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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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. 2Go, 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. 2Go, 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 (C–E) 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.

 
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. 1Go) 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. 2DGo). 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. 2CGo and 3Go and Table 1Go). 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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Table 1. Effect of GnRH Sequence Deletion upon Transgene Expression in non-GnRH Neurons in the Adult Male Mouse

 
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. 2Go, C and E, and Fig. 4Go). These included the two populations in the LS and pBNST (Fig. 2EGo), 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. 2EGo), suprachiasmatic nucleus, thalamus, piriform cortex and, in 5.2-GNLZ-0B mice, the caudate putamen (Fig. 4Go).



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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 5–10 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.

 
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. 1Go). 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. 3Go). 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 1Go).

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 3–4 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 2Go). 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)

 
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. 5Go).



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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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 85–90% 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 10–15% 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 88–90% 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
DNA Constructs
GNZ construct
The pGnRHSmaI cassette (gift of Dr. A. Mason. Prince Henry’s 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. 1Go 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 (6–10 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 15–20 ml of 4% paraformaldehyde in PBS (pH 7.4). Brains were removed and postfixed for 1–2 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 6–10 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. 2DGo) 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 Henry’s 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. Back

Received for publication July 7, 1999. Revision received August 13, 1999. Accepted for publication August 18, 1999.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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