp, corresponding to a segment (nucleotides 94 to 551) of the xative solution described above, then washed with
a recently registered partial cDNA sequence (GenBank PBS. Next, slides were acetylated with 0.25% acetic
accession number AJ400622). For Northern-blot analysis, anhydrate in 0.1 M triethanolamine for 10 min, washed
32
the amplied 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

amplied 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-labeled
were determined using an ABI377 autosequencer (Perkin probes were diluted to 1 mg/ ml with hybridization buffer
Elmer). This plasmid was linearized with NotI (for sense (0.3 M NaCl, 20 mM TrisHCl pH 8.0, 50% formamide,
probe) or NcoI (for antisense probe). Digoxigenin (DIG)- 13Denharts 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) and
and SP6 polymerases, respectively, using the DIG RNA pre-heated at 808C for 5 min; the hybridization mixture
Labeling Kit (Roche Molecular Biochemicals). was applied to each slide and incubated overnight at 558C.
After hybridization, slides were washed with 43SSC at
608C, followed by washing in high-stringency wash solu-
2.2. Northern-blot analysis
tion (50% formamide, 23SSC) at 608C. Next, slides were
rinsed with RNase buffer (0.5 M NaCl, 10 mM TrisHCl
Gene expression was evaluated using a mouse multiple
pH 8.0 and 0.5 M EDTA) three times for 10 min each at
tissue northern (MTN) blot (Clontech). After prehybridiza-
32
378C, incubated with 2 mg/ ml RNase in RNase buffer for
tion, the membrane was hybridized with a-[ P] dCTP-
30 min at 378C, then rinsed twice in RNase buffer for 10
labeled mouse TMEFF1 and b-actin cDNAs according to
min. Sections were washed again in high-stringency wash
the manufacturers procedure. Washed membranes were
buffer at 608C for 30 min, and color detection of the
autoradiographed for 18 h at 2808C.
hybridized probe was performed using the DIG Nucleic
Acid Detection Kit (Roche Molecular Biochemicals) ac-
2.3. Tissue preparation
cording to the manufacturers instructions. Anatomical
localizations were veried using the atlas of Franklin and
2.3.1. Brains
Paxinos [7].
Two-month-old (129/ SvEv3C57BL/ 6j)F male mice
1
were anesthetized with pentobarbital, and fresh brains were
removed quickly and immediately frozen on powdered dry
ice. Tissue sections (1420 mm thick) were cut using a
3. Results
cryostat 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 brain
was embedded in parafn, 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). Parafn sections and and moderate expression was observed in the lung and
frozen sections were cut to 5 mm- and 10 mm-thickness, heart (Fig. 1). However, the predominant expression in the
respectively, and mounted onto glass slides coated with brain implicates involvement of TMEFF1 in the central
3-aminopropyltriethoxysilane. nervous system.
50 N. Kanemoto et al. / Molecular Brain Research 86 (2001) 4855
3.2. In situ hybridization
3.2.1. Gross examination of TMEFF1 expression sites
Distribution of TMEFF1 mRNA was examined grossly
in sagittal brain sections. DIG-labeled antisense probe gave
prominent signals in the forebrain olfactory region, cere-
bral cortex and hippocampus (Fig. 2A), while a sense
probe gave no signal (Fig. 2B).
3.2.2. Precise examination of TMEFF1 expression sites
Precise expression sites of TMEFF1 were investigated
using serial coronal brain sections and intermittently cut
sagittal brain sections; sites where expression was detected
are summarized in Table 1. In the olfactory bulb, strong
signals were observed in cells of the mitral cell layer and
the external plexiform layer (Fig. 3A). As the positive cells
in the external plexiform layer are large in size and located
in the outer part of the layer, they are thought to be
periglomerular cells. The anterior olfactory nucleus, tenia
tecta, pirform cortex (Fig. 3B), basolateral nucleus and
basomedial nucleus of the amygdaloid nuclear complex
(Fig. 3C), and entorhinal cortex (Fig. 3D) also express
TMEFF1. Expression of TMEFF1 was detected through-
out the cerebral neocortex; in particular, the pyramidal
cells of fth layer of the cingulate cortex, motor cortex
(Fig. 3E), somatosensory cortex and posterior parietal
association area gave signicant signals. In the hippocam-
Fig. 1. Expression of the TMEFF1 gene as determined by Northern blot pus, expression of TMEFF1 appeared stronger in the CA3
32
analysis. 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 specicity. 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.
N. Kanemoto et al. / Molecular Brain Research 86 (2001) 4855 51
Table 1
a
Localization of TMEFF1 and TMEFF2 mRNA in the mouse central nervous system
b b
Locus TMEFF1 Remarkable portions TMEFF2 Remarkable portions
Olfactory bulb 111 Mi, Peri 111 Mi, GrO
Anterior 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, BMP
Entorhinal 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 III
Corpus 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, DG
Subiculum 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, Gr
Lateral (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 the
basomedial amygdaloid nucleus; Co, cortical amygdaloid nucleus; Vand III, layer fth and third of cerebral cortex; CA1-3, CA1-3 eld; DG, dentate gyrus;
Pij, Purkinje cell; Gr, granular layer.
expression was detected in the paraventricula nucleus (Fig. Leydigs cells, which synthesize testosterone. In the lung,
3G) and ventromedial hypothalamic nucleus (Fig. 3H) of expression of TMEFF1 was detected in all types of
the 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, including
motor trigeminal nucleus. A prominent signal was also not only areas containing cardiac myocytes but also heart
seen 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
52 N. Kanemoto et al. / Molecular Brain Research 86 (2001) 4855
Fig. 3. Localization of TMEFF1 mRNA in the mouse brain and hypophysis. Brain and pituitary sections from 2-month-old male mice were hybridized
with DIG-labeled TMEFF1 antisense probe. (A) coronal section of the olfactory bulb. EPl, external plexiform layer; Mi, mitral cell layer; Gl, glomerular
layer; GrO, grannule layer. (B) coronal section of the forebrain olfactory region. DTT, dorsal tenia tecta; VTT, ventral tenia tecta; AO, anterior olfactory
nucleus; 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 association
area. (F) sagital section of the hippocampus. CA1-3, CA1-3 eld; 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 the
hypophysis. AL, anterior lobe; IL, intermediate lobe; PL, posterior lobe. (J) coronal section of the locus coeruleus. LC, locus coeruleus. (K) coronal section
of the cerebellar deep nucleus. Lat, lateral (dentate) cerebellar nucleus. Bar5100 mm.
N. Kanemoto et al. / Molecular Brain Research 86 (2001) 4855 53
Fig. 3. (continued)
also presented in Table 1. In the olfactory pathway, both of the cortex, whereas strong signals for TMEFF2 were
genes are strongly expressed, while periglomerular cells of limited to the third layer of the retrosplenial agranular
the olfactory bulb express TMEFF1 specically, and the cortex. In the hippocampus, stronger expression of
cortical nucleus of the amygdala and entorhinal cortex TMEFF1 was found in the subiculum and the elds of
express TMEFF2 predominantly. The pyramidal cells of CA3 and CA1. In contrast, TMEFF2 is more highly
the fth layer of the fore part of the cerebral cortex gave expressed in the elds of CA2 and CA3, and the dentate
more remarkable signals for TMEFF1 than the other layers gyrus. Specic or predominant signals of TMEFF1 were
54 N. Kanemoto et al. / Molecular Brain Research 86 (2001) 4855
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 of
dial hypothalamic nucleus of the hypothalamus, and the the vagus, and the hypoglossal nucleus, all of which are
anterior 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 deciency in
habenular, cranial nerve nuclei (III, IV, V, VII, X and XII), homozygous mice may impair the ability to feed, resulting
ambiguus 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 ber tracts, e.g. corpus which is a major center for the noradrenergic neurons [13],
callosum, mbria hippocampus, cingulum, external cap- and in the wide area of cerebral neocortex (e.g. motor
sule, 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 fth
indicates that both TMEFF1 and TMEFF2 genes are layer. No TMEFF2 signal is detected in the locus
expressed in glial as well as neuronal cells. In particular, coeruleus, and strong signals in the cerebral neocortex are
prominent expression in these populations appears to be a limited to the third layer of the retrosplenial agranular
characteristic of the TMEFF2 gene. cortex. Given the expression patterns of TMEFF1 and
TMEFF2, our previous observation that the recombinant
TMEFF2 protein failed to support survival of cortical
4. Discussion neurons is intriguing; it is possible that TMEFF1 exerts a
trophic 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 the
like protein family; both proteins contain two follistatin- hypothalamus and the anterior lobe of the hypophysis. As
like domains and a single EGF-like domain, and are indicated by experiments by Eib and colleagues in
structurally 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 and
and brevican [16] have EGF-like domains. Notably, agrin, small transcripts in the brain, possibly the result of
which plays a key role in the aggregation of acetylcholine alternative splicing or different polyA signal usage. Since
receptors during synaptogenesis in the neuromuscular our in situ hybridization probe is unable to discriminate
junction, also contains follistatin-like domains that share between the two transcripts, further study is needed to
considerable 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 and
proteins also possess neurotrophic acitvities. We recently TMEFF2 exhibit different patterns of expression. These
demonstrated that a puried recombinant TMEFF2 poly- results indicate that expression of TMEFF1 and TMEFF2
peptide containing the putative extracellular domain can is differentially regulated, and that both genes play region-
function as a survival factor for hippocampal and mesence- specic roles in the central nervous system.
phalic neurons, but not for cortical neurons [10]. These
results indicate that TMEFF2 is a novel trophic factor for
some types of neurons. Furthermore, the activation of
Acknowledgements
erbB4 tyrosine kinase receptor by TMEFF2 has been
shown [15]. The high amino acid sequence homology
We thank Drs E. Kondo and H. Tsujino for help with the
shared by TMEFF1 and TMEFF2 implies that TMEFF1
technical instruction and T. Iwanaga and M. Okano for
may possess similar trophic activity, possibly for different
technical assistance.
types of neurons. Therefore, our comparison of TMEFF1
and TMEFF2 mRNA expression provides clues to the
specic roles of TMEFF1 and TMEFF2 in the central
nervous system.
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