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The Journal of Neuroscience, September 15, 2001, 21(18):7079-7088
Transforming Growth Factor  : A Promoter of Motoneuron Survival
of Potential Biological Relevance
Séverine
Boillée,
Josette
Cadusseau,
Muriel
Coulpier,
Gaël
Grannec, and
Marie-Pierre
Junier
Institut National de la Santé et de la Recherche
Médicale Unité 421, Faculté de Médecine, 94010 Créteil, France
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ABSTRACT |
Expression of transforming growth factor  (TGF), a member of
the epidermal growth factor (EGF) family, is a general response of
adult murine motoneurons to genetic and experimental lesions, TGF
appearing as an inducer of astrogliosis in these situations. Here we
address the possibility that TGF expression is not specific to
pathological situations but may participate to the embryonic development of motoneurons. mRNA of TGF and its receptor, the EGF
receptor (EGFR), were detected by ribonuclease protection assay in the
ventral part of the cervical spinal cord from embryonic day 12 (E12)
until adult ages. Reverse transcription-PCR amplification of their
transcripts from immunopurified E15 motoneurons, associated with
in situ double-immunohistological assays, identified
embryonic motoneurons as cellular sources of the TGF-EGFR couple.
In vitro, TGF promoted the survival of immunopurified
E15 motoneurons in a dose-dependent manner, with a magnitude similar to
BDNF neuroprotective effects at equivalent concentrations. In a
transgenic mouse expressing a human TGF transgene under the control
of the metallothionein 1 promoter, axotomy of the facial nerve provoked
significantly less degeneration in the relevant motor pool of
1-week-old mice than in wild-type animals. No protection was
observed in neonates, when the transgene exhibits only weak expression
levels in the brainstem. In conclusion, our results point to TGF as
a physiologically relevant candidate for a neurotrophic role on
developing motoneurons. Its expression by the embryonic motoneurons,
which also synthesize its receptor, suggests that this chemokine is
endowed with the capability to promote motoneuron survival in an
autocrine-paracrine manner.
Key words:
EGF; EGFR; erbB1; development; facial nucleus; spinal
cord
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INTRODUCTION |
TGF is one of the members of the
epidermal growth factor (EGF) family, which share an EGF-like domain
functioning as their receptor binding domain, and act through the erbB
family of tyrosine kinase receptors (Hackel et al., 1999 ). This family
can be subdivided into two groups according to the capability of the
EGF-like factors to bind or not the EGF receptor (EGFR or erbB1) (Lee
et al., 1995). TGF, the first member of the EGF family to have been
discovered after EGF (Todaro et al., 1980), is one of the EGFR ligands
that constitute the first group. Products of the neuregulin 1 gene are
part of a second group of molecules that do not bind EGFR but do bind
erbB3 and erbB4 and profoundly affect motoneuronal metabolism. They
have been shown to be synthesized by embryonic motoneurons and to be
necessary for proper motoneuronal maturation and, in particular, for
the establishment of adequate interactions between the motoneuron and
its glial and muscular environment (for review, see Gassmann and Lemke,
1997; Burden, 1998). In contrast, links between members of the
first EGF family group and motoneuronal metabolism have been
established only under pathological conditions up to this day.
Motoneuronal TGF upregulation is thus the only example of enhanced
synthesis of an EGF family factor by neuronal cells submitted to an
aggression. It was found in wobbler mutant mice undergoing
degeneration of their cervical spinal motoneurons, in
muscle-deficient murine mutants affected by a degenerative process targeting their lumbar spinal motoneurons, and in hypoglossal motoneurons after axonal crush and cut (Junier et al., 1994, 1998; Lisovoski et al., 1997). The tightly correlated time courses of TGF
motoneuronal production with that of astrogliosis development and EGFR
expression by reactive astrocytes designated these glial cells as
TGF primary targets. TGF capability to induce astrogliosis when
overexpressed in the CNS was subsequently demonstrated (Rabchevsky et
al., 1998). Although TGF expression was not observed in intact adult
motoneurons, the possibility of an involvement of TGF in motoneuronal metabolism during development remains to be evaluated. The
expression of several members of a given growth factor family by
developing motoneurons (Elde et al., 1991; Schecterson and Bothwell,
1992; Krieglstein et al., 1998; Kanda et al., 1999; Garces et al.,
2000) and of other EGF family members by immature motoneurons support
such a possibility. We thus studied whether TGF is expressed by
embryonic motoneurons and sought for its possible function in the
developing spinal cord. We report that embryonic motoneurons are
cellular sources for both the factor and its receptor and show that
TGF has the capability to promote motoneurons survival during
embryonic and early postnatal development.
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MATERIALS AND METHODS |
Mice and rats were housed in an air-conditioned room with access
to water and food ad libitum. Sprague Dawley rats (Janvier, Le Genest St. Isle, France) were used for experiments on embryos and
motoneuron cultures. Mice were used to assay in vivo TGF effects on motoneuronal survival. Metallothionein 1 (MT1)-hTGF transgenic mice from the MT-42 line were a generous gift from Dr. G. Merlino of the National Cancer Institute (Bethesda, MD) and bred in our
animal room. This transgenic line has been described in detail by
Jhappan et al. (1990). It carries a 917 bp human TGF cDNA under the
control of the zinc-inducible mouse metallothionein 1 promoter. These
transgenes have been obtained through injection of the construct
directly into CD1 one-cell mouse embryos (Jhappan et al., 1990). Their
age-matched controls corresponded to the parental CD1 strain (Charles
River, Saint-Aubin les Elbeuf, France).
RNA extraction from tissue samples. Total RNA was obtained
by the method of Chomczynski and Sacchi (1987) from the ventral part of
the cervical spinal cord of embryonic day 12 (E12) to postnatal day 60 (P60) rats and the brainstem of postnatal mice. Pregnant females were
anesthetized with chloral hydrate (400 mg/kg, i.p.), and the fetuses
were retrieved surgically. A longitudinal incision was made dorsally
from the neck to the tail of the fetus, and the spinal cord was removed
and its ventral part was trimmed out after flattening the spinal cord
on its ventral surface. The dorsal columns were discarded, and the
ventral halves were collected. The entire length of the spinal cord was
dissected out from E12 embryos. At all other embryonic ages and in
postnatal animals, only the cervical part of the spinal cord was
collected. Postnatal and adult animals received an overdose of
pentobarbital (60 mg/kg, i.p.), and their cervical spinal cord was
rapidly removed and the ventral part was dissected out as described
above. For RNA extraction from the cervical spinal cords, the tissue
samples were pooled before the extraction as follows: five animals per tube from E12-E21, four animals per tube from P2-P6, and three animals per tube from P14-P60. For brainstem collection, the mice were
decapitated under pentobarbital anesthesia, the brains were rapidly
removed, and the brainstem was dissected out under a binocular microscope. An anterior cut was made rostrally to the trapezoid bodies
and a posterior cut rostrally to the lateral reticular nucleus. The
resulting tissue piece encompassed the entire extent of the facial
nuclei, and tissues from two different animals were pooled per tube.
All tissues were immediately frozen on dry ice and stored at  80C
until further use.
Ribonuclease protection assay. The antisense TGF cRNA
probe used was obtained by in vitro transcription of a 400 bp EcoRI/HindIII cDNA fragment corresponding to
the coding region of the rat TGF cDNA, subcloned into the pGEM3Z
vector (Junier et al., 1991). The transcription was performed as
described previously (Junier et al., 1991) in the presence of
[32P]CTP. The template was linearized
with EcoRI to obtain a 430 nucleotide (nt)
[32P]CTP-labeled antisense RNA using SP6
RNA polymerase, 30 of the nucleotides corresponding to the vector
multiple cloning site. The antisense EGFR RNA probe was derived from a
578 bp Sau3A1 cDNA fragment of the rat EGFR cDNA (Petch et al., 1990),
linearized with AvaII, and transcribed with T7 polymerase.
Only 160 bp of this cDNA fragment corresponds to the sequence of the
extracellular domain of the full-length EGFR mRNA (Junier et al.,
1993). The 169 nt antisense cyclophilin probe was obtained from a
PstI/XmnI 111 bp fragment of the rat cyclophilin
cDNA (Danielsson et al., 1988), subcloned into the pBluescript vector.
The template, linearized with EcoRI and transcribed with T7
RNA polymerase, yielded a 169 nt probe, 58 of which corresponding to
the vector multiple cloning site. Levels of the mRNA of interest were
assayed as described previously (Junier et al., 1991). Briefly, the
tissue total RNA (5 µg) was simultaneously hybridized to the
antisense TGF probe (500,000 dpm) or the antisense EGFR probe and to
the cyclophilin antisense RNA probe (5000 dpm) to assess for procedural
losses. Cyclophilin mRNA is expressed constitutively in brain tissue
(Danielsson et al., 1988). The purified RNA-RNA duplexes were
electrophoresed through a 7 M urea-5%
acrylamide gel, and the gel was exposed to XAR-5 films for either 18 hr
(for cyclophilin signal analysis) or 4-8 d (for TGF and EGFR signal
analysis). Autoradiograms were analyzed by laser densitometry using the
cyclophilin mRNA signal to standardize TGF mRNA values. Results are
expressed as the ratio of the TGF mRNA signal to the cyclophilin
mRNA signal.
Immunohistochemistry. E17 and E18 embryos were anesthetized
on ice before being perfused transcardially with warm heparinized saline followed by 0.1 M PBS, pH 7.4, containing
4% paraformaldehyde. The spinal cord was immediately removed and
post-fixed for 4 hr at 4°C in the same fixative. Tissues were frozen
after a 24 hr incubation in a 30% sucrose cryoprotective medium.
Cryostat sections (10 µm thickness) were cut in the frontal plane.
Immunohistochemistry was performed on sections mounted on gelatinized
slides. The sections were incubated overnight at 4°C in the
appropriate dilution of the primary antibody in 0.05 M Tris, 154 mM NaCl, 0.02%
BSA, and 0.3% Triton X-100, pH 7.5. Immunohistochemical detection was
achieved using either the avidin-biotin complex immunoperoxidase
technique and the diaminobenzidine chromogen (Vector Laboratories,
Burlingame, CA) or the tyramide signal amplification system coupled to
the fluorochrome cyanine 3 (Cy3) or fluorescein (FITC; NEN, Paris, France). TGF-immunoreactive cells were identified with rabbit polyclonal antibody 1296 (1:5000), which recognizes a peptide sequence
(amino acids 137-151) contained within the cytoplasmic domain of the
TGF precursor (pro-TGF) (Gentry et al., 1987). The use of an
antiserum that recognizes the intracellular portion of the precursor
ensured the identification of the cells that synthesize TGF as
opposed to cells susceptible to bind it. Adjacent sections were stained
for the EGF receptor using the rabbit polyclonal antibodies 29.1 (1:250; Sigma, St. Quentin Fallavier, France) or sc-03 [1:100; Santa
Cruz Biotechnology (Santa Cruz, CA) and Tebu (Le Perray en Yvelines,
France)], which recognize a peptide sequence (amino acids 1005-1016)
localized within the intracellular C terminal tail of the EGFR.
Alternate sections were stained for choline acetyltransferase (ChAT)
using the goat polyclonal antibody AB144P [1:500; Chemicon (Temecula,
CA) and Euromedex (Mundolsheim, France)]. Control experiments included
omission of either the primary antiserum or the secondary antibody.
Specificity of the pro-TGF and EGFR antibodies in the nervous
tissues, including preadsorption of the antibodies with their antigens,
has been reported previously (Junier et al., 1991, 1994; Lisovoski et
al., 1997; Rabchevsky et al., 1998). Immunofluorescence was observed with a fluorescent microscope (Axioplan 2; Zeiss, Le Pecq, France). Images were acquired on a Cool SNAP camera using the Cool SNAP software
(Zeiss). For confocal immunofluorescence, slides were observed using a
Zeiss LSM 410 confocal microscope. Two lasers were used depending on
the fluorochrome. The excitation wavelengths were 543 nm for Cy3
and 488 nm for FITC, and the emission wavelengths were 570 and 510-525
nm for Cy3 and FITC, respectively. Tissue sections were observed with
the 63×, 1.4 numerical aperture lens (Plan-Apochromat; Zeiss,
Oberkochen, Germany), the pinhole was set at 20, and the resolution on
x-y was of 0.2 and 0.7 µm for z. Images were
prepared for printing using Adobe Photoshop software (Adobe Systems,
San Jose, CA).
Facial nerve cut. Cuts of the facial nerve were performed as
described previously (Coulpier et al., 1996) on 2- and 8-d-old homozygous MT1-hTGF transgenic mice and their age-matched controls corresponding to the parental CD1 strain (Charles River). Animals were
anesthetized on ice and subjected to a unilateral transection of the
right facial nerve. The small branch that innervates the caudal
auricular muscle and corresponds to motoneurons in the ventromedial
part of the facial nucleus was sparred. The success of the axotomy was
verified by the sagging of the right facial muscles before the animals
were killed. Seven days after the operation, mice were killed and
perfused with 4% paraformaldehyde as described above. Sections (30 µm thick) of the brainstem were obtained throughout the entire extent
of the facial nucleus and stained with cresyl violet. The nucleoli of
the motoneurons were counted every 2 sections over the entire surface
of the right and left facial nuclei in each animal. Results are
expressed as percentage of total motoneurons counted in the left,
unlesioned nucleus (mean ± SEM). Statistical analysis was done
using ANOVA, followed by a Fisher's test. Immunofluorescent detection
of EGFR in the facial nucleus was performed as described previously.
p75NTR immunodetection was done using the
rabbit polyclonal anti-p75NTR antibodies
(1:500; Promega, Madison, WI) and diaminobenzidine as a chromogen
(Vector Laboratories). Counting of the numbers of
p75NTR-immunoreactive motoneurons was
performed and analyzed as described above.
Northern blot analysis. Total RNA (10 µg/lane) was size
fractionated in agarose-formaldehyde gels and blotted onto
nitrocellulose membranes. The hTGF transgene and cyclophilin mRNAs
were successively identified by hybridization to the following cDNA
probes labeled using the random primer method and
[32P]dCTP: 316 bp hTGF cDNA fragment
(Jhappan et al., 1990) and 1 kb rat cyclophilin cDNA (Danielsson et
al., 1988). Signals were detected with a PhosphorImager (Molecular
Dynamics, Orsay, France) and analyzed with the ImageQuant software
(Molecular Dynamics) using the cyclophilin mRNA signal to standardize
hTGF mRNA values (Rabchevsky et al., 1998).
Motoneuron cultures. Cell suspensions of fetal motoneurons
were prepared from 15-d-old rat fetuses as described previously (Camu
and Henderson, 1992). Motoneurons were isolated and purified using the
immunopanning method. The cells were first separated on a metrizamide
gradient that segregates large cells. The motoneurons were then
isolated using 192 IgG attached previously to a substrate coated with
anti-mouse antibodies. Determination of the neuronal enrichment of the
cell suspension was performed on aliquots of the cell suspension
smeared on a gelatin-coated glass slide. Immunohistochemical detection
of the neuronal-specific microtubule-associated protein-2 (MAP-2) and
of the motoneuronal-specific transcription factor islet-1 indicated a
92 and 96% enrichment in neurons, respectively, whereas only 2% of
the cell suspension was immunolabeled with an antibody recognizing the
astrocyte-specific marker GFAP. Two thousand five hundred purified
motoneurons were seeded in 100 µl of serum-free L15 medium into
96-well plates coated previously with merosin (2 µg/ml; Life
Technologies, Cergy Pontoise, France) as described previously (Hanson
et al., 1998). One hour after plating, 100 µl of L15 medium alone or
containing rat synthetic TGF (0.1-50 ng/ml; Sigma) or recombinant
human brain-derived neurotrophic factor (BDNF) (10 ng/ml; Chemicon and
Euromedex) was added to each well. Cultures were maintained at 37°C
under a 5% CO2 atmosphere for 3 d. At that
time, 88 and 87% of the cells corresponded to motoneurons, as assayed
with islet-1/2 and ChAT immunoreactivity, respectively. The vast
majority of the remaining cells displayed a neuronal morphology. The
cells were then fixed with 4% paraformaldehyde and 0.1%
glutaraldehyde in PBS. Immunolabeling with goat anti-ChAT antibody
(1:200) was performed as described above using an initial 30 min
blocking step in PBS containing 0.2% Triton X-100 and 5% normal
rabbit serum and diaminobenzidine as a chromogen. The number of
motoneurons was determined over the entire culture dish area.
Motoneurons had to exhibit ChAT immunoreactivity and at least three
neurites with a length at least twice as long as the soma diameter to
be included in the counts. Results are expressed as mean ± SD
motoneurons of three independent experiments. Statistical analysis were
done using ANOVA, followed by a Fisher's test.
Reverse transcription-PCR amplification. Rat embryonic (E15)
motoneurons were purified as described above, pelleted, and immediately frozen on dry ice. RNA was extracted from
106 cells using Trizol following the
instructions of the manufacturer (Life Technologies). After DNase
digestion, 200 ng of total RNA was reverse transcribed using oligo-dT
as a primer and the Omniscript reverse transcriptase following the
instructions of the manufacturer (Qiagen, Courtaboeuf, France).
Substitution of RNA with H2O served as control of
the reverse transcription (RT). PCR amplification was performed using
the Taq polymerase from Life Technologies and the following
primers: TGF upper primer, TGG AGA ACA GCA CGT CC; TGF lower
primer, GCG CTG GGC TTC TCG TG; EGFR upper primer, GAG TCT AGA CAC CAG
AGT GAT GTG TGG AG; and EGFR lower primer, GAG CTG CAG CGC TGG GGG TCT
CGG GCC AT. The amplified cDNAs corresponded to nt 212-575 of the rat
TGF cDNA and to nt 2863-3116 of the rat EGFR cDNA. PCR was done
using a hot-start procedure and the following amplification program: 15 sec at 94°C, 1 min at 55°C, and 2 min at 72°C, 35 cycles. PCR
products were purified on a 2% agarose gel containing ethidium bromide
before being transferred onto a Nytran membrane (Amersham Pharmacia
Biotech, Orsay, France). The membranes were prehybridized for 4 hr and hybridized overnight at 37°C with either a
[32P]dCTP-labeled TGF or
[32P]dCTP-labeled EGFR cDNA probe
(106 cpm/ml) corresponding to nt 1-400
and nt 2863-3116 of the TGF and EGFR rat cDNAs, respectively. The
prehybridization and hybridization solutions were composed of 50%
formamide, 5× SSC, 25 mM
Na2HPO4, pH 6.8, 1× Denhardt's solution, 5%
dextran sulfate, 1% SDS, and 20 µg/ml denaturated ssDNA. The
membranes were washed twice for 15 min in 1× SSC and 0.1% SDS at room
temperature and twice in 0.25× SSC and 0.1% SDS at 37C for 15 min
before be exposed to BIOMAX-MS Kodak films (Eastman Kodak, Rochester, NY).
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RESULTS |
Developmental expression of TGF and EGFR mRNA in the ventral
horn of the rat cervical spinal cord
TGF mRNA levels were evaluated by ribonuclease protection assay
at different developmental stages in the ventral part of the rat
cervical spinal cord, in which motoneurons are located. Ribonuclease
protection assay of TGF and cyclophilin mRNAs yielded a 400 and 111 bp protected band, respectively (Fig.
1A). TGF mRNA signal
was detected at 12 d of gestation (E12), the earliest embryonic
age examined. TGF mRNA levels increased progressively throughout the
gestation to reach adult levels at birth (Fig. 1B).
Ribonuclease protection of the EGFR and cyclophilin mRNA yielded a 160 and 111 bp protected band, respectively (Fig.
2A). Like TGF mRNA,
EGFR mRNA was detected in the spinal cord of E12 embryos. Its levels
increased at subsequent embryonic ages and exhibited little variations
up to the second week of postnatal life, before decreasing at 3 postnatal weeks (Fig. 2B).
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Figure 1.
TGF mRNA levels in the ventral horn of the
cervical spinal cord. A, Autoradiogram of TGF
ribonuclease protection assay (4 d exposure, 5 µg/lane total RNA).
TGF, Undigested TGF cRNA probe;
Cyclo, undigested cyclophilin cRNA probe;
Digested probes, digested TGF and cyclophilin cRNA
probes; E15 csc, 5 µg of total RNA from the ventral
horn of an E15 rat cervical spinal cord. B,
Densitometric analysis of TGF mRNA signals. Each
point represents five (E12-E21), four (P2-P6), and
three (P14-P60) animals. Mean ± SD; n = 3-4.
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Figure 2.
EGFR mRNA levels in the ventral horn of the
cervical spinal cord. A, Autoradiogram of EGFR
ribonuclease protection assay (8 d exposure, 5 µg/lane total RNA).
EGFR, Undigested EGFR cRNA probe; Cyclo,
undigested cyclophilin cRNA probe; Digested probes,
digested EGFR and cyclophilin cRNA probes; E17 csc, 5 µg of total RNA from the ventral horn of a E17 rat cervical spinal
cord. B, Densitometric analysis of EGFR mRNA signals.
Each point represents five (E12-E21), four (P2-P6),
and three (P14-P22) animals. Mean ± SD; n = 3-5.
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RT-PCR amplification of TGF and EGFR transcripts from E15
immunopurified motoneurons
To determine whether motoneurons could be one of the cellular
sources of TGF and/or EGFR mRNA detected by ribonuclease protection assay in the ventral spinal cord, RT-PCR amplification of TGF and
EGFR transcripts was performed on total RNA extracted from immunopurified E15 motoneurons. Immunohistochemical detection of the
neuronal-specific protein MAP-2 and of the motoneuronal-specific transcription factor islet-1 indicated a 92 and 96% enrichment in
neurons, respectively, whereas only 2% of the cell suspension was
immunolabeled with an antibody recognizing the astrocyte-specific marker GFAP. RT-PCR amplification yielded a 363 and 255 bp band corresponding to the size expected for TGF and EGFR cDNAs,
respectively. The identity of the RT-PCR-derived products was verified
through Southern blot hybridization with the rat TGF and EGFR cDNA
probes (Fig. 3).
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Figure 3.
Southern blot hybridization of the RT-PCR
amplification of the TGF and EGFR transcripts from E15 purified
motoneuron total RNA. The hybridization probes corresponded to the rat
TGF or the EGFR cDNA probes. RT, The reverse
transcriptase solution was omitted from the PCR reaction.
+RT, PCR reaction in the presence of the reverse
transcriptase solution.
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Cellular sources of pro-TGF and its receptor in the embryonic
rat cervical spinal cord
Immunohistochemical procedures were used to verify the presence of
TGF and EGFR proteins in the cervical spinal cord and to identify
their cellular sources in 17- and 18-d-old embryos. In the 17-d-old
embryo, pro-TGF-immunoreactive cells were observed in the
mediolateral and ventral parts of the spinal cord (Fig. 4A) in which
motoneurons, identified by their immunoreactivity for the motoneuron
marker ChAT, were observed (Fig. 4G). In addition, some
pro-TGF-immunoreactive cells were gathered along the medial axis of
the cervical spinal cord (data not shown). Fluorescent pro-TGF
immunohistochemistry yielded similar results in E18 spinal cord
embryos. In the ventral part of the spinal cord, TGF-immunoreactive cells appeared as large-sized neurons (Fig.
4B,C). In the 17-d-old embryo,
EGFR-immunoreactive cells displayed a localization similar to
pro-TGF-immunoreactive cells (Fig. 4D).
Fluorescent EGFR immunohistochemistry yielded similar results in E18
spinal cord embryos, EGFR-immunoreactive cells appearing as large-sized
neurons in the ventral part of the spinal cord (Fig.
4E,F). Motoneuronal identity
of the pro-TGF- and EGFR-immunoreactive cells observed in the
ventral horn of the embryonic spinal cord was further confirmed with
double-immunofluorescent staining using either TGF and ChAT
antibodies or EGFR and ChAT antibodies (Fig.
5). Confocal microscopy showed that
neurons immunoreactive for TGF were also immunoreactive for ChAT
(Fig. 5A-C). ChAT immunolabeling appeared diffused
throughout the cytoplasm (Fig. 5A), whereas TGF
immunostaining exhibited a punctated aspect (Fig. 5B).
Likewise, EGFR-immunoreactive neurons were also ChAT immunoreactive
(Fig. 5D-F), with the EGFR signal being
preferentially localized at the plasma membrane (Fig.
5E,F).
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Figure 4.
Immunohistochemical localization in the cervical
spinal cord of 17- and 18-d-old rat embryos of the TGF precursor,
its receptor EGFR, and ChAT, a motoneuron marker. A,
TGF precursor immunolabeling in 17-d-old cervical spinal cord. The
arrow points to some of the labeled cells in the ventral
region of the spinal cord. B, C,
Immunofluo- rescent staining of the TGF precursor in the
ventralmost part of the cervical spinal cord gray matter of 18-d-old
rat embryos at low (B) and high
(C) magnifications. D, EGFR
immunolabeling in 17-d-old cervical spinal cord. The
arrow points to some of the EGFR-immunoreactive cells
localized in the ventral portion of the spinal cord. E,
F, Immunofluorescent staining in the ventralmost part of
the cervical spinal cord gray matter of 18-d-old rat embryos of the
EGFR at low (E) and high
(F) magnifications. Note that EGFR-immunoreactive
neurons appear more numerous in E than TGF
precursor-immunoreactive neurons in F. G,
ChAT immunohistochemical labeling. Immunolabeled neurons are localized
in the mediolateral and ventral regions of the spinal cord in 17-d-old
cervical spinal cord. The arrow points to some of the
labeled cells in the ventral region of the spinal cord. In
B, C, E, and
F, the original immunofluorescent signal appeared
red (cyanine 3 fluorochrome). Scale bar:
A, D, G, 150 µm;
B, E, 140 µm; C,
F, 56 µm.
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Figure 5.
Confocal visualization of ChAT and TGF
precursor (A-C) and of ChAT and EGFR
(D-F) immunofluorescent labelings in the ventral
part of the cervical rat spinal cord of an 18-d-old embryo. TGF
precursor-immunoreactive neurons (B) are also
ChAT immunolabeled (A). C, Overlay
of ChAT (green) and TGF (red)
immunofluorescent signals depicted in A and
B. Likewise, EGFR-immunoreactive neurons
(E) are also ChAT immunoreactive
(D). F, Overlay of ChAT
(green) and EGFR (red)
immunofluorescent signals depicted in D and
E. Scale bar, 10 µm.
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TGF effects on embryonic rat motoneuron survival cultured in
serum-free medium
The observation of TGF and EGFR expressing motoneurons during
the period of developmental cell death led us to evaluate the effects
of this growth factor on the survival of motoneurons immunopurified from E15 embryonic spinal cord. The percentage of motoneurons in the
cellular preparation was assessed with the 4D5 monoclonal antibody,
which recognizes the motoneuron-specific transcription factors
islet-1/2 (Ericson et al., 1992), and with an anti-ChAT polyclonal
antibody. The purification procedure yielded a 88 and 87% enrichment
in motoneurons, as assayed with islet-1/2 and ChAT immunoreactivity,
respectively. Motoneurons were seeded at low density in serum-free
medium to avoid accumulation of motoneuron-derived growth factors and
addition of EGF-like factors known to be abundant in serum.
Motoneuronal survival was assessed after 3 d in culture. At that
time, glia-like cells were rare (zero to five cells per well),
including in the presence of TGF. Well developed ChAT-immunoreactive cells displaying a large soma and at least three neurites with a length
at least twice as long as the largest soma diameter were taken into
account (Fig. 6). BDNF, a neurotrophin
well known to be trophic for cultured embryonic motoneurons (Henderson
et al., 1993), was used as a positive control. A statistically
significant positive effect of TGF was observed on motoneurons
survival. TGF promoted motoneurons survival within a concentration
range of 0.1-50 ng/ml, the maximal effect being observed at 50 ng/ml, the highest concentration tested. At 10 ng/ml, TGF effect on motoneuron survival was similar to the survival-promoting effect of 10 ng/ml BDNF (Fig. 7).
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Figure 6.
ChAT-immunoreactive neurons after 3 d in
culture in the presence of 1 ng/ml TGF. Scale bar, 100 µm.
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Figure 7.
Effect of TGF on spinal motoneuron survival.
Number of motoneurons was determined after 3 d in culture in
serum-free medium (white bar), in the presence of
0.1-50 ng/ml TGF (gray and black bars), or in
the presence of 10 ng/ml BDNF (hatched bar). Cells were
taken into account when exhibiting ChAT immunoreactivity and displaying
a large soma and at least three neurites with a length at least twice
as long as the largest soma diameter. The number of motoneurons was
determined over the entire culture dish area. Means ± SD;
n = 7-10; three independent experiments.
*p < 0.005; **p < 0.0005;
***p < 0.0001; ANOVA followed by a Fisher's test.
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Axotomy-induced motoneuronal death in 1-week-old
MT1-hTGF mice
The ability of TGF to promote motoneuron survival in
vivo was explored using the paradigm of facial nerve cut, which
triggers motoneuronal death in young postnatal mice. Mice
overexpressing an hTGF transgene in the CNS were used. In this mouse
strain, the transgene expression is under the control of the
metallothionein 1 promoter. Because metallothionein 1 is expressed at
barely detectable levels in embryonic and early postnatal rodents
brains (Waalkes and Klaassen, 1984; Choudhuri et al., 1996; Penkowa et
al., 1999), hTGF mRNA levels were first evaluated by Northern blot
assays at 1, 8, and 15 postnatal days in the mid-brainstem, in which facial nuclei are localized (Fig.
8A, left).
hTGF mRNA levels were low at birth and increased through postnatal
development to reach six times higher levels at postnatal day 15, the
oldest age examined (Fig. 8A, right).
Vulnerability of facial motoneurons to axotomy-induced death has been
reported to decrease with aging. It has been shown that axotomy induces
death of 90% of the motoneurons in 2-d-old mice, of 50% in 1-week-old
mice, and of 10% in 2-week-old mice (Kuzis et al., 1999). Facial nerve
cuts were therefore performed on 8-d-old animals, a time at which half
of the facial motoneurons is sensitive to axotomy-induced death and
significant amounts of hTGF mRNA are detected in the MT1-hTGF
transgenic mice brainstem. In addition, facial motoneuron response to
axotomy was monitored in 2-d-old animals, a time at which the TGF
transgene is expressed at low levels. The examination of facial nuclei
7 d after the transection of the facial nerve at 2 d of age
revealed a dramatic reduction in the number of neurons in control mice,
and, as expected, similar observations were made in transgenic mice.
Eighty percent of the facial motoneurons were lost (mean ± SEM of
motoneurons corresponding to 1377 ± 28 in intact nucleus vs
325 ± 19 in axotomized nucleus in control mice and 1367 ± 25 vs 273 ± 19 in transgenic mice; n = 4) (Fig.
8B). Most surviving motoneurons were observed in the
ventromedial part of the nucleus, which corresponds to the nonsectioned
caudal auricular branch of the facial nerve. In 8-d-old control mice,
axotomy also triggered facial motoneuron death 7 d after lesion
but to a lesser extent than in 2-d-old animals. Fifty percent of the
motoneurons were lost (mean ± SEM of motoneurons corresponding to
1359 ± 22 in intact nucleus vs 666 ± 39 in axotomized
nucleus in control mice; n = 4) (Fig.
8B). In 8-d-old transgenic mice, surviving facial
motoneurons observed 7 d after axotomy appeared more numerous than
in control mice. Only 18% of the motoneurons were lost (mean ± SEM of motoneurons corresponding to 1193 ± 21 in intact nucleus
vs 978 ± 33 in axotomized nucleus; n = 4) (Fig.
8B). Observation of numerous EGFR-immunoreactive motoneurons in the facial nucleus of these mice indicated that these
cells are endowed with the capability to be directly affected by TGF
(Fig. 8B, inset). Such a low percentage of
motoneurons death observed after the axotomy of the facial nerve of
8-d-old transgenic mice is, in control mice, attained only when the
axotomy is performed after 2 weeks of postnatal life (Kuzis et al.,
1999). The possibility, nevertheless, remained that motoneuron
protection observed in the 8-d-old transgenic mice resulted from an
accelerated maturation of the facial motoneurons. To address this
possibility, motoneuronal expression of the low-affinity neurotrophin
receptor p75NTR was used as an index of
motoneuron maturation in the intact facial nucleus.
p75NTR motoneuronal expression is indeed
known to decrease progressively during the first 2 weeks of postnatal
life before disappearing in the adults (Eckenstein, 1988; Yan and
Johnson, 1988; Ernfors et al., 1989). The numbers of
p75NTR-immunoreactive motoneurons in
2-week-old transgenes and controls were similar and represented
2.4-3.4% of the population of facial motoneurons (control mice
32 ± 9.5 vs transgenic mice 40.7 ± 6.6 p75NTR-immunoreactive motoneurons;
mean ± SEM; n = 3). In 1-week-old transgenes,
p75NTR-immunoreactive facial motoneurons
were 4.4 times more numerous than in 2-week-old control animals but
represented only 10.4% of the population of facial motoneurons instead
of 16.2% in 1-week-old controls (transgenic mice 142.3 ± 5.7 vs
control mice 223 ± 14.5; mean ± SEM; n = 3;
p = 0.0001).
View larger version (31K):
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Figure 8.
Effect of TGF on axotomy-induced motoneuronal
death in the facial nucleus of controls and MT1-hTGF transgenic
mice. A, Human TGF transgene expression in the
ventral brainstem of 1-, 8-, and 15-d-old MT1-hTGF mice.
Left, Northern blot analysis of hTGF mRNA levels.
Cyclophilin mRNA served as an internal standard. Right,
Densitometric analysis of hTGF mRNA signals. Note the increase in
hTGF mRNA levels as the animals age. Each point
represents two animals. B, Quantification of facial
motoneurons on the unlesioned and lesioned facial nuclei 7 d after
lesion in control (CD1) and MT1-hTGF transgenic mice operated at
either 2 or 8 d of age. Results are represented as percentages of
surviving motoneurons in the lesioned facial nucleus 7 d after
axotomy relative to motoneuron numbers in the contralateral, unlesioned
facial nucleus. Unhatched bars, Unlesioned nucleus.
Hatched bars, Lesioned nucleus. Mean ± SEM;
n = 4; ANOVA followed by the Fisher's test;
***p < 0.001. Inset,
Immunohistochemical localization of EGFR in the facial nucleus of
1-week-old MT1-hTGF transgenic mice. Scale bar, 75 µm.
|
|
| |
DISCUSSION |
Studies over the past 8 years have revealed that developing
motoneurons synthesize at least one class of the EGF family of growth
factors, the neuregulin1 gene products. Our results show that, in rat
embryonic motoneurons, this family extends to TGF. They also
indicate that this expression might have functional consequences by
providing evidences in vitro and in vivo of a role for TGF as a promoter of motoneuron survival.
A functional autocrine-paracrine loop indicated by TGF and
EGFR coexpression
Localization of TGF in embryonic nervous tissues is only known
in the forebrain (Kornblum et al., 1997). Our results show that TGF
and EGFR transcripts can be detected in the rat spinal cord from
12 d postcoitum until adult ages. Most cells immunoreactive for
the TGF precursor and its receptor were localized in the ventral and
mediolateral part of the cervical spinal cord. This distribution
matches that observed for motoneurons identified by their ChAT
immunoreactivity and their large soma size (Altman and Bayer, 1985;
Phelps et al., 1990). Double-immunohistological assays showing that
pro-TGF- and EGFR-immunoreactive cells are indeed ChAT
immunoreactive, associated with RT-PCR amplification of the TGF and
EGFR transcripts from suspensions of purified motoneurons containing at
most 2% of GFAP-immunoreactive cells, confirmed motoneurons as
cellular sources of the TGF-EGFR couple.
TGF promotes motoneuron survival
TGF and EGFR coexpression in embryonic motoneurons during the
period of developmental cell death raised the possibility that TGF
affects regulation of neuronal survival through an autocrine-paracrine mode of action. The increase in motoneuron survival observed in culture
in the presence of TGF indicates that this factor has the capability
to promote survival of embryonic motoneurons, an observation similar to
that reported by Hanson et al. (1998) with a single TGF
concentration of 50 ng/ml. TGF effect is dose dependent and, at the
highest concentrations, similar in magnitude to the survival
stimulatory effect of equivalent concentrations of BDNF in our culture
conditions. EGFR expression by motoneurons and the lack of significant
glial contamination in the cultures (zero to five cells per culture
well, including in the presence of TGF) indicate that astrocytes do
not mediate TGF effects on motoneurons. This singles out motoneurons
among the neuronal populations known to be responsive to TGF
neurotrophic effects, which, up to this day, have been shown to occur
in most cases through the astrocytes intermediacy (for review, see
Junier, 2000).
TGF is not the only growth factor endowed with the capability to act
in an autocrine-paracrine manner on motoneurons. The growth factors
TGF 2 and TGF3, as
well as neurotrophin-3, are synthesized by embryonic motoneurons
together with their receptor and promote embryonic motoneuronal
survival (Flanders et al., 1991; Henderson et al., 1993; Gouin et al.,
1996; Krieglstein et al., 1998). To examine whether TGF
promoting-survival action on motoneurons extends to in vivo
situations, we used the paradigm of facial nerve cut in early postnatal
mice. This model is widely used to test the ability of growth factors
to promote motoneuron survival in situ (Houenou et al.,
1994). In addition, axotomized motoneurons die in an apoptotic manner,
as during physiological developmental death (de Bilbao et al., 2000;
Vanderluit et al., 2000), and are protected by the same molecules
(Sendtner et al., 1990, 1992; Yan et al., 1992, 1995; Henderson et al.,
1993, 1994; Koliatsos et al., 1993; Dubois-Dauphin et al., 1994; Kato
and Lindsay, 1994; Martinou et al., 1994; Farlie et al., 1995; Vejsada et al., 1995; Coulpier et al., 1996). In the MT1-hTGF mice, the transgene expression is driven by the metallothionein 1 promoter, and
metallothionein 1 synthesis appears only during late postnatal development (Waalkes and Klaassen, 1984; Choudhuri et al., 1996; Penkowa et al., 1999). Accordingly, low amounts of the transgene were
expressed in the mid-brainstem of 2-d-old transgenic mice. In contrast,
facial motoneurons are protected from axotomy-induced cell death in
1-week-old mice when expression of human TGF transgene increases in
the brainstem. Approximately 60-65% of the facial motoneurons
disappearing in control mice are rescued from death in MT1-hTGF
mice. The possibility exists that TGF overexpression leads to an
accelerated maturation of the motoneurons and, hence, to an enhanced
resistance to the deleterious effects of axotomy. Although a reduced
amount of p75NTR-immunoreactive
motoneurons was observed in the transgenic mice facial nucleus (10% of
the facial motoneuron population in 1-week-old transgenic mice vs 16%
in age-matched controls), it is not sufficient to account for the
increase in motoneuron survival observed in the transgenic mice. Hence,
if accelerated motoneuronal maturation indeed occurs in these
transgenic mice, it accounts only partly for the TGF neuroprotective
effects obtained in this in vivo model. In addition,
detection of numerous EGFR-immunoreactive facial motoneurons in
1-week-old intact facial nucleus suggests that facial motoneurons can
be directly affected by TGF. The possibility remains, however, that
other EGFR-expressing cells, not clearly detectable in our assay, also
mediate part of the TGF neuroprotective effects. Altogether, the
present results indicate that TGF, like other factors trophic for
motoneurons (Oppenheim, 1996; Sendtner et al., 1996), is available
in situ in the developing spinal cord, its receptor is
expressed by embryonic motoneurons, and its survival-promoting effects
can be disclosed in both in vitro and in vivo
assays. These data give credence to the existence of a biological
relevant role of TGF in motoneuronal development.
However, the extent of this biological relevance remains to be
evaluated. Resolution of this issue depends in part on examination of
mice lacking TGF or its receptor. The EGFR null mice exhibit embryonic or early postnatal lethality depending on the genetic background, essentially as a result of placental or lung defects (Miettinen et al., 1995; Sibilia and Wagner, 1995; Threadgill et al.,
1995). They display severe impairment of brain development (Threadgill
et al., 1995; Kornblum et al., 1998; Sibilia et al., 1998), but
alterations in motor nuclei have not been explored. In contrast, TGF
null mice develop normally (Luetteke et al., 1993; Mann et al., 1993),
and the only CNS defect reported to date corresponds to the lack of a
nigral dopaminergic subpopulation (Blum, 1998). To our knowledge, no
exploration of the TGF / motoneuronal systems has been
undertaken. The absence of major CNS defect, at least at the clinical
level, may stem from the existence of functional redundancies between
the different EGFR ligands. Indeed, at least two other EGFR ligands,
heparin-binding-EGF and amphiregulin, have been detected in the spinal
cord (Hayase et al., 1998) (MPJ, unpublished results). It may also
result from the existence of subtle interventions for TGF, the
factor mediating in part the action of other promoters of motoneuron
survival. Its absence would thus lead only to discreet functional
modifications. Development of TGF-blocking antibodies should allow
to definitely evaluate whether TGF is physiologically involved in
the control of motoneuron development.
TGF: another member of the EGF family to be expressed by
embryonic motoneurons
Our results show that embryonic motoneurons express another EGF
family member in addition to neuregulin1. However, marked differences
exist between these factors. Neuregulin1 ensures the adequate
interactions between motoneurons and their environment. It affects
Schwann cell metabolism at all stages of their development and the
establishment of the neuromuscular junction, both Schwann and muscle
cells expressing neuregulin1 receptors (Burden, 1998; Mirsky and
Jessen, 1999). In contrast, our results indicate that the motoneurons
themselves constitute TGF targets in the embryo and that the factor
fulfils a trophic role toward the motoneurons. An effect of TGF on
Schwann and muscle cells cannot, however, be excluded. Its receptor has
been described in these cells that, at least in vitro, may
be sensitive to its mitogenic and/or differentiative actions (Lim and
Hauschka, 1984; Olwin and Hauschka, 1988; Toma et al., 1992; Sastry et
al., 1996; Halse et al., 1999). This, associated with the capability of
the different EGF family members to interact through
heterodimerizations of their respective receptors (Beerli and Hynes,
1996; Graus-Porta et al., 1997; Lenferink et al., 1998), allows
interactions between neuregulin1 and TGF to be envisioned. Another
difference stems from the switch in TGF cellular sources during
development. Neuregulin1 products are synthesized by motoneurons
throughout life (Chen et al., 1994) and, in the adult, participate to
the maintenance of the neuromuscular junction (Sandrock et al., 1997).
In contrast, TGF source changes from motoneurons to white matter
astrocytes and few small-sized neurons in mature animals. At this
stage, TGF motoneuronal expression may be observed only under
pathological situations, and reactive astrocytes and Schwann cells
constitute then its potential targets (Toma et al., 1992; Junier et
al., 1994; Lisovoski et al., 1997). Inversely to TGF, motoneuronal
neuregulin1 expression declines in adults after axotomy
(Bermingham-McDonogh et al., 1997), suggesting that TGF could
compensate in part neuregulin1 loss.
Conclusion
Our results allow TGF to be added to the list of growth factors
capable of acting on developing motoneurons. It is now acknowledged that factors trophic for motoneurons may derive not only from their
muscle targets but also from local sources (Oppenheim, 1996; deLapeyrière and Henderson, 1997). In this context, TGF
appears as a putative autocrine-paracrine growth factor for motoneurons.
| |
FOOTNOTES |
Received Dec. 29, 2000; revised June 25, 2001; accepted June 29, 2001.
This research was supported by the Association Française Contre
les Myopathies (a fellowship to S.B. and a grant to M.P.J.). We are
grateful to Dr. Glenn T. Merlino for the generous donation of the
transgenic mice used to breed for our experiments, Dr. Yin J. Ma for
the design of the TGF and EGFR PCR primers, and Dr. Larry E. Gentry
for his generous gift of the antibody 1296. We thank Jean-Pierre
Bellier for his participation to preliminary experiments and Dr. Marc
Peschanski for critical reading of this manuscript.
Correspondence should be addressed to Marie-Pierre Junier at her
present address: Institut National de la Santé et de la Recherche
Médicale U114, Collège de France, 11 place Marcelin Berthelot, 75231 Paris cedex 05, France. E-mail:
marie-pierre.junier{at}college-de-france.fr.
M. Coulpier's present address: Molecular Neurobiology, Department of
Neurosciences, Karolinska Institute, S17177 Stockholm, Sweden.
| |
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