 |
 Previous Article | Next Article 
The Journal of Neuroscience, July 1, 2000, 20(13):5001-5011
Glial Cell Line-Derived Neurotrophic Factor and Developing
Mammalian Motoneurons: Regulation of Programmed Cell Death Among
Motoneuron Subtypes
Ronald W.
Oppenheim1,
Lucien J.
Houenou1,
Alexender S.
Parsadanian2,
David
Prevette1,
William D.
Snider3, and
Liya
Shen4
1 Department of Neurobiology and Anatomy and
Neuroscience Program, Wake Forest University School of Medicine,
Winston-Salem, North Carolina 27157-1010, 2 Department of
Neurology Washington University School of Medicine, St. Louis,
Missouri 63110, 3 University of North Carolina Neuroscience
Center, University of North Carolina, Chapel Hill, North Carolina
27599, and 4 Laboratory of Cellular Carcinogenesis and
Tumor Promotion, National Cancer Institute, National Institutes of
Health, Bethesda, Maryland 20892
 |
ABSTRACT |
Because of discrepancies in previous reports regarding the role of
glial cell line-derived neurotrophic factor (GDNF) in motoneuron (MN)
development and survival, we have reexamined MNs in GDNF-deficient mice
and in mice exposed to increased GDNF after in utero
treatment or in transgenic animals overexpressing GDNF under the
control of the muscle-specific promoter myogenin (myo-GDNF). With the exception of oculomotor and abducens MNs, the survival of all other
populations of spinal and cranial MNs were reduced in GDNF-deficient embryos and increased in myo-GDNF and in utero treated
animals. By contrast, the survival of spinal sensory neurons in the
dorsal root ganglion and spinal interneurons were not affected by any of the perturbations of GDNF availability.
In wild-type control embryos, all brachial and lumbar MNs appear to
express the GDNF receptors c-ret and GFR 1 and the MN markers
ChAT, islet-1, and islet-2, whereas only a small subset express
GFR2. GDNF-dependent MNs that are lost in GDNF-deficient animals
express ret/GFR1/islet-1, whereas many surviving GDNF-independent MNs express ret/GFR1/GFR2 and islet-1/islet-2. This indicates that many GDNF-independent MNs are characterized by the presence of
GFR2/islet-2. It seems likely that the GDNF-independent population represent MNs that require other GDNF family members (neurturin, persephin, artemin) for their survival. GDNF-dependent and -independent MNs may reflect subtypes with distinct synaptic targets and afferent inputs.
Key words:
motoneurons; cell death; GDNF; spinal cord; embryo; mouse
GDNF receptors; knock-out; transgenic
| |
INTRODUCTION |
Glial cell line-derived neurotrophic
factor (GDNF) was the first member of the GDNF family of
neurotrophic factors to be identified and was originally isolated based
on its ability to promote the survival and differentiation of
dopaminergic neurons in primary cultures from embryonic ventral
midbrain (Lin et al., 1993 ). Since the initial discovery of GDNF,
several other family members with neurotrophic actions have been
reported, and receptors for each of these have also now been described
(Rosenthal, 1999). The GDNF family of receptors are composed of a
complex involving the transmembrane Ret receptor tyrosine kinase and
one or more of at least four glycosyl phosphatidyl-inositol (GPI)
membrane-anchored ligand-binding components, GFR1- GFR4.
The four GDNF family ligands all use Ret but have their own preferred
co-receptors (GDNF/1; neurturin/2; artemin/3; persephin/4),
although some members may also interact with nonpreferred co-receptors
(e.g., GDNF/2 and neurturin/1), albeit with lower efficiency.
One neuronal population that is responsive to some of the GDNF family
members, including GDNF, are somatic motoneurons (MNs). GDNF and
neurturin are expressed in skeletal muscle and Schwann cells of
developing and adult mammals (Henderson et al., 1994; Suter-Crazzolara and Unsicker, 1994; Choi-Lundberg and Bohn,
1995; Springer et al., 1995; Sánchez et al., 1996; Wright and
Snider, 1996; Nguyen et al., 1998a; Suzuki et al., 1998; Golden et al., 1999), and Ret and GFR1 are expressed in all populations of spinal and cranial MNs that have been examined so far (Pachnis et al., 1993;
Nakamura et al., 1996; Jing et al., 1996; Treanor et al., 1996;
Bisgrove et al., 1997; Trupp et al., 1997, 1998; Glazner et al.,
1998; Golden et al., 1998; Yu et al., 1998).
These data indicate that GDNF is expressed in cells that closely
interact with developing MNs (skeletal muscle, Schwann cells) and that
receptors for GDNF are expressed on developing spinal and cranial MNs.
GDNF family members can also promote the survival of MNs in
vitro and in vivo, and mouse mutants deficient in GDNF or GFR1 are reported to have reduced MN numbers (Henderson et al.,
1994; Soler et al., 1999; Oppenheim et al., 1995; Li et al., 1995; Yan
et al., 1995; Moore et al., 1996; Sánchez et al., 1996; Cacalano
et al., 1998). Although these various lines of evidence are consistent
with the role of GDNF as a neurotrophic survival factor for
subpopulations of somatic MNs, several nagging questions remain.
For example, muscle-specific overexpression of GDNF in transgenic mice
during embryonic development is reported to result in normal MN numbers
in neonatal mice (Nguyen et al., 1998b), and there are even
discrepancies in the literature as to whether there is any MN loss at
all in either GFR1-deficient mice (cf. Enomoto et al., 1998 with
Cacalano et al., 1998) or in GDNF-deficient mice (cf. Moore et al.,
1996 and Sánchez et al., 1996 with Pichel et al., 1996).
It is also not clear whether the apparent loss of MNs in GDNF- or
GFR1-deficient mice involves GDNF regulation of survival versus
proliferation, migration, or differentiation of MNs. For these various
reasons, we have undertaken a detailed analysis of the role of GDNF in
the development of mouse MNs.
| |
MATERIALS AND METHODS |
GDNF-deficient embryos. The GDNF / mutants were
derived from natural or timed mating of GDNF+/ × GDNF+/ mice (from
an R29 ES clone on a CD1 background.) The age of the embryos was
determined by the presence of vaginal plug in the pregnant mothers and
indicated as embryonic day 0.5. Embryos were dissected and immediately
immersed in either Bouin's fixative for a few days (for neuronal
counting and pyknotic analysis) or 4% paraformaldehyde in 1×
PBS for 8-16 hr at 4°C and cryopreserved in 30% sucrose in
PBS and sectioned by cryostat at 15 µm. Some of the embryos were
freshly frozen on dry ice powder and sectioned by cryostat at 15 µm.
All embryos were genotyped by PCR with the following primer sets. To
detect the GDNF knock-out allele of a 255 bp PCR
product, the upper and lower primers used were
5'-CGGAGCCGGTTGGCGCTACCGG and 5'-ACGACTCGGACCGCCATCGGTG. To detect the
GDNF wild-type allele of 337 bp PCR product, we used the following
primer set: 5'-GAGAGGAATCGGCAGGCTGCAGCTG and 5'-CAGATACATCCACATCGTTTAGCGG.
Myo-GDNF transgenic mice. To examine the effect of increased
target-derived GDNF (and NT-3 or NT-4) on MN survival, we used embryonic and neonatal animals that overexpress these factors under a
muscle-specific myogenin promoter that drives transgene expression in
mice embryos beginning before the onset of either muscle innervation or
the programmed cell death (PCD) of MNs and which continues to be
expressed postnatally (Nguyen et al., 1998b). Embryos and neonates from
the myo-GDNF transgenic lines examined here express high amounts of
GDNF in muscle (Nguyen et al., 1998b). Brain and spinal cord from
transgenic and wild-type mice were immersion-fixed in Carnoy's or
Bouin's solution, paraffin-embedded, sectioned serially (12 µm), and
stained with either thionin or hematoxylin/eosin. Cell counts of
healthy surviving and dying pyknotic MNs were performed as described below.
In utero treatment with GDNF. BALB/c ByJ mice (The
Jackson Laboratory, Bar Harbor, ME) were bred in Wake Forest Medical
School animal facility. On gestation day 14.5 (the morning a vaginal plug was observed is designated E0.5), pregnant females were
anesthetized with ether, and partial laparotomy was performed under
sterile conditions. One uterus (3-5 embryos) was exposed, and each
embryo was injected with 5 µg of GDNF in 5 or 10 µl of saline
(0.9% NaCl, pH 7.2) using a modified 10 µl gauge Hamilton
microsyringe, as described previously (Houenou et al., 1990, 1994).
Injections were made into the amniotic fluid, embryos were replaced in
the uterus, and the mother was allowed to recover after the abdomen was
sutured. Fetuses from the contralateral uterus were used as controls
(saline only). On E18.5, mice were killed with an overdose of ether,
and fetuses were collected by cesarean section. Spinal cords from
control and trophic factor- or saline-treated embryos were dissected
out, fixed in Bouin's solution, and processed as described above.
Neuronal counts. Motoneurons were counted in every fifth or
tenth section through each population examined, and the totals were
multiplied by five or ten to give an estimate of total cell numbers.
Cell counts were done blind as to the treatment condition of the
embryo/neonate (control vs experimental) using a well established counting method that effectively eliminates the possibility of counting
the same cell (healthy or pyknotic) twice (Clarke and Oppenheim, 1995).
Data for pyknotic MNs are expressed as per 1000 surviving MNs.
For assessing sensory neurons, cells in every fifth section of the
fourth or fifth lumbar (L4-L5) dorsal root ganglion (DRG) were
counted. Spinal interneurons were counted in every
20th section through the entire lumbar
enlargement. One lateral half (hemisection) of the spinal cord was used
for counts of interneurons, and neurons located in the dorsal horn and
ventral horn were excluded from these counts. Cranial MNs in the
following motor nuclei were also assessed quantitatively: oculomotor
(III), trigeminal (V), abducens (VI), facial (VII), spinal accessory
(XI), and hypoglossal (XII). Although a major focus in the spinal cord
was on lumbar and brachial MNs, thoracic MNs were also counted in
some of the embryos/neonates. Because the rostrocaudal boundaries of
the XI motor nucleus was difficult to define precisely, MN counts
were confined to the first 1 or 2 cervical segments (C1-C2), and MN numbers were expressed as the number per section for both XI MNs and
cervical spinal MNs.
In situ hybridization.
35S-UTP-labeled riboprobes
complementary to c-ret, GFR1, GFR2, isl-1, isl-2, and ChAT were
synthesized according to the supplier's protocol (Stratagene, La
Jolla, CA) and used for in situ hybridization as described
previously (McMahon et al., 1992). A 0.627 kb antisense riboprobe for a
mouse c-ret cDNA at the 3' untranslated region (3'-UTR) was derived
from pMCRET7 cDNA linearized at the Nsi I position (Pachnis et al.,
1993). Antisense riboprobe to mouse GFR1 was complementary to
position 720-1415 (GenBank accession number AB000800). Two independent riboprobes were used for detection of GFR2 transcripts: a 633 bp
fragment complementary to rat GFR2 at amino acid positions 217-247
(Wang, 1998) or a 190 bp mouse GFR2 riboprobe corresponding to nucleotide position 1-190 (GenBank accession number AF002701). Probes complementary to full-length mouse isl-1 (1.1 kb), rat isl-2
(1.6 kb), and rat ChAT (2.2 kb) cDNAs were obtained from Drs. Sam Pfaff
and Terry Rabbitts. In situ hybridization studies were
performed on cryosections (transverse or sagittal) of E15.5 and E18.5
embryos. For quantitative analysis of the in situ
hybridization, adjacent transverse sections on a single slide were
hybridized separately with two different riboprobes, and slides of
GDNF+/+ and / sections were processed simultaneously under
identical conditions (e.g., the same stringency of wash and exposure
time on emulsion, etc.). Images were imported into the computer
directly using a CG-7 frame grabber to avoid distortion of signal
intensity using the same exposure for all the sections quantified
(Scion Corporation). A stage micrometer was used to calibrate the scale before the data analysis of images. The values for in situ
signals are expressed as total pixels (area) in the selected region
(ventral horn) and as the average density of pixels in the same region and were quantified using NIH Image 1.60 based on signals from at least
five images from each of two or three embryos per region per age. The
data are presented as mean pixels or density ± SD. Statistical
significance (p value) was calculated by two-tailed Student's t test.
| |
RESULTS |
Spinal and cranial MNs are reduced in GDNF-deficient mice
Cell counts of spinal MNs on E18.5 revealed a significant loss in
homozygous GDNF-deficient embryos compared to either wild-type controls
or heterozygotes (Figs. 1,
2A). Brachial,
thoracic, and lumbar MNs were reduced by 34, 22, and 37%,
respectively. A detailed developmental analysis of lumbar MNs between
E12.5 and E18.5 indicates that initially GDNF-deficient embryos have MN
numbers comparable to controls but that during the major period of
normal cell death (E13.5-E18.5), they lose more MNs than controls, and
this loss is associated with increased numbers of degenerating
(pyknotic) MNs (Table 1). Although not
examined in as much detail, the number of brachial MNs were also
comparable in wild-type and GDNF-deficient embryos on E12.5, whereas by
E15.5 and E18.5 the GDNF-deficient embryos had 24 and 34% fewer MNs,
respectively (data not shown). The number of sensory neurons in the DRG
and interneurons in the spinal cord on E18.5 were unaffected in the
GDNF-deficient embryos (Fig. 2B).
View larger version (98K):
[in this window]
[in a new window]
|
Figure 1.
Transverse sections of E18.5 spinal cord
of GDNF-deficient (/) and wild-type (+/+) control embryos.
A-D, Brachial; E-H, thoracic;
I-L, lumbar. Dotted lines delineate the
ventral horn. Scale bars: B (applies to A, B, E,
F, I, J), 200 µm; D (applies to
C, D, G, H, K, L), 100 µm.
|
|
View larger version (22K):
[in this window]
[in a new window]
|
Figure 2.
A, Spinal motoneuron numbers
(mean ± SD) on E18.5 in GDNF-deficient (/) and wild-type
control (+/+) embryos. Values in the bars are sample size (embryos).
B, The number (mean ± SD) of lumbar spinal
interneurons and L4 DRG sensory neurons on E18.5 in GDNF-deficient
(/) and control (+/+) embryos. C, The number
(mean ± SD) of cranial MNs on E18.5 in GDNF-deficient (/) and
control (+/+) embryos. 1p < 0.05;
2p < 0.01;
3p < 0.005, t
tests.
|
|
An analysis of MN numbers along the rostrocaudal axis of the lumbar
spinal cord on E18.5 revealed that most of the MN loss occurred in the
rostral three-fifths of the lumbar enlargement (Fig.
3A). Because a small amount of
normal MN death continues for a few days after E18.5 (Oppenheim et al.,
1986) and because most of this loss occurs in caudal lumbar regions
(Fig. 3), it seems likely that the apparent restriction of MN loss on
E18.5 in GDNF-deficient embryos to the rostral and intermediate (vs caudal) lumbar segments is an artifact of not being able to assess MN
numbers postnatally in these embryos because of their
embryonic/perinatal death. Although the analysis of MN numbers shows
that there is a significant loss in the GDNF-deficient embryos, single
Nissl-stained transverse sections of the spinal cord do not always
accurately reflect this (Fig. 1) because the loss is distributed along
the entire spinal cord (Fig. 3). However, these sections do accurately reflect the normal histology and morphology of the spinal cord in
GDNF-deficient embryos.
View larger version (17K):
[in this window]
[in a new window]
|
Figure 3.
A, The number (mean ± SD, SD
not shown for illustrative clarity) of motoneurons per section for five
equal lengths of lumbar spinal cord along the rostrocaudal axis on
E18.5 (+/+, /) and P2 (+/+). B, The number
(mean ± SD) of pyknotic motoneurons per section along the
rostrocaudal axis of lumbar spinal cord in wild-type control (+/+)
animals on E18.5 and P2. In A, E18.5 +/+ versus E18.5
/; 1p < 0.005; E18.5 +/+ versus P2
+/+; 2p < 0.01. In B,
1p < 0.05;
2p < 0.01, t
tests.
|
|
Because cranial somatic MNs express c-ret and GFR1, we have also
examined MN numbers in several of these nuclei. With the exception of
the abducens and oculomotor nuclei, all other cranial motor nuclei
examined here exhibit a significant loss of MNs (trigeminal 25%,
hypoglossal 21%, and facial 18%; Fig. 2C). Taken together, these data indicate that most populations of spinal and cranial somatic
MNs exhibit decreased cell numbers in GDNF-deficient mice and that this
loss is attributable to increased naturally occurring cell death.
Reductions in c-ret-, GFR1-, and GFR2-expressing cells in
GDNF-deficient embryos
Although it is clear from the data on surviving and degenerating
MNs that the loss of GDNF results in increased cell death, because not
all MNs die, it was of interest to determine the phenotype of surviving
versus dying MN subpopulations. Because GDNF is believed to signal
primarily via c-ret and GFR1 (or less effectively via c-ret and
GFR2), we have examined cells expressing these receptor mRNAs using
in situ hybridization. Additionally, to further characterize the surviving MNs in GDNF-deficient embryos, we have examined mRNA
expression for ChAT and for the LIM family homeobox nuclear transcription factors islet-1 and islet-2.
The expression of c-ret in presumptive MNs in the ventral horn was
reduced in the brachial, thoracic, and lumbar regions of E15.5
GDNF-deficient mice (Fig. 4). Because of
the normal rostrocaudal gradient of MN death in the mouse embryo spinal
cord (Yamamoto and Henderson, 1999), the decrease in c-ret expression
is more apparent in the brachial region at this age. However, by E18.5 all three regions exhibit a clear loss of c-ret expression in ventral
cells (Fig. 5), and consistent with the
cell counts (Fig. 2), the thoracic region shows less of a loss than
brachial or lumbar. Although regions dorsal to the ventral horn
that normally express c-ret, GFR1, and isl-1 (see below) also
exhibit reduced mRNA signals, based on our quantification of
interneuron numbers at E18.5 (Fig. 2), this reduction is unlikely to
reflect cell death.
View larger version (163K):
[in this window]
[in a new window]
|
Figure 4.
Expression of c-ret in spinal motoneurons of
embryos on E15.5. Transverse sections of c-ret in situ
hybridization at brachial, thoracic, and lumbar levels.
Arrowheads indicate ventral horn region;
arrows indicate vertebra. Scale bar, 100 µm.
|
|
View larger version (128K):
[in this window]
[in a new window]
|
Figure 5.
Expression of c-ret in brachial, thoracic, and
lumbar spinal cord on E18.5. Scale bar, 200 µm. Note the reduced mRNA
signal in surviving MNs compared to E15.5 (Fig. 4).
|
|
In an attempt to quantify the loss of c-ret expression, we have
averaged the total area (pixels) expressing c-ret in the
ventral spinal cord from several embryos in five independent in
situ hybridization experiments. The relative reduction in c-ret
expression in the ventral horn region of E15.5, GDNF-deficient embryos
was 50, 26, and 35% for brachial, thoracic, and lumbar segments,
respectively (Table 2). Except for
the lumbar region, the density of the c-ret signal was also reduced
(Table 3). We have no explanation for the
discrepancy in the area versus density data in the lumbar region at
this age. However, by E18.5 both measures were also significantly
reduced in the lumbar segments (data not shown, but see Fig. 5). These
data indicate that the loss of GDNF results in a significant reduction
in c-ret expression in ventral neurons in all three spinal regions
examined. However, some of the reduction of the c-ret signal in
GDNF-deficient embryos appears to reflect a loss of (or reduced)
expression in surviving MNs. This is especially obvious on E18.5 (Fig.
5). Therefore, the reduced density measure likely represents both cell
loss and a reduced in situ signal on the remaining
MNs.
In vitro biochemical studies have demonstrated that GDNF binds
preferentially to GFR1 and less efficiently to GFR2, but that all
GDNF family members can signal through c-ret (Baloh et al., 1997;
Creedon et al., 1997; Trupp et al., 1997). The retention of many c-ret
expressing MNs in GDNF-deficient mice suggests that their survival may
be mediated by other GDNF ligands or by GDNF receptor components other
than GFR1. We first examined whether the loss of c-ret-expressing
cells was correlated with the loss of GFR1 or GFR2 in brachial
segments of E15.5 GDNF-deficient embryos. Most MNs in the brachial and
lumbar regions appear to express GFR1, whereas only a subset express
GFR2 (Fig. 6). Although reduced
significantly, many GFR1-expressing MNs remained in the ventral
brachial spinal cord of GDNF-deficient animals (Fig. 6). Based on area
measurements there was a close correlation between the loss of GFR1
(50%) and c-ret (50%) expression in GDNF-deficient mice (Table 2). A
similar relationship was also observed in the thoracic region (26%
loss of c-ret and GFR1). Although the normal expression of GFR2
in the brachial ventral horn of wild-type mice is much less than that
of GFR1 (Fig. 6), there is an apparent increased expression of
GFR2 in the ventral horn of GDNF-deficient mice (Fig. 6, Table 2).
The low level of GFR2 expression in wild-type mice is not
attributable to poor hybridization of the probe because the weak
expression signal was confirmed using two independent riboprobes, and
both probes give strong signals in other embryonic tissues (data not
shown).
View larger version (84K):
[in this window]
[in a new window]
|
Figure 6.
Expression (in situ hybridization
using antisense cRNA probes) of multiple markers in transverse sections
of E15.5 brachial spinal cord of GDNF-deficient and control embryos.
Arrowheads indicate the ventral horn;
arrow indicates dorsal population of isl-1-expressing
neurons. Scale bar, 100 µm.
|
|
To determine whether the surviving neurons in the ventral horn of
GDNF-deficient mice were, in fact, differentiating MNs, we examined the
expression of two MN-specific markers, ChAT and islet (isl-1 and
isl-2), by in situ hybridization. The expression of ChAT
appeared to be reduced in tissue sections from E15.5 GDNF-deficient embryos (Fig. 6), and this was confirmed by the area measurements showing a 50 and 37% reduction in brachial and thoracic segments, respectively (Table 4). Because this
reduction in ChAT is comparable in magnitude to the reduction in c-ret,
this indicates that most, if not all, of the surviving
c-ret/ChAT-positive ventral neurons in the GDNF-deficient embryos are
differentiating MNs.
To further determine the phenotype of surviving MNs in the
GDNF-deficient mice, we examined the expression of isl-1 and isl-2. Whereas isl-2 is MN-specific, isl-1 is strongly expressed in MNs as
well as in a subset of dorsal interneurons in the spinal cord. There
was a reduction in isl-1 expression in the ventral horn of brachial
(30%) and lumbar (50%) segments, but no apparent loss in the thoracic
region of GDNF-deficient embryos (Table 2). By contrast, there was no
apparent reduction in brachial isl-2 expression on E15.5 (Table 4) or
in any segmental region of the ventral horn of E18.5 GDNF-deficient
embryos (data not shown); isl-2 area measures in the ventral horn at
all spinal levels on E18.5 were also comparable to wild-type embryos
(data not shown). Because the area measurements for isl-2, c-ret,
isl-1, and ChAT mRNA expression were similar in the brachial ventral
spinal cord of GDNF-deficient embryos, this indicates that the
surviving GDNF-independent MN population expresses isl-2, whereas the
GDNF-dependent MNs are isl-2-negative and isl-1-positive. All surviving
c-ret/GFR1-expressing brachial MNs in the GDNF-deficient mice also
appear to coexpress isl-1 and isl-2 as well as show an apparent
upregulation of GFR2 expression (Table 2).
In utero GDNF treatment rescues MNs from
cell death
In previous studies, we found that the administration of a single
injection of different putative neurotrophic factors to mouse embryos
in utero on E14 was sufficient to significantly reduce the
normal death of spinal MNs when assessed on E18 (Houenou et al., 1994).
In the present study, we observed that GDNF is also able to rescue both
spinal and cranial MNs from cell death when administered in
utero on E14 (Fig. 7). On average,
GDNF treatment increased MN numbers on E18.5 by ~21%. Except for the
abducens and oculomotor nuclei (data not shown), all populations of
somatic MNs were rescued from cell death by GDNF. Lumbar spinal
interneurons and sensory (L4 DRG) neuron numbers were also unaffected
(data not shown). These data suggest that the survival of
subpopulations of most somatic MNs is regulated by limiting amounts of
endogenous GDNF. As described below, however, MN numbers can be
increased even more when endogenous levels of muscle-derived GDNF are
constitutively increased throughout the period of normal MN cell death.
View larger version (20K):
[in this window]
[in a new window]
|
Figure 7.
The number (mean ± SD) of spinal and cranial
motoneurons on E18.5 in mouse embryos treated with GDNF in
utero on E14. 1p < 0.01;
2p < 0.05. E,
GDNF-treated; C, Control saline-treated.
Numbers in bars are sample size
(embryos).
|
|
Muscle-specific overexpression of GDNF (myo-GDNF) in transgenic
mice promotes MN survival
Neonatal mice from the transgenic lines expressing high levels of
GDNF under a muscle-specific (myogenin) promoter (Nguyen et al., 1998b)
were found to have a significant increase in spinal and cranial MNs
(Table 5, Fig.
8). By contrast, transgenic mice that
over express NT-3 or NT-4 (Myo-NT-3, myo-NT-4) have MN numbers comparable to controls. Based on cell counts in cervical spinal segments (C1-C2) both cervical spinal MNs (control spinal MNs = 3.6 ± 0.5 per section, n = 5, vs 8.3 ± 2.3 per section, n = 5, for myo-GDNF animals;
p < 0.002) and XI cranial MNs (control = 4.7 ± 0.6 per section, n = 5 vs 7.5 ± 0.9 per
section, n = 5, for myo-GDNF; p < 0.005) were increased in P1 transgenic mice.
View larger version (90K):
[in this window]
[in a new window]
|
Figure 8.
Transverse sections of P1 spinal cord from
myo-GDNF transgenic animals (A, C, E, G, L), controls
(B, D, F, H, K), and myo-NT-3
(I) and myo-NT-4 (J)
animals. A-D, Brachial; E-J, lumbar;
K, L, cervical. Scale bars:
A (applies to A, B, E, F), 150 µm; C (applies to C, D, G-J), 80 µm; K (applies to K, L), 70 µm.
Dotted lines in A-J delineate the
ventral horn, and in K and L, the
cervical (arrows) and spinal accessory
(XI) MNs.
|
|
Consistent with the results from both the GDNF-deficient mice and the
mice after in utero administration of GDNF, MN numbers in
the abducens and oculomotor nuclei were not affected in the myo-GDNF
transgenic mice. Similarly, sensory (DRG) and spinal interneurons were
also unaffected in the myo-GDNF mice (Table 5). The increased number of
spinal MNs (lumbar 45%, brachial 42%) in the myo-GDNF mice was
considerably greater than that seen after in utero GDNF
treatment (lumbar 22%, brachial 23%) but was close to the MN loss
observed in GDNF-deficient mice (e.g., 37% loss of lumbar MNs vs a
45% increase in myo-GDNF mice). Myo-GDNF embryos examined on E15.5,
during the normal cell death period for spinal MNs, had increased
numbers of surviving lumbar MNs (3667 ± 194, n = 4 vs 3110 ± 183, n = 4 for controls;
p < 0.01) and fewer degenerating (pyknotic) lumbar MNs
(48 ± 12, n = 4 vs 67 ± 18, n = 4; p < 0.05). This indicates that
the overexpression of GDNF increases MN numbers by preventing
programmed cell death.
| |
DISCUSSION |
Using three different approaches to perturb GDNF levels in the
developing mouse embryo, we have shown that each of the three results
in an alteration in the number of MNs that survive during the period of
naturally occurring PCD. GDNF-deficient embryos exhibit reductions in
brachial, thoracic, and lumbar MNs and in MNs in the III, V, VII, XI,
and XII cranial motor nuclei, whereas oculomotor and abducens MNs were
unaffected. The overexpression of GDNF in developing muscle (myo-GDNF
transgenics) and the injection of exogenous GDNF in utero
increased MN survival in all of the same MN populations whose numbers
were reduced in the GDNF-deficient embryos, whereas again oculomotor
and abducen MNs were unaffected. The perturbation of GDNF availability
specifically affected MNs, in that the survival of neither sensory
(DRG) nor spinal interneurons were altered by any of the three
approaches. The effects of overexpression of GDNF in the myo-GDNF
transgenics was also trophic factor-specific because MN numbers in
myo-NT-3 and myo-NT4/5-transgenic animals were comparable to wild-type
control values. When considered together, these results provide
compelling evidence for the role of GDNF as a physiologically relevant
MN survival factor in the developing murine nervous system. However,
because some of these results differ from previous reports they also
raise several questions.
Facial MNs
After neonatal axotomy, mammalian facial MNs exhibit a robust
survival response to treatment with exogenous GDNF (Henderson et al.,
1994; Zurn et al., 1994; Yan et al., 1995; Oppenheim et al., 1995). By
contrast, genetic deletion of GDNF or its primary co-receptor GFR1
are reported to have no effect on the number of surviving facial MNs
when examined in embryonic, neonatal, and adult animals (Moore et al.,
1996; Sánchez et al., 1996; Cacalano et al., 1998). Using three
different approaches to perturb GDNF availability, we have consistently
observed an ~20% increase or decrease (depending on whether GDNF was
increased or decreased) in the number of facial MNs of embryonic and
neonatal animals. This result is based on the evaluation of >20
experimental and 30 control animals in which cell counts were made
blind to treatment. Accordingly, we are confident that the modest but
significant alteration of facial MNs observed here is real. Despite
this discrepancy, however, there is agreement among all the different
studies regarding the role of GDNF in the survival of lumbar and
trigeminal MNs, and our results now extend the MN populations that have
been shown to respond to GDNF to include cervical, brachial, and
thoracic spinal MNs and facial, hypoglossal, and spinal accessory
cranial MNs.
Other non-MN populations
None of the three approaches used by us to alter GDNF availability
affected the number of surviving sensory DRG neurons, spinal interneurons, or oculomotor or abducens MNs. From this, we conclude that at the embryonic ages examined here GDNF is not likely to be
required for the survival of these populations even though all three
populations express GDNF receptors. Although a previous study of
GDNF-deficient animals reported finding a 20% loss of DRG neurons on
P0 (Moore et al., 1996), neonatal animals deficient in the preferred
GDNF co-receptor GFR1 have normal numbers of DRG neurons (Cacalano
et al., 1998; Enomoto et al., 1998). Because the excess GDNF in the
myo-GDNF transgenic animals is not available to most DRG neurons, the
lack of an effect on sensory neuron numbers in these animals is to be
expected. One likely explanation for the discrepancy between our
results and those of Moore et al. (1996) regarding sensory neurons is
the different ages of the GDNF-deficient animals examined in the two
studies (E18.5 vs P0).
The failure of both oculomotor and abducens MNs to exhibit a
responsiveness to GDNF in any of the three approaches used here to
perturb GDNF availability is surprising in light of the fact that these
MNs express both c-ret and GFR receptors in the adult (Trupp et al.,
1997; Golden et al., 1998; Glazner et al., 1998). However, receptor
expression has not yet been examined during development.
GDNF-dependent and independent MNs
There is considerable evidence supporting the idea that the
survival of developing MNs depends on multiple neurotrophic factors produced by different cell types (Oppenheim, 1996; Hanson et al., 1998;
Arce et al., 1998). This is perhaps not surprising given that MNs
differ phenotypically depending on their location, peripheral targets,
and afferent inputs (Tsuchida et al., 1994; Lin et al., 1998). The fact
that treatment with specific neurotrophic factors in vitro
and in vivo and genetic deletion of specific neurotrophic factors and their receptors only affect a proportion of all MNs in a
given population (e.g., lumbar MNs) is consistent with the idea of MN
subpopulations that require distinct trophic factors for their
survival. A striking example of this is the case of MNs that are
responsive to the muscle-derived neurotrophic factor hepatocyte growth
factor/scatter factor (HGF/SF). In both chicken and rat, only
limb-innervating MNs are rescued from PCD by HGF/SF (Yamamoto et al.,
1997; Novak et al., 2000).
The present results are also consistent with this idea in that
although the survival of virtually all MN populations examined by us
(spinal and cranial) are affected by GDNF availability, only a
subpopulation (20-40%) in each region or motor nucleus are dependent
on GDNF for their survival. Although alternative explanations of this
observation are possible (Oppenheim, 1996), we favor the notion that
these represent distinct MN phenotypes that, based on their unique
pattern of receptor expression, relative to other MNs, are dependent on
GDNF for their survival. This is not meant to exclude the possibility
that these same MNs can also respond to or are dependent on other
neurotrophic factors (Oppenheim et al., 1993; Oppenheim, 1996; Arce et
al., 1998; Hanson et al., 1998).
We find that c-ret and GFR1 are expressed on most, if not all,
spinal MNs in the developing mouse embryo, whereas only a subset of
these are lost in GDNF-deficient animals, and only a proportion of the
MNs lost during naturally occurring PCD are rescued by increased
availability of GDNF (myo-GDNF and in utero treatment).
Thus, there are GDNF-dependent and GDNF-independent subpopulations of
spinal MNs. Based on our similar findings for cranial MNs (i.e.,
partial cell losses in each nucleus), these also appear to be composed
of GDNF-dependent and independent subpopulations. In a recent study
that examined the death of spinal MNs in GFR1 and GFR2-deficient
mice, a selective vulnerability of subpopulations was also observed
(Garcés et al., 2000). Few if any MNs were lost in
GFR2-deficient mice (Rossi et al., 1999), whereas distinct subpopulations were missing in GFR1-deficient animals. Examination of the spinal cord of our GDNF-deficient embryos revealed a loss of
c-ret expression comparable to the amount of cell loss. Although there
was a close correlation between the loss of c-ret and GFR1 expression in spinal MNs, many GFR1-expressing MNs remained in GDNF-deficient embryos. Additionally, the apparent reduction in c-ret
expression in surviving MNs of GDNF-deficient mice on E18.5 suggests
that the maintenance of c-ret expression between E15 and E18 may
require GDNF. The expression of GFR2 was considerably less than
GFR1 in spinal MNs of wild-type control embryos (Garcés et
al., 2000), and GFR2 expression was increased in GDNF-deficient embryos. Accordingly, GFR2-expressing MNs represent only a small subset of all MNs and do not appear dependent on GDNF for their survival.
Our observation that a subpopulation of c-ret/GFR1-positive MNs are
lost in GDNF-deficient embryos is consistent with the reported loss of
only subsets of spinal MNs in GFR1-deficient animals (Cacalano et
al., 1998; Garcés et al., 2000) and with previous reports of
partial MN losses in GDNF-deficient embryos (Moore et al., 1996;
Sánchez et al., 1996). Our failure to observe a loss of
GFR2-expressing MNs in GDNF-deficient embryos is also consistent
with GFR2 being the preferred co-receptor for neurturin (Klein et
al., 1997; Heuckeroth et al., 1999). Although the survival of cultured
rat MNs is promoted by neurturin (Klein et al., 1997), a quantitative
analysis of MNs in Neurturin or GFR2-deficient animals has not yet
been reported (Rossi et al., 1999; Heuckeroth et al., 1999).
Interestingly, however, cultured MNs from GFR2-deficient embryos
retain their responsiveness to both GDNF and Neurturin (Garcés et
al., 2000), suggesting that mouse spinal MNs may actually respond to
Neurturin via receptors other than GFR2.
To confirm that the surviving cells in the ventral horn of the
GDNF-deficient embryos are in fact MNs, we have used two specific MN
markers, ChAT and islet. There was a reduction in ChAT expression in
the spinal cord of GDNF-deficient embryos consistent with the cell loss
and with the reduced expression of c-ret/GFR1. Using separate probes
for isl-1 and isl-2, we found expression of both mRNAs in spinal MNs,
whereas isl-1 was also strongly expressed in a subset of dorsal
interneurons. Interestingly, there was a reduction of isl-1 expression
in GDNF-deficient embryos comparable to the observed MN cell loss and
to the reduction in c-ret/GFR1/isl-1 expression in brachial and
lumbar MNs, whereas there was no apparent loss of isl-2 expression.
From this, we can conclude that the GDNF-dependent MNs represent a
subset of c-ret/GFR1/isl-1-expressing MNs, whereas the
GDNF-independent MNs are an isl-2-positive subpopulation. The fact that
all surviving c-ret/GFR1-expressing MNs also appear to coexpress
isl-1 and isl-2 (and show an upregulation of GFR2) suggests that
these represent a subset of MNs that may be dependent on other GDNF
family members for their survival. Both neurturin and persephin have
been reported to promote the survival of mammalian and avian MNs (Klein
et al., 1997; Oppenheim et al., 1998; Soler et al., 1999) and therefore
may be trophic factors for these GDNF-independent MNs. We have also
found that another GDNF family member, artemin (Baloh et al., 1998),
can also promote the survival of subpopulations of avian MNs in
vitro and in vivo (R. Oppenheim, unpublished data).
The expression of LIM homeobox genes, including isl-1 and isl-2, by
avian embryo MNs is organized topographically in the spinal cord such
that the combinatorial expression of these genes define distinct
subsets of MNs that reflect their position in the spinal cord and their
peripheral targets (Tsuchida et al., 1994). Although the expression of
LIM homeobox genes in mammalian MNs is not as well characterized as in
the chick, based on the pattern of expression in the chick, one might
predict that the differential expression of isl-1 and isl-2 among
GDNF-dependent and GDNF-independent mouse MNs may reflect different
phenotypes regarding peripheral targets or afferent inputs.
| |
FOOTNOTES |
Received Jan. 11, 2000; revised April 5, 2000; accepted April 11, 2000.
This work was supported by Grants NS20402 and NS31380 from the National
Institutes of Health to Ronald W. Oppenheim, a grant from the Muscular
Dystrophy Association to Lucien J. Houenou, and by Grant NS37273 from
the National Institutes of Health to William D. Snider. We thank Dr. H. Westphal for his advice and support.
Correspondence should be addressed to Dr. Liya Shen, Laboratory of
Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute,
National Institutes of Health, Building 37, Room 2B15, 9000 Rockville
Pike, Bethesda, MD 20892. E-mail: shenl{at}mail.nih.gov.
| |
REFERENCES |
-
Arce V,
Pollock RA,
Philippe J-M,
Pennica D,
Henderson CE,
deLapeyriére D
(1998)
Synergistic effects of Schwann- and muscle-derived factors on motoneuron survival involved GDNF and cardiotrophin-1 (CT-1).
J Neurosci
18:1440-1448[Abstract/Free Full Text].
-
Baloh RH,
Tansey MG,
Golden JP,
Creedon DJ,
Heuckeroth RO,
Keck CL,
Zimonjic DB,
Popescu NC,
Johnson EM,
Millbrandt J
(1997)
TrnR2, a novel receptor that mediates neurturin and GDNF signaling through ret.
Neuron
18:793-802[Web of Science][Medline].
-
Baloh RH,
Tansey MG,
Lampe PA,
Fahrner TJ,
Enomoto H,
Simburger KS,
Leitner ML,
Araki T,
Johnson EM,
Millbrandt J
(1998)
Artemin, a novel member of the GDNF ligand family, supports peripheral and central neurons and signals through the GFR3-ret receptor complex
Neuron
21:1291-1302[Web of Science][Medline].
-
Bisgrove BW,
Raibl DW,
Walter V,
Eisen JS,
Grunwald DJ
(1997)
Expression of c-ret in the zebrafish embryo: Potential roles in motoneuronal development.
J Neurobiol
33:749-768[Web of Science][Medline].
-
Cacalano G,
Fariñas I,
Want L-C,
Hagler K,
Forgie A,
Moore M,
Armanini M,
Phillips H,
Ryan AM,
Reichardt LF,
Hynes M,
Davies A,
Rosenthal A
(1998)
GFR1 is an essential receptor component for GDNF in the developing nervous system and kidney.
Neuron
21:53-62[Web of Science][Medline].
-
Choi-Lundberg DL,
Bohn MC
(1995)
Ontogeny and distribution of glial cell line-derived neurotrophic factor (GDNF) mRNA in rat.
Dev Brain Res
85:80-88[Medline].
-
Clarke PGH,
Oppenheim RW
(1995)
Neuron death in vertebrate development: in vivo methods.
Methods Cell Biol
46:277-321[Web of Science][Medline].
-
Creedon DJ,
Tansey MG,
Baloh RH,
Osborne PA,
Lampe PA,
Fahrner TJ,
Heuckeroth RO,
Millbrandt J,
Johnson EM
(1997)
Neurturin shares receptors and signal transduction pathways with glial cell line-derived neurotrophic factor in sympathetic neurons.
Neurobiol
94:7018-7023.
-
Enomoto H,
Araki T,
Jackman A,
Heuckeroth RO,
Snider WD,
Johnson EM,
Millbrandt J
(1998)
GFR1-deficient mice have deficits in the enteric nervous system and kidneys.
Neuron
21:317-324[Web of Science][Medline].
-
Garcés A,
Haase G,
Airaksinen MS,
Livet J,
Filippi P,
deLapeyriére O
(2000)
Signaling by GDNF and NTN through GFR1 and GFR2 in distinct subpopulations of spinal motoneurons.
J Neurosci
20:4992-5000[Abstract/Free Full Text].
-
Glazner GW,
Mu X,
Springer JE
(1998)
Localization of glial cell line-derived neurotrophic factor receptor alpha and c-ret mRNA in rat central nervous system.
J Comp Neurol
391:42-49[Web of Science][Medline].
-
Golden JP,
Baloh RH,
Kotzbauer PT,
Lampe PA,
Osborne PA,
Millbrandt J,
Johnson EM
(1998)
Expression of neurturin, GDNF and their receptors in the adult mouse CNS.
J Comp Neurol
398:139-150[Web of Science][Medline].
-
Golden JP,
DeMaro JA,
Osborne PA,
Millbrandt J,
Johnson EM
(1999)
Expression of neurturin, GDNF and GDNF family-receptor mRNA in the developing and mature mouse.
Exp Neurol
158:504-528[Web of Science][Medline].
-
Hanson MG,
Shen S,
Wiemelt AP,
McMorris FA,
Barres BA
(1998)
Cyclic AMP elevation is sufficient to promote the survival of spinal motor neurons in vitro.
J Neurosci
18:7361-7371[Abstract/Free Full Text].
-
Henderson CE,
Phillips HS,
Pollock RA,
Davies AM,
Lemeulle C,
Armanini M,
Simpson LC,
Moffet B,
Vandlen RA,
Koliatsos VE,
Rosenthal A
(1994)
GDNF: a potent survival factor for motoneurons present in peripheral nerve and muscle.
Science
266:1062-1064[Abstract/Free Full Text].
-
Heuckeroth RO,
Enomoto H,
Grider JR,
Golden JP,
Hanke JA,
Jackman A,
Molliver DC,
Bardgett ME,
Snider WD,
Johnson EM,
Millbrandt J
(1999)
Gene targeting reveals a critical role for neurturin in the development and maintenance of enteric, sensory and parasympathetic neurons.
Neuron
22:253-263[Web of Science][Medline].
-
Houenou LJ,
Pincon-Raymond M,
Garcia L,
Harris AJ,
Rieger F
(1990)
Neuromuscular development following tetrodotoxin-induced inactivity in mouse embryos.
J Neurobiol
21:1249-1261[Web of Science][Medline].
-
Houenou LJ,
Li L,
Lo AC,
Yan Q,
Oppenheim RW
(1994)
Naturally occurring and axotomy-induced motoneuron death and its prevention by neurotrophic agents: a comparison between chick and mouse.
Prog Brain Res
102:217-226[Web of Science][Medline].
-
Jing S,
Wen D,
Yu Y,
Holst PL,
Luo Y,
Fang M,
Tamir R,
Antonio L,
Hu Z,
Cupples R,
Louis J-C,
Hu S,
Altrock BW,
Fox GM
(1996)
GDNF-induced activation of the ret protein tyrosine kinase is mediated by GDNF-, a novel receptor for GDNF.
Cell
85:1113-1124[Web of Science][Medline].
-
Klein RD,
Sherman D,
Ho WH,
Stone D,
Bennett GL,
Moffat B,
Vandlen R,
Simmons L,
Gu Q,
Hongo J-A,
Devaux B,
Poulsen K,
Armanini M,
Nozaki C,
Asai N,
Goddard A,
Phillips H,
Henderson CE,
Takahashi M,
Rosenthal A
(1997)
A GPI-linked protein that interacts with ret to form a candidate neurturin receptor.
Nature
387:717-721[Medline].
-
Li L,
Wu W,
Lin L-F,
Lei M,
Oppenheim RW,
Houenou LJ
(1995)
Rescue of adult mouse motoneurons form injury-induced cell death by glial cell line-derived neurotrophic factor.
Proc Natl Acad Sci USA
92:9771-9775[Abstract/Free Full Text].
-
Lin LH,
Doherty DH,
Lile JD,
Becktesh S,
Collins F
(1993)
GDNF: A glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.
Science
260:1130-1132[Abstract/Free Full Text].
-
Lin JH,
Saito T,
Anderson DJ,
Lance-Jones C,
Jessell TM,
Arber S
(1998)
Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression.
Cell
95:393-407[Web of Science][Medline].
-
McMahon A,
Joyner A,
Bradley A,
McMahon J
(1992)
The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expression cells by 9.5 days post coitum.
Cell
69:581-595[Web of Science][Medline].
-
Moore MW,
Klein RD,
Fariñas,
Sauer H,
Armanini M,
Phillips H,
Reichardt LF,
Ryan AM,
Carver-Moore K,
Rosenthal A
(1996)
Renal and neuronal abnormalities in mice lacking GDNF.
Nature
382:76-79[Medline].
-
Nakamura M,
Ohta K,
Hirokawa K,
Fukushima M,
Uchino M,
Ando M,
Tanaka H
(1996)
Developmental and denervation changes in c-ret proto-oncogene expression in chick motoneurons.
Mol Brain Res
39:1-11[Medline].
-
Nguyen QT,
Keller-Peck CR,
Lichtman JW,
Silos-Santiago I,
Snider WD
(1998a)
GDNF expression is restricted to a subset of muscles and muscle fibers.
Soc Neurosci Abstr
24:2002.
-
Nguyen QT,
Parsadanian AS,
Snider WD,
Lichtman JW
(1998b)
Hyperinnervation of neuromuscular junctions caused by GDNF overexpression in muscles.
Science
279:1725-1730[Abstract/Free Full Text].
-
Novak KD,
Prevette D,
Wang SW,
Gould T,
Oppenheim RW
(2000)
Hepatocyte growth factor/scatter factor (HGF/SF) is a neurotrophic survival factor for lumbar but not for other somatic motoneurons in the chick embryo.
J Neurosci
20:326-337[Abstract/Free Full Text].
-
Oppenheim RW
(1996)
Neurotrophic survival molecules for motoneurons: an embarrassment of riches.
Neuron
17:195-197[Web of Science][Medline].
-
Oppenheim RW,
Houenou LJ,
Rieger F,
Pincon-Raymond M,
Powell JA,
Standish JL
(1986)
The development of motoneurons in the embryonic spinal cord of the mouse mutant, muscular dysgenesis (mdg/mdg).
Dev Biol
114:426-436[Web of Science][Medline].
-
Oppenheim RW,
Prevette D,
Haverkamp LJ,
Houenou L,
Yin QW,
McManaman J
(1993)
Biological studies of a putative avian muscle-derived neurotrophic factor that prevents naturally occurring motoneuron death in vivo.
J Neurobiol
24:1065-1079[Web of Science][Medline].
-
Oppenheim RW,
Houenou LJ,
Johnson JE,
Lin L-FH,
Li L,
Lo AC,
Newsome AL,
Prevette DM,
Wang SW
(1995)
Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF.
Nature
373:344-346[Medline].
-
Oppenheim RW,
Wang SW,
Prevette D
(1998)
A comparison between GDNF, neurturin and persephin in promoting motoneuron survival.
Soc Neurosci Abstr
24:807.
-
Pachnis V,
Mankoo B,
Costantini F
(1993)
Expression of the c-ret proto-oncogene during mouse embryogenesis.
Development
119:1005-1017[Abstract].
-
Pichel JG,
Shen L,
Sheng HZ,
Granholm A-C,
Drago J,
Grinberg A,
Lee EJ,
Huang SP,
Saarma M,
Hoffer BJ,
Sariola H,
Westphal H
(1996)
Defects in enteric innervation and kidney development in mice lacking GDNF.
Nature
382:73-76[Medline].
-
Rosenthal A
(1999)
The GDNF protein family: gene ablation studies reveal what they really do and how.
Neuron
22:201-207[Web of Science][Medline].
-
Rossi J,
Luukko K,
Poteryaev D,
Laurikainen A,
Sun YF,
Laakso T,
Eerikäinen S,
Tuominen R,
Lakso M,
Rauvala H,
Arumäe U,
Pasternack M,
Saarma M,
Airaksinen MS
(1999)
Retarded growth and deficits in the enteric and parasympathetic nervous system in mice lacking GFR2, a functional neurturin receptor.
Neuron
22:243-252[Web of Science][Medline].
-
Sánchez MP,
Silos-Santiago I,
Frisén J,
He B,
Lira SA,
Barbacid M
(1996)
Renal agenesis and the absence of enteric neurons in mice lacking GDNF.
Nature
382:70-73[Medline].
-
Soler RM,
Dolcet X,
Encinas M,
Eges J,
Bayascas JR,
Comella JX
(1999)
Receptors of the glial cell line derived neurotrophic factor family of trophic factors signal cell survival through the phosphatidylinositol 3-kinase pathway in spinal cord motoneurons.
J Neurosci
19:9160-9169[Abstract/Free Full Text].
-
Springer JE,
Seeburger JF,
He J,
Gabrea A,
Blankenhorn EP,
Bergman LW
(1995)
cDNA sequence and differential mRNA regulation of two forms of glial cell line-derived neurotrophic factor in Schwann cells and rat skeletal muscle.
Exp Neurol
131:47-52[Web of Science][Medline].
-
Suter-Crazzolara C,
Unsicker K
(1994)
GDNF is expressed in two forms in many tissues outside the CNS.
Mol Neurosci
5:2486-2488.
-
Suzuki H,
Hase A,
Miyata Y,
Arahata K,
Akazawa C
(1998)
Prominent expression of glial cell line-derived neurotrophic factor in human skeletal muscle.
J Comp Neurol
402:303-312[Medline].
-
Treanor JS,
Goodman L,
deSauvage F,
Stone DM,
Poulsen KT,
Beck CD,
Gray C,
Armanini MP,
Pollock RA,
Hefti F,
Phillips HS,
Goddard A,
Moore MW,
Buj-Bello A,
Davies AM,
Asai N,
Takahashi M,
Vandlen R,
Henderson CE,
Rosenthal A
(1996)
Characterization of a multicomponent receptor for GDNF.
Nature
382:80-84[Medline].
-
Trupp M,
Belluardo N,
Funakoshi H,
Ibáñez CF
(1997)
Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene and GDNF receptor- indicates multiple mechanisms of trophic actions in the adult rat CNS.
J Neurosci
17:3554-3567[Abstract/Free Full Text].
-
Trupp M,
Raynoschek C,
Belluardo N,
Ibanez CF
(1998)
Multiple GPI-anchored receptors control GDNF-dependent and independent activation of the c-Ret receptor tyrosine kinase.
Mol Cell Neurosci
11:47-63[Web of Science][Medline].
-
Tsuchida T,
Ensini M,
Morton SB,
Baldassare M,
Edlund T,
Jessell TM,
Pfaff SL
(1994)
Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.
Cell
79:957-970[Web of Science][Medline].
-
Wang CY,
Ni J,
Jiang H,
Hsu TA,
Dugich-Djordjevic M,
Feng L,
Zhang M,
Mei L,
Gentz R,
Lu B
(1998)
Cloning and characterization of glial cell line-derived neurotrophic factor receptor-B: a novel receptor for members of glial cell line-derived neurotrophic factor family of neurotrophic factors.
Neuroscience
83:7-14[Medline].
-
Wright DE,
Snider WD
(1996)
Focal expression of glial cell line-derived neurotrophic factor in developing mouse limb bud.
Cell Tissue Res
286:209-217[Web of Science][Medline].
-
Yamamoto Y,
Henderson CE
(1999)
Patterns of programmed cell death in populations of developing spinal motoneurons in chicken, mouse and rat.
Dev Biol
214:60-71[Medline].
-
Yamamoto Y,
Livit J,
Pollock RA,
Garces A,
Arce V,
deLapeyrière O,
Henderson CE
(1997)
Hepatocyte growth factor (HGF/SF) is a muscle-derived survival factor for a subpopulation of embryonic motoneurons.
Development
124:2903-2913[Abstract].
-
Yan Q,
Matheson C,
Lopez OT
(1995)
In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons.
Nature
373:341-344[Medline].
-
Yu T,
Scully S,
Yu Y,
Fox GM,
Jing S,
Zhou R
(1998)
Expression of GDNF family receptor components during development: implications in the mechanisms of interaction.
J Neurosci
18:4684-4696[Abstract/Free Full Text].
-
Zurn AD,
Baetge EE,
Hammang JP,
Tan SA,
Aebischer P
(1994)
Glial cell line-derived neurotrophic factor (GDNF), a new neurotrophic factor for motoneurons.
NeuroReport
6:113-118[Web of Science][Medline].
Copyright © 2000 Society for Neuroscience 0270-6474/00/20135001-11$05.00/0
This article has been cited by other articles:
|
|
|
|
 
P.-Y. Wang, A. Protheroe, A. N. Clarkson, F. Imhoff, K. Koishi, and I. S. McLennan
Mullerian inhibiting substance contributes to sex-linked biases in the brain and behavior
PNAS,
April 28, 2009;
106(17):
7203 - 7208.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. W. Gould and H. Enomoto
Neurotrophic Modulation of Motor Neuron Development
Neuroscientist,
February 1, 2009;
15(1):
105 - 116.
[Abstract]
[PDF]
|
|
|
|
|
|
|
T. W. Gould, S. Yonemura, R. W. Oppenheim, S. Ohmori, and H. Enomoto
The Neurotrophic Effects of Glial Cell Line-Derived Neurotrophic Factor on Spinal Motoneurons Are Restricted to Fusimotor Subtypes
J. Neurosci.,
February 27, 2008;
28(9):
2131 - 2146.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Baudet, E. Pozas, I. Adameyko, E. Andersson, J. Ericson, and P. Ernfors
Retrograde Signaling onto Ret during Motor Nerve Terminal Maturation
J. Neurosci.,
January 23, 2008;
28(4):
963 - 975.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. R. Buss, T. W. Gould, J. Ma, S. Vinsant, D. Prevette, A. Winseck, K. A. Toops, J. A. Hammarback, T. L. Smith, and R. W. Oppenheim
Neuromuscular Development in the Absence of Programmed Cell Death: Phenotypic Alteration of Motoneurons and Muscle
J. Neurosci.,
December 27, 2006;
26(52):
13413 - 13427.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Rothermel, K. Volpert, M. Burghardt, C. Lantzsch, A. A. Robitzki, and P. G. Layer
Knock-Down of GFR{alpha}4 Expression by RNA Interference Affects the Development of Retinal Cell Types in Three-Dimensional Histiotypic Retinal Spheres.
Invest. Ophthalmol. Vis. Sci.,
June 1, 2006;
47(6):
2716 - 2725.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P.-Y. Wang, K. Koishi, A. B. McGeachie, M. Kimber, D. T. MacLaughlin, P. K. Donahoe, and I. S. McLennan
Mullerian Inhibiting Substance acts as a motor neuron survival factor in vitro
PNAS,
November 8, 2005;
102(45):
16421 - 16425.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
G. B. Banks, R. Kanjhan, S. Wiese, M. Kneussel, L. M. Wong, G. O'Sullivan, M. Sendtner, M. C. Bellingham, H. Betz, and P. G. Noakes
Glycinergic and GABAergic Synaptic Activity Differentially Regulate Motoneuron Survival and Skeletal Muscle Innervation
J. Neurosci.,
February 2, 2005;
25(5):
1249 - 1259.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. B. Rind, R. Butowt, and C. S. von Bartheld
Synaptic Targeting of Retrogradely Transported Trophic Factors in Motoneurons: Comparison of Glial Cell Line-Derived Neurotrophic Factor, Brain-Derived Neurotrophic Factor, and Cardiotrophin-1 with Tetanus Toxin
J. Neurosci.,
January 19, 2005;
25(3):
539 - 549.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Jain, C. K. Naughton, M. Yang, A. Strickland, K. Vij, M. Encinas, J. Golden, A. Gupta, R. Heuckeroth, E. M. Johnson Jr, et al.
Mice expressing a dominant-negative Ret mutation phenocopy human Hirschsprung disease and delineate a direct role of Ret in spermatogenesis
Development,
November 1, 2004;
131(21):
5503 - 5513.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Briscoe and D. G. Wilkinson
Establishing neuronal circuitry: Hox genes make the connection
Genes & Dev.,
July 15, 2004;
18(14):
1643 - 1648.
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. M. Myers and L. M. Mulligan
The RET Receptor Is Linked to Stress Response Pathways
Cancer Res.,
July 1, 2004;
64(13):
4453 - 4463.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. R. Arenkiel, P. Tvrdik, G. O. Gaufo, and M. R. Capecchi
Hoxb1 functions in both motoneurons and in tissues of the periphery to establish and maintain the proper neuronal circuitry
Genes & Dev.,
July 1, 2004;
18(13):
1539 - 1552.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. W. Gould and R. W. Oppenheim
The Function of Neurotrophic Factor Receptors Expressed by the Developing Adductor Motor Pool In Vivo
J. Neurosci.,
May 12, 2004;
24(19):
4668 - 4682.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. G. Forger, D. Prevette, O. deLapeyriere, B. de Bovis, S. Wang, P. Bartlett, and R. W. Oppenheim
Cardiotrophin-Like Cytokine/Cytokine-Like Factor 1 is an Essential Trophic Factor for Lumbar and Facial Motoneurons In Vivo
J. Neurosci.,
October 1, 2003;
23(26):
8854 - 8858.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Sun, T. W. Gould, S. Vinsant, D. Prevette, and R. W. Oppenheim
Neuromuscular Development after the Prevention of Naturally Occurring Neuronal Death by Bax Deletion
J. Neurosci.,
August 13, 2003;
23(19):
7298 - 7310.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. P. Rakowicz, C. S. Staples, J. Milbrandt, J. E. Brunstrom, and E. M. Johnson Jr
Glial Cell Line-Derived Neurotrophic Factor Promotes the Survival of Early Postnatal Spinal Motor Neurons in the Lateral and Medial Motor Columns in Slice Culture
J. Neurosci.,
May 15, 2002;
22(10):
3953 - 3962.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C.-Y. Wang, F. Yang, X.-P. He, H.-S. Je, J.-Z. Zhou, K. Eckermann, D. Kawamura, L. Feng, L. Shen, and B. Lu
Regulation of Neuromuscular Synapse Development by Glial Cell Line-derived Neurotrophic Factor and Neurturin
J. Biol. Chem.,
March 15, 2002;
277(12):
10614 - 10625.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. R. Keller-Peck, G. Feng, J. R. Sanes, Q. Yan, J. W. Lichtman, and W. D. Snider
Glial Cell Line-Derived Neurotrophic Factor Administration in Postnatal Life Results in Motor Unit Enlargement and Continuous Synaptic Remodeling at the Neuromuscular Junction
J. Neurosci.,
August 15, 2001;
21(16):
6136 - 6146.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. W. Oppenheim, S. Wiese, D. Prevette, M. Armanini, S. Wang, L. J. Houenou, B. Holtmann, R. Gotz, D. Pennica, and M. Sendtner
Cardiotrophin-1, a Muscle-Derived Cytokine, Is Required for the Survival of Subpopulations of Developing Motoneurons
J. Neurosci.,
February 15, 2001;
21(4):
1283 - 1291.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Garces, G. Haase, M. S. Airaksinen, J. Livet, P. Filippi, and O. deLapeyriere
GFRalpha 1 Is Required for Development of Distinct Subpopulations of Motoneuron
J. Neurosci.,
July 1, 2000;
20(13):
4992 - 5000.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
U. Schweizer, J. Gunnersen, C. Karch, S. Wiese, B. Holtmann, K. Takeda, S. Akira, and M. Sendtner
Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult
J. Cell Biol.,
January 21, 2002;
156(2):
287 - 298.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
Gene
