Brain somatostatin receptors are up-regulated in somatostatin-deficient mice

The peptide somatostatin (SST) is widely synthesized in the brain and periphery and acts through a family of five receptors (SSTR1-5) to exert numerous effects. A gene product related to SST, cortistatin (CST), also interacts with SSTR1-5. Here we have investigated the regulation of SSTR1-5 and of CST in SST knockout (SSTKO) mice. The five SSTRs were quantitated individually by subtype-selective binding analysis, by immunocytochemistry, and by mRNA measurement and showed, in the brain of SSTKO mice, up-regulation of subtypes 1, 2, 4, and 5, and down-regulation of SSTR3. Peripheral tissues displayed both subtype- and tissue-specific changes in SSTR1-5 mRNA levels of expression. Lack of SST did not up-regulate normal CST expression in brain nor did it induce its expression in the periphery. SST-like immunoreactivity, however, was induced in the proximal midgut in SSTKO animals, suggesting intestinal expression of a novel SST-like gene.Fil: Ramírez, José L.. McGill University; CanadáFil: Mouchantaf, Rania. McGill University; CanadáFil: Kumar, Ujendra. McGill University; CanadáFil: Otero Corchon, Veronica. Oregon Health and Science University; Estados UnidosFil: Rubinstein, Marcelo. Oregon Health and Science University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Low, Malcolm J.. Oregon Health and Science University; Estados UnidosFil: Patel, Yogesh C.. McGill University; Canad

Transgenic mice engineered to target Cre/LoxP-mediated DNA recombination into catecholaminergic neurons

To introduce restricted DNA recombination events into catecholaminergic neurons using the Cre/loxP technology, we generated transgenic mice carrying the Cre recombinase gene driven by a 9 kb rat tyrosine hydroxylase (TH) promoter. Immunohistochemistry performed on transgenic mouse brain sections revealed a high number of cells expressing Cre in areas where TH is normally expressed, including the olfactory bulb, hypothalamic and midbrain dopaminergic neurons, and the locus coeruleus. Double immunohistochemistry and immunofluorescence indicated that colocalization of TH and Cre is greater than 80%. Cre expression was also found in TH-positive amacrine neurons of the retina, chromaffin cells of the adrenal medulla, and sympathetic ganglia. We intercrossed TH-Cre mice with the floxed reporter strain Z/AP and observed efficient Cre-mediated recombination in all areas expressing TH, indicating that transgenic Cre is functional. Therefore, we have generated a valuable transgenic mouse strain to induce specific mutations of "floxed" genes in catecholaminergic neurons.Fil: Gelman, Diego Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Noain, Daniela Maria Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Avale, Maria Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Otero Corchon, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Low, Malcolm J.. Oregon Health and Science University; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Centro de Estudios Científicos; Chil

Partially redundant enhancers cooperatively maintain Mammalian Pomc expression above a critical functional threshold

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.Fil: Lam, Daniel D.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Yamashita, Miho. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: López Leal, Rodrigo. Centro de Estudios Científicos; ChileFil: Otero Corchon, Veronica. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Meece, Kana. Columbia University. College of Physicians and Surgeons. Department of Medicine; Estados UnidosFil: Sampath, Harini. Oregon Health & Science University. Center for Research on Occupational and Environmental Toxicology; Estados UnidosFil: Mercer, Aaron J.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Wardlaw, Sharon L.. Columbia University. College of Physicians and Surgeons. Department of Medicine; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados Unidos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Low, Malcolm J.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados Unido

Thrittene, homologous with somatostatin-28(1-13), is a novel peptide in mammalian gut and circulation

Preprosomatostatin is a gene expressed ubiquitously among vertebrates, and at least two duplications of this gene have occurred during evolution. Somatostatin-28 (S-28) and somatostatin-14 (S-14), C-terminal products of prosomatostatin (ProS), are differentially expressed in mammalian neurons, D cells, and enterocytes. One pathway for the generation of S-14 entails the excision of Arg13-Lys14 in S-28, leading to equivalent amounts of S-28(1-12). Using an antiserum (F-4), directed to the N-terminal region of S-28 that does not react with S-28(1-12), we detected a peptide, in addition to S-28 and ProS, that was present in human plasma and in the intestinal tract of rats and monkeys. This F-4 reacting peptide was purified from monkey ileum; and its amino acid sequence, molecular mass, and chromatographic characteristics conformed to those of S-28(1-13), a peptide not described heretofore. When extracts of the small intestine were measured by RIA, there was a discordance in the ratio of peptides reacting with F-4 and those containing the C terminus of ProS, suggesting sites of synthesis for S-28(1-13) distinct from those for S-14 and S-28. This was supported by immunocytochemistry, wherein F-4 reactivity was localized in gastrointestinal (GI) endocrine cells and a widespread plexus of neurons within the wall of the distal gut while immunoreactivity to C-terminal domains of S-14 and S-28 in these neurons was absent. Further, F-4 immunoreactivity persisted in similar GI endocrine cells and myenteric neurons in mice with a targeted deletion of the preprosomatostatin gene. We believe that these data suggest a novel peptide produced in the mammalian gut, homologous with the 13 residues of the proximal region of S-28 but not derived from the Pros gene. Pending characterization of the gene from which this peptide is derived, its distribution, and function, we have designated this peptide as thrittene. Its localization in both GI endocrine cells and gut neurons suggests that thrittene may function as both a hormone and neurotransmitter.Fil: Ensinck, John W.. University of Washington; Estados UnidosFil: Baskin, Denis G.. University of Washington; Estados Unidos. Department of Veterans Affairs Puget Sound Health Care System; Estados UnidosFil: Vahl, Torsten P.. University of Cincinnati; Estados UnidosFil: Vogel, Robin E.. University of Washington; Estados UnidosFil: Laschansky, Ellen C.. University of Washington; Estados UnidosFil: Francis, Bruce H.. University of Washington; Estados UnidosFil: Hoffman, Ross C.. ZymoGenetics; Estados UnidosFil: Krakover, Jonathan D.. ZymoGenetics; Estados UnidosFil: Stamm, Michael R.. ZymoGenetics; Estados UnidosFil: Low, Malcolm J.. Oregon Health Sciences University; Estados UnidosFil: Rubinstein, Marcelo. Oregon Health Sciences University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Otero Corchon, Veronica. Oregon Health Sciences University; Estados UnidosFil: D'Alessio, David A.. University of Cincinnati; Estados Unido

<i>Pomc</i> expression in adult nPE mutant mice.

<p>(A) Representative <i>Pomc in situ</i> hybridization with a digoxigenin-labeled riboprobe in coronal sections through the hypothalamic arcuate nucleus of adult male mice (age 8 wk). 3V, third ventricle. Scale bar, 100 μm. (B) Histogram of integrated density of the cellular colorimetric <i>in situ</i> hybridization signal. Images from a minimum of 4 sections per biological replicate were thresholded for minimum object size and intensity, and automated cell counts, together with their individual integrated optical densities, were performed by the NIS Elements software (Nikon). (C-E) <i>Pomc</i> expression measured by qRT-PCR in hypothalamus (C), anterior pituitary (D), and brainstem (E) of adult male mice (age 8 wk). n = 8 per genotype. All quantitative data are presented as mean + 1 S.E.M. Genotype means were compared by two-tailed t-tests. * <i>P</i> < 0.05, *** <i>P</i> < 0.001 compared to +/+.</p

The <i>Pomc</i> neuronal enhancers nPE1 and nPE2 Share a common <i>cis</i>-regulatory code.

<p>(A) Evolutionary tree of placental mammalian lineages. The relative lengths of DNA sequences in kilobases (kb) separating nPE1 from nPE2 and nPE2 from exon 1 are illustrated by purple and green bars, respectively. (B) Alignment between an almost palindromic sequence carrying canonical homeodomain binding sites (HDBS) present in nPE1core and a remarkably similar sequence present within nPE2 region 1. (C) Scheme of nPE1core and regions 1 and 3 of nPE2 showing the relative positions of conserved HDBS. nPE1core carries a pair of inverted HDBS similar to another present in nPE2 region 1 (green boxes, full sequences depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1B</a>). Another pair of HDBS is present in region 3 of nPE2 (purple boxes). Canonical HDBS of the NKX subfamily are shown (blue boxes). Red letters indicate the mutated nucleotides. Grey boxes denote the critical enhancer regions determined previously in transgenic mice. (D) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE1core. The control nPE1core<i>Pomc</i>-EGFP carries the wild-type (wt) enhancer sequence whereas nPE1core(mut)<i>Pomc</i>-EGFP (mut) carries six nucleotide substitutions covering all HDBS (red letters in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1C</a>). Coronal brain sections showing EGFP expression in hypothalamic arcuate nucleus of nPE1core<i>Pomc</i>-EGFP (Left) but not in nPE1core(mut)<i>Pomc</i>-EGFP transgenic founder newborn mice (Right). (E) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE2. The control transgene nPE2<i>Pomc</i>-EGFP carries the wild-type (wt) enhancer whereas nPE2(mut)<i>Pomc</i>-EGFP (mut) carries twelve nucleotide substitutions covering all HDBS (red letters in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1C</a>). EGFP is expressed in the arcuate nucleus of founder transgenic mice carrying nPE2<i>Pomc</i>-EGFP (Left) but not nPE2(mut)<i>Pomc</i>-EGFP (Right). 3V, third ventricle.</p

Hypothalamic POMC-derived peptides in nPE mutant mice.

<p>(A) Representative ACTH immunohistochemistry in coronal hypothalamic sections. 3V, third ventricle. Scale bar, 200 μm. (B) Hypothalamic α-MSH and (C) β-endorphin content measured by radioimmunoassay. All data are from adult male mice (age 8 wk). n = 6–18. All quantitative data are presented as mean + 1 S.E.M. Genotype means were compared by two-tailed t-tests. *** <i>P</i> < 0.001 compared to +/+.</p

Somatostatin is required for masculinization of growth hormone–regulated hepatic gene expression but not of somatic growth

Pulsatile growth hormone (GH) secretion differs between males and females and regulates the sex-specific expression of cytochrome P450s in liver. Sex steroids influence the secretory dynamics of GH, but the neuroendocrine mechanisms have not been conclusively established. Because periventricular hypothalamic somatostatin (SST) expression is greater in males than in females, we generated knockout (Smst(–/–)) mice to investigate whether SST peptides are necessary for sexually differentiated GH secretion and action. Despite marked increases in nadir and median plasma GH levels in both sexes of Smst(–/–) compared with Smst(+/+) mice, the mutant mice had growth curves identical to their sibling controls and retained a normal sexual dimorphism in weight and length. In contrast, the liver of male Smst(–/–) mice was feminized, resulting in an identical profile of GH-regulated hepatic mRNAs between male and female mutants. Male Smst(-/-) mice show higher expression of two SST receptors in the hypothalamus and pituitary than do females. These data indicate that SST is required to masculinize the ultradian GH rhythm by suppressing interpulse GH levels. In the absence of SST, male and female mice exhibit similarly altered plasma GH profiles that eliminate sexually dimorphic liver function but do not affect dimorphic growth