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Mu¨ llerian inhibiting substance contributes to
sex-linked biases in the brain and behavior
Pei-Yu Wang, Anna Protheroe, Andrew N. Clarkson, Floriane Imhoff, Kyoko Koishi, and Ian S. McLennan
1
Department of Anatomy, University of Otago, Dunedin, New Zealand
Communicated by Patricia K. Donahoe, Massachusetts General Hospital, Boston, MA, March 2, 2009 (received for review October 29, 2008)
Many behavioral traits and most brain disorders are common to
males and females but are more evident in one sex than the other.
The control of these subtle sex-linked biases is largely unstudied
and has been presumed to mirror that of the highly dimorphic
reproductive nuclei. Sexual dimorphism in the reproductive tract is
a product of Mu¨ llerian inhibiting substance (MIS), as well as the sex
steroids. Males with a genetic deficiency in MIS signaling are
sexually males, leading to the presumption that MIS is not a neural
regulator. We challenge this presumption by reporting that most
immature neurons in mice express the MIS-specific receptor
(MISRII) and that male Mis
ⴚ/ⴚ
and Misrii
ⴚ/ⴚ
mice exhibit subtle
feminization of their spinal motor neurons and of their exploratory
behavior. Consequently, MIS may be a broad regulator of the
subtle sex-linked biases in the nervous system.
anti-Mu¨ llerian hormone 兩exploratory behavior 兩motor neuron 兩
sexual dimorphism
Many behavioral traits are more evident in one sex than the
other, but they do not define the biological sex of an
individual. Empathy, for example, has a female bias, but some of
the greatest men are empathetic. Likewise, girls engage in less
rough-and-tumble play than boys, but a boy who shuns rough-
and-tumble play is still a boy. With traits such as these, the
characteristics of individual males and females overlap, with the
sex-linked bias existing only within the population as a whole.
This subtle form of sexual dimorphism is pervasive throughout
the nervous system and involves nuclei whose function is not
directly related to reproduction. In this paper, we refer to this
form of sexual dimorphism as sex-linked bias, to distinguish it
from the sexual dimorphism of the primary reproductive tissues,
which is binary in nature.
The nervous system also contains nuclei where the male and
female forms do not overlap. Such nuclei control primary sexual
function, with their dimorphic nature being generated by tes-
tosterone, or its active metabolites (1). The regulation of sex-
linked bias in the brain has been widely assumed to mirror that
of the sexually dimorphic nuclei. However, testosterone is only
episodically present during male development, with third-
trimester male fetuses and boys older than 1 year having minimal
levels of testosterone (2–4). Extensive brain development occurs
during these periods, with some regions having increased ana-
tomical dimorphism during the period when testosterone is
minimal (5, 6). Thus, although the sex steroids contribute to
sex-linked biases, other factors must also be important. We
present evidence here that Mu¨llerian inhibiting substance (MIS)
(synonym, anti-Mu¨llerian hormone) is one of these factors.
MIS is a testicular hormone that triggers the regression of the
uterine precursor (Mu¨llerian ducts) in males around the ninth
week of gestation (7). However, the level of MIS in the blood of
males does not decline after the loss of the Mu¨llerian ducts,
remaining high until puberty, at which stage it falls by an order
of magnitude (8–10). The low level of ovarian production of
MIS, in contrast, begins only postnatally (8 –10). High levels of
plasma MIS are thus a unique feature of developing males. This
hormonal MIS may affect the maturation of the fetal lung (11),
but there is currently no explanation for why MIS is present in
male blood for such an extended period of development. We
report here that most developing neurons express the MIS-
specific receptor, MISRII, and that male mice with null muta-
tions in either Mis or Misrii have partial feminization of their
sex-linked biases, even though they are phenotypically and
sexually male. This finding implicates MIS as a regulator of
sex-linked bias.
Results
The Testes Is the Sole Embryonic Source of MIS. The gonads have
historically been considered to be the sole source of MIS, but we
have recently shown that mature motor neurons in both sexes
also produce it (12). We therefore determined whether produc-
tion of MIS also occurred in the developing brain. In confirma-
tion of the historic understanding, only trace levels of MIS
mRNA were detected in the embryonic head, which by mass is
predominantly the brain. The only proportion of the embryo that
contained significant levels of MIS mRNA was the urogenital
region (Fig. S1A). Similarly, MIS protein was detected by
immunohistochemistry in the testes (Fig. S1B), but not in the
developing brain (Fig. S1C). This finding contrasts with the
mature brain, where motor (12) and various other neurons
contain readily detectable levels of MIS.
MIS Regulates the Number of Spinal Motor Neurons. The numbers of
motor neurons that innervate the limbs have been extensively
studied, but the possibility that they exhibit subtle sex-linked bias
has only rarely been considered (13). Our recent observation
that MIS promotes the survival of embr yonic motor neurons in
vitro (12) suggests that they should be. Simply, over one-half of
the motor neurons present in the spinal cords of mouse embryos
die between the 13th day of gestation and birth (14), with the
extent of this cell death being controlled by multiple survival
factors. MIS is produced in the embryonic testes from the 12th
day of gestation onward (15), which should attenuate motor
neuron loss in male fetuses, provided that MIS is a neuronal
survival factor in vivo. Consistent with this prediction, the
number of motor neurons in the lumbar lateral motor column
(LMC) of wild-type male and female mice had almost no overlap
in both the Mis and Misrii colonies (Fig. 1A). The magnitude of
the male bias was 16–17% in both colonies. This sex-linked
difference depended on MIS, because MIS-deficient (Mis
⫺/⫺
)
males had female numbers of motor neurons (Fig. 1B), with
heterozygous males (Mis
⫹/⫺
) being intermediate between wild-
type males and females (Fig. 1B). The effect of Mis gene dose on
motor neuron number was significant (P⫽0.03) by regression
analysis.
Author contributions: P.-Y.W., A.P., A.N.C., F.I., K.K., and I.S.M. designed research; P.-Y.W.,
A.P., A.N.C., F.I., and K.K. performed research; I.S.M. analyzed data; and I.S.M. wrote the
paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed at: Department of Anatomy, University of
Otago, P.O. Box 913, Dunedin, New Zealand. E-mail: ian.mclennan@stonebow.
otago.ac.nz.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0902253106/DCSupplemental.
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MIS is a member of the TGF

superfamily, which signals
through a complex consisting of type I and type II receptors.
MISRII is thought to be a unique and obligatory receptor for
MIS, because null mutations of Mis and Misrii both lead to
persistent Mu¨llerian duct syndrome (retention of the uterine
precursor in males) (7). MISRII mRNA in neurons is abundant
compared with the mRNA for other growth factor receptors but
is an order of magnitude less abundant than that present within
the classical MIS targets (Mu¨llerian ducts, testes) (12, 16). This
observation raises the issue of whether MISRII is essential for
MIS signaling in neurons. Consistent with this possibility, male
Misrii
⫺/⫺
mice had feminized numbers of LMC motor neurons
(Fig. 1C), a malformation that mirrored that seen in the Mis
⫺/⫺
male mice (Fig. 1B). Female Mis
⫺/⫺
and Misrii
⫺/⫺
mice both had
wild-type numbers of LMC motor neurons (Fig. 1 Band C),
which was expected given that female embryos do not produce
MIS (15, 17). Consequently, MISRII is a useful marker for
MIS-sensitive neurons (see below).
Androgen-Dependent Dimorphism Is Independent of MIS. The motor
neurons involved in primary sexual function are highly dimor-
phic, under the control of testosterone acting through the
androgen receptor (18, 19). Some LMC motor neurons express
androgen receptors (18), creating the possibility that the effect
of MIS on LMC neurons is secondary to perturbation of
testosterone function. If this was occurring, then testosterone-
dependent dimorphism should be diminished in the Mis
⫺/⫺
mice.
We therefore examined the spinal bulbocavernosus motor nu-
cleus, which is involved in the function of the penis (18, 19). In
marked contrast to the LMC motor neurons, the numbers of
motor neurons in the bulbocavernosus nucleus of male Mis
⫺/⫺
mice were similar to those of a wild-type male and were overtly
different from the female number (Fig. 2).
Most Neurons Express MIS Receptors. All of the classical neuronal
survival factors affect multiple types of neurons. We therefore
examined whether MIS was a specific regulator of limb-innervating
motor neurons or a broad regulator of the nervous system. Con-
sistent with the latter, the levels of mRNA for the MIS-specific
receptor in the embryonic brain and spinal cord were similar. The
cellular lineages that produce MISRII were identified by using
genetically modified mice that produce lacZ in MISRII-expressing
cells (MISRII-Cre-lacZ) (Fig. S2). Most neurons in the brain, spinal
cord, and peripheral nervous system of adult MISRII-Cre-lacZ
mice were lacZ-positive, whereas the glia, blood vessels, and
fibroblasts were lacZ-negative (Fig. 3 A–C). Once lacZ is induced
in a cell in the MISRII-Cre-lacZ mice, all daughter cells will express
it (Fig. S2). The immediate precursor of neurons (neuroepithelia)
undergo a series of stem cell-like divisions, which give rise to
postmitotic neurons during the early stages of the lineage and
various glia in the terminal divisions. The absence of lacZ in the glia
thus indicates that postmitotic neurons are the only cells within the
neuronal lineage to express significant levels of MISRII. This
contention was verified by examining the brains of MISRII-Cre-
lacZ embryos. The neuroepithelium of the spinal cord and brain
were lacZ-negative, whereas lacZ-positive neurons were abundant
in the brains of embryonic day 12 (E12) MISRII-Cre-lacZ embr yos.
This finding indicates that lacZ reporter is activated within 24 h of
neurons becoming postmitotic and beginning to differentiate.
Sections of embryonic brains were then stained with an
antibody to MISRII to determine whether the expression of
MISRII in neurons is continuous during development and
whether all developing neurons expressed MISRII at a similar
level. The antibody is specific to MISRII as it immunoprecipi-
tates a single protein from the spinal cord (12) and does not stain
sections of Misrii
⫺/⫺
brains (Fig. 3G). The anti-MISRII antibody
stained most neurons in the developing brain and spinal cord but
with differing intensities. The motor neurons in the spinal cord,
for example, were more intensely stained than adjacent inter-
neurons, although this may merely be because motor neurons are
comparatively large and are the most mature neurons in the
developing brain. This differential staining of motor and inter-
neurons persisted throughout development and is also evident in
the mature spinal cord (12). The staining of neurons in the
ventral spinal cord preceded that of the dorsal cord, with dorsal
spinal neurons having only trace levels of MISRII protein at
E12 (Fig. 3D). This dorsal–ventral difference was lost by E14
(Fig. 3E).
Exploratory Behavior Is MIS Dependent. We then examined whether
the expression of MISRII in the brain leads to a male bias in a
Fig. 1. The LMC of the lumbar spinal cord has MIS-dependent dimorphism. (A) Number of motor neurons in male (blue) and female (pink) wild-type mice derived
from matings of either Mis⫹/⫺(circles) or Misrii⫹/⫺parents (squares). (Band C) Effect of Mis (B)orMisrii (C) genotype on the sexual dimorphism illustrated in A.
The bars are the mean ⫾standard error of the mean of 6–9 mice. *, Significant difference (Student’s t) from the wild-type males: 1, P⫽0.002; 2, P⬍0.001; 3,
P⫽0.013; 4, P⫽0.010. #, A significant effect (P⫽0.030) of gene dose by regression analysis.
Fig. 2. The dimorphism of the spinal bulbocavernosus (SNB) motor neurons
is normal in mice that lack the Mis gene. The bars are the mean ⫾standard
error of the mean of 4– 6 mice. *, Both female groups are significantly
different from both of the male groups, P⬍0.001.
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behavioral trait. Male mice have larger territories and a greater
tendency to explore than females. When mice were placed in a
nocturnal open-field chamber, the males and female traversed
the floor to similar extents (Fig. 4A), but the wild-type males
reared twice as often as the wild-type females, in an attempt to
move beyond the confines of the chamber (Fig. 4B). In marked
contrast to the wild-type males, the behavior of Misrii
⫺/⫺
males
was indistinguishable from that of the Misrii
⫹/⫹
and Misrii
⫺/⫺
females (Fig. 4 Aand B). This is consistent with the male bias in
this behavior being generated by MIS. In other tests, such as
running on a rotarod, Misrii
⫹/⫹
and Misrii
⫺/⫺
male mice were
indistinguishable (Fig. 4C). This is also consistent with MIS
regulating the subtle sex-linked differences in the brain, while
being entirely dispensable for acquisition of the nondimorphic
features of the brain.
Discussion
MIS Contributes to Sex-Linked Biases in the Brain. Sex-linked biases
are pervasive throughout the nervous system, as evidenced by
quantitative anatomical, pharmacological, fMRI, and behavioral
studies (6, 20, 21). The current report provides evidence that the
testicular hormone MIS contributes to these sex-linked biases.
The natural expression of MIS in the current study was
sufficient to increase the number of neurons in the LMC in male
mice to beyond the female range (Fig. 1) and to produce male
exploratory behavior that was overtly different than that of
female mice (Fig. 4). The clarity of these sex-linked differences
reflects the fact that the mice had minimal genetic variation and
had been raised in controlled conditions. In human populations
and outbred colonies, the size of motor nuclei varies greatly
between individuals (22). In such populations, a 16% male –
female difference (Fig. 1) would present as a subtle male bias,
with the male and female populations having extensive overlap.
MIS May Contribute to Variation in the Male Population. The ex-
pression of MIS varies between individual males, which may
contribute to diversity within the male population. In 1-year-old
boys, for instance, the plasma concentration of MIS ranges from
10 to 444 ng/ml (n⫽40; girl range, 1–4 ng/ml) (ref. 8; see also
ref. 23). The effect of MIS on neurons is dose dependent, both
in vitro (12) and in vivo (Fig. 1B). Hence, MIS would be ex pected
to produce a population of males that contains individuals who
are little different than females for a particular trait and other
individuals who exhibit an extreme male phenotype for that trait.
The incidences and/or severities of most brain disorders
exhibit a sex-linked bias (24). This observation suggests that the
sex-linked biases in the brain are able to either attenuate or
amplify the consequences of underlying brain pathology. If so,
then individuals with a high expression of MIS may be predis-
posed to develop certain conditions with a male bias (e.g.,
attention-deficit hyperactivity disorder, autism, motor neuron
disease) and to be relatively protected against some female-bias
conditions (e.g., anorexia).
MIS May Be a Broad Regulator of Neurons. The neural basis of the
sex-linked difference in the exploratory behavior of mice is
unknown. However, the initiation of movement is controlled in
the brain proper, indicating that the observed MIS-dependent
bias in exploratory behavior is unlikely to be secondar y to the
effect of MIS on spinal motor neurons. The MIS receptor is
broadly present in brain neurons (Fig. 3) (25), thus providing
multiple theoretical pathways by which MIS could regulate
exploratory and other behaviors. Furthermore, the physiological
actions of neural regulators are often context dependent, vari-
ably regulating neuronal number, pattern of synaptic connection,
neurotransmitter type, and neurotransmitter expression in a
neuron-specific and stage-specific manner (26). Thus, although
the current data implicate MIS as a regulator of neural networks,
the definition of the breadth of its action will require detailed
investigations of many different types of neurons.
The Neural Actions of MIS Are Cryptic. Mis
⫺/⫺
and Misrii
⫺/⫺
boys
and male mice have been extensively studied with respect to the
persistence of their Mu¨llerian ducts. This fact raises the issue of
why MIS has not previously been recognized as a regulator of the
nervous system. Two issues may be relevant here.
First, the role of MIS in the brain appears to relate to
sex-linked biases, rather than to binary sexual dimorphism.
Sex-linked biases are evident only at the level of the population
and would not be apparent unless groups of MIS-deficient
individuals were compared with their peers. In humans, Mis
⫺/⫺
and Misrii
⫺/⫺
males have a persistent Mu¨llerian duct, which
prevents the descent of their testes (7). This malformation is
readily detected, but Mis
⫺/⫺
and Misrii
⫺/⫺
men are very rare and
have not been systematically studied. MIS has both positive and
ABC
DEFG
Fig. 3. Localization of MISRII in the nervous system. (A–C) MISRII-Cre-lacZ lineage tracing. (A) Whole brain of a MISRII-Cre-lacZ E16 embryo. (B) Littermate control
(Misrii⫹/⫹, ROSA26-lacZ Cre). (C) Section of the cerebellum of a MISRII-Cre-lacZ adult mouse showing lacZ stain in neurons (arrowheads) but not the white matter
(arrow). (D–G) Immunohistochemical detection of MISRII. Sections of lumbar spinal cord of E12 (D) and E14 (E) embryos stained with an anti-MISRII antibody.
The asterisk indicates the neuroepithelium, with the dorsal–ventral (d–v) axis indicated. (F) Section of E14 spinal cord stained with control IgG. (G) Section of the
cerebellum from a Misrii⫺/⫺mouse stained with the anti-MISRII antibody. (Scale bars: B,10mm;C, 1 mm; F, 100
m.)
Wang et al. PNAS
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NEUROSCIENCE
negative effects on the production of testosterone, with the
consequence that Mis
⫺/⫺
mice have grossly normal levels of
testosterone (27), and are sexually male (28). Hence, the partial
feminization of Mis
⫺/⫺
males may have been overlooked because
of their overt male features, which have been generated by the
normality of their sex steroids and sex chromosomes.
Second, MIS signaling in neurons may be partially different
than that which occurs in the classical target of MIS (Mu¨llerian
ducts and the gonads). The neural and reproductive forms of
MIS signaling are both dependent on MISRII, because the
phenotype of Mis
⫺/⫺
and Misrii
⫺/⫺
exhibit the same neural (Fig.
1) and reproductive defects (28). The abundance of MISRII in
the different cell types, however, is quite different: MISR II is
one of the most abundant growth factor receptors produced by
motor neurons, but, paradoxically, the level of MISRII mRNA
and protein in neurons is ⬍1% of that in the testes (12, 16).
Consequently, historic studies of MISRII have overlooked the
presence of MISRII in the brain, because the levels of MISRII
in nonreproductive tissues were normalized to that of the testes.
The reason for the extraordinary abundance of MISRII in
reproductive tissues is unknown. In this context, however, it is
worth noting that the MIS-induced regression of the Mu¨llerian
ducts is insensitive to the abundance of MIS, with partial
retention of the ducts occurring only if the level of MIS falls
below a low threshold (29). In contrast, Mis
⫹/⫺
male mice exhibit
a diminished sex bias in their lateral motor column (Fig. 1), and
MIS-induced survival of motor neurons in vitro has a log-linear
dose–response curve (12) that spans the entire physiological
range for MIS (8).
The Neural Actions of MIS May Be Hormonal. MIS is generally
considered to be a hormone during development, because MIS
from a twin male in some species can remove the uterus from his
twin sister, if their placental blood supplies anastomose (7). The
neural actions of MIS described here also appear to be hor-
monal. First, the embryonic brain lacked significant levels of
MIS mRNA and none of its cells was MIS immunoreactive (Fig.
S1C). The only detectable source of MIS was the testes. Hence,
the neural actions of MIS must be either hormonal or indirect.
Second, purified embryonic motor neurons respond to MIS in
vitro, indicating their MIS receptors are functional (12). Fur-
thermore, the observed actions of MIS in vitro (neuronal
survival) (12) and in vivo (increased neuronal number) (Fig. 1)
are analogous, which is consistent with MIS acting directly on
neurons. Third, the normality of the testosterone-dependent
dimorphism in Mis
⫺/⫺
mice (Fig. 2 and above discussion) indi-
cates that the phenotype of Mis
⫺/⫺
mice is not secondary to
altered production or function of testosterone.
If testicular MIS acts directly on neurons, then it must be able
to reach the brain and spinal cord. MIS is a protein and will
therefore be unable to passively pass through the blood–
cerebrospinal fluid (CSF) barrier. The blood–CSF barrier de-
velops early, but its embryonic and postnatal characteristics
differ from each other and from the mature state (30, 31). Many
protein hormones are actively transported into the CSF (32, 33),
and it will be important to determine whether MIS is so
transported. If it is, then the testes may be directly affecting the
brain of boys, via MIS, and male puberty may involve the
diminishment of one male signal (MIS) as well as the reemer-
gence of another (testosterone).
In conclusion, the primary reproductive tissues have binary
sexual dimorphism, because the roles of males and females in
reproduction are fundamentally different. Binar y sex differences
are predominantly generated via testosterone and its active
metabolites, with the role of MIS being limited to preventing
males from developing a uterus. However, there are many traits
that are variable in both male and female populations, but which
are more common in one sex or the other. Sex-linked biases in
nonreproductive tissues appear to result from multiple mecha-
nisms, including sex chromosomes, gonadal hormones, and the
environment (1). Consequently, the various traits with sex-linked
biases are not coordinately regulated, creating rich diversity
within the male and female populations. The current study
implicates MIS as one of the factors that generates sex-linked
biases and variability within the male lineage.
Materials and Methods
Animals. The University of Otago’s Animal Ethics Committee approved all
experiments. The Misrii⫹/⫺mice have Cre recombinase knocked into the Misrii
coding region, leading to a null Misrii allele and to the expression of Cre
recombinase under the control of the endogenous Misrii promoter (34) (Fig.
S2). The Misrii⫹/⫺mice were generated from AB-1 embryonic stem cells and
were initially maintained on a C57BL/6 ⫻129/SvEv background (34). These
mice were a generous gift from Richard R. Behringer (University of Texas,
Fig. 4. MISRII-dependent behavior. Mice were placed in an open-field
apparatus under nocturnal conditions for 20 min and their movement was
recorded by sensors. (A) Rearing: the data are the number of times each mouse
reared during the second 10 min of the test, to explore beyond the confines
of the apparatus. (B) Distance traveled: the data are the distance traveled by
the mouse in the first 10 min of the test. (C) Rotarod run time: the data are the
maximum length of time that the mouse ran on an accelerating rotarod, from
3 trials. The results are the mean ⫾standard error of the mean and are
presented as a percentage of the mean for Misrii⫹/⫹male mice. The Misrii⫹/⫹
and Misrii⫺/⫺mice from 11 litters were tested (40 mice; group size, 9–11) on
the open field. Ten of the litters were also tested on the rotarod (34 mice;
group size, 7–10). *, Significant difference (Student’s t) from the Misrii⫹/⫹
males, P⬍0.01.
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Houston, TX). The Mis⫹/⫺mice were also generated from AB-1 stem cells (35)
and backcrossed to C57BL/6 by The Jackson Laboratory, from whom they were
purchased. Both the Mis and Misrii colonies have been maintained by brother–
sister matings at the University of Otago. The ROS26-lacZ Cre reporter mice
(36) were obtained from a colony at the University of Otago. The housing of
the mice was as described in ref. 37, with the genotype determined by PCRs
using the primers listed in Table S1.
Neuronal Counts. Wild-type, heterozygous, and null-mutant neonates from
Mis⫹/⫺⫻Mis⫹/⫺and Misrii⫹/⫺⫻Misrii⫹/⫺matings were killed at birth by
decapitation and fixed by immersion in 4% paraformaldehyde in 0.01 M
sodium phosphate buffer, pH 7.4 (PB). The caudal spinal cords from the
midthoracic region were removed for analysis of the lumbar LMC. Cell death
in the murine LMC is complete by birth (14), and this age is the standard for
estimation of LMC motor neurons (38). The spinal bulbocavernosus (SNB)
motor neurons develop later than LMC motor neurons and may be dually
influenced by prenatal and postnatal testosterone (18). The numbers of motor
neurons in this nucleus were therefore determined by using spinal cords from
6-week-old mice that have been fixed by cardiac perfusion with 4% parafor-
maldehyde. The spinal cords were embedded in Technovit resin, serially
sectioned at either 40
m (LMC) or 30
m (SNB) and stained with cresyl violet.
The numbers of motor neurons were estimated by using an optical dissector
(39), with the counting particle being the nucleolus. Every fifth section was
examined through the entire LMC, whereas all sections containing the SNB
were analyzed. Examples of LMC and bulbocavernosus motor neurons are
illustrated in Fig. S3. The cell counts were done blind with respect to the sex
and the genotype of the mice.
MISRII-Cre-lacZ. Misrii⫹/⫺dams were time mated with a ROSA26-lacZ Cre
reporter stud, and the brains from the resulting MISRII-Cre-lacZ fetuses (Fig.
S2) were collected at 13, 14, 16, or 20 days of gestation, or after the pups had
matured to adulthood (6– 8 weeks). The fetal brains were fixed on ice for 2 h
in a solution containing 2% paraformaldehyde, 0.2% glutaraldehyde, 0.02%
Nonidet P-40, and 0.01% sodium deoxycholate, and after washing they were
stained overnight in 5 mM potassium ferricyanide, 5 mM potassium ferrocya-
nide, 1 mM MgCl2, 0.02% Nonidet P-40, 0.01% sodium deoxycholate, and 0.1
M PB (40). The tails of the fetuses were used for genotyping, with the
ROSA26-lacZ Cre, Misrii⫹/⫹pups serving as controls. The adult brains were
snap-frozen in isopentane and serially sectioned in a cryostat, and the result-
ing sections were fixed for 10 min at 4 °C in 2% paraformaldehyde in 0.1 M PB
containing 2 mM MgCl2, pH 7.3, after which they were washed in PBS con-
taining 2 mM MgCl2, and stained overnight at room temperature with 2 mM
MgCl2,20mMK
3Fe(CN)6,20mMK
4Fe(CN)6, and 4 mg/ml X-Gal in PBS.
Immunohistochemistry. Transverse sections of lumbar spinal cords and testes
from E12, E14, E16, and E18 embryos, neonates, and adult wild-type mice were
cut in a cryostat at a thickness of 10
m. The sections were stained by
immunohistochemistry as described in ref. 41, using either goat anti-MISRII or
goat anti-MIS (R&D Systems), with nonimmune IgG (Sigma) used as a control
antibody. Sections of the brains of adult Misrii⫺/⫺mice were used as an
additional negative control. The immunoreactivity was visualized by using
3-amino-9-ethylcarbamide (Sigma).
Real-Time PCR. Five 12-day-old C57BL/6 fetuses were dissected into the por-
tions listed in Fig. S1, and total RNA fractions were prepared for each portion
by using TRIzol reagent (Invitrogen). The isolated RNA fractions were treated
with DNase I (Promega) to remove genomic DNA contamination and cDNA
was synthesized by using SuperScript II RNase H⫺(Invitrogen) and oligo(dT)20.
The real-time PCRs were performed by using an ABI Prism 7000 (Applied
Biosystems), SYBR Green Master Mix (Applied Biosystems), and gene-specific
primers (12). A 2-step PCR was carried out with denaturation at 95 °C for 15 s,
annealing and extension combined at 60 °Cfor 1 min in a total of 40 –50 cycles.
The uniqueness of amplicons was analyzed by using dissociation curves and by
sequencing. Standard curves were generated for each gene, and the copy
number of the mRNA transcripts was calculated and normalized relative to the
level of GAPDH.
Behavioral Analysis. The 9-week-old wild-type and null-mutant mice from 11
litters from Misrii⫹/⫺studs and dams were placed in an open-field apparatus
(Opto-varimex; Columbus Instruments) under nocturnal lighting conditions
(37) (40 mice; 10 male and 9 female Misrii⫹/⫹, plus 10 male and 11 female
Misrii⫺/⫺). The mice from 10 of the 11 litters were subsequently tested on a
Rotamex rotarod (Columbus Instruments) when 10 –12 weeks of age (10 male
and 7 female Misrii⫹/⫹, plus 8 male and 7 female Misrii⫺/⫺). The mice were
tested by their regular caregiver in an anteroom attached to the room that
housed the mice. The apparatus was cleaned after each mouse.
With the open-field test, each mouse was placed in the center of the 43 ⫻
43 cm chamber and its movements were tracked for 20 min by an array of
infrared beams. The movement of the mice across the floor of the apparatus
was recorded by a computer, along with a record of the number of times the
mice reared on their hindlimbs. As the test progressed, the mice spent less time
traversing the floor of the apparatus and were more likely to rear in an
attempt to move beyond the chamber. The data are presented as the distance
traveled in the first 10 min and the number of rearings in the second 10 min
of the test, because these correspond to the periods when the behavior was
predominantly occurring. The observed effects of sex and genotype were
similar when the data for the entire 20 min were analyzed.
Mice were trained to run on the rotarod for 3 consecutive days and tested
on the fourth day. The initial speed of the rotarod was 5 rpm, with an
acceleration of 0.5 rpm every 5 s, and with the mouse’s presence on the rod
being automatically detected by infrared beams. Each mouse was tested 3
times, and the average and maximum performances of the mice were ana-
lyzed. Both methods of analysis gave comparable results and only the maxi-
mum performance of the mice is presented here.
Statistical Analysis. The data were examined by 1-way ANOVA for sex differ-
ences (Fig. 1 A) or by 2-way ANOVA for sex and genotype (Fig. 1 Band C) (2, 4).
Significant effects were confirmed by Student’s ttests. The effect of gene dose
on LMC motor neuron number was examined by linear regression (Fig. 1B).
ACKNOWLEDGMENTS. We thank Mrs. N. Batchelor and J. McLay for expert
technical assistance. The work was supported by The Marsden Fund (Royal
Society, New Zealand) and by an Otago Research Grant. A.N.C. was supported
by a postdoctoral fellowship for the Neurological Foundation (New Zealand).
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