Transgenics Identify Distal 5'- and 3'-Sequences Specifying Gonadotropin-Releasing Hormone Expression in Adult Mice
Jean-Rémi Pape1,
Michael J. Skynner1,
Nicholas D. Allen and
Allan E. Herbison
Laboratories of Neuroendocrinology (J.-R.P., M.J.S., A.E.H.)
and Developmental Neurobiology (N.D.A.) The Babraham
Institute Cambridge, CB2 4AT, United Kingdom
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ABSTRACT
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GnRH neurons play a critical role in regulating
gonadotropin secretion, but their scattered distribution has prevented
detailed understanding of their molecular and cellular properties
in vivo. Using GnRH promoter-driven transgenics we have
examined here the role of 5'- and 3'-murine GnRH sequences in
specifying GnRH expression in the adult mouse. Transgenic mice bearing
a lacZ construct incorporating 5.5 kb of 5'-, all the introns and
exons, and 3.5 kb of 3'-murine GnRH sequence were found to express
ß-galactosidase (ßgal) immunoreactivity in approximately 85% of
all GnRH neurons. Deletion of GnRH sequence 3' to exon II had no effect
upon transgene expression in the GnRH population (89%) but resulted in
the appearance of ectopic ßgal immunoreactivity in several regions of
the brain. The production of additional mice in which 5'-elements were
deleted to leave only -2.1 kb of sequence resulted in an approximately
40% reduction in the number of GnRH neurons expressing ßgal. Mice in
which further deletion of 400 bp allowed only -1.7 kb of 5'-sequence
to remain exhibited a complete absence of ßgal immunoreactivity
within GnRH and other neurons. These results suggest that elements 3'
to exon II of the GnRH gene have little role in enabling GnRH
expression within the GnRH phenotype but, instead, are particularly
important in repressing the GnRH gene in non-GnRH neurons. In contrast,
elements located between -2.1 and -1.7 kb of distal 5'-sequence
appear to be critical for the in vivo activation of GnRH
expression within GnRH neurons in the adult brain.
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INTRODUCTION
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The GnRH neurons represent the final output cells of the neuronal
network controlling mammalian fertility. As such, an understanding of
the mechanisms through which the GnRH gene is regulated within these
neurons is critical. However, investigation of the GnRH neuron and GnRH
gene regulation in vivo has proven difficult due to the
unusual embryonic origin of the GnRH phenotype and their relatively
small number and scattered distribution within the medial septum (MS)
and hypothalamus of the postnatal brain (1, 2, 3).
In contrast to the paucity of in vivo data, a wealth of
information exists on the regulation of the rat and human GnRH gene
in vitro using immortalized GnRH-secreting cell lines
derived from the mouse (4, 5). These investigations have identified
several 5'-regions and mapped multiple cis-acting elements
of the rat and human GnRH promoter that are involved in specifying and
regulating GnRH gene expression in vitro. For example, an
evolutionarily well conserved 160-bp proximal promoter is thought to
play a role in the determination of neuron-specific GnRH gene
expression as well as its regulation in vitro (4, 5, 6, 7).
Further, the analysis of immortalized cell lines combined with DNA
footprinting and site-directed mutagenesis have demonstrated that
transcription factors such as Oct-1, GATA, and SCIP/Oct-6/Tst1 bind to
elements within the rat GnRH promoter and regulate its expression
in vitro (8, 9, 10, 11).
Although in vitro studies in immortalized cell lines have
been of great use in defining transcriptional complexes involved in
regulating rat and human GnRH gene expression, they are unlikely to
address the true complexity and mechanisms of gene regulation in
vivo in the adult brain (12). The GnRH neurons are a dynamic
neuronal phenotype receiving multiple neurochemical inputs and
additionally subject to a variety of different neurohumoral signals
throughout development and during adulthood (3, 13, 14). Furthermore,
there are species differences in the structure and function of 5' GnRH
gene elements (7, 16, 17), including that of the conserved proximal
promoter (18). In this light, it may be relevant that critical rat and
human GnRH gene elements have been elucidated within a murine
transcriptional environment in vitro (5, 9) and in
vivo (15).
In the present study, we have attempted to circumvent some of these
problems and address the issue of in vivo GnRH gene
specification by generating transgenic mice carrying various deletions
of the murine GnRH gene (mGnRH) linked to a lac-Z reporter gene
cassette. We have shown previously that a 13.5-kb mGnRH-lacZ (GNZ)
construct is sufficient to drive the correct temporal and spatial
expression of transgene in native GnRH neurons in the mouse (19). In
this study, deletions based upon the GNZ construct have addressed the
role of 3'- and distal 5'-sequences in specifying GnRH gene expression
within the GnRH phenotype. Our findings indicate that 3'-elements are
important in restricting the expression of GnRH to the GnRH phenotype
while a 400-bp region between -1.7 and -2.1 kb in 5'-flanking
sequence is critical for activating GnRH gene expression within adult
GnRH neurons in vivo.
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RESULTS
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Expression of ß-Galactosidase (ßgal) in GNZ Transgenic Mice
The GNZ construct spanned the mGnRH coding sequence including the
full intron/exon structure with 5.5 kb of 5'- and 3.5 kb of 3'-sequence
and had a lacZ reporter cassette inserted into exon II sequences
encoding the GnRH decapeptide (Fig. 1
).
As previously described (19), three independent lines of GNZ mice
(3210, 3235, 3252) were generated, which showed identical spatial and
temporal patterns of transgene expression. In the present study, the
analysis of homozygous 3252 mice (n = 5, homozygous males)
revealed the presence of the lacZ gene product ßgal throughout the
cytoplasm of cells (Fig. 2
, A and B)
located within the classic distribution of the septohypothalamic GnRH
neurons; MS, diagonal band of Broca/rostral preoptic area (rPOA), and
ventrolateral anterior hypothalamic area (AHA). Numerous cells located
within the lateral septum (LS) and posterior division of the bed
nucleus of the stria terminalis (pBNST), and a small number in the
region of the lateral olfactory nucleus (LON), were also found to
express the transgene. In this case, immunoreactivity existed as small
intracytoplasmic donut-like organelles; a subcellular pattern of
transgene expression common in lacZ neuronal transgenics (20). Dual
labeling immunofluorescence experiments (Fig. 2
, A and B) demonstrated
that 85.2 ± 3.0% of GnRH neurons located throughout the
septohypothalamic continuum expressed ßgal immunoreactivity in GNZ
mice (MS, 79.0 ± 4.4%; rPOA, 86.2 ± 2.5%; AHA, 84.5
± 2.8%). Perikaryal GnRH immunoreactivity was not found in the LS,
pBNST, or LON and ßgal immunofluorescence was not present in
nontransgenic mice.

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Figure 1. Structure of the Different GnRH-lacZ Constructs
The GNZ construct contains 5.5 kb of 5'-, all 4 exons, and introns and
3.5 kb of 3'-murine GnRH gene sequence with lacZ
(triangle) incorporated into exon II sequences (see
Materials and Methods). Shading indicates
different regions removed to make the various deletion
constructs.
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Figure 2. Expression of ßgal Immunoreactivity in GNZ
(A and B) and 5.2-GNLZ-0 (CE) Male Mice
A and B, Dual immunofluorescence for ßgal (panel A, FITC) and
GnRH (panel B, Texas Red) in an rPOA section. Note that the five GnRH
neurons (arrowheads in panel B) all express ßgal while
GnRH-immunoreactive fibers do not. Scale bar represents 40 µm. C,
Low-power view of an rPOA section dual labeled for ßgal (black
nuclei) and GnRH (brown staining). Note that
ßgal is present in all of the GnRH neurons as well as non-GnRH
neurons. Scale bar represents 100 µm. D, High-power view of a
dual-labeled ßgal (black) immunoreactive GnRH
(brown) neuron showing nuclear localized transgene as
well as the intracytoplasmic donut (arrow). Scale bar
represents 10 µm. E, ßgal staining at the level of the anterior
hypothalamus showing transgene expression in the pBNST and within
distinct groups of anterior hypothalamic cells (arrow).
Scale bar represents 150 µm. 3V, Third ventricle.
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Effect of 3'-Deletion on ßgal Expression in Transgenic Mice
To assess the role of 3'-sequences in specifying GnRH expression,
additional lines of transgenic mice were generated bearing GNZ-like
constructs (Fig. 1
) in which approximately 6.5 kb of 3'-sequence was
deleted from either the start of sequences encoding the GnRH
decapeptide (5.2-GNLZ-0A), or the start of exon II (5.2-GNLZ-0B). The
remaining 5'-sequence was linked to a nuclear localizing signal-lacZ
cassette. The two different constructs were made because preliminary
work had suggested a possible role for exon II sequences in GnRH
expression. Thus, the 5.2-GNLZ-0A and 5.2-GNLZ-0B constructs were
identical except for the latter missing the first 69 bp of coding
sequence in exon II. During the linearization and subsequent
purification, a 300-bp region at the extreme 5' terminus of the GNZ
construct was deleted in both of the 5.2-GNLZ-0 transgenes.
Four independent lines of transgenic mice were generated bearing the
5.2-GNLZ-0A construct, of which two (481, 496) expressed the transgene.
In the 481 line, immunostaining revealed the presence of
nuclear-located ßgal transgene in the great majority of GnRH neurons.
All cells displaying nuclear ßgal also expressed intracytoplasmic
donut-like ßgal immunoreactivity (Fig. 2D
). A quantitative analysis
of transgene expression in heterozygous mice of the 481 line (n =
5, heterozygous males) demonstrated that approximately 89% of GnRH
neurons expressed the transgene (MS, 83.9 ± 3.5%; rPOA,
90.1 ± 2.0%; AHA, 89.9 ± 1.5%). A similar analysis in
heterozygous mice of the 496 line (n = 5, heterozygous males)
revealed much lower levels of transgene expression with approximately
30% of GnRH neurons containing ßgal immunoreactivity throughout the
GnRH continuum (MS, 21.0 ± 4.4%; rPOA, 29.2 ± 5.3%;
AHA, 31.1 ± 6.2%). A further five lines of transgenic mice were
generated bearing the 5.2-GNLZ-0B construct, of which four (1311, 1314,
1324, 1325) were found to express the transgene although one (1311) was
lost with breeding. Analysis of the 1311 founder and the three other
lines (n = 5, heterozygous males each) detected high levels of
transgene expression with 90%, 92%, 82%, and 90% of all GnRH
neurons immunoreactive for ßgal in the 1311, 1314, 1324, and 1325
lines, respectively (Figs. 2C
and 3
and Table 1
). The pattern of transgene expression
was independent of the location of the GnRH neurons and 3 within the
septohypothalamic continuum in all lines (e.g. 1314 line:
MS, 90.8 ± 4.2%; rPOA, 93.2 ± 2.6%; AHA, 86.7 ±
6.1%).

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Figure 3. Percentage of Adult Male GnRH Neurons with
Detectable ßgal Immunoreactivity in the Different Groups of GnRH-lacZ
Transgenic Mice
Expression in the GNZ-3252 mice was 85%, which is identical to that of
the other GNZ-3210 line (19 ). Mean (±SEM) values for the
5.2-GNLZ-0 mice come from five different pedigrees (excluding the
5.2-GNLZ-0A-496 line, see text), two pedigrees for the 2.1-GNZ-0 mice,
and three lines for the 1.7-GNZ-0 mice.
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In lines from both of the 5.2-GNLZ-0 pedigrees, substantial
nuclear-located and donut-like ßgal immunoreactivity was consistently
observed in cells that lay outside the GnRH neuron distribution (Fig. 2
, C and E, and Fig. 4
). These
included the two populations in the LS and pBNST (Fig. 2E
), as found in
GNZ mice, in addition to cells in the anterior BNST, organum
vasculosum of the lamina terminalis, diagonal band of Broca,
rPOA, and anterior hypothalamus (Fig. 2E
), suprachiasmatic nucleus,
thalamus, piriform cortex and, in 5.2-GNLZ-0B mice, the caudate putamen
(Fig. 4
).

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Figure 4. Camera Lucida Diagrams Showing the Distribution of
Transgene-Expressing Cells at Three Levels of the Mouse Forebrain in
5.2-GNLZ-0B
Each black dot represents 510 ßgal immunoreactive
neurons. LS, Lateral septum; MS, medial septum; rPOA, rostral preoptic
area; HDB, horizontal diagonal band; CP, caudate putamen; PIR, piriform
cortex; Tu, olfactory tubercle; aBNST and pBNST, anterior and posterior
bed nucleus stria terminalis; SCN, suprachiasmatic nucleus; SFN,
septofimbrial nucleus; PVN, thalamic paraventricular nucleus; LON,
lateral olfactory tract nucleus.
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Effect of 5'-Deletions upon ßgal Expression in Transgenic
Mice
Lines of transgenic mice were subsequently made in which the
5'-elements of the GNZ construct were deleted so as to leave 2.1 kb
(2.1-GNZ-0) or 1.7 kb (1.7-GNZ-0) of 5'-sequence with the lacZ cassette
inserted at the beginning of exon II sequences encoding GnRH (Fig. 1
).
These deletions carried no 3'-sequence distal to the exon II transgene
insertion site.
Two lines of mice (3419, 3439) were generated carrying the 2.1-GNZ-0
construct and both expressed the transgene. However, the male 3419
founder was found to be infertile. Single-labeling immunocytochemistry
for ßgal demonstrated the presence of transgene throughout the
cytoplasm of cells located in the distribution of the septohypothalamic
GnRH neurons in both lines. Dual-labeling immunofluorescence in the
3419 founder and five male heterozygous 3439 offspring confirmed the
presence of transgene in GnRH neurons and showed that 61% and 58% of
GnRH neurons expressed ßgal immunoreactivity, respectively (Fig. 3
). Again, the pattern of transgene
expression in GnRH neurons was independent of their location within the
GnRH continuum (MS, 59.3 ± 11.8%; rPOA, 58.8 ± 4.3%; AHA,
56.0 ± 5.5% in 3439 line). Transgene expression outside of the
GnRH neurons existed in the form of the intracytoplasmic ßgal
immunoreactive donuts and was identified within cells of the brain
distributed in an identical manner to that of the 5.2-GNLZ-0 lines
(Table 1
).
Three transgenic lines (2604, 2616, 2624) were generated bearing the
1.7-GNZ-0 construct but no ßgal immunoreactivity, or Xgal signal, was
detected in GnRH or other cells of the brain in any of the three lines
or their founders (n = 5 males and 34 females, each line).
Endogenous GnRH Expression in Transgenic Lines
Although a marker other than GnRH would be preferable for the
in vivo analysis of the likely heterogeneous GnRH
population, it remains the only known distinguishing characteristic of
the phenotype. The distribution and number of GnRH-immunoreactive
neurons detected in all transgenic lines were the same as that seen in
nontransgenic mice and not different between lines (Table 2
). The GnRH constructs used in this
study were designed so as to not produce the GnRH decapeptide.
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Table 2. Comparison between Each Transgenic Line of the
Number of GnRH Neurons Detected per Section (mean ±
SEM) at Three Levels of the GnRH Continuum in Adult Male
Mice (Numbers Given in
Brackets)
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Transgene Expression as a Function of Sex and Allelic Copy
Number
To examine the potential for differences in transgene expression
as a result of allelic copy number, the expression of the transgene was
examined in heterozygous and homozygous 5.2-GNLZ-0B mice of the 1314
line. Additionally, to determine whether sex differences may exist in
transgene expression, male and female mice (n = 5, each sex) from
both the 1314 and 2.1-GNZ-0 lines were evaluated. No differences in
transgene expression within GnRH neurons, or elsewhere in the brain,
were found between homozygous and heterozygous mice or between males
and females (Fig. 5
).

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Figure 5. Comparison of the Percentage of GnRH Neurons
Expressing ßgal Immunoreactivity in Male and Female Mice of the
5.2-GNLZ-0B (1314 line) and 2.1-GNZ-0 (3439) Pedigrees and an Analysis
of Heterozygous (Htz) and Homozygous (Hmz) 1314 Mice
Values represent mean ± SEM.
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DISCUSSION
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We demonstrate here that a 13.5-kb GNZ construct containing 5.5 kb
of 5'-, all the introns and exons, and 3.5 kb of 3'-murine GnRH
sequence is sufficient to target a lacZ transgene to the GnRH phenotype
with a high degree of specificity in transgenic mice. As reported
previously for both existing GNZ lines (19), we were able to detect
ßgal within 8590% of GnRH neurons using dual-labeling
immunofluorescence. It is presently unclear why the transgene is not
detected in 100% of GnRH neurons. One possibility is that the residual
1015% of GnRH neurons express ßgal at a level below detectability
or, alternatively, that a significant degree of transcriptional
heterogeneity may exist within the GnRH population. We also found
substantial transgene expression within the LS and pBNST in GNZ mice.
Recent studies have shown that cells in both the LS and pBNST
synthesize authentic GnRH during embryogenesis and early postnatal
development (19). The persistence of transgene expression in the cells
of the LS and pBNST of the adult, when GnRH peptide is absent, is
likely, therefore, to reflect the lack of a critical repressor element
within the GNZ construct (19). Thus, the GNZ construct contains
sufficient cis-acting elements to faithfully specify
transgene expression to the GnRH phenotype in the mouse.
Role of 3'-Elements in GnRH Expression within the Brain
The deletion of GnRH sequence 3' to exon II was found to have no
effect upon the presence of the transgene within the GnRH phenotype.
Regardless of whether the 3'-elements were deleted from the ATG or GnRH
coding sequence of exon II, the percentage of GnRH neurons expressing
transgene (89%) was not found to be different from that of the GNZ
line (85%). Five of the six 5.2-GNLZ-0 lines exhibited transgene
expression in 8890% of the GnRH neurons while only approximately
30% of GnRH neurons expressed ßgal in the remaining 496 line. The
discrepancy between the 496 and the other five lines is most likely due
to an effect of flanking DNA at the site of transgene integration,
which is most commonly that of repression (12, 21). A major problem
with the analysis of gene expression through promoter-driven
transgenesis is that of the potential variability resulting from
position effect. This example serves to highlight the importance of
obtaining several lines with consistent expression profiles.
Our finding that 3'-elements are not necessary for the neuron-specific
expression of the GnRH gene within the GnRH phenotype is in good
agreement with data from the hpg mouse in which a 33.5-kb
deletional mutation involving the distal half of the mouse GnRH gene
does not prevent GnRH transcription in the GnRH neurons (22). While
3'-gene elements have similarly been shown to have no role in targeting
POMC expression to the pituitary gland (23), the present results would
appear to be the first evaluating directly the effect of 3'-gene
deletion on neuropeptide gene targeting within the brain.
Although we do not find a role for 3'-elements in targeting the GnRH
phenotype, they do appear to have a function in repressing GnRH gene
expression in other neurons. In all six of the 5.2-GNLZ-0 lines, we
observed transgene expression within multiple hypothalamic and limbic
sites in addition to the thalamus and caudate putamen. While some of
these brain regions have been reported to contain GnRH immunoreactivity
in the embryonic monkey (24), we have been unable to provide evidence
for authentic GnRH expression in any of these regions in the developing
mouse (our unpublished observations). Thus, unlike the LS and
pBNST populations revealed by the GNZ mice, we believe these further
sites of transgene synthesis represent true ectopic expression
unrelated to the GnRH gene. Although we cannot exclude the possibility
that the 300-bp deletion of the extreme 5'-elements in these
transgenics may play a role, its seems most likely that important
repressor sequences exist within introns B/C, exons 3/4, and/or 3.5 kb
of 3'-sequence of the murine GnRH gene. We saw no further ectopic
expression after deletion of 5'-elements suggesting, at least, that
tissue-specific repressor elements are unlikely to exist between -5.2
and -2.1 kb of the GnRH enhancer. The location of repressor sequences
is highly variable with neuron-specific genes (25), and their precise
location within the 6.5 kb of 3'-GnRH sequence identified here remains
to be determined.
Role of Distal 5'-Elements in GnRH Gene Expression in
Vivo
Transgenic mice bearing lacZ constructs with 2.1 kb of 5'-flanking
sequence were found to have lower levels of transgene expression in
GnRH neurons compared with either the 5.5 kb or 5.2 kb 5'-transgenics,
which express equivalently. This lower level of transgene expression
was evidenced by the 40% reduction in the number of GnRH neurons
expressing detectable ßgal immunoreactivity in 2.1-GNZ-0 lines. These
observations suggest that important enhancer element(s) exist between
-5.2 and -2.1 kb of the mouse GnRH gene. Although very little is
known about the mouse GnRH gene, extensive studies of rat GnRH
sequences in vitro have identified an important distal
300-bp enhancer between -1863 and -1571 (5, 9, 26) where both Oct-1
and GATA family transcription factors are known to act (8, 11).
Preliminary work has shown that the Oct-1 binding element at -1788 in
the rat enhancer is perfectly conserved in the mouse and has a similar
critical role in enabling basal murine GnRH gene expression in
vitro (27). This element lies at -2245 (from the transcription
start site) in the mouse GnRH gene and is, therefore, very likely to be
one of the elements contributing to the reduced levels of basal
transgene expression we observe in the 2.1-GNZ-0 transgenics.
More dramatically, however, we found that further deletion of 5'-
sequences to -1.7 kb resulted in a complete absence of detectable
transgene within the GnRH neurons or elsewhere in the brain. In this
case, it is important to note that none of the three founder lines
exhibited detectable ßgal immunoreactivity. In the complete series of
transgenic mice used in this study, 65% (11/17) of founder lines were
found to have the transgene incorporated into a region of the genome
enabling its expression. As such, the chance that all three of the
1.7-GNZ-0 lines had the transgene located in silent regions of DNA is
very small. It is most likely, therefore, that the 1.7-GNZ-0 construct
contains insufficient enhancer elements to enable detectable levels of
transgene transcription (12). Our inability to detect ßgal using
immunocytochemistry or Xgal histochemistry does not, however, exclude
the possibility that low levels of transgene transcription do occur in
GnRH neurons carrying the 1.7-GNZ-0 construct.
This study indicates that the 400-bp region between -2.1 and -1.7 kb
is a critical enhancer region for the mGnRH gene in vivo. It
is of interest that the equivalent region in the rat GnRH gene has not
been similarly identified in vitro within mouse cell lines.
Because of the species differences between rodent and human GnRH
enhancer sequence (7, 16, 17), it is perhaps less surprising to find
that human 5'-elements distal to -1.1 kb are of little significance to
basal GnRH expression in the mouse in vivo (15). However,
like the human gene (15), our present data would suggest that the
conserved proximal promoter, which mediates a number of hormonal and
second messenger responses (5), may be insufficient on its own to
enable significant basal GnRH gene expression.
In summary, we report here a series of transgenic mice in which
deletions of mGnRH-lacZ constructs have enabled us to examine the role
of 5'- and 3'-elements in specifying GnRH expression in
vivo. The results indicate that elements 3' to exon II have little
role in targeting GnRH expression to the GnRH phenotype but do contain
elements used to repress gene expression in non-GnRH neurons. In
contrast, a clear role was demonstrated for distal 5'-elements in
activating expression within GnRH neurons in the mouse. While
5'-elements within sequences spanning -5.2 to -1.7 kb are shown to be
important, a 400-bp region between -1.7 and -2.1 kb, in particular,
was demonstrated to be critical for the expression of mGnRH. Together,
these observations provide the first information on the functional
organization of the mGnRH gene in vivo and provide the basis
for future experiments aimed at elucidating the critical transcription
factors and elements responsible for GnRH expression in the adult
brain.
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MATERIALS AND METHODS
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DNA Constructs
GNZ construct
The pGnRHSmaI cassette (gift of Dr. A. Mason. Prince Henrys
Institute, Melbourne, Australia) contained all the introns and exons of
the GnRH gene, 5.5 kb of upstream 5'-, and 3.5 kb of downstream
3'-sequence in addition to a SmaI restriction site
engineered by site-directed mutagenesis into exon II sequences encoding
amino acids 2 and 3 of the GnRH decapeptide. A lac-Z cassette was
inserted at the SmaI site to produce the GNZ construct (Fig. 1
and Ref. 19). The GNZ construct was released before microinjection
using BamHI and BstEII double digestion and
purified as described previously (19). To produce subsequent
constructs, appropriate regions of mGnRH sequence were subcloned into
plasmid vectors before the insertion of lac-Z cassettes.
3'-Deletion Constructs
5.2-GNLZ-0A, the region 5' to the SmaI site of exon II, was
subcloned into BglII-SmaI cut pEGFP-1
(CLONTECH Laboratories, Inc., Palo Alto, CA) using
BamHI and SmaI double digestion of pGnRH to
produce p1068. Subsequently, nuclear localized ßgal (nls-lac-Z) was
inserted from pZ (28) using SmaI-NotI double
digestion. 5.2-GNLZ-0B, a construct in which the transgene was inserted
into the start of exon II (immediately 5' to the ATG), was constructed
from p1068 as follows: PCR primers ExIFor
5'-CGGAAGGTCGACATCCCTTTGACTTTC-3' and ExIIRev
5'-GGAATTCGTCCACTCTAAGGGACATCA-3' were used to amplify a 1049-bp region
of exon I-intron A-exon II from pGnRH using Pfu DNA polymerase
(Stratagene, La Jolla, CA). This blunt-ended PCR amplicon
was cut using SalI (which cuts within the sequence of the
ExIFor primer) and ligated into XhoI-SmaI cut
p1068 to produce p1168. nls-lacZ was introduced into this vector using
XmaI-NotI digestion of pZ, to release the
nls-lac-z cassette, which was ligated into the
AgeI-NotI sites of p1168. The 5.2-GNLZ-0
transgenes were released using NsiI and NotI
double digestion before purification and injection.
5'-Deletion Constructs
These constructs were produced by removing the nls sequence of the
5.2-GNLZ-0A construct by replacing the SmaI-EcoRV
with SmaI-EcoRV from the GNZ construct, and using
either BglII (2.1-GNZ-0) or AflII (1.7-GNZ-0) to
remove 5'-sequences and NotI to remove vector sequences. For
all deletion constructs, integration sites were sequenced to verify
correct alignment.
Transgenic Animals
Transgenic mice were produced by pronuclear injection (29).
Briefly, fertilized mouse eggs from superovulated F1 mice (CBA x
C57/B16) mated to F1 males were visualized using differential
interference contrast optics on an inverted microscope
(Nikon, Melville, NY). DNA was introduced into the male
pronucleus using manual micromanipulators and glass capillary
micropipettes (Leitz, Rockleigh, NJ). 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 PCR analysis of genomic DNA isolated
from tail biopsies after weaning as reported previously (19). All mice
were bred and housed at the Babraham Institute according to UK Home
Office requirements under projects 80/972 and 80/1005.
Analysis of Transgene Expression
Transgenic mice were maintained under conditions of 12 h of
light (lights on at 0600 h) with constant access to food and water
and treated in accordance with UK Home Office protocols. Male and
female transgenic mice (610 weeks of age) were administered an
overdose of Avertin (tribromoethanol and 2-methylbutan-2-ol in
10% ethanol; 0.2 ml/20 g, ip) and perfused directly through the left
ventricle of the heart with 1520 ml of 4% paraformaldehyde in PBS
(pH 7.4). Brains were removed and postfixed for 12 h at room
temperature before being immersed in a 30% sucrose, Tris-buffered
saline (TBS) solution at 4 C. The following day three sets of 40 µm
thick coronal sections were cut through the rostral forebrain,
including the preoptic area and hypothalamus, using a freezing
microtome. In the 1.7-GNZ-0, 2.1-GNZ-0 and GNZ lines, analysis was
undertaken by processing one set of sections for ßgal using
peroxidase-based immunocytochemistry and a further set of sections
using dual GnRH-ßgal immunofluorescence. In the 5.2-GNLZ-0 lines,
with nuclear localized transgene, a single set of sections underwent
dual peroxidase-based immunocytochemistry for ßgal and then GnRH.
Peroxidase-Based Immunostaining
Free floating, single-labeling ßgal immunocytochemistry was
undertaken as reported previously (19). In brief, sections were placed
in a 1% H2O2, 40% methanol, TBS solution for
5 min followed by three washes in TBS. Sections were then incubated in
a polyclonal rabbit antisera specific for ß-gal (1:8,000; ICN Biomedicals, Inc. GmbH, Postfach, Germany) for 48 h at 4 C
on a shaking platform followed by washing with TBS and incubation in
biotinylated goat antirabbit IgGs (1:200; Vector Laboratories, Inc., Peterborough, UK) for 90 min at room temperature. Sections
were then washed in TBS and placed in the Vector Elite avidin
peroxidase substrate (1:100; Vector Laboratories, Inc.)
for a further 90 min before identification with the
nickel-diaminobenzidine tetrahydrochloride chromagen using glucose
oxidase. In the case of the GNLZ lines, ßgal-stained sections were
processed further for GnRH immunoreactivity by treatment with 40%
methanol/TBS/1% H2O2 and incubation in a
polyclonal rabbit antisera recognizing amino acids 610 of the GnRH
decapeptide (1:40,000; LR1 antibody, gift of R. Benoit, McGill
University, Montreal, Quebec, Canada) for 40 h at 4 C. After
washing, sections were placed in peroxidase-conjugated goat antirabbit
IgGs (1:400; Vector Laboratories, Inc.) for 4 h at
room temperature and immunoreactivity was revealed by diaminobenzidine
tetrahydrochloride without nickel. All immunoglobulins were dissolved
in TBS containing 0.3% Triton X-100, 0.3% BSA, and primary antibody
solutions also contained 2% normal goat serum. The specificity of both
antibodies in the mouse brain has been established (3, 19), and
controls consisted of the omission of either ßgal or GnRH antisera
from the immunostaining protocol. Sections were examined under a BIOMED
microscope (Leitz) and individual cells were examined at
25x objective magnification. For each GNLZ mouse, three to four
sections containing the MS, three to four sections containing the rPOA,
and two to three sections containing the AHA were selected, and the
number of brown GnRH-immunostained cells with and without black
ßgal immunoreactivity (Fig. 2D
) were counted in each animal. These
counts provided an average value for each area in each animal and were
used to determine mean (±SEM) values.
Dual Labeling Immunofluorescence
Free-floating dual labeling for ßgal and GnRH was undertaken as
reported previously (19). In brief, one set of sections was incubated
in an antibody mixture comprised of the polyclonal rabbit ßgal
antibodies (1:4,000) and a polyclonal sheep antisera detecting
C-terminal epitopes of GnRH (1:1,000; gift of A. Caraty, Nouzilly,
France) for 40 h at 4 C. Sections were then placed in fluorescein
isothiocyanate (FITC) donkey antirabbit (1:50, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and
biotinylated horse antigoat (1:200; Vector Laboratories, Inc.) immunoglobulins for 90 min at room temperature. After
washing, sections were placed in Texas Red Avidin (2.6 µl/ml;
Vector Laboratories, Inc.) for an additional 90 min at
room temperature. Controls consisted of the omission of one of the
primary antibodies from the immunostaining protocol. Sections were
viewed under a DM-RB fluorescent microscope (Leica Corp.),
and individual cells were examined at 40x or 100x objective
magnification with switching between Texas Red and FITC filter sets to
determine whether or not cells were double labeled. For each mouse,
three to four sections containing the MS, three to four sections
containing the rPOA, and two to three sections containing the AHA were
selected and the number of GnRH only and dual
GnRH-ßgal-immunoreactive cells were 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.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. A. Mason (Prince Henrys
Institute, Melbourne, Australia) for providing the GnRH-SmaI
plasmid, and Drs. A. Caraty (INRA, Nouzilly, France) and R. Benoit
(McGill University, Montreal, Quebec, Canada) for the generous gifts of
their antibodies. The authors thank members of the Babraham Institute
Small Animal Facility for their valued assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Allan E. Herbison, Laboratory of Neuroendocrinology, Babraham Institute, Cambridge CB2 4AT, United Kingdom.
Work was supported by the Wellcome Trust (J.-R.P.) and Biotechnology
and Biological Sciences Research Council (UK).
1 These authors contributed equally. 
Received for publication July 7, 1999.
Revision received August 13, 1999.
Accepted for publication August 18, 1999.
 |
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