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The Journal of Neuroscience, July 15, 1999, 19(14):5955-5966
Promoter Transgenics Reveal Multiple Gonadotropin-Releasing
Hormone-I-Expressing Cell Populations of Different Embryological Origin
in Mouse Brain
Michael J.
Skynner,
Ruth
Slater,
Joan A.
Sim,
Nick D.
Allen, and
Allan E.
Herbison
Laboratories of Neuroendocrinology and Developmental Neurobiology,
The Babraham Institute, Cambridge CB2 4AT, United Kingdom
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ABSTRACT |
Gonadotropin-releasing hormone-I (GnRH-I) is thought to be
expressed by a single, highly spatially restricted group of neurons, which originate in the olfactory placode and migrate through the nose
into the medial septum and hypothalamus from where they control fertility. Transgenic mice bearing a 13.5 kb GnRH-I-lacZ reporter construct were derived and found to express high levels of
 -galactosidase mRNA and protein within the septohypothalamic GnRH
neurons in a correct temporal and spatial manner. Unexpectedly, low
levels of -galactosidase were also present in three further
populations of cells within the lateral septum, bed nucleus of the
stria terminalis, and tectum. Analysis of wild-type mice with three
different GnRH-I antibodies revealed distinct and transient patterns of
GnRH-I peptide expression during development in all three of these
populations revealed by transgenics. The synthesis of GnRH by cells of
the lateral septum was the most persistent and remained until the third
postnatal week. Embryonic "small eye" Pax-6 null mice, which fail
to develop an olfactory placode, were also examined and shown to have
equivalent populations of GnRH-I-immunoreactive cells in the lateral
septum, tectum, and bed nucleus of the stria terminalis but none of the
migrating cells that form the septohypothalamic GnRH population. These
results prove that so-called "ectopic" expression in promoter
transgenic lines can reflect authentic developmental patterns of gene
expression. They further provide the first demonstration in mammalian
brain that multiple neuronal populations of different embryological
origin express GnRH-I peptide during embryonic and postnatal development.
Key words:
-galactosidase; development; gonadotropin-releasing
hormone; luteinizing hormone-releasing hormone; mouse; Pax-6; promoter
transgenics; septum
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INTRODUCTION |
A variety of experimental approaches
have demonstrated that the neuropeptide gonadotropin releasing-hormone
(GnRH) is expressed in a highly restricted group of neurons within the
mammalian brain (Silverman et al., 1994 ). These cells are known to
originate in the olfactory placode and migrate through the nasal septum
and forebrain to reside, principally, within the medial septum and hypothalamus (Schwanzel-Fukuda and Pfaff; 1989; Wray et al., 1989). It
is equally well established that the majority of these
septohypothalamic GnRH neurons project to the median eminence, from
where they control the activity of pituitary gonadotrophs and, thus,
represent the final output neurons of the neural network regulating
fertility (Silverman et al., 1994). Evidence also suggests a role for
GnRH in the regulation of mammalian sexual behavior (Moss and McCann, 1973; Pfaff, 1973).
Although an equivalent septohypothalamic population of GnRH neurons is
present in nonmammalian taxa, a second form of GnRH was discovered in
some of these species and shown to be expressed by a population of
cells located in the midbrain (Muske, 1993; Sherwood et al., 1993;
Tobet et al., 1997). The conservation and expression of this second
form, now termed GnRH-II, have recently been demonstrated in the
midbrain of the monkey, but curiously not the rodent, and the human
gene has been cloned (Lescheid et al., 1997; White et al., 1998). Thus,
at least two forms of GnRH exist in the mammalian brain; GnRH-I, which
is expressed by a restricted group of midline septohypothalamic neurons
of olfactory origin that control reproduction, and GnRH-II, which is
synthesized in midbrain cells of likely local origin but unknown function.
One of the great practical difficulties in trying to characterize
GnRH-I neurons is that they do not lie within a specific region of the
brain but, rather, lie scattered throughout the medial septum, preoptic
area, and basal hypothalamus of the mammalian brain (Silverman et al.,
1994). To try and address this problem, we have initiated a program of
research that involves the targeting of the GnRH-I neurons with
promoter-driven transgenics in vivo in the mouse. We report
here the first surprising result of these studies in that not one but
four cell populations in the brain are found to express the
-galactosidase (gal) reporter when driven by 13.5 kb of GnRH-I
genomic sequences. Follow-up investigations in transgenic and wild-type
mice have demonstrated that all four populations of neurons synthesize
authentic GnRH-I, and work in Pax-6 null mice has revealed a
differing embryological origin for the three newly identified
populations compared with that of the classic septohypothalamic GnRH-I neurons.
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MATERIALS AND METHODS |
Production of GnRH-LacZ transgenic mice
A GnRH reporter construct was assembled by inserting a 3 kb
-galactosidase cassette from pGeo into the XbaI site
of a 13.5 kb genomic clone (pGnRHSmaI; gift of Dr. A. Mason,
Prince Henry's Institute, Melbourne, Australia), which contained all
introns and exons as well as 5 kb of 5'- and 3.5 kb of 3'-flanking
sequences of murine GnRH. The ATG start site for GnRH transcription of
this plasmid had been mutated to TTG and a SmaI restriction
site introduced between sequences coding for amino acids 2 and 3 of the
GnRH decapeptide. For microinjection, the construct was excised from
the vector (pSP65) using SalI and BstEII double
digestion, separated by agarose gel electrophoresis, and gel-purified
using Glassmilk (Qiagen, Hilden, Germany). DNA was further
purified using an Amicon (Beverly, MA) 30 micopure/microcon
microconcentrator. DNA concentration was estimated by reference to DNA
concentration standards after agarose gel electrophoresis and diluted
to a final injection concentration of 2.5 ng/µl in 10 mM
Tris-HCl, pH 7.4, and 0.1 mM EDTA.
All mice were bred and housed at The Babraham Institute according to UK
Home Office requirements under projects 80/972 and 80/1005, and
transgenic mice were produced by pronuclear injection (Hogan et al.,
1994). Briefly, fertilized mouse eggs from superovulated F1 mice
(CBA × C57/Bl6) mated to F1 males were visualized using differential interference contrast optics on an inverted Nikon (Tokyo,
Japan) microscope. DNA was introduced into the male pronucleus using
Leitz (Wetzlar, Germany) manual micromanipulators and glass capillary
micropipettes. Eggs were cultured overnight to the two-cell stage and
subsequently transferred to the oviducts of pseudopregnant F1 recipient
female mice.
Transgenic mice were identified by either Southern blot or PCR analysis
of genomic DNA isolated from tail biopsies. For the former, 0.5 cm of
tail was removed from mice after weaning under general anesthesia and
digested overnight at 55°C in lysis buffer containing 100 mM Tris-HCl, pH 8.5, 5 mM EDTA, pH 8.0, 0.2%
SDS, 200 mM NaCl, and 100 µg/ml Proteinase K (Sigma,
Poole, UK). Undigested material was removed by centrifugation, and the
supernatant was extracted with an equal volume of phenol-chloroform.
Genomic DNA was precipitated by the addition of an equal volume of
isopropanol and spooled into 200 µl of Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA). Thirty
microliters of DNA were then digested overnight with EcoRI,
and fragments were resolved on horizontal 0.8% agarose gels. After
transfer to a Hybond N+ membrane, by alkali capillary blotting,
membranes were hybridized in Church buffer (0.5 M phosphate
buffer, pH 7.5, 7% SDS, and 1 mM EDTA) overnight with a
random-primed [ -32P]dCTP (ICN-Flow)-labeled 3.0 kb
SmaI fragment spanning the entire -galactosidase gene,
isolated from p-Geo. Hybridization was performed at 65°C in a
rotary oven (Hybaid). After hybridization, the filters were washed in
Church wash (40 mM phosphate buffer and 1% SDS) three
times for 10 min each, wrapped in Saran wrap (DuPont, Wilmington, DE),
and exposed to x-ray film (X-OMAT; Kodak, Rochester, NY) using
intensifying screens at  70°C.
For PCR identification, Lac-ZMF (5'-CCACGGCCACCGATATTATTTGCCCG-3')
and Lac-ZMR (5'-TTTTGCTTCCGTCAGCGCTGGATGCG-3') primers were used to
amplify a 410 bp fragment from the -galactosidase gene in individual
25 µl reactions containing 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 1.5 mM MgCl2, 0.2 µM dNTPs, 1 µM each, 0.6 U of
Taq DNA polymerase (Pharmacia), and 0.5 µl of genomic DNA. PCR was performed in a Robocycler (Stratagene, La Jolla, CA) as follows: 95°C for 3 min, 60°C for 1 min, and 72°C for 2 min for one cycle, followed by 24 cycles of 95°C for 40 sec, 60°C for 35 sec, and 72°C for 50 sec. Amplicons were resolved on 1.5% horizontal agarose gels, visualized using ethidium bromide staining, and photographed using a gel documentation system (Bio-Rad, Hercules, CA).
Immunocytochemical analysis
Antibodies. A polyclonal rabbit antibody (55976; ICN
Biomedicals GmbH, Postfach, Germany) raised against gal from
Escherichia coli was used to detect transgene expression
(Min et al., 1994). Three different polyclonal rabbit antibodies
specific for GnRH-I were used for peroxidase immunostaining: LR1 (a
gift from R. Benoit, McGill University, Montreal, Canada) raised
against [D-lys6]-GnRH-I, which detects
amino acids 6-10 of both the precursor and amidated decapeptide
(Silverman et al., 1990); SW1 (a gift from S. Wray, National Institute
of Neurological Diseases and Stroke) raised against mammalian
GnRH-I (Wray et al., 1988); and GF6 (a gift from Dr. N. Sherwood,
University of Victoria, Victoria, Canada) raised against
synthetic salmon
[Trp7-Leu8]-GnRH-I, which
detects mammalian GnRH in addition to other vertebrate GnRH-I
decapeptides (Quanbeck et al., 1997). A polyclonal sheep antibody, BDS
037 (a gift from Dr. A. Caraty, INRA, Nouzilly, France), raised
against mammalian GnRH-I, which detects C-terminal epitopes of GnRH-I
(Caraty et al., 1995), was used for the dual-labeling immuofluorescence
study. Adsorption control experiments were undertaken by incubating the
respective GnRH antibody with GnRH-I1-10 peptide
(Sigma) at a concentration of 5 µg/ml overnight at 4°C before use
on brain sections.
Peroxidase-based immunostaining in adult mice. Adult (60- to
90-d-old) transgenic GnRH-LacZ (GNZ) and wild-type (CBA × C57/Bl6) mice were administered an overdose of tribromoethanol
(Avertin; 0.2 ml/20 gm, i.p.) and perfused directly through the left
ventricle of the heart with 15-20 ml of 4% paraformaldehyde in PBS,
pH 7.4. Brains were removed and post-fixed for 1-2 hr at room
temperature before being placed in a 30% sucrose, Tris-buffered saline
(TBS) solution at 4°C. The following day, 30-µm-thick coronal
sections were cut into four sets of sections through the rostral
forebrain, including the preoptic area and hypothalamus, using a
freezing microtome. Free-floating immunocytochemistry was then
undertaken by placing sections in a 1%
H2O2, 40% methanol, TBS solution for 5 min followed by three washes in TBS. Sections were then incubated in
one of the polyclonal rabbit primary antibodies (gal, 1:8000; LR1,
1:20,000; SW1, 1:5000; and GF6, 1:8000) for 40-64 hr at 4°C followed
by TBS washing at room temperature and incubation in biotinylated goat
anti-rabbit Igs (1:200; Vector Laboratories, Peterborough, UK) for 90 min. Sections were then washed and placed in the Vector Elite
avidin-peroxidase substrate (1:100) for a further 90 min before
reacting with the nickel-diaminobenzidene tetrahydrochloride chromagen
using glucose oxidase. The cytoarchitecture of selected coronal brain
sections was determined by counterstaining with hematoxylin and eosin.
All Igs were dissolved in TBS containing 0.3% Triton-X-100 and 0.3%
bovine serum albumin, and primary antibody solutions also contained 2%
normal goat serum. Controls consisted of the omission of one of the
primary antibodies from the immunostaining protocol and the use of GnRH
antibodies preadsorbed with GnRH peptide.
Dual-labeling immuofluorescence in adult mice. A set of
coronal sections from adult mice were processed for dual gal-GnRH fluorescence by incubation in an antibody mixture comprising the polyclonal rabbit gal (1:4000) and polyclonal sheep GnRH-I (1:1000) Igs for 40 hr at 4°C. After washing in TBS at room temperature, the
sections were placed in FITC donkey anti-rabbit (1:50; Jackson ImmunoResearch, West Grove, PA) and biotinylated horse anti-goat (1:200; Vector) Igs for 90 min at room temperature. After washing, sections were placed in Texas Red-avidin (2.6 µl/ml; Vector) for a
further 90 min at room temperature and then mounted onto slides and
coverslipped with Vectashield (Vector). Controls consisted of the
omission of one of the primary antibodies from the immunostaining protocol. Sections were viewed under a Leica (Nussloch, Germany) DM-RB
fluorescent microscope, and individual cells were examined at 40 or
100× objective magnification where switching between Texas Red (TX;
Leica) and FITC (I3; Leica) filter sets determined whether cells were
double-labeled. For each mouse, three or four sections containing the
medial septum (see Fig. 2B) and three or four
sections containing the rostral preoptic area (see Fig. 2C)
were selected, and the number of GnRH-only and dual
GnRH-gal-immunoreactive cells was counted in each animal. These
counts provided an average value for each animal, which were used to
determine mean ± SEM values for each cell population.
Peroxidase-based immunostaining in embryonic and neonatal
mice. Transgenic GNZ, Pax-6 mutant small eye (Sey) and
wild-type adult female mice were time-mated with stud males at the
Babraham Institute, and pregnancy was determined by the presence of
vaginal plugs (morning of plug detection = gestational day 0.5).
These females were killed by cervical dislocation to provide embryonic day (E) 11.5, 12.5, 13.5, 14.5, 16.5, and 18.5 fetuses. Embryos were
dissected and decapitated, and their whole heads either snap-frozen on
dry ice or immersed in the 4% paraformaldehyde fixative, pH 7.4, for 2 hr at room temperature before being placed in a 40% sucrose-TBS
solution overnight at 4°C. Snap-frozen embryonic brains were cut in
the coronal plane on a cryostat at 15 µm thickness into four sets,
whereas fixed brains were cut in either the coronal or sagittal plane
at 30 µm thickness into three sets on a cryostat and freeze-thawed
onto gelatinized slides. Snap-frozen sections were kept at 80°C
until processed for immunocytochemistry, whereas sections from
immersion-fixed brains were processed immediately after sectioning.
Brain sections from both protocols were brought to room temperature,
immersed in 4% paraformaldehyde fixative for 30 min, washed in TBS,
placed in 1%H2O2, 40% methanol, and
TBS for 5 min, and washed again in TBS, and then 150-200 µl of
primary antibody (gal, 1:2000; LR1, 1:4000; SW1, 1:3000; and GF6,
1:5000) in the Triton-X-BSA-TBS buffer with 2% normal goat serum added
was placed on each slide so that all brain sections were covered.
Slides were maintained in humidified chambers at 4°C for 72 hr, with an additional 50 µl of respective antibody applied to each section each day. Slides were then washed several times in TBS before placing
150-200 µl of 1:200 biotinylated goat anti-rabbit Igs (Vector) on
each slide for 90 min at room temperature. Slides were washed, and
150-200 µl of 1:100 Vector Elite reagent was placed on each slide
for a further 90 min. Slides were washed again in TBS and finally
reacted with the nickel-diaminobenzidene tetrahydrochloride chromagen
using glucose oxidase. Control experiments consisted of the incubation
of slides in buffer missing a primary antibody and the use of adsorbed
primary antibodies. Mice were also examined at postnatal (P) days 3 and
4 and at day 16. In the case of the P3/4 mice, neonates were
decapitated, and their whole heads were immersed in the fixative
solution and processed as above for the immersion-fixed embryonic
brains. The P16 mice were treated in the same manner as the adults
described above.
Dual X-gal-immunocytochemistry in embryonic mice. Homozygous
GNZ 3252 E16.5 (n = 2) and E17.5 (n = 2) embryos were immersion fixed and cut in the coronal plane as
described above. Slides were then placed in a X-gal solution [2
mM MgCl2, 4 mM
K3Fe(CN)6, 4 mM
K4Fe(CN)6, and 4 mg/ml
5-bromo-4-chloro-3-indoyl--D-galactosidase in TBS]
overnight at room temperature to reveal transgene-expressing cells in
the brain. Sections were rinsed several times in TBS and processed for
GF6 immunostaining exactly as described above. Slides were mounted with
glycerin and viewed on a Leica DM RB microscope.
In situ hybridization for -galactosidase mRNA in
adult mice
Adult male and female GNZ mice were killed by cervical
dislocation, and their brains were rapidly removed and frozen on dry ice. Brains were stored at 80°C until being cut on a cryostat in
the coronal plane at 15 µm thickness through the septum and rostral
hypothalamus and thaw-mounted onto gelatinized slides. Cut sections
were stored at 80°C until processed for in situ hybridization using the same procedure as that reported previously (Herbison and Fenelon, 1995). In this case, 40- and 45-mer
oligonucleotides complimentary to nucleotides 1256-1295 and 2822-2866
of gal cDNA, respectively, were synthesized and labeled with
35S to a specific activity of 109
cpm/µg. Slides were then hybridized with each probe individually or
both. After hybridization, sections were coated with emulsion and left
to develop for 8-10 weeks (dual probes) or 12 weeks (single probes)
before being lightly counterstained with methylene blue and
coverslipped with DPX. Competition experiments were undertaken by hybridizing slides with both probes but in the presence of a 50-fold
excess of unlabeled oligonucleotides.
Analysis of relative mRNA expression was undertaken using an image
analyzer (Seescan, Cambridge, UK), which enabled the number of silver
grains clustered over individual cells to be determined (Herbison and
Fenelon, 1995). To determine the relative gal mRNA expression in the
lateral septum and the GnRH neurons, the 20 highest expressing lateral
septal cells found within two coronal brain sections were analyzed and
compared with silver grain counts from 10-15 medial septal and 20 preoptic area GnRH neurons in each of the male and female mice. Silver
grain counts from individual cells were combined to provide an average
for each mouse, and these values were used to provide mean ± SEM
data. Statistical analysis was undertaken using the nonparametric
Mann-Whitney U test.
In situ hybridization for GnRH-I-associated peptide
mRNA in embryonic mice
To ensure that the GnRH expressed in the new populations was in
fact derived from the GnRH-I gene, fresh-frozen sections from E14.5
embryonic wild-type mice were obtained as described above. In
situ hybridization using a 48 mer oligonucleotide complementary to
sequences encoding the last 16 amino acids of GnRH-I exon 3 (nucleotides 3095-3143), which are responsible for GnRH-associated peptide-I (GAP; Mason et al., 1986) was undertaken using the method described above for adult brain sections. Whereas relatively small differences exist in the sequences encoding the GnRH-I and GnRH-II decapeptides, the GAP sequence of GnRH-I and GnRH-II is highly divergent (White et al., 1998). After hybridization with a
35S-labeled probe of specific activity of
109 cpm/µg, sections were coated with emulsion and
left to develop for 7-10 d before being lightly counterstained with
methylene blue and coverslipped with DPX. Competition experiments were
undertaken by hybridizing slides with the GAP probe but in the presence
of a 50-fold excess of unlabeled oligonucleotide.
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RESULTS |
Six transgenic founder mice (four male and two female) were
derived, and three mice (male 3210, female 3235, and female 3252) were
found to have offspring expressing the transgene. Blot analysis indicated that the 3252 line was likely to result from a single copy of
the transgene, the 3235 line from a dual copy, and the 3210 from
multiple ~10 copy insertion. Mice from the three lines appeared
normal in all respects and exhibited normal levels of fertility. The
3252 line was bred to homozygosity, whereas transgene expression in the
3235 line was found to decline with generation number and the line
culled. Results from offspring of the 3235 founder are included in the
adult gal expression section.
Transgene expression in adult mice
-Gal immunocytochemistry
The three lines of GNZ mice exhibited identical patterns of
transgene expression. This consisted of cells displaying two different patterns of gal immunoreactivity; the most obvious population was a
continuum of mostly bipolar-type immunoreactive neurons (Fig.
1A,B), which began in
the medial septum and passed ventrally through the midline regions of
the rostral preoptic area to reach the base of the lateral hypothalamus
(Figs. 1-3). The second pattern of gal immunostaining consisted of
a very light cytoplasmic staining coupled with the presence of an
intensely staining intracytoplasmic "donut-like" organelle (Fig.
1D,E) commonly seen in neuronal LacZ transgenics
(Friedrich et al., 1993). Cells exhibiting this pattern of
immunoreactivity were found throughout the rostrocaudal length of the
lateral septum, including its septohippocampal, dorsal, intermediate,
and septofimbral nuclear divisions (Fig.
2A-C). Transgene-expressing cells in the intermediate division extended down
its ventrolateral projections on either side of the diagonal band of
Broca (Franklin and Paxinos, 1997; Fig. 2B,C). A few
gal-immunoreactive cells were also detected laterally in the region
of the olfactory tubercle (Fig. 2B-D). The second
major population of cells expressing this "donut" pattern of gal
immunoreactivity was found in, and adjacent to, the ventral-most part
of the posterior division of the bed nucleus of the stria terminalis
(pBNST; Fig. 2D). An identical pattern of transgene
expression was observed using the chromogenic substrate Xgal to detect
-galactosidase (results not shown).
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Figure 1.
Gal immunoreactivity (A, B, D,
E) and mRNA expression (C, F) in the
rostral preoptic area (A-C) and lateral septum
(D-F) of adult female GNZ mice. Note that gal
immunoreactivity is present throughout the cytoplasm of GnRH neurons
(A, B) but located principally within circular
donut-like structures (E, arrow) in the cytoplasm of
lateral septal cells (D, E). Silver grain density after
gal in situ hybridization is substantially greater in
GnRH neurons (C) compared with lateral septal
cells (F). Scale bars: A, D, 50 µm; B, C, E, F, 5 µm.
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Figure 2.
Camera lucida diagram of transgene expression in
rostral (A) to caudal (D)
coronal sections of a female GNZ mouse. Triangles
represent gal-immunoreactive cells with intense cytoplasmic staining
(GnRH neurons), whereas dots represent cells exhibiting
a light gal-immunoreactive soma with distinct donuts (see Fig.
1E). aBST, Anterior bed nucleus of
the stria terminalis; ac, anterior commissure;
AHA, anterior hypothalamic area; AcbC,
accumbens nucleus core; AcbS, accumbens nucleus shell;
AD, anterodorsal thalamic nucleus; AVPV,
anteroventral periventricular nucleus; cc, corpus
callosum; Cpu, caudate-putamen; DB,
DBB, diagonal band of Broca; f, fornix;
Icj, islands of Calleja; LV, lateral
ventricle; LSI, intermediate division of the lateral
septum; LSV, ventral division of the lateral septum;
MnPN, median preoptic nucleus; MPN,
medial preoptic nucleus; MS, medial septum;
oc, optic chiasm; OT, olfactory tubercle;
OVLT, organum vasculosum of the lamina terminalis;
pBNST, principal encapsulated division of bed nucleus of
the stria terminalis; SCN, suprachiasmatic nucleus;
SHi, septohippocampal nucleus; T,
thalamus; TT, tenia tecta; sm, stria
medularis; 3V, third ventricle.
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No differences were detected between males and females in any of the
three lines, and breeding the 3252 line to homozygosity had no effect
on the distribution or apparent intensity of gal immunoreactivity.
In control experiments, in which the gal antibody was omitted from
the procedure, no staining was evident in the sections. Equally, when
gal immunostaining was undertaken on nontransgenic littermates, no
staining resulted.
Dual-labeling immunocytochemistry
The relationship of gal immunoreactivity to GnRH neurons was
determined by staining adjacent sections for gal and GnRH and also
undertaking dual-labeling immuofluorescence for gal and GnRH. The
staining of adjacent sections from both male and female GNZ mice
revealed an identical pattern of cytoplasmic GnRH and gal
immunoreactivity within the medial septum and hypothalamus (Fig.
3A,B). Gal staining was not
evident in the axons of neurons. Although GnRH-immunoreactive fibers
and the very occasional bipolar cell body were detected in the lateral
septum and BNST, we could find no evidence for cytoplasmic GnRH
immunoreactivity within the distribution of the transgenic cells of the
lateral septum and pBNST. The distribution of GnRH immunoreactivity
observed was in complete agreement with previous studies in the mouse
(Jennes and Stumpf, 1986; Schwanzel-Fukuda et al., 1987).
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Figure 3.
Coronal rostral preoptic area sections of male
3252 mice immunostained for GnRH (A, C) and gal
(B, D). A, B, Immunoperoxidase staining
on consecutive coronal sections for GnRH (A) and
gal (B) shows a similar distribution of cell
body staining. OC, Optic chiasm; OVLT,
organum vasculosum of the lamina terminalis. Scale bar, 250 µm.
C, D, Dual-labeling immuofluorescence for GnRH
(C, red) and gal (D, green). Note that
the GnRH neurons in view express different levels of gal
immuofluorescence and that, as seen in B, GnRH axons
rarely express gal immunoreactivity. Scale bar, 40 µm.
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Dual-labeling immunofluorescence was undertaken on brain sections from
four adult heterozygous 3210 males and five adult homozygous 3252 male
mice. The pattern of immunofluorescence for gal and GnRH was
identical to that observed with immunoperoxidase detection, and the
great majority of GnRH neurons (red fluorescence; Fig. 3C)
were found to contain green gal immunofluorescence (Fig. 3D). Cell counts revealed that 85 ± 3% of medial
septal and 86 ± 3% of rostral preoptic GnRH-immunoreactive
neurons were also immunoreactive for gal in 3210 mice and that
81 ± 4% of medial septal and 87 ± 3% of rostral preoptic
GnRH-immunoreactive neurons were positive for gal in 3252 mice. In
total, 85 ± 3% (3210) and 86 ± 3% (3252) of all GnRH
neurons were found to express gal immunoreactivity. No GnRH
immunoreactivity was detected in gal immunofluorescent cells of the
lateral septum or pBNST. Omission of either the gal or GnRH antibody
resulted in an absence of green or red immunofluorescence,
respectively, and dual labeling immunostaining on nontransgenic 3210 littermates resulted in only red immuofluorescence.
-Gal mRNA in situ hybridization
Coronal brain sections of male (n = 5) and female
(n = 5) adult homozygous 3252 mice underwent gal
in situ hybridization and analysis in parallel. Sections
hybridized with either of the single oligonucleotides or the two probes
together as a mixture displayed a distribution of silver grain clusters
identical to that of the gal immunostaining. However, the numbers of
silver grains clustered over individual septal cells (Fig.
1F) were much lower than those observed over the
midline-positioned cells of the medial septum and rostral preoptic
(Fig. 1C). The distinct GnRH distribution pattern of the
highly expressing cells in the rostral preoptic area and absence of
non-GnRH transgenic cells in this area (Figs. 2C, 3) make it
reasonable to assume that gal mRNA-expressing cells in the rostral
preoptic area represent GnRH neurons.
The analysis of sections from male and female mice hybridized with the
mixture of two gal oligonucleotides revealed that cellular gal
mRNA expression was at least threefold greater in rostral preoptic GnRH
neurons compared with cells located in the intermediate division of the
lateral septum (males, lateral septum 65 ± 5 silver grains per
cell vs 186 ± 8 in preoptic GnRH neurons; females, lateral septum
71 ± 6 silver grains per cell vs 238 ± 16 in preoptic GnRH
neurons). A significant sex difference in cellular silver grain counts
was evident for rostral preoptic area GnRH neurons
(p = 0.03, Mann-Whitney U test) but
not lateral septal cells. Because the hybridization signal was
relatively weak in the lateral septum (Fig. 1F), only
the most highly expressing lateral septal cells were selected and, as
such, this is likely to underestimate the difference in gal mRNA
levels between the two populations. Sections hybridized in the presence
of excess unlabeled oligonucleotides displayed no silver grain
clusters, and, equally, no signal was detected in coronal brain
sections from nontransgenic mice.
Transgene expression in embryonic and neonatal GNZ mice
The analysis of transgene expression by gal immunocytochemistry
in fresh-frozen and immersion-fixed embryonic brains revealed the
presence of gal-immunoreactive cells in the classical distribution of the migrating GnRH neurons, as well as the lateral septum, pBNST,
and, additionally, the developing tectum.
Transgene expression in the nose
Brains from heterozygous 3210 mice were analyzed at E11.5, 12.5, 13.5, and 15.5 (n = 3-5 at each stage), and brains
from homozygous 3252 mice were assessed at E11.5, 12.5, 13.5, 14.5, 16.5, and 18.5 (n = 3-5 at each stage) using gal
and GnRH immunocytochemistry. In both lines of mice,
gal-immunoreactive cells were detected in the olfactory
placode-derived medial olfactory pit and vomeronasal organ and in
clusters or cords of cells passing through the nasal septum to converge
on a localized point at the base of the telencephalic hemispheres (Fig.
4A). Cells located in
the medial olfactory pit were small and ovoid in shape with few stained
processes (Fig. 4B), whereas immunoreactive cells
clustered in the cords of cells within the nasal septum were elongated
with unipolar or bipolar process (Fig. 4C). An analysis of
the numbers of gal- and GnRH-immunoreactive cells detected in the
nasal septum as a whole was undertaken in a series of snap-frozen
immunostained E12.5-18.5 embryonic 3252 brains. This revealed an
identical number of gal and GnRH cells in the nasal septum at each
embryonic stage and, as reported previously for GnRH (Schwanzel-Fukuda
and Pfaff, 1989; Wray et al., 1989), a decline in cells numbers during
development (Fig. 5).
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Figure 4.
Gal immunoreactivity in parasagittal sections
of embryonic GNZ 3252 mice. In all photomicrographs the nose is to the
right. A-C, Increasingly high-power
views of gal immunoreactivity in the nose and developing septum of
an E12.5 embryo. Note the large numbers of immunoreactive cells within
the vomeronasal organ (vno) with tracks of cells passing
back to the developing forebrain where gal-immunoreactive cells are
already present. A high-power view of a cell in the nasal septum is
shown in C. Scale bars: A, 100 µm;
B, 50 µm; C, 5 µm. D,
E, Low- and high-power views of gal-immunoreactive cells
within the developing septum of an E15.5 mouse. Two types of
immunoreactive cells are present; one with strong, often bipolar,
cytoplasmic staining (E, arrow), which were found in the
most rostral part of the septum (D, arrows), and another
displaying fainter immunoreactivity in smaller-diameter cells often
without processes (E), which were scattered
throughout the septum (D). Scale bars:
D, 100 µm; E, 10 µm. F,
G, High- and low-power views of gal-immunoreactive cells
within the developing preoptic area and bed nucleus of the stria
terminalis of an E15.5 mouse. Multiple small and faintly immunoreactive
cells (F) are observed clustered between the
preoptic recess (por) and the lateral ventricle
(lat V) separate from the septal population
(G). Scale bars: F, 10 µm;
G, 100 µm. H, Gal-immunoreactive
cells are also present in a distinct band within the inner half of the
developing tectum. Scale bar, 120 µm.
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Figure 5.
Mean ± SEM numbers of GnRH- and
gal-immunoreactive neurons detected in the three most midline
parasagittal sections of the nasal septum of E12.5, 13.5, 14.5, 16.5, and 18.5 3252 GNZ mice (n = 4-5 at each
stage).
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Transgene expression in the brain
Cells immunoreactive for gal were detected in the developing
septum from E11.5 onward (Fig. 4A,D). Between E11.5
and 13.5, they were found to be small in diameter (6.1 ± 0.2 µm; range, 5.8-7.8 µm) and usually displayed an ovoid ring of
cytoplasmic immunoreactivity without any stained processes. After E13.5
a second type of cell was encountered in the septum, which was larger in diameter, often exhibiting a clear unipolar or bipolar morphology, and more intensely stained (Fig. 4D,E). Although the
bipolar-type cell was restricted to the more medial and frontal aspects
of the septum (Fig. 4D), the small cells remained
localized within the lateral septum. Coronal sections in E14.5 and
neonatal (P3/4 and P16) mice confirmed that the small ovoid cells were
restricted to the lateral septum. The staining of consecutive
immersion-fixed parasagittal sections for GnRH and gal in
E13.5-18.5 embryos revealed a similar distribution of immunoreactivity
with numerous small ovoid cells also being identified with the LR1
antibody in the lateral septum (Fig.
6A).
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Figure 6.
GnRH-I in parasagittal (A-E; nose
is to the right) and coronal
(F-J) sections of wild-type embryonic and
postnatal mice. A, GnRH-immunoreactive (LR1) cells in
the nasal septum and developing telenchephalon of an E12.5 mouse. Note
the typical appearance of GnRH neurons migrating through the nose in
tracks and also the immunoreactive cells present along the rostral edge
of the brain. Inset (scale bar, 10 µm), Typical ovoid
morphology of these cells. B, C, GnRH immunoreactivity
(GF6) in a midline parasagittal section (B) and a
section 300 µm lateral to it (C) of an E14.5
mouse. Note in B the typical distribution of the
migrating GnRH neurons as they enter the brain and pass down through
the septum. In the more lateral section (C), some
of these bipolar migrating GnRH neurons can be seen leaving the septum
(arrow), whereas a number of fainter and smaller
immunoreactive neurons are evident scattered throughout the lateral
septum. Scale bars, 200 µm. D, GnRH immunoreactivity
(LR1) in the septum of an off-midline parasagittal section from an
E15.5 mouse. Note the presence of intensely stained bipolar neurons
alongside small round and multipolar-shaped cells. Scale bar, 40 µm.
E, GAP-I mRNA in situ hybridization in
septum of E15.5 mouse. Note the presence of both very high
(arrowheads) and low (arrows) expressing
cells. Scale bar, 15 µm. F, G, Coronal sections
showing GnRH-immunoreactive (GF6) cells in the ventral pBNST of an E15.5 mouse. Note in
F the presence of two "normal" intensely stained
bipolar cells near the third ventricle (3V) and
the population of fainter cells magnified in G.
OC, Optic chiasm. Scale bar: F, 200 µm;
G, 15 µm. H-J, Coronal views through
the medial septum (MS) and lateral septum (LS) after GnRH
immunocytochemistry (GF6) in E14.5 (H), P3
(I), and P16 (J)
mice. Note that the intensely staining bipolar neurons reside within
the medial septum at all stages, whereas the number of more faintly
staining, small-sized cells in the lateral septum become markedly less
in number at P16. J, inset, Typical multipolar
morphology of lateral septal GnRH neurons in postnatal mice. Scale
bars: H, I, 80 µm; J, 10 µm.
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Cells immunoreactive for gal were also detected between the septum
and the preoptic recess streaming up toward the lateral ventricle (Fig.
4G). These cells, which were similar in morphology to those
identified in the lateral septum (Fig. 4F), were
first detected on E14.5 and, in coronal E16.5 sections, observed to lie
within the ventral pBNST and preoptic area.
Dual labeling X-gal-GnRH (GF6) immunostaining in coronal sections of
E16.5-17.5 embryos demonstrated co-expression of the transgene and
GnRH immunoreactivity through the presence of blue X-gal reaction
product in the great majority of brown GF6-immunoreactive cells.
Dual-labeled cells were observed in the classical distribution of GnRH
neurons as well as in the lateral septum and pBNST. A third population
of transgene-expressing cells was observed in the inner half of the
tectum (Fig. 4H). In this case, gal-immunoreactive cells were not detected until E15.5 but had disappeared by P3/4. Immunoreactive cells were found to display a regular ovoid morphology with few processes. Staining consecutive parasagittal sections with the
LR1 antibody revealed the same pattern of immunoreactivity.
In all cases, the staining of nontransgenic embryonic and postnatal
mice resulted in an absence of gal immunostaining, and the
adsorption of the LR1 antibody with GnRH peptide abolished staining.
GnRH-I in wild-type mice
Because work detailed above in the GNZ mice suggested that
multiple GnRH-I-expressing cell populations existed, a series of studies was undertaken in nontransgenic (CBA × C57/Bl6) embryonic and neonatal (E11.5, E12.5, E14.5, E15.5, E19.5, P3/4, and P16; n = 3-5 at each stage, except E19.5, n = 2) and adult mice (n = 4, each sex), which underwent
the immersion-fixation (E11.5-P3/4) or perfusion-fixation (P16 and
adult) immunocytochemistry procedure using three different GnRH-I antisera.
In addition to the classical pattern of GnRH-I immunoreactivity in the
nasal septum and forebrain (Fig. 6A,B), all three
antibodies detected a large number of relatively faintly stained,
small-diameter (6-8 µm), circular or ovoid-shaped cells within the
lateral septum from E11.5 onward. At E11.5 and 12.5, immunoreactive
cells were located within the rostral edge of the developing
telencephalon (Fig. 6A), whereas in older embryos
they were distributed throughout the lateral septum (Fig.
6B,C,H-J). Parasagittal sections cut through
the boundary of the medial and lateral septum revealed the presence of
both types of cell side by side in embryonic animals (Fig.
6D). In all cases, the immunoreactivity exhibited by
the bipolar GnRH neurons of the medial septum and hypothalamus was much
stronger than that observed in the small immunoreactive neurons of the
lateral septum (Fig. 6A-D). Counts of
GF6-immunoreactive cell profiles in serial sagittal sections from three
E14.5 mice revealed that ~600 small-sized, circular or ovoid-shaped
GnRH cells existed within the lateral septum at this age (individual embryo estimates of 408, 618, and 711 cells). Fine immunoreactive cell
processes were sometimes associated with these small cells, but it was
not until after birth that their multipolar nature was readily apparent
in all cells (Fig. 6J). The numbers of
GnRH-immunoreactive cells in the lateral septum were reduced after
birth and no longer detectable with the LR1 or SW1 antibody at P3/4
(Figs. 6H-J, 7). Thus, by P16 only a few
GF6-immunoreactive cells were detectable in the lateral septum, where
they were found alongside the classical GnRH neurons in the ventral
projections of the intermediate division of the lateral septum (Fig.
6J). These cells could not be detected in adult mice
of either sex.
A similar developmental sequence was found for GnRH in the pBNST, where
a small number of faintly stained immunoreactive cells were first
observed at E14.5, but thereafter, these cells were only detected with
the GF6 antibody (Fig. 6F,G). These cells displayed a
small ovoid-type of morphology, similar to those of the lateral septum,
and many exhibited fine immunoreactive processes (Fig. 6G).
Counts of GF6-immunoreactive cell profiles in serial coronal sections
from three E15.5 mice revealed that ~200 GnRH-expressing cells formed
the pBNST population at this age (individual embryo estimates of 174, 180, and 276 cells). Using the GF6 antibody, these cells persisted
until P3/4 but were greatly reduced in number by P16 and absent in
adults of either sex (Fig. 7).
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Figure 7.
Schematic indicating the developmental profile of
gal and GnRH immunoreactivity in the septohypothalamic, lateral
septum, pBNST, and tectal cell populations of the wild-type and GNZ
mouse. The gray shading indicates whether GnRH
immunoreactivity was detected with all three GnRH antibodies (LR1, SW1,
GF6; light gray) or GF6 alone (dark
gray).
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A third pattern of GnRH staining was found in the tectum. Here, all
three GnRH antibodies detected the same population of ovoid-shaped
cells from E15.5 through E19.5. Thereafter, all three equally failed to
detect any cells within the tectum of postnatal mice (Fig. 7). The mean
diameter of immunoreactive cells at E16.5 was 7.3 µm (range, 5.8-9.6
µm). In all cases, the immunostaining of sections with LR1, SW1, or
GF6 antibody preadsorbed with GnRH-I peptide resulted in a complete
absence of immunoreactivity. Furthermore, no staining was evident with
any of the three antibodies in the midbrain of mice. Although GnRH-II
has not, as yet, been detected in rodents, all other species show
GnRH-II in this location (Muske, 1993; Sherwood et al., 1993; Lescheid
et al., 1997; Tobet et al., 1997).
One set of E15.5 brains from five wild-type mice underwent in
situ hybridization with the GAP-I probe. Dense clusters of silver grains (Fig. 6E) were found over cells extending from
the nasal septum through to the preoptic area in a distribution
identical to that observed previously (Wray et al., 1989) for the
migrating GnRH neurons. In addition, a number of cells with a lot fewer silver grains per cluster were detected throughout the lateral septum
(Fig. 6E), in the developing BNST, and also in the
tectum with the same distribution as that found after GnRH-I
immunostaining. Two control sections hybridized in the presence of
excess unlabeled GAP-I probe displayed no silver grain clusters.
GnRH immunoreactivity in Small eye (Sey) mice
Because Sey mice fail to develop an olfactory placode (Thieler et
al., 1978; Hogan et al., 1986; Hill et al., 1991), they represented a
useful model for examining whether any of the new GnRH-I populations
identified in these studies share a common embryological origin with
the septohypothalamic GnRH-I neurons, which are born in the olfactory
epithelium (Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989).
Immersion-fixed immunocytochemistry was undertaken on six E14.5-16.5
homozygous Sey embryos taken from four different litters, which were
cut in coronal and parasagittal planes. Homozygous Sey mice were
obtained by crossing heterozygous Sey mice and selecting embryos
without any visual pigment. No GnRH immunoreactivity was detected in
the nose of Sey mice with any of the three GnRH antibodies (Fig.
8A). However, LR1-,
SW1-, and GF6-immunostained cells were present in the lateral septum (Fig. 8B-D) and tectum (Fig.
8E,F) of Sey mice, whereas staining in the
pBNST was only detected with the GF6 antibody. The distribution and
morphology of immunoreactive cells in all three areas was identical to
that observed in wild-type and GNZ mice (Fig. 8). In comparison with
wild-type mice, the GnRH staining intensity in the lateral septum
appeared to be stronger in Sey mice, and a few cells were detected that
were small in size but exhibited a bipolar-type morphology (Fig.
8D). The removal of primary antibody or preadsorbtion
of the LR1 antibody with GnRH resulted in an absence of
immunoreactivity in Sey brains.
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Figure 8.
GnRH immunoreactivity in parasagittal sections
(nose to the right) of the embryonic Sey mouse. A, B,
Low- and high-power views of GnRH immunoreactivity (GF6) in an E14.5
Sey mouse. Note the complete absence of GnRH staining within the nose
but presence, magnified in B, within the septum. Scale
bars: A, 250 µm; B, 40 µm. C,
D, High-power views of GnRH-immunoreactive (LR1) cells in the
septum. Scale bars, 10 µm. E, F, GnRH immunoreactivity
(E, GF6; F, LR1) in the tectum of an
E16.5 Sey mouse. Scale bars: E, 140 µm;
F, 10 µm.
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DISCUSSION |
We report here that a 13.5 kb murine GnRH construct is sufficient
to direct the correct temporal and spatial expression of the lacZ
reporter to native GnRH neurons in three independent lines of GNZ
transgenic mice. Using dual immuofluorescence we demonstrate that
~85% of GnRH neurons in the medial septum and hypothalamus of the
adult mouse express gal. In other transgenic lines derived using the
same construct, but in which gal protein is directed toward the
nucleus of the cell to enable dual-labeling peroxidase-based
immunocytochemistry, 95-100% of the GnRH neurons are found to express
gal (J. R. Pape, M. J. Skynner and A. E. Herbison, unpublished
observations). Thus, it seems most likely that the 10-15% of GnRH
neurons in GNZ mice without gal immunoreactivity are expressing
gal at a level below our detection limit with immuofluorescence. It
has been notoriously difficult to achieve cell-specific expression in
the brain using neuropeptide gene promoter-driven reporters (Waschek,
1995). The almost perfect targeting of GnRH neurons reported here is in
good agreement, however, with the functional study by Mason and
colleagues (1986) in which the same 13.5 kb of GnRH sequence was used
to restore reproductive fertility to the GnRH null hypogonadal mouse.
Because this construct contains sufficient cis-acting
regulatory elements with which to specify expression in GnRH-I neurons,
the GNZ lines described here will provide a useful "baseline" for
future in vivo promoter analysis and the targeting of the
GnRH phenotype.
"Ectopic" neural populations with
promoter-driven transgenics
The presence and extent of unexpected transgene-expressing
populations observed in the brain of GNZ mice were somewhat surprising. Previous research using a similar GnRH construct (Mason et al., 1986)
did not report extensive expression outside of the classic septohypothalamic GnRH neurons. Equally, transgenic mice carrying a
human GnRH promoter driving the luciferase reporter were not described
to exhibit substantial expression outside the hypothalamus (Wolfe et
al., 1996). We show here, with quantitative gal in situ
hybridization, that the level of transgene mRNA expression in lateral
septal cells is at least fourfold lower than that of septohypothalamic
GnRH neurons. A preliminary report also suggests that relatively low
levels of gal expression exist in the lateral septum of transgenic
mice bearing a 3kb murine GnRH promoter-lacZ construct (Spergel et al.,
1997). Thus, it seems likely that this reduced level of mRNA
expression, employment of luciferase as a reporter, and/or use of the
human GnRH promoter in the mouse are likely to underlie the failure to
identify these newly identified GnRH populations in other transgenic lines.
The presence of so-called ectopic populations of
transgene-expressing cells in the brain has been reported frequently in
both neuronal and non-neuronal promoter-driven transgenic lines
(Gunzburg et al., 1991; Rubenstein et al., 1992; Ang et al.,
1993; Min et al., 1994; Waschek, 1995). The pattern of transgene
expression in GNZ mice was found to be consistent within the three
lines and, therefore, is unlikely to be a consequence of a transgene "position effect." In light of the necessity for neurons to
maintain a diverse, yet cell-specific, pattern of gene regulation, the presence of previously unrecorded transgene expression in just three,
apparently disparate neuronal cell populations was intriguing. Previous
investigators had speculated that so-called ectopic transgene expression in the brain may reflect developmental patterns of gene
regulation (Min et al., 1994; Waschek, 1995). We now demonstrate conclusively that this is indeed the case, because each of the three
apparent ectopic populations is shown here to express authentic GnRH-I
during development. This finding raises the likelihood that transgene
expression described previously as ectopic in other pedigrees may
actually represent activation of the transgene in developmentally
relevant neuronal cell populations.
Our immunocytochemical studies clearly show that the endogenous GnRH-I
gene is not expressed in the lateral septum and BNST of the adult
despite the presence of transgene mRNA and protein in these populations
throughout life. Thus, it seems most likely that cis- or
trans-acting repressor elements not present in the transgene
are brought into play in late development to suppress GnRH-I gene
transcription in the lateral septum and BNST. This suggests that
distinct groups of cells are programmed to activate the GnRH-I gene but
that, as in other neuronal phenotypes (Goodman and Mandel, 1998),
transcriptional repressors are important in restricting GnRH-I
expression to just the septopreoptic GnRH neurons in the adult. The
identity of the repressors maintaining the adult pattern of GnRH-I
expression is unknown. However, we note that transgene and GnRH-I
expression starts and stops in parallel within the developing tectum,
suggesting that tectal cells use a unique repressor complex which is
present within the 13.5 kb construct.
Presence of multiple GnRH-expressing neurons in
mammalian brain
It is established in a wide range of species that GnRH-I and
GnRH-II are expressed in distinct, spatially restricted regions within
the brain; the GnRH-I population is distributed in a specific septohypothalamic distribution, whereas GnRH-II neurons are located within the midbrain (Muske, 1993; Sherwood et al., 1993; Lescheid et
al., 1997). Taking insight from the distribution of
transgene-expressing populations in GNZ mice, we now demonstrate the
existence of four distinct populations of GnRH-I neurons in the normal
mouse. One represents the neuroendocrine septohypothalamic GnRH
neurons, whereas the other three are anatomically and developmentally
distinct neuronal populations, which express GnRH-I in a transient
manner during development. All three of the newly identified
populations are synthesizing authentic GnRH-I rather then GnRH-II,
because (1) they are detected by three different GnRH-I antibodies; (2) each population was found to express mRNA transcribed from GnRH-I exon
III, a highly divergent region of the GnRH-I and GnRH-II genes (White
et al., 1998); (3) the tectal population has been shown previously to
be GnRH-I in nature (Wu et al., 1995); and (4) the cells were all
targeted by 13.5 kb of GnRH-I sequence.
Although the identification of GnRH-I populations in the lateral septum
and BNST is new, transient GnRH-I immunoreactivity had been detected
previously in the tectum by Wu and colleagues (1995) and is in
excellent agreement with the present findings. Furthermore, recent work
in the primate indicates that these newly identified GnRH populations
may not simply be a phenomenon of rodents. N-terminal GnRH
immunoreactivity was recently revealed in neurons within a number of
different brain regions, including the lateral septum, amygdala,
claustrum, and globus pallidus of embryonic monkeys (Quanbeck et al.,
1997). Indeed, the distribution and morphology of such cells within the
developing lateral septum of the monkey are highly reminiscent of those
reported here.
We also demonstrate that these new populations of GnRH neurons do not
originate from the olfactory placode, the birth site of the
septohypothalamic neurons (Schwanzel-Fukuda and Pfaff, 1989; Wray et
al., 1989). The Sey phenotype arises from a point mutation in the Pax-6
gene and results in mice with failed development of the eye and
olfactory placodes (Thieler et al., 1978; Hogan et al., 1986; Hill et
al., 1991). As found previously (Dellovade et al., 1998), none of the
migrating septohypothalamic GnRH-I neurons was present in homozygous
Sey mice. However, in striking contrast, we observed normal populations
of GnRH-I-immunoreactive cells in the lateral septum, BNST, and tectum
of Sey mice. This indicates that these cells must have a nonplacodal
origin and that, equally, their development is independent of Pax-6.
Although studies in chicken have shown that GnRH-II, and possibly
thalamic GnRH-I, neurons originate outside of the olfactory system
(Norgren and Gao, 1994; Northcutt and Muske, 1994), the present work is the first to prove a nonplacodal origin of GnRH-I neurons in mammals.
The function of these newly identified GnRH populations is unknown.
However, the identification of distinct brain regions that express
GnRH-I, and one that continues to synthesize GnRH-I into the third
postnatal week, suggests that this neuropeptide may have previously
unsuspected roles in neural development. Our studies also indicate that
a unique form of GnRH-I processing occurs within the lateral septal and
BNST cell populations. Although immunoreactivity in these structures
was detected initially with all three GnRH-I antibodies, both LR1 and
SW1 immunoreactivity ceased at specific developmental time points only
to leave the GF6 immunoreactivity before it too disappeared (Fig. 7).
The septopreoptic GnRH neurons, which are able to fully process the
pro-GnRH precursor by E14.5 (Livine et al., 1993), show equivalent GF6
immunoreactivity throughout embryonic and postnatal development.
Because the GF6 antibody is directed against the N-terminal region of
GnRH-I (Quanbeck et al., 1997), it is possible that this pattern of
staining represents cell-specific patterns of GnRH cleavage within late
embryonic septal and BNST neurons.
In summary, we report that 13.5 kb of GnRH sequence is sufficient to
direct transgene expression to the classical GnRH neurons throughout
development. Cells expressing transgene were also detected in the
lateral septum, pBNST, and tectum of GNZ mice, and subsequent immunocytochemical and in situ studies in wild-type mice
have proven that these cells express authentic GnRH-I for differing periods during embryonic and postnatal development. Further
investigations in Pax-6 mutant mice have shown that none of these newly
identified populations of GnRH neurons originates from the olfactory
placode. Together, these observations represent the identification of
multiple GnRH neuronal populations of diverse embryological origin
within the mammalian brain and indicate that so-called ectopic reporter expression in transgenic animals may reveal novel patterns of developmental gene expression within the brain.
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FOOTNOTES |
Received Jan. 19, 1999; revised April 9, 1999; accepted May 7, 1999.
This work was supported by the Lister Institute of Preventive Medicine
and the Biotechnology and Biological Sciences Research Council.
We thank Dr. A Mason for providing the GnRH-SmaI plasmid and Drs. A. Caraty, R. Benoit, N Sherwood, and S. Wray for generous gifts of antibodies. We thank members of the Babraham Institute Small
Animal Facility for their valued assistance and Dr. A. Woodhouse for
early discussions. This work was presented in part at the 27th Annual
Meeting of the Society for Neuroscience, New Orleans, 1997.
Correspondence should be addressed to Dr. Allan E. Herbison, Laboratory
of Neuroendocrinology, Babraham, Cambridge CB2 4AT, UK.
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