GPR54 is a G-protein-coupled receptor, which binds kisspeptins and is widely expressed throughout the brain. Kisspeptin-GPR54 signaling has been implicated in the regulation of pubertal and adulthood gonadotropin-releasing hormone (GnRH) secretion, and mutations or deletions of GPR54 cause hypogonadotropic hypogonadism in humans and mice. Other reproductive roles for kisspeptin-GPR54 signaling, including the regulation of developmental GnRH secretion or sexual behavior in adults, have not yet been explored. Using adult wild-type (WT) and GPR54 knock-out (KO) mice, we first tested whether kisspeptin-GPR54 signaling is necessary for male and female sexual behaviors. We found that hormone-replaced gonadectomized GPR54 KO males and females displayed appropriate gender-specific adult sexual behaviors. Next, we examined whether GPR54 signaling is required for proper display of olfactory-mediated partner preference behavior. Testosterone-treated WT males preferred stimulus females rather than males, whereas similarly treated WT females and GPR54 KO males showed no preference for either sex. Because olfactory preference is sexually dimorphic and organized during development by androgens, we assessed whether GPR54 signaling is essential for sexual differentiation of other sexually dimorphic traits. Interestingly, adult testosterone-treated GPR54 KO males displayed "female-like" numbers of tyrosine hydroxylaseimmunoreactive and Kiss1 mRNA-containing neurons in the anteroventral periventricular nucleus and likewise possessed fewer motoneurons in the spino-bulbocavernosus nucleus than did WT males. Our findings indicate that kisspeptin-GPR54 signaling is not required for male or female copulatory behavior, provided there is appropriate adulthood hormone replacement. However, GPR54 is necessary for proper male-like development of several sexually dimorphic traits, likely by regulating GnRH-mediated androgen secretion during "critical windows" in perinatal development.

The human ␤-globin locus contains five actively transcribed genes that are arranged in their developmental order of expression. High-level expression of the ␤-globin gene cluster is dependent on the presence of the locus control region (LCR) (18), an element characterized by a series of five DNase I-hypersensitive sites (HSs) located 6 to 22 kb upstream of the ε-globin gene (9,10,18,44). Naturally occurring deletions of this element result in changes in chromatin structure that extend at least 200 kb 3Ј of the deletion, transcriptional silencing of the ␤-globin locus, and a phenotype of ␤ thalassemia (4,5,12,22). Functional properties of the LCR include activation of the ␤-globin locus (10, 18), restriction of globin gene expression to cells of the erythroid cell lineage (18, 45), enhancement of globin gene expression (11,18,39), and protection from position effects of globin genes transferred in transgenic mice (13,18,25,41).Transgenic mice have been extensively used to study the developmental control of the ␤-globin genes, the function of the LCR, and the role of individual HSs in ␤-globin gene regulation. Linkage of individual HSs to individual globin genes have shown that HS2, HS3, and HS4 are capable of conferring position-independent expression of globin genes, with stronger activation of expression at a specific stage of development (14, 25). Several observations have led to a model suggesting that the HSs form a complex that directly interacts with globin gene promoters by looping of the intervening DNA (7, 28, 46). HS2, HS3, and HS4 have 200-to 400-bp core regions that are able to provide position-independent expression in transgenic mice (27,34,35,37,42). These HS core regions may be indispensable components of the LCR complex; deletions of the HS3 or HS4 core elements result in disruption of HS function and reduction of globin gene expression (3).Discernment of the function of individual HSs and analysis of how the LCR interacts with individual genes during development require studies in the context of intact, native ␤-globin loci. Entire ␤-globin loci have been used to generate transgenic mice, by ligating two cosmids to produce a 70-kb fragment (40) or by using 248-kb (30) or 150-kb (15, 36) yeast artificial chromosomes harboring the ␤-globin locus (␤-YACs). Mice carrying ␤-YACs show correct regulation of the human globin genes, presumably because all the human cis-regulatory elements are present in the transferred sequences of the ␤-globin locus and are properly recognized by the murine transacting environment. In ␤-YAC transgenic mice, the ε-globin gene is expressed during the embryonic stage of development and is confined to primitive erythropoiesis in the yolk sac. The ␥-globin genes are also expressed in the embryonic yolk sac, but unlike their murine homologous gene, ␤h1, ␥-globin gene expression continues in the fetal liver stage of erythropoiesis. Human ␤-globin gene expression occurs only in the cells of definitive erythropoiesis.To delineate the role of HS3 in LCR function and globin gene ...

The neuropeptide galanin has been implicated in anxiety-related behaviors, cognition, analgesia, and feeding in rodents. Neuromodulatory actions of galanin are mediated by three G-protein coupled receptors, GalR1, GalR2, and GalR3. The present study investigates the role of the GalR2 receptor by evaluating behavioral phenotypes of mice with a targeted mutation in the GalR2 gene. A threetiered behavioral phenotyping approach first examined control measures of general health, body weight, neurological reflexes, sensory abilities and motor function. Mice were then assessed on several tests for cognitive and anxiety-like behaviors. GalR2 null mutants and heterozygotes were not significantly different from wildtype littermates on two cognitive tests previously shown to be sensitive to galanin manipulation: acquisition of the Morris water maze spatial task, and trace cued and contextual fear conditioning, an emotional learning and memory task. Two independent cohorts of GalR2 null mutant mice demonstrated an anxiogenic-like phenotype in the elevated plus-maze. No genotype differences were detected on several other measures of anxiety-like behavior. The discovery of an anxiogenic phenotype specific to the elevated plus-maze, similar to findings in GalR1 null mutants, highlights the potential therapeutic efficacy of targeting GalR1 and GalR2 receptors in treating anxiety disorders. Keywords galanin; GalR2 null mutant; anxiogenic-like; mice; learning; memory; nociception; neuropeptide Converging evidence from many laboratories implicates galanin and galanin receptors in anxiety-like and depression-related behaviors, via modulation of neuroendocrine and noradrenergic systems (Barrera et al., 2005;Echevarria et al., 2005;Holmes et al., 2002Holmes et al., , 2003 Khoshbouei et al., 2002a,b). Rats administered galanin intracerebroventricularly (ICV) showed a significant increase in punished responding in the Vogel punished drinking test (Bing et al., 1993). Conversely, intra-amygdala administration of galanin produced a dose dependent decrease in punished drinking without affecting unpunished drinking or behavior in a second conflict-based test, the elevated plus-maze (Möller et al., 1999). Restraint stress in rats induced anxiogenic-like behavior in a social recognition task and in the elevated plus-maze

Neuromedin U (NMU) is a highly conserved neuropeptide with a variety of physiological functions mediated by two receptors, peripheral NMUR1 and central nervous system NMUR2. Here we report the generation and phenotypic characterization of mice deficient in the central nervous system receptor NMUR2. We show that behavioral effects, such as suppression of food intake, enhanced pain response, and excessive grooming induced by intracerebroventricular NMU administration were abolished in the NMUR2 knockout (KO) mice, establishing a causal role for NMUR2 in mediating NMU's central effects on these behaviors. In contrast to the NMU peptide-deficient mice, NMUR2 KO mice appeared normal with regard to stress, anxiety, body weight regulation, and food consumption. However, the NMUR2 KO mice showed reduced pain sensitivity in both the hot plate and formalin tests. Furthermore, facilitated excitatory synaptic transmission in spinal dorsal horn neurons, a mechanism by which NMU stimulates pain, did not occur in NMUR2 KO mice. These results provide significant insights into a functional dissection of the differential contribution of peripherally or centrally acting NMU system. They suggest that NMUR2 plays a more significant role in central pain processing than other brain functions including stress/anxiety and regulation of feeding.Neuromedin U (NMU) is a highly conserved neuropeptide, present in various species from amphibians to mammals (reviewed in reference 3). In humans, NMU is a 25-amino-acid (aa) peptide (NMU-25), and in rodents, it is a 23-aa peptide (NMU-23), whereas in some other mammalian species an 8-aa peptide (NMU-8) has also been found. NMU-8 is identical to the C terminus of NMU-25, which is the most highly conserved region of the entire peptide, and has receptor affinity in vitro similar to that of NMU-25. NMU is widely distributed in the body, with the most abundant expression in the gastrointestinal tract, anterior pituitary, spinal cord, brain, and genitourinary tract (6, 42). Correspondingly, NMU has been implicated in regulating a variety of physiological functions, including smooth-muscle contraction, blood pressure regulation, stress response, feeding and energy homeostasis, nociception, and circadian rhythm (reviewed in reference 3).Two G-protein-coupled receptors, NMUR1 and NMUR2, have been identified as the receptors for NMU (8,18,20,21,37,41,42). The two receptors belong to the rhodopsin-like class A G-protein-coupled receptors family and share ϳ50% identity with each other in the seven-transmembrane region. The tissue distribution of the two receptors is quite distinct and complementary to each other: NMUR1 is expressed predominantly in the periphery, with highest levels in the gastrointestinal tract (8,10,18,42), whereas NMUR2 is predominantly expressed in the central nervous system, with greatest expression in regions of hypothalamus, medulla, and spinal cord (9,10,14,20,21,41).In the brain, NMU is expressed in hypothalamic regions associated with regulation of food intake and energy homeostasi...

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.