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Endocrinology Vol. 145, No. 12 5798-5806
Copyright © 2004 by The Endocrine Society

Transcriptional Regulation of Agouti-Related Protein (Agrp) in Transgenic Mice

Christopher B. Kaelin, Allison Wanting Xu, Xin-Yun Lu and Gregory S. Barsh

Departments of Genetics and Pediatrics (C.B.K., A.W.X., G.S.B.), Stanford University School of Medicine, Stanford, California 94305; and Department of Pharmacology (X.-Y.L.), University of Texas Health Science Center, San Antonio, Texas 78229

Address all correspondence and requests for reprints to: Greg Barsh, Beckman Center B271A, Stanford University School of Medicine, Stanford, California 94305-5323. E-mail: gbarsh{at}cmgm.stanford.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Agouti-related protein (Agrp) encodes a hypothalamic neuropeptide that promotes positive energy balance by stimulating food intake and reducing energy expenditure. Agrp expression in the brain is restricted to neurons within the arcuate nucleus of the hypothalamus, and expression levels are elevated as a consequence of food deprivation. We tested a series of bacterial artificial chromosome reporter constructs with varying amounts of sequence flanking the Agrp transcription unit in transgenic mice to identify and refine a region of DNA capable of recapitulating characteristics of Agrp expression. We report that a 42.5-kb region upstream of Agrp, containing three distinct regions that are evolutionarily conserved between mouse and human, is necessary and sufficient to consistently drive reporter expression specifically within AgRP neurons in a fasting-responsive manner. In addition, we demonstrate that this region allows for the stable expression of Cre recombinase in transgenic mice, providing a genetic tool for studying anabolic neural circuits that control energy balance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
ENERGY HOMEOSTASIS IS the process whereby body weight and fat mass are maintained at relatively stable levels over long periods despite day-to-day fluctuations in food intake and energy expenditure and depends on the ability of the central nervous system (CNS) to sense and respond to changes in adiposity. Over the last decade, identification of neuropeptide genes that affect feeding and analysis of their expression has provided valuable insight into the organization and regulation of the central network controlling body weight (reviewed in Ref.1). The underlying mechanisms that regulate the transcription of these genes, however, remain unknown. Their investigation is critical for a more complete understanding of how the brain maintains energy balance.

Neurons in the arcuate nucleus of the hypothalamus, a key region involved in the central regulation of energy homeostasis, produce two types of neuropeptides with opposite effects on food intake. Anabolic neuropeptides, which include Agouti-related protein (AgRP) and neuropeptide Y (NPY), stimulate feeding and promote weight gain, whereas catabolic neuropeptides, which include {alpha}-MSH and cocaine and amphetamine-regulated transcript (CART), inhibit feeding and promote weight loss. Anabolic and catabolic neuropeptide genes are expressed in different neuronal populations, providing spatially distinct and opposing circuits (reviewed in Refs.2 , 3).

Leptin signaling provides a link between body energy stores and these neuronal circuits. Leptin is an adipocyte-derived peptide hormone that circulates at levels corresponding to adipose tissue mass (4, 5, 6, 7). Leptin administration suppresses food intake and decreases body weight (8), whereas leptin deficiency causes hyperphagia and obesity (7). At a cellular level, the effects of leptin are mediated by an isoform of the leptin receptor (Lepr-b) which is expressed prominently in the arcuate nucleus (9, 10, 11), including many neurons that also express either Agrp (12) or proopiomelancortin (Pomc) (13), the gene encoding {alpha}-MSH. Perturbations of leptin signaling alters various aspects of these neuronal subtypes, including neuropeptide gene expression (reviewed in Ref.14), membrane potential and firing rate (15, 16, 17), and synaptic plasticity (18). Taken together, these data suggest that neuronal circuits in which Agrp, Pomc, Npy, and Cart are expressed play a critical role in communicating changes in the levels of body adipose stores to the rest of the brain.

With regard to hypothalamic neuropeptide gene expression, a reduction of leptin signaling brought about by starvation or by mutations of Leptin or the Lepr cause increased expression of Npy (19, 20) and Agrp (21, 22, 23), but decreased expression of Pomc (24) and Cart (25). Conversely, pharmacological administration of leptin causes decreased expression of Npy (26) and Agrp (2) but increased expression of Pomc (24, 27). Among these responses, up-regulation of Agrp in response to food deprivation provides a useful system for studying neuropeptide gene regulation for two reasons. First, in the CNS, Agrp expression is restricted to a discrete population of neurons in the arcuate nucleus of the hypothalamus (21); by contrast, Npy is expressed in many regions of the brain (28). Second, the magnitude of the Agrp expression response to fasting tends to be larger than the other indicators, with mRNA levels increasing more than 10-fold after 48 h of food deprivation in mice (23), thereby providing a robust indicator of leptin signaling.

To help define the molecular and cell biological circuitry underlying the central control of energy balance, we used a strategy based on comparative genome sequence analysis and transgenic reporter experiments to identify key regulatory elements from the Agrp gene. Dissecting cis-acting regulatory sequences using recombinant genomic clones and reporter genes has been carried out mostly in cultured cell systems rather than whole animals because transgenic systems are relatively inefficient and because regulatory regions required for proper expression in vivo often lie a considerable distance from their transcriptional target and cannot be included on conventional plasmid-sized pieces of DNA. An important advance in the last several years that addresses both hurdles is the ability to modify bacterial artificial chromosome (BAC) clones using homologous recombination in Escherichia coli (29), followed by introduction of the modified BAC into animals by pronuclear microinjection (30). This approach, often referred to as BAC recombineering, has now been applied and/or modified by several groups to place a wide variety of reporter genes under control of cis-acting regulatory elements present on BAC clones (reviewed in Ref.31).

Indeed, previous studies of Agrp regulation identified a 700-bp region just upstream of the transcriptional initiation site that is capable of activating luciferase reporter expression in cultured cells (32). Single-nucleotide polymorphisms within this region have been used in human association studies (33, 34), but the results have been difficult to evaluate without an in vivo assay for Agrp regulation. As described below, our results define a region of DNA that is both necessary and sufficient to regulate the spatial expression and fasting response of Agrp and that contains evolutionarily conserved sequences that lie more than 40 kb upstream of the transcriptional start site. These results provide both an additional region in which to search for human sequence variants that may contribute to pathological variation in energy balance and the basis for developing transgenic strategies to explore the role of various gene products in AgRP neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Isolation and characterization of BACs containing Agrp
A 228-bp DNA fragment from exon 4 of mouse Agrp was used to screen two mouse BAC genomic libraries, a C57BL/6J library (Genome Systems, St. Louis, MO) and a 129/Sv library (CITB-CJ7-B, Invitrogen, Carlsbad, CA), for BAC clones containing the Agrp locus. Isolated clones were mapped with respect to each other using BAC endpoint mapping techniques. Restriction mapping was performed by pulse-field gel electrophoresis and Southern hybridization using standard techniques. Upon public availability of mouse genomic sequence, precise measurement of BAC insert size was assessed by PCR analysis. BAC165, BAC167, BAC171, and BAC172 correspond to clones isolated from the Genome Systems C57BL/6J library at filter positions 44c11, 93k01, 171n11, and 201b05, respectively.

BAC reporter construction
BAC clones were modified by inserting an internal ribosomal entry site (IRES)-ßgeo reporter into the 3' untranslated region (UTR) of Agrp using a BAC recombination strategy developed by Yang et al. (29). Initially, a targeting cassette, consisting of the IRES-ßgeo cassette flanked on either side by BAC homology arms, was inserted into pBluescript II KS (Stratagene, La Jolla, CA), through a successive series of cloning steps to create pATV1–4. A 752-bp upstream homology arm and a 626-bp downstream homology arm were generated by PCR, whereas the 4.7-kb IRES-ßgeo cassette was isolated from pGT1.8Iresßgeo (35) by XbaI restriction digestion. The targeting cassette was subsequently excised from pATV1–4 by SalI restriction digestion and cloned into the SalI site on the pASV1 shuttle vector (29). Homologous recombination between the shuttle vector and Agrp BAC clones was carried out as described previously (29) and resulted in a 2-bp deletion within the 3' UTR of Agrp at the integration site, corresponding to the positions 107087233 and 107087234 in the mouse genomic sequence of chromosome 8 (mm5, May 2004, NCBI build 33). Correctly targeted BAC molecules were confirmed by both PCR and Southern hybridization. Also, restriction analysis confirmed that no rearrangement of the BAC inserts occurred during the modification process.

The primer sets used to generate the homology arms are as follows: for the upstream homology arm, Agrp201 (5'-GATCCGGTCGACGCCAGGCCATGCTGACTG-3') and Agrp202 (5'-GATCGTCTAGAATCCATTGGCTAGGTGCG-3'); and for the downstream homology arm, Agrp203 (5'-GATCGTCTAGATGTTTGGGCAAAGGCAGG-3') and Agrp204 (5'-GATCGGAGCTCGTCGACACGCATGCGTTACTCTGG-3').

For the Tg.AgrpCre line, a different approach was used that will be described in detail elsewhere (Xu, A. W., C. B. Kaelin, X. Y. Lu, K. Takeda, S. Akira, M. W. Schwartz, and G. S. Barsh, submitted for publication). In brief, coding sequences for Cre recombinase were inserted at the Agrp translational initiation site, and a linear fragment was prepared that carried 47.5 kb of 5' flanking sequence and 0.7 kb of 3' flanking sequence.

Establishing BAC transgenic mouse lines
BAC transgene constructs were prepared from bacterial cultures grown under chloramphenicol selection using the QIAGEN Large Construct Kit (QIAGEN, Valencia, CA). Circular (Tg.BACßgeo) or linear (Tg.AgrpCre) BAC molecules were injected into pronuclei of either FVB or CBA/C57BL6 F1 fertilized eggs following standard microinjection procedures by the Stanford Transgenic Research Facility. Transgenic founder (F0) mice were verified by Southern blot hybridization (using a lacZ probe) and by PCR using primers derived from the IRES sequence: Ires101 (5'-CTAACGTTACTGGCCGAAGC-3') and Ires102 (5'-TACGCTTGAGGAGAGCCATT-3').

To establish transgenic lines, F0 mice were mated with either FVB mice in the case of FVB founders or C57BL6 mice in the case of CBA/C57BL6 founders. Transgenic lines were maintained by mating transgenic offspring of a single founder. All experiments were carried out under a protocol approved by the Stanford Administrative Panel on Laboratory Animal Care, in which mice were housed in filter-top cages in a specific pathogen-free facility and provided free access to standard lab chow before experimentation.

5-Bromo-4-chloro-3-indolyl-ßD-galactoside (X-gal) staining
Except where indicated, Tg.BACßgeo mice were fasted for 48 h before reporter expression analysis. Food was removed from cage setups between 1600 and 1800 h. After CO2 euthanasia, the brain was immediately dissected and 1-mm coronal brain slices were cut through the hypothalamic region using a prechilled mouse brain matrix (ASI Instruments, Warren, MI). Brain slices were fixed at 4 C for at least 1 h in PBS containing 4% paraformaldehyde (PFA), washed three times for 30 min in PBS containing 0.1% Triton X-100, and incubated for 24 h at 37 C in X-gal staining solution (5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg/ml X-gal, and 2 mM MgCl2 in PBS).

Alternatively, transgenic mice were deeply anesthetized with an ip injection of Avertin (1.25% tribromoethanol) and fixed by transcardial perfusion with PBS containing 4% PFA. After fixation, brains were immediately dissected, incubated at 4 C for 4 h in PBS containing 4% PFA, infiltrated with 30% sucrose in PBS overnight at 4 C, and then frozen in Tissue-Tek OCT solution (Sakura Finnetek, Torrance, CA). The 20-µm-thick coronal sections through the hypothalamus were cut with a cryostat and incubated for 24 h at 37 C in X-gal staining solution. The sections were subsequently fixed in PBS containing 4% PFA and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA).

X-gal staining intensity was scored both within the hypothalamic arcuate nucleus and in other brain regions by visual inspection under a dissecting microscope. Scoring ranged from 0–5, where 0 indicated no detectable staining and 5 indicated the highest level of staining observed. In experiments comparing staining intensity between free-fed and fasted mice, transgenic littermates were used and all steps were carried out in parallel to minimize experimental variability.

In situ hybridization
Double-labeling in situ hybridization was performed according to the procedure described previously (36). Briefly, antisense riboprobes for lacZ mRNA and mouse Agrp mRNA were synthesized and labeled with [35S]CTP and [35S]UTP (Amersham, Arlington Heights, IL) or digoxigenin-UTP (Roche Molecular Biochemicals, Indianapolis, IN), respectively. Fresh-frozen mouse brain sections were incubated with a mixture of [35S]lacZ and digoxigenin-labeled Agrp probes in a hybridization buffer overnight at 55 C. Sections were rinsed in twice in standard saline citrate solution (SSC; 1x SSC contains 150 mM sodium chloride and 15 mM sodium citrate), treated with RNase A (1 h at 37 C), and washed in SSC with decreasing stringency (2–0.5x SSC) at room temperature. Sections were then washed in 0.1x SSC at 65 C for 1 h followed by immunohistochemical staining for visualization of digoxigenin-labeled Agrp probe. Briefly, brain sections were first treated with a blocking solution (0.1 M phosphate buffer containing 0.5% Triton X-100 and 0.25% carageenan, pH 7.5) for 4 h and then incubated overnight with an alkaline phosphatase-conjugated antibody against digoxigenin (Roche Molecular Biochemicals), diluted to 1:15,000. After rinsing in 0.1 M phosphate buffer and 0.1 M Tris buffer (30 min each), sections were incubated with color reaction buffer containing 0.45% nitroblue tetrazolium chloride (Roche Molecular Biochemicals), 0.35% 5-bromo-4-chloro-3-indoylphosphate 4-toluidine salt (Roche Molecular Biochemicals), 5% polyvinyl alcohol, and 0.24% levamizole. Color reaction was completed overnight at 4 C. Sections were rinsed in water and incubated with 0.1 M glycine buffer containing 0.5% Triton X-100 for 10 min. Finally, sections were fixed in 2.5% glutaraldehyde for 2 h. After rinsing in water and dehydrating in a graded series of alcohol, sections were dipped in liquid emulsion (Ilford KD-5; Polysciences, Warrington, PA), air dried, and stored in a sealed dark box at 4 C. After 4 wk of exposure to emulsion, sections were developed, fixed, dehydrated, and coverslipped in a xylene-based mounting medium (Permount; Fisher Scientific, Houston, TX). Agrp mRNA labeled with digoxigenin-labeled probe was visualized as brown-purple precipitate, and lacZ mRNA labeled with 35S-labeled probe was visualized as silver grains. Signal specificity was ensured either by hybridization with sense-strand probes or pretreatment of brain sections with RNase A (200 µg/ml at 37 C for 60 min).

Sequence analysis
Human and mouse genomic sequences used for comparative sequence analyses in this study were obtained from publicly available assemblies (NCBI human build 34 and NCBI mouse build 32) using the Ensembl genome browser (http://www.ensembl.org). Comparative sequence analysis was performed using PipMaker (37) and VISTA (38) after masking repetitive sequences with RepeatMasker (Smit, AFA & Green, P RepeatMasker at http://repeatmasker.org).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Recapitulating Agrp expression with a BAC transgene
Agrp mRNA is encoded by four exons that lie on a 2-kb region on mouse chromosome 8 (21), immediately upstream from the Atp6v0d1 gene. We identified an Agrp-containing BAC clone that contained approximately 75 kb of 5' flanking sequence and 37 kb of 3' flanking sequence and modified this BAC using homologous recombination in Escherichia coli (29) to insert a ß-galactosidase reporter cassette (35) into the 3' UTR of Agrp (Fig. 1AGo). Insertion of the reporter cassette, which consists of an IRES (39) and ßgeo (40), results in a 2-bp deletion within the 3' UTR but does not otherwise alter the BAC sequence or the exon-intron structure of Agrp; instead it lengthens the 3' UTR of Agrp by 4.7 kb to create a dicistronic mRNA approximately 5.1 kb in length (Fig. 1AGo).



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  		FIG. 1. BAC reporter construction and analysis in transgenic lines. A, The precise insertion of a 4.7-kb IRES-ßgeo cassette into the 3' UTR of Agrp on BAC167 and BAC171 by homologous recombination in E. coli. Numbers indicate the approximate lengths (in kb) of flanking genomic sequences relative to the Agrp transcriptional start site. B, X-gal staining in the medial arcuate nucleus of a 20-µm brain section from a Tg.BAC171ßgeo mouse indicating ß-galactosidase activity as a result of transgenic reporter expression. C–F, X-gal staining in brain slices from Tg.BAC171ßgeo mice (C and D) or Tg.BAC167ßgeo mice (E and F) that were either free-fed (C and E) or fasted for 48 h (D and F). Fasting increased the intensity of X-gal staining in the hypothalamic arcuate nucleus of transgenic mice.

 
The modified BAC (BAC171ßgeo) was microinjected into fertilized mouse eggs as a circular molecule to make transgenic mice. Of four lines established from independent founders, two lines expressed the reporter as determined by X-gal staining of mouse brains. In both Tg.BAC171ßgeo lines, reporter expression in the CNS was restricted to the medial arcuate nucleus of the hypothalamus, and no reporter expression was observed in other brain regions (Fig. 1BGo). The effect of food deprivation on reporter expression was also tested in one of the lines. The intensity of X-gal staining in the arcuate nucleus of transgenic mice fasted for 48 h was markedly elevated when compared with staining in the arcuate nucleus from transgenic mice fed ad libitum (Fig. 1Go, C and D). We also examined mRNA expression in one of the Tg.BAC171ßgeo lines by Northern hybridization (data not shown). As predicted, an mRNA fragment of approximately 5 kb was detected with either an Agrp probe or a ßgeo probe, but the level of expression was considerably reduced compared with the endogenous 0.4-kb Agrp transcript, possibly because the dicistronic transcript was less stable. Consistent with this observation, none of our BAC transgenic mice developed obesity or increased growth, as occurs in an Agrp cDNA transgene driven by the Actb promoter (22).

Mapping Agrp regulatory sequences with additional BAC transgenes
Because BAC171 defined a region sufficient to recapitulate the spatial and fasting-responsive aspects of Agrp expression, we tested a series of BAC reporter constructs that contained varying amounts of the flanking regions represented on BAC171 in transgenic assays to refine the position of sequences controlling Agrp expression. From 11 BACs that contained Agrp coding sequences, we identified three (BAC165, BAC167, and BAC172) whose endpoints mapped within the genomic region contained on BAC171. These BACs were modified in a manner identical with that described above for BAC171ßgeo, and transgenic lines were established and tested for expression of ß-galactosidase by X-gal staining of adult brain slices.

Individual transgenic lines from all three constructs were capable of driving lacZ expression in the arcuate nucleus (Fig. 2Go and Table 1Go), although the frequency of positive lines with the smallest construct, BAC165ßgeo, was low (2 of 12) compared with the other lines (12 of 16; Tables 1Go and 2Go). As described below, this may be explained by the presence of critical Agrp regulatory sequences that lie close to the 5' end of BAC165.



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  		FIG. 2. Mapping of Agrpregulatory regions using additional BAC transgenes. A, BACßgeo and AgrpCre constructs used to drive reporter expression in transgenic mouse lines. The numbers within the gray barsindicate the approximate length (in kb) of flanking genomic sequence relative to the transcriptional start site of Agrp. The dashed line distinguishes ßgeo constructs from the Cre construct. B, X-gal staining indicating ß-galactosidase expression in 1-mm coronal brain slices at the level of the hypothalamic arcuate nucleus, isolated from transgenic mice of the indicated lines after 48 h of food deprivation (C) and from a Tg.AgrpCre; R26R/+ mouse that was free-fed. D, Double in situ hybridization for lacZ mRNA (punctate black, 35S) and Agrp mRNA (brown, digoxigenin) in the hypothalamic arcuate nucleus from a Tg.AgrpCre; R26R/+ mouse.

 

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  		TABLE 1. Arcuate nucleus and CNS ectopic expression in transgenic reporter lines

 

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  		TABLE 2. Comparative analysis of reporter expression in transgenic reporter lines

 
Including BAC171, a total of 14 transgenic lines exhibited a characteristic pattern of X-gal staining in the arcuate nucleus. For eight of these lines (including a line generated with BAC165), we tested the response to fasting and observed increased intensity of X-gal staining in every case (Fig. 1Go, C–F, and Table 2Go). These results suggest that regulatory elements responsible for up-regulation of Agrp expression in response to fasting either are coincident with regulatory elements that mediate the specific pattern of expression in the arcuate nucleus or else reside in a more proximal position relative to the transcription unit.

Expression of Cre recombinase under control of Agrp regulatory elements
To further investigate the requirement of Agrp flanking sequences for arcuate-specific expression, and to develop a tool that would be useful for manipulating gene expression specifically in AgRP neurons, we inserted protein coding sequences for Cre recombinase into an Agrp-containing BAC, generated transgenic mice, and then tested for expression of the Cre transgene by crossing to animals carrying the Gt(Rosa)26Sortm1Sor allele (R26R), in which lacZ transcription is driven by the widely expressed Rosa26 promoter after Cre-mediated excision of a loxP-flanked polyadenylation cassette (41).

Using this approach, a Tg.AgrpCre/+; R26R/+ line was established in which the pattern of lacZ expression driven by the Rosa26 promoter was identical with that observed in Tg.BAC171ßgeo lines, where lacZ expression is driven by Agrp regulatory elements (Fig. 2Go, B and C). The Cre transgene was constructed using a different approach from the BACßgeo transgenes (Xu, A. W., C. B. Kaelin, X. Y. Lu, K. Takeda, S. Akira, M. W. Schwartz, and G. S. Barsh, submitted for publication), such that it carried 47.5 kb of 5' flanking sequence and 0.7 kb of 3' flanking sequence (Fig. 2AGo); thus, regulatory elements responsible for specific expression of Agrp in the arcuate nucleus are likely to lie in the 5' flanking region.

To refine the localization of lacZ expression more precisely, we examined tissue sections from Tg.AgrpCre/+; R26R/+ mice by double in situ hybridization using a digoxigenin-labeled probe for Agrp mRNA and a radiolabeled probe for lacZ mRNA. LacZ mRNA-expressing cells were also positive for Agrp mRNA (Fig. 2CGo), which indicates that the pattern of X-gal staining observed in tissue slices corresponds to that of Agrp on a cellular level.

Ectopic reporter expression in transgenic lines
Several of the BAC transgenic lines exhibited X-gal staining in regions of the brain outside the arcuate nucleus of the hypothalamus (Fig. 2Go and Table 1Go). In most cases, this ectopic lacZ expression was restricted to distinct brain regions, including the amygdala, the piriform cortex, the lateral walls of the third ventricle, the dorsal third ventricle and surrounding organs, the lateral ventricle, the granular layer of the dentate gyrus, and the pyramidal cell layer of the hippocampus.

In lines made using the smallest amount of Agrp flanking sequence (Tg.BAC165ßgeo), ectopic brain expression was always observed in lines demonstrating arcuate nucleus reporter expression and was also observed in lines that never demonstrated arcuate nucleus reporter expression. In contrast, ectopic brain expression in Tg.BAC167ßgeo and Tg.BAC172ßgeo lines was observed only in lines demonstrating arcuate nucleus reporter expression. Importantly, reporter expression in a subset of Tg.BAC167ßgeo and Tg.BAC172ßgeo lines (5 of 12) was specific for the arcuate nucleus of the hypothalamus with no detectable expression in other brain regions. These results suggest that a distal regulatory region contained on BAC167 and BAC172 but absent from BAC165 may be necessary to restrict Agrp expression to the arcuate nucleus.

In lines demonstrating both arcuate nucleus and ectopic brain expression, we did not observe fasting-induced increases in X-gal staining at ectopic sites of expression, even when staining within the arcuate nucleus was markedly elevated by fasting (Fig. 1Go, E and F). This suggests that the signals and/or cellular machinery required for inducing the response to fasting are localized to neurons within the arcuate nucleus.

We also examined Tg.AgrpCre/+; R26R/+ mice for evidence of ectopic lacZ expression and found that approximately 30% of these animals exhibited widespread X-gal staining that affected multiple brain regions as well as mesenchymal and epithelial tissues throughout the body (data not shown). Widespread brain expression of lacZ in Tg.AgrpCre/+; R26R/+ animals was always accompanied by expression in multiple nonneural tissues. This suggests that Cre recombinase is expressed at low levels in the early embryo of Tg.AgrpCre/+; R26R/+ mice, and occasionally reaches a threshold above which recombination takes place in a cell that gives rise to tissues in multiple germ layers. Indeed, low levels of Agrp mRNA expression in the early embryo have also been reported previously (42).

In adult tissues, expression of Agrp mRNA has been reported in the adrenal gland (21, 22), where a potential role has been suggested for modulation of glucocorticoid production (43). We did not observe significant adrenal gland X-gal staining in either Tg.BACßgeo/+ or Tg.AgrpCre/+; R26R/+ mice; however, these experiments were complicated by a low level of background X-gal staining in nontransgenic animals.

Transgene silencing
The results described above and summarized in Tables 1Go and 2Go are based on transgenic lines rather than individual animals. A transgenic line was scored as positive if at least one transgenic animal exhibited X-gal staining in the arcuate nucleus of the hypothalamus and/or in other brain regions. However, for BACßgeo transgenes, we consistently observed variation in staining within individual transgenic lines, even though the genetic background remained constant. In general, littermates assayed in parallel typically had identical levels of staining, but we noticed a trend toward reduced staining over successive generations of breeding, usually resulting in the complete loss of ß-galactosidase activity. We attributed the variability and eventual loss of staining in Tg.BACßgeo lines to transgene silencing. By contrast, the Tg.AgrpCre/+; R26R/+ line did not exhibit susceptibility to gene silencing, and the strength and extent of X-gal staining remained stable among different transgenic animals and across multiple generations.

A potential explanation for the apparent difference between Tg.BACßgeo and Tg.AgrpCre lines is the presence of lacZ, IRES, or vector sequences on BACßgeo constructs, because these sequences were not included in the AgrpCre construct (in Tg.AgrpCre/+; R26R/+ mice, the lacZ sequence is present in a different genomic context). It has been suggested that inclusion of bacterial sequences on a transgene may increase its susceptibility to transcriptional silencing. In particular, several studies have reported transcriptional silencing or variegated expression of lacZ reporter constructs in transgenic mice that is overcome when the same construct is used to drive the expression of a different reporter (44, 45, 46, 47, 48). In addition, genetic heterogeneity in the Tg.AgrpCre/+ animals (in which there are contributions from several different inbred strains) may act to buffer against reporter gene silencing.

Comparative sequence analysis of mouse and human Agrp flanking regions
As described above, the ability of the BACßgeo and AgrpCre transgenes to activate reporter expression specifically in the arcuate nucleus of the hypothalamus suggests that critical regulatory elements lie within a region surrounding the Agrp transcription unit, which extends from 42.5 kb of 5' flanking sequence to 0.7 kb of 3' flanking sequence. As a parallel approach to refine Agrp regulatory regions within this segment of DNA, we compared human and mouse genomic sequences using the Pipmaker (data not shown) and VISTA alignment tools (Fig. 3Go). We identified three regions of 5' sequence conservation, referred to below as distal conserved regions I and II (DCR I and DCR II) and the proximal conserved region. As depicted in Fig. 3Go, the conserved regions were defined as a series of similarity peaks that met the VISTA criteria of more than 75% sequence identity over a 100-bp window; however, similar results were obtained with window sizes varying from 50–200 bp.



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  		FIG. 3. Comparative analysis of mouse and human genomic sequences upstream of the Agrp transcription unit. Percent identity plot (VISTA) showing the level of mouse/human sequence conservation in the Agrp genomic region. The percentage of nucleotide identity is plotted as a function of position along the mouse sequence. Numbers along the x-axis indicate distance (in kb) from the transcriptional start site of mouse Agrp. Peaks of evolutionary conservation that overlap with exons are shaded blue. Aligned noncoding regions displaying more than 75% sequence identity over 100 bp are shaded pink. Solid arrows indicate the position of known genes or clusters of spliced ESTs. Broken arrows indicate the breakpoint for genomic sequences contained on the transgenes. Regions with multiple, adjacent peaks of sequence conservation that map within the Agrp regulatory region defined by transgenic experiments are highlighted with red boxes.

 
The proximal conserved region lies immediately upstream of Agrp exon 1, spans 760 bp, and contains a number of potential transcription factor binding sites that may contribute to gene regulation. By contrast, DCR I and DCR II lie much further upstream, 26.3 kb and 37.6 kb, respectively, and span larger genomic distances. DCR I spans 3189 bp and exhibits three distinct similarity peaks, whereas DCR II spans 4538 bp and exhibits five distinct similarity peaks.

Mapping the positions of BAC171, BAC167, BAC172, and BAC165 endpoints onto the diagram of evolutionarily conserved regions flanking Agrp revealed that BAC165ßgeo, which produced both a low proportion of transgenic lines with specific hypothalamic expression and a high incidence of ectopic brain expression, contains the proximal conserved region and DCR I but lacks DCR II. By contrast, the other BACßgeo transgenes and the AgrpCre transgene contain all three regions and produced relatively high proportions of transgenic lines with specific hypothalamic expression and less ectopic expression than did BAC165ßgeo.

A potential explanation for these findings speculates that key regulatory sequences for Agrp lie within DCR I and help bring about the arcuate nucleus-specific expression observed in BAC165ßgeo lines but that additional regions within DCR II further enhance and restrict this expression in lines constructed from the other transgenes. An alternative explanation, that specific expression observed in Tg.BAC165ßgeo lines occurred as a result of a fortuitous insertion event, seems less likely, because enhancer trap expression patterns similar to that of Agrp have not been reported previously and because arcuate nucleus expression was observed in two independent Tg.BAC165ßgeo lines.

In either case, the evolutionarily conserved sequences in DCR II might function as an enhancer-like positive element that acts over a relatively long distance to increase Agrp expression specifically in the arcuate nucleus and/or as a boundary element that helps to insulate Agrp transcription from the effects of surrounding chromatin. Some insight is apparent from the position of additional genes in the region (Fig. 3Go). The nearest gene downstream of Agrp is Atp6v0d1 and encodes a subunit of the vacuolar proton pump (49). Atp6v0d1 is widely expressed in embryonic and adult tissues (50) and lies in the same orientation and immediately adjacent to Agrp, with a transcriptional initiation site 0.7 kb away from the Agrp polyadenylation site. The nearest gene upstream of Agrp lies on the opposite strand and is represented by multiple expressed sequence tags (ESTs) as UniGene Mm.41261, which is predicted to encode a protein of unknown function weakly similar to a subunit of RNA polymerase II. More than 150 ESTs for Mm.41261have been isolated from a wide range of tissues, but there is considerable heterogeneity in their 5' ends, with one group of ESTs beginning in DCR I, a second beginning in DCR II, and a third group beginning another 10 kb upstream (Fig. 3Go).

Given the high level of conservation and the absence of predicted protein-coding sequences, DCR I and DCR II are likely to act as cis-acting regulatory regions for Agrp or Mm.41261. Determining whether DCR I and DCR II function as positive regulatory elements, as boundary elements, or both will require additional experiments. However, given the gene-rich landscape, it would not be surprising if Agrp, which is extremely tissue- and cell-type specific, made use of boundary elements to insulate itself from the potential influence of neighboring genes that are widely expressed.

Concluding remarks
Experimental dissection of cis-acting transcriptional regulatory elements using reporter genes in transgenic mice can provide basic insight into eukaryotic gene regulation as well as new experimental tools for manipulating gene expression. Moreover, the identification of evolutionarily conserved sequences within regulatory regions defined by transgenic reporter experiments can provide a more specific context in which to direct searches for potential transcription factor binding sites or sequence variants involved in human disorders. In the case of Agrp, it is not yet clear how the conserved regions depicted in Fig. 3Go act to restrict mRNA expression to anabolic neurons in the arcuate nucleus of the hypothalamus. However, the approximately 45-kb region of DNA we identified should be useful for modulating the expression of other genes in Agrp neurons, either indirectly using the Cre-loxP system or directly by constructing new transgenes using the same strategy.

Interestingly, previous studies have identified regulatory sequences that are important for neuronal subtype-specific expression of two other hypothalamic neuropeptide genes, Prepro-orexin (51) and GnRH (52). In both studies, the deletion of sequences from a reporter transgene that conferred spatially restricted expression patterns caused ectopic reporter expression in transgenic mice. As in this study, Prepro-orexin and GnRH deletion transgenes directed ectopic reporter expression to specific brain regions in multiple lines, indicating that the ectopic expression pattern was inherent to the transgene and was not caused by the position of transgene insertion. By contrast to Agrp, regulatory regions for Prepro-orexin and GnRH lie within several kilobases of the transcriptional initiation site; however, all three neuropeptide genes have specific expression patterns resulting in their localization to discrete neuronal subtypes within the hypothalamus. The observation that a characteristic pattern of ectopic expression results from the absence of a particular regulatory region could be explained by mechanisms that normally restrict expression by relying on multiple interacting transcription factors expressed in overlapping patterns.

The identification of Agrp regulatory regions offers a system to manipulate gene expression in an AgRP neuron-specific manner. Because Agrp expression in the brain is limited to a specific population of arcuate nucleus neurons, its regulatory regions provide an optimal tool for genetic strategies aimed toward understanding the regulation and role of anabolic circuitry in the arcuate nucleus.


   Acknowledgments
 
We thank Dr. Yanru Chen and the Stanford Transgenic Facility for microinjection.


   Footnotes
 
This investigation was supported by a grant from the National Institutes of Health (DK-48506).

Abbreviations: AgRP, Agouti-related protein; BAC, bacterial artificial chromosome; CART, cocaine and amphetamine-regulated transcript; CNS, central nervous system; DCR, distal conserved region; EST, expressed sequence tag; IRES, internal ribosomal entry site; NPY, neuropeptide Y; PFA, paraformaldehyde; UTR, untranslated region; X-gal, 5-bromo-4-chloro-3-indolyl-ß-D-galactoside.

Received July 23, 2004.

Accepted for publication August 26, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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