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Volume 17, Number 24,
Issue of December 15, 1997
Sexual Dimorphism in the Spinal Cord Is Absent in Mice Lacking
the Ciliary Neurotrophic Factor Receptor
Nancy G. Forger1,
Michelle L. Howell1,
Lynn Bengston1,
Laura MacKenzie1,
Thomas M. DeChiara2, and
George D. Yancopoulos2
1 Department of Psychology and Center for
Neuroendocrine Studies, University of Massachusetts, Amherst,
Massachusetts 01003, and 2 Regeneron Pharmaceuticals, 777 Old Saw Mill River Road, Tarrytown, New York 10591
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Ciliary neurotrophic factor (CNTF) has potent survival-promoting
effects on motoneurons in vitro and in
vivo. We examined knockout mice with null mutations of the gene
for either CNTF itself or the  -subunit of the CNTF receptor
(CNTFR) to assess whether CNTF and/or its receptors are involved in
the development of a sexually dimorphic neuromuscular system. Male
rodents have many more motoneurons in the spinal nucleus of the
bulbocavernosus (SNB) than do females. This sex difference is caused by
hormone-regulated death of SNB motoneurons and their target muscles.
Sexual dimorphism of SNB motoneuron number developed completely
normally in CNTF knockout (CNTF  /) mice. In contrast, a sex
difference in the SNB was absent in CNTFR / animals: male mice
lacking a functional CNTF -receptor had fewer than half as many SNB
motoneurons than did wild-type males and no more than did their female
counterparts. Size of the bulbocavernosus and levator ani muscles, the
main targets of SNB motoneurons, was not affected in either CNTF or CNTFR knockout males. These observations suggest that signaling through the CNTF receptor is involved in sexually dimorphic development of SNB motoneuron number and that target muscle survival per se is not
sufficient to ensure motoneuron survival in this system. In addition,
our observations are consistent with the suggestion that CNTF itself is
not the only endogenous ligand for the CNTF receptor. A second, as yet
unknown, ligand may be important for neural development, including
sexually dimorphic motoneuron development.
Key words:
motoneuron;
androgen;
sexual dimorphism;
ciliary
neurotrophic factor;
knockout mice;
androgen
INTRODUCTION
Naturally occurring death of
motoneurons is a widespread phenomenon in the developing vertebrate
nervous system. Approximately one-half of all motoneurons generated in
the spinal cord die during a prenatal period of cell death (Hamburger,
1975 ; Chu-Wang and Oppenheim, 1978; Lance-Jones, 1982; Harris and
McCaig, 1984). Cell death begins after motoneurons have made contact
with their target muscles, and signals from muscles appear to be
important for controlling the magnitude of motoneuron loss (Hamburger,
1975; Oppenheim, 1991). Several recently purified neurotrophic factors can enhance motoneuron survival in developing animals when supplied exogenously, although it is not known which, if any, of these trophic
molecules are the physiologically relevant signals normally regulating
motoneuron death (for review, see Ip and Yancopoulos, 1996; Oppenheim,
1996).
A special case of naturally occurring motoneuron death is seen among
sexually dimorphic motor pools, such as the spinal nucleus of the
bulbocavernosus (SNB) of rodents. The SNB is a cluster of motoneurons
in the lower lumbar spinal cord that innervates striated perineal
muscles including the bulbocavernosus (BC), levator ani (LA), and
external anal sphincter (Schröder, 1980; McKenna and Nadelhaft,
1986). The BC/LA complex wraps around the rectum and the base of the
penis and mediates sexual reflexes such as erection and ejaculation
(Sachs, 1982; Wallach and Hart, 1983).
BC/LA muscles and SNB motoneurons initially form in prenatal rats of
both sexes. The persistence of this neuromuscular system is
androgen-dependent, however, and the muscles and motoneurons degenerate
in females during a perinatal cell death period (Cihak et al., 1970;
Nordeen et al., 1985). As a result, adult male rats have ~4 times as
many SNB motoneurons as do females (Breedlove and Arnold, 1980).
Although this neuromuscular system has been studied most intensively in
rats, a significant sex difference in the number of motoneurons
innervating the bulbocavernosus muscle (male > female) exists in
many mammals including mice, gerbils, hyenas, monkeys, dogs, and humans
(Ueyama et al., 1985; Forger and Breedlove, 1986; Wee and Clemens,
1987; Ulibarri et al., 1995; Forger et al., 1996).
BC/LA muscle size and SNB motoneuron number can be permanently
masculinized in female rats and mice by treating them with the androgen
testosterone during perinatal development (Breedlove and Arnold, 1983;
Nordeen et al., 1985; Wagner and Clemens, 1989a). Testosterone
apparently acts directly at the BC/LA muscles, with sparing of SNB
motoneurons an indirect consequence of hormone action at the muscle
(Fishman and Breedlove, 1988; Fishman et al., 1990; Freeman et al.,
1997; Jordan et al., 1997). We have reported recently that ciliary
neurotrophic factor (CNTF) administered to perinatal female rats can
also slow or prevent SNB motoneuron death (Forger et al., 1993;
Bengston et al., 1996). Moreover, CNTF receptors are expressed by both
SNB motoneurons and their target muscles (Forger et al., 1997; Xu and
Forger, 1997). This raises the possibility that some effects of
testosterone on the SNB system may be mediated via a neurotrophic
factor such as CNTF. To determine whether CNTF or the -component of
its receptor, CNTFR, is normally involved in sexually dimorphic
development of the SNB, we have examined SNB motoneuron number and
BC/LA muscle size in CNTF and CNTFR +/+ and / mice.
MATERIALS AND METHODS
Animals. CNTF knockout mice (CNTF /) and their
wild-type siblings (CNTF +/+) were generously provided by Dr. Hans
Thoenen (Max-Planck Institute). CNTF gene expression was abolished in 129/SV-C57BL/6 mice by homologous recombination as described previously (Masu et al., 1993). CNTF / mice develop normally and exhibit only
modest neuromuscular deficits in adulthood (Masu et al., 1993). In the
present study, adult mice, 3-21 months of age, were examined.
CNTF acts through a three-part, cell surface receptor complex
consisting of a ligand-binding component (CNTFR) and the signal
transducing components LIFR and gp130 (Davis et al., 1993a; Stahl
and Yancopoulos, 1994). Expression of CNTFR is restricted primarily
to cells of the nervous system and to striated muscle, and expression
of CNTFR is required for direct CNTF action (Ip et al., 1993). Mice
heterozygous for a mutation of CNTFR (CNTFR +/) were generated
as a cross between 129 and C57BL/6 strains (DeChiara et al., 1995). The
progeny of heterozygous matings are ~25% wild-type (CNTFR +/+),
50% heterozygous (CNTFR +/), and 25% homozygous knockouts
(CNTFR /). Only wild-type and homozygous knockouts were examined
in the present study, except where indicated. Although apparently
normal in size and appearance at birth, mice lacking expression of
CNTFR fail to suckle and die within 24 hr (DeChiara et al., 1995).
All CNTFR +/+ and / mice in the present study, therefore, were
killed on postnatal day 1. The sex of each pup was confirmed by
inspection of the gonads, and the CNTFR mutant genotype was
determined by Southern blotting of tail DNA as in DeChiara et al.
(1995).
Motoneuron counts. Motoneurons were counted in the three
major motor pools of the lower lumbar spinal cord: the SNB, the
dorsolateral nucleus (DLN), and the retrodorsolateral nucleus (RDLN).
The SNB and DLN innervate sexually dimorphic muscles associated with
the penis, and both of these nuclei undergo androgen-regulated cell death during perinatal development. The RDLN innervates primarily muscles of the foot, which are present and similar in both sexes. RDLN
motoneuron number is not sexually dimorphic or altered by early hormone
treatments (Jordan et al., 1982). In rats, SNB motoneurons reside in a
relatively tight cluster just below the central canal. As has been
reported previously for house mice (Wagner and Clemens, 1989b), we
found that many SNB motoneurons in the mouse strains used in this study
extended more ventrally and ventro-laterally along the gray-white
border of the medial ventral horn. Retrograde tracing (see below)
confirmed that these motoneurons innervate the perineal muscles, and we
therefore included these cells in our counts of the SNB.
Thirty-two adult CNTF +/+ and / mice were perfused and post-fixed
with 4% paraformaldehyde in PBS. The lumbosacral portion of the spinal
cord was removed, cryoprotected by soaking in a 10% sucrose solution
overnight, and frozen-sectioned in the coronal plane at a thickness of
40 µm. Alternate sections were mounted on separate slides and stained
with thionin. SNB motoneurons were counted bilaterally, and the RDLN
was counted unilaterally, in alternate sections. Only those motoneurons
in which the nucleus was visible were included in the counts. SNB raw
counts were multiplied by 2 and RDLN counts were multiplied by 4 to
estimate the total bilateral number of motoneurons in each pool. SNB
motoneuron size was determined for all CNTF +/+ and / animals; cell
size in the RDLN was determined for 14 randomly chosen CNTF / and
wild-type males. The soma and nucleus of all SNB and RDLN motoneurons
were traced by camera lucida from four sections equally spaced
throughout the rostro-caudal extent of each cell group. Cross-sectional
areas were determined from the tracings using a digitizing pad linked to a computer.
Thirty-six CNTFR +/+ and / newborns were decapitated and
immersion fixed in Bouin's solution for at least 2 d. The
lumbosacral spinal cords were then embedded in paraffin,
cross-sectioned at 12 µm, and stained with thionin. Motoneuron number
in the SNB and RDLN of all mice was counted bilaterally in alternate
sections. We later counted the DLN bilaterally in alternate sections in a cohort of 14 mice (see Results). Only cells in which both a nucleus
and a nucleolus were clearly present were included in the motoneuron
counts of newborns. The size of the somata, nuclei, and nucleoli of SNB
motoneurons was determined for 20 randomly selected CNTFR +/+ and
/ animals, as described above. All motoneuron counts and cell size
measurements were performed on slides coded to conceal the subject's
sex and genotype.
Determination of muscle size. The BC/LA muscle complex was
dissected out of adult CNTF +/+ and / males, trimmed, and weighed by an individual blind to the mutant genotype of the animal. Size of
the BC/LA muscle complex of CNTFR +/+ and / newborns of both
sexes was determined from serial sections through the perineum. The
perineal region was embedded in paraffin and cut in cross section with
respect to the rectum at a thickness of 7 µm. Sections were stained
with trichrome (modified Gomori's method), and camera lucida tracings
of the LA and BC muscles were made from every 8th section. Muscle
cross-sectional areas were determined on a digitizing pad linked to a
computer, and cross-sectional areas throughout the extent of both
muscles were summed to determine total BC and LA size in each
animal.
Retrograde labeling of motoneurons. Cholera toxin conjugated
to horseradish peroxidase [CT-HRP; 1 µl of a 0.2% (w/v) aqueous solution, List Biological Labs] was injected into the BC muscles, or
was simply injected subcutaneously in the region of the BC/LA and
ischiocavernosus muscles, of newborn CNTFR +/+, CNTFR +/, and
CNTFR / mice. In several cases, a second, 0.5 µl, injection was made into the flexor digitorum brevis muscle of the foot. Pups were
returned to dams and, after a 4.5-6.0 hr survival time, overdosed with
chlorapent anesthesia. A tail sample was taken for Southern blot
analysis of the CNTFR genotype, and the animals were perfused with
PBS followed by 1% paraformaldehyde/1.25% glutaraldehyde. Spinal
cords were removed, post-fixed in paraformaldehyde/glutaraldehyde for 3 hr, and then immersed in 10% sucrose overnight. Cords were frozen-sectioned at 30 µm, and free-floating sections were incubated with tetramethylbenzidine according to the method of Mesulam
(1978).
Statistical analyses. Means ± SEM are presented
throughout. Motoneuron number in each motor pool (SNB, DLN, RDLN) was
analyzed by two-way ANOVA (sex by knockout status). Mass of the BC/LA
muscle complex in male CNTF +/+ and / males, as well as BC and LA
muscle sizes of CNTFR +/+ and / animals, was compared by
independent two-tailed t tests. Differences in motoneuron
size between groups were evaluated in two ways. First, in accord with
all previously published papers on the SNB neuromuscular system, the
mean motoneuron size in the RDLN and SNB of each animal was determined,
and for the subsequent ANOVA, n = number of animals.
The means and SEs reported in Table 1
have been calculated in this way. Next, to compare our results with
previous studies on CNTF and CNTFR knockout mice (Masu et al., 1993;
DeChiara et al., 1995), we analyzed cell size data with the individual
motoneuron as the unit of measure, and n = number of motoneurons traced. Correlations between age and motoneuron
number were determined by Pearson's correlation coefficient.
RESULTS
Motoneuron number is not affected by deletion of the CNTF gene
The number of SNB motoneurons in CNTF +/+ and / mice is shown
in Figure 1A. As
expected, wild-type males had many more SNB cells than did females
(p < 0.0001). The normal sex difference in SNB
motoneuron number was also observed in the CNTF / animals (p < 0.0001). Thus, deletion of the CNTF gene
did not affect the development of sexual dimorphism in the SNB. There
was no main effect of the gene disruption on SNB motoneuron number, nor
was there a sex × genotype interaction (p > 0.50 in both cases). In other words, deletion of the CNTF gene did
not affect SNB cell number when sex was ignored as a variable, and the
gene deletion was equally without effect in males and females.
Fig. 1.
Motoneuron counts in adult CNTF +/+ and / mice
of both sexes. A, The number of motoneurons in the SNB
is sexually dimorphic in both wild-type and knockout animals
(*p < 0.0001), and there is no effect of the gene
knockout on the magnitude of the sex difference. B,
There is no effect of sex or of genotype on the number of motoneurons
in the RDLN.
[View Larger Version of this Image (23K GIF file)]
Motoneuron counts in the RDLN exhibited no sex difference, no effect of
the CNTF gene knockout, and no sex × genotype interaction (Fig.
2B). There was a trend
toward slightly lower RDLN cell counts in females of both genotypes,
but this was not statistically significant (p = 0.12). Masu et al. (1993) have reported that the number of motoneurons
in the brainstem facial nucleus of CNTF / mice declines with age
and that by 28 weeks of age CNTF / mice exhibit a statistically significant reduction in motoneuron number. The mice in the present study ranged from 3 to 21 months of age, and mean age did not differ
between the CNTF / and CNTF +/+ groups (CNTF +/+: 16.8 ± 1.2 months; CNTF /: 14.6 months ± 1.7 months; p > 0.25). Confirming the observation that motoneuron number declines
with age in CNTF / mice (Masu et al., 1993), age was negatively
correlated with motoneuron number in the RDLN and SNB of CNTF /
mice, although this was statistically significant only for the SNB
(RDLN: r = 0.368, p = 0.15; SNB:
r = 0.552, p < 0.03). However, when
analysis of motoneuron number is restricted to animals over 6 months of age the pattern of results was the same as that described above, i.e.,
there was no significant effect of the CNTF gene deletion on motoneuron
number in the RDLN or SNB (p > 0.80 in both
cases).
Fig. 2.
Motoneuron counts in CNTFR +/+ and / mice
of both sexes. A, SNB motoneuron number is sexually
dimorphic in newborn wild-type mice (*p < 0.001),
but CNTFR / males have no more SNB motoneurons than do / or
+/+ females. B, The number of RDLN motoneurons was
reduced by ~20% in both knockout males and females
(p < 0.0001 for main effect of the gene
knockout). C, The ratio SNB motoneuron number to RDLN
motoneuron number was significantly greater in wild-type males than in
CNTFR / males or in females (*p < 0.01 in
each case). Knockout males and females did not differ on this measure.
[View Larger Version of this Image (16K GIF file)]
Motoneuron and muscle size is not affected in CNTF
knockout mice
As expected from previous studies (Breedlove and Arnold, 1981;
Wagner and Clemens, 1989a), adult males had larger SNB somata (p < 0.005) and nuclei
(p < 0.05) than did females (somata males: 654 ± 18 µm2; somata females: 543 ± 32 µm2; nuclei males: 216 ± 9 µm2; nuclei females: 180 ± 13 µm2). However, there was no effect of the CNTF
gene deletion on the size of motoneuronal somata or nuclei in the SNB
(p > 0.70 in both cases; data not shown).
Similarly, RDLN soma size was not different in CNTF / and CNTF +/+
animals (616 ±49 versus 618 ± 59 µm2,
respectively), nor was there an effect of the gene knockout on mean
RDLN nuclear size (206 ± 26 and 199 + 37 µm2
for / and +/+ males, respectively). This was true regardless of
which of the two methods was used for cell size analysis (see Materials
and Methods). Thus, our observations in the SNB and RDLN differ from
the previous report of a decrease in soma size, and a transient
increase in cell nuclear size, in spinal motoneurons of CNTF / mice
(Masu et al., 1993).
The BC/LA muscle complex was compared in 7 CNTF +/+ and 13 CNTF /
animals. The muscles were well developed and appeared grossly normal in
both groups; BC/LA wet weight was not significantly different (CNTF
+/+: 151 ± 12 gm; CNTF /: 140 ± 7 gm; p = 0.40).
Deletion of CNTFR eliminates the sex difference in SNB
motoneuron number
Motoneuron number in CNTFR +/+ and / animals was analyzed
in two separate cohorts of animals. The pattern of results was the
same, and the data from these two replications have been combined in
the following discussion. SNB cell number was sexually dimorphic on the
day of birth in CNTFR +/+ mice, with males possessing 62% more
motoneurons than females (p < 0.001) (Fig.
2A). The magnitude of this sex difference is not as
large as was observed in the adult mice described above, presumably
because females continue to lose motoneurons in the SNB during the
first several postnatal days (Nordeen et al., 1985; Wagner and Clemens,
1989a). In contrast, there was no sex difference in CNTFR /
newborns. Male CNTFR / mice had fewer than half as many SNB
cells than did wild-type males (p < 0.0001) and
did not differ from CNTFR / females on this measure
(p > 0.20) (Fig. 2A).
There was no effect of sex on the number of RDLN motoneurons of
CNTFR +/+ and / animals (p > 0.10).
Motoneuron number in the RDLN was reduced by ~20% in knockout mice
of both sexes (Fig. 2B) (p < 0.0001), in agreement with the previous report of a significant reduction in the number of motoneurons throughout the lumbar spinal cord in newborn CNTFR / animals (DeChiara et al., 1995). We reasoned that if the reduction in SNB motoneuron number seen in CNTFR / males was caused simply by generalized motoneuron death in CNTFR knockouts, then the mean ratio of SNB motoneuron number to
RDLN motoneuron number would be the same in the knockout and wild-type
animals. This was in fact true for CNTFR +/+ and / females
(p > 0.40) (Fig. 2C). However,
reductions in SNB cell number of CNTFR / males were
not proportional to reductions in the RDLN, and the SNB/RDLN
ratio was significantly lower in CNTFR / males than in CNTFR
+/+ males (p = 0.001). The SNB/RDLN ratio of
knockout males did not differ significantly from that of wild-type or
knockout females.
Motoneuron size in CNTFR +/+ and / mice
The mean size of SNB motoneurons was determined for five animals
in each of the four groups (see Table 1). There was no significant effect of sex or knockout status on soma, nuclear, or nucleolar size
when the mean cell size of each animal was used as the unit of
analysis. Using individual motoneurons as the unit of analysis, DeChiara et al. (1995) observed a reduction in the soma size of spinal
motoneurons in CNTFR / animals on postnatal day 1. When the
present data are analyzed as in DeChiara et al. (1995), the 7.5%
reduction in SNB soma size of CNTFR / males relative to their
wild-type counterparts is statistically significant
(p < 0.05), although we do not see a similar
effect in females.
BC/LA size is normal in CNTFR / males, but the LA is
reduced in CNTFR / females
Size of the BC and LA muscles, targets of SNB motoneurons, was
determined from sections through the perineal area of 8 males and 13 females (Fig. 3). There was no difference
in LA or BC muscle size between CNTFR wild-type and / males
(p > 0.50 in both cases) (Figs 3,
4A). Thus, the BC/LA
muscles of knockout males appear to develop normally, despite the fact
that motoneuron number is greatly reduced in the SNB of these animals.
The BC and LA muscles were much smaller in females than in males (Fig.
4; note the difference in scale on the ordinates in A and
B), and very little BC muscle could be identified
definitively in any female. Size of the BC did not differ between
CNTFR +/+ and / females (p > 0.50),
whereas the LA muscle was significantly larger in CNTFR +/+ than in
CNTFR / females (Fig. 4B)
(p < 0.005).
Fig. 3.
Cross sections through the perineums of CNTFR
+/+ (A) and CNTFR /
(B) males. These sections were taken from mice
with overall LA and BC muscle sizes close to their group means and are
the single section from each animal with the greatest area of combined
BC and LA. Dorsal is down. P, Base of
penis; rec, rectum; LA, levator ani;
BC, bulbocavernosus. Scale bar, 300 µm.
[View Larger Version of this Image (150K GIF file)]
Fig. 4.
Size of the BC and LA muscles in CNTFR +/+ and
/ animals. A, There was no effect of the gene
knockout on the size of the LA or BC of males. B, The LA
was significantly larger in CNTFR +/+ than in / females
(*p < 0.005).
[View Larger Version of this Image (18K GIF file)]
CT-HRP labels the same motor pools in CNTFR +/+, +/, and
/ animals
We next asked whether SNB motoneurons of CNTFR / males are
in contact with the BC/LA muscles and whether motoneurons innervating the BC/LA are restricted to the expected SNB position. Injections of
CT-HRP into the perineal muscles labeled motoneurons in both the SNB
and the DLN, with the DLN exhibiting particularly heavy labeling (Fig.
5). This pattern of CT-HRP accumulation
presumably reflects uptake by motor nerve terminals in the BC/LA and
ischiocavernosus (IC) muscles, because the BC/LA and IC are immediately
adjacent to one another and together form a complex of muscles attached to the base of the penis. The pattern of labeled motoneurons was the
same for CNTFR +/+, +/, and / animals. In no case did we
observe an anomalous projection to the perineal muscles in the
knockouts. Injections of CT-HRP into the flexor digitorum brevis
labeled motoneurons in the ipsilateral RDLN and, in some cases, nearly
all RDLN motoneurons were labeled (Fig. 5A). Thus, as in the
rat, the RDLN of mice appears to innervate primarily intrinsic muscles
of the foot.
Fig. 5.
Photomicrographs of the ventral horn of the spinal
cord of CNTFR +/+ (A) and CNTFR /
(B) male mice, demonstrating retrograde labeling
of motoneurons. CT-HRP was injected on the day of birth, and animals
were killed 6 hr later. Injections were made into the perineums of both
animals, labeling motoneurons in the DLN and
SNB. The flexor digitorum brevis of the right foot of
the CNTFR +/+ male was also injected, resulting in the labeling of motoneurons in the ipsilateral RDLN
(A). Dorsal is up.
cc, Central canal. Scale bar, 250 µm.
[View Larger Version of this Image (85K GIF file)]
The "missing" SNB motoneurons in CNTFR / males are not
in the DLN
In the rat, motoneurons that eventually form the SNB originate in
the region of the DLN. They then undergo a late, secondary migration
into the more medial, SNB position (Sengelaub and Arnold, 1986). Under
certain perinatal hormone conditions, the BC/LA muscles can become
innervated by motoneurons in the DLN (Breedlove, 1985; Sengelaub and
Arnold, 1989). Because of the common developmental history of SNB and
DLN motoneurons, and the robust labeling of the DLN after perineal
CT-HRP injections observed above, we evaluated the possibility that the
"missing" SNB motoneurons of CNTFR / males might be located
in the DLN. However, counts of the DLN in thionin-stained sections
reveal that CNTFR / males have significantly fewer DLN
motoneurons than CNTFR +/+ males (206 ± 30 vs 310 ± 25;
p < 0.02) and have no more DLN motoneurons than CNTFR / or +/+ females (238 ± 22 and 232 ± 23, respectively; p > 0.40 in both cases). Thus, the
pattern of motoneuron number in the SNB and DLN, two androgen-dependent
cell groups, is the same. The reduction of SNB cell number in CNTFR
/ males cannot be explained by the inability of motoneurons to
migrate from the DLN.
DISCUSSION
The sexually dimorphic SNB neuromuscular system appeared
completely normal in mice lacking CNTF. In contrast, the expected sex
difference in SNB motoneuron number was not seen in newborn mice
lacking the CNTF receptor. Normal, sexually dimorphic
development of the BC and LA target muscles was observed in both CNTF
and CNTFR / males. These observations have at least three
implications for understanding the development of sexual dimorphism in
this system: (1) the maintenance of elevated numbers of SNB motoneurons in male mice depends on a functional CNTFR; (2) the survival of the
BC/LA target muscles is not sufficient to ensure maintenance of SNB
motoneurons; and (3) CNTF itself is not the relevant ligand for CNTF
receptor-dependent development of sexual dimorphism in the SNB. Each of
these points will be considered in turn below.
Fewer SNB motoneurons were present in CNTFR / males than in
CNTFR +/+ males, and this difference greatly exceeded generalized motoneuron loss in the knockouts. The most straightforward explanation of our results is that many SNB cells of males died in the absence of
CNTFR. Because the survival of SNB motoneurons is normally regulated
by testosterone, these results indicate that hormone effects on SNB
cell death could involve a pathway that requires signaling through the
CNTF receptor. This might be true if, for example, testosterone
normally maintains the production of a CNTF-like trophic molecule from
BC/LA muscles; in the absence of CNTF receptors on SNB nerve terminals,
the normal response to testosterone then would be blocked. CNTF
receptors also might be required to mediate the reception of trophic
support from neural afferents to SNB motoneurons.
Alternatively, it could be argued that as many SNB motoneurons survive
in CNTFR / males as in +/+ males, but that many of the SNB cells
in the knockouts are not distinguishable as motoneurons and, therefore,
were missed in our counts. This might be true if, for example, SNB
motoneurons of knockout males were severely shrunken or altered in
phenotype. We might also fail to accurately assess SNB cell number if
deletion of the CNTFR gene affected motoneuronal migration and the
motoneurons were not in their usual location within the spinal cord.
These explanations seem unlikely because those SNB motoneurons that
were counted in knockout males were 93% as large as those in wild-type
animals. In addition, CT-HRP injections into the perineum retrogradely
labeled motoneurons in the SNB and DLN in both the CNTFR +/+ and the
/ mice. At least within the confines of the lower lumbar cord, we
did not observe labeled motoneurons in an anomalous position, nor was there evidence of greater numbers of motoneurons in the DLN of knockout
males, which might indicate an effect on migration.
On the other hand, we cannot rule out the possibility that fewer SNB
motoneurons were initially generated in CNTFR / animals. It will
be of interest in future experiments to examine fetal mice to determine
whether the same number of SNB and DLN motoneurons are present
before the cell death period in CNTFR / and wild-type animals.
Previous studies have established that manipulations that rescue the
developing BC/LA muscles generally also spare SNB motoneurons. Treatment of female rats or mice with testosterone around the time of
birth leads to permanent masculinization of BC/LA muscle size and a
concomitant masculinization of SNB motoneuron number (Cihak et al.,
1970; Breedlove and Arnold, 1983; Wagner and Clemens, 1989a). Mutations
that render the androgen receptor nonfunctional result in the absence
of BC muscles and a feminine number of SNB motoneurons in affected male
rats and mice (Breedlove and Arnold, 1981; Olsen et al., 1988).
Finally, in perinatal female rats, injections of CNTF prevent the death
of both the SNB motoneurons and their target muscles (Forger et al.,
1993, 1995). In each case, rescue of BC/LA muscles is accompanied by
rescue of SNB motoneurons, and it is not clear whether muscle
persistence per se is sufficient for SNB motoneuron survival or whether
specific signals from the BC/LA muscles are required for the sparing of SNB motoneurons.
In the present study, the BC/LA muscles of CNTFR / males were
indistinguishable in size and appearance from those of wild-type controls, yet fewer than half as many SNB motoneurons were observed in
the mutants. At least some of those SNB motoneurons present on
postnatal day 1 in CNTFR / mice were in contact with the perineal muscles, as demonstrated by the ability to label them retrogradely by CT-HRP injections delivered to the perineum. CNTFR / mice do not survive beyond postnatal day 1, and it is possible that the BC/LA muscle would eventually degenerate in the knockout males
if the animals' survival could be prolonged. The BC/LA muscles of
CNTFR / males also may have subtle defects in morphology or
physiology not evident from a relatively crude analysis of muscle size.
Nonetheless, it seems clear that the simple presence of the BC/LA
target muscles does not ensure normal SNB development. Rather,
specific signals from the BC/LA muscles apparently are required for SNB
motoneuron survival, and the CNTF receptor may play a necessary part in
the signaling cascade.
Although targeted disruption of the CNTFR gene did not affect
development of the BC/LA muscles in male mice, the size of the LA was
significantly reduced in CNTFR / females. This suggests a role
for CNTFR in development of the female LA. Possibly, signaling through the CNTF receptor ameliorates the atrophy of the
androgen-dependent LA muscle when androgen levels are low, as in
perinatal females; but CNTFR is not required for normal LA
development provided that androgen levels are adequately high. It is
not clear whether CNTFR might also affect the timing of BC muscle
degeneration, because even in wild-type females the BC was nearly
absent on the day of birth.
CNTFR expression in skeletal muscle is markedly upregulated after
denervation (Davis et al., 1993b), and administration of CNTF to adult
rats can slow the muscle atrophy induced by denervation (Helgren et
al., 1994). CNTF also prevents the atrophy of the BC/LA muscles that
normally occurs in perinatal female rats (Forger et al., 1993), and
CNTFR message is upregulated in the perinatal BC/LA after androgen
blockade (Xu and Forger, 1997). These findings suggest that CNTF
receptor expression in striated muscles plays an important trophic role
in times of muscle atrophy, such as after denervation or during
androgen deprivation of androgen-dependent muscles. In normal,
innervated striated muscle, however, CNTF can actually promote
catabolism (Henderson et al., 1994; Martin et al., 1996). The reason
for these different effects is not at all clear.
Several observations have led to the suggestion that CNTF itself is not
a developmentally relevant ligand for the CNTF receptor, but that
another, as yet unidentified ligand for CNTFR exists and regulates
motoneuron survival in development. The most compelling evidence for
this view is that deletion of the CNTF gene in mice does not cause
measurable motor defects until well into adulthood, whereas the
deletion of CNTFR results in a significant reduction of motoneuron
number at birth and early postnatal mortality (Masu et al., 1993;
DeChiara et al., 1995). The fact that CNTF lacks a classical signal
sequence calls into question whether CNTF can even be secreted as a
target-derived trophic factor (Lin et al., 1989; Stöckli et al.,
1989) and has led to the suggestion that CNTF may instead have a role
in the response to neural injury (Sendtner et al., 1992). In the
present study, deletion of the CNTFR gene had a detrimental effect
on SNB motoneuron number in male mice, whereas deleting the gene for
CNTF did not. These findings support the existence of a second ligand
for CNTFR and indicate an involvement of this second ligand in the
sexually dimorphic development of perineal motoneurons.
FOOTNOTES
Received July 15, 1997; revised Sept. 15, 1997; accepted Sept. 26, 1997.
This work was supported by National Institutes of Health Grant HD33044
and the Whitehall Foundation. We thank Anthony Lucarelli for excellent
technical assistance and Elizabeth Connor for helpful comments on an
earlier version of this manuscript.
Correspondence should be addressed to Nancy G. Forger, Department of
Psychology, University of Massachusetts, Amherst, MA 01003-7710.
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