Cardiometabolic Consequences of Deleting the Regulator of G protein Signaling-2 (Rgs2) From Cells Expressing Agouti-Related Peptide or the ANG (Angiotensin) II Type 1A Receptor in Mice.

BACKGROUND: RGS (regulator of G protein signaling) family members catalyze the termination of G protein signaling cascades. Single nucleotide polymorphisms in the RGS2 gene in humans have been linked to hypertension, preeclampsia, and anxiety disorders. Mice deficient for Rgs2 (Rgs2Null) exhibit hypertension, anxiety, and altered adipose development and function. METHODS: To study cell-specific functions of RGS2, a novel gene-targeted mouse harboring a conditional allele for the Rgs2 gene (Rgs2Flox) was developed. These mice were bred with mice expressing Cre-recombinase via the Agouti-related peptide locus (Agrp-Cre) to cause deletion of Rgs2 from all cells expressing Agrp (Rgs2Agrp-KO), or a novel transgenic mouse expressing Cre-recombinase via the ANG (angiotensin) type 1A receptor (Agtr1a/ AT1A) promoter encoded in a bacterial artificial chromosome (BAC-AT1A-Cre) to delete Rgs2 in all Agtr1a-expressing cells (Rgs2AT1A-KO). RESULTS: Whereas Rgs2Flox, Rgs2Agrp-KO, and BAC-AT1A-Cre mice exhibited normal growth and survival, Rgs2AT1A-KO exhibited pre-weaning lethality. Relative to littermates, Rgs2Agrp-KO exhibited reduced fat gains when maintained on a high fat diet, associated with increased energy expenditure. Similarly, surviving adult Rgs2AT1A-KO mice also exhibited increased energy expenditure. Surprisingly, given the hypertensive phenotype previously reported for Rgs2Null mice and evidence supporting a role for RGS2 in terminating AT1A signaling in various cell types, Rgs2AT1A-KO mice exhibited normal blood pressure, ingestive behaviors, and renal functions, both before and after chronic infusion of ANG (490 ng/kg/min, sc). CONCLUSIONS: These results demonstrate the development of a novel mouse with conditional expression of Rgs2 and illustrate the role of Rgs2 within selected cell types for cardiometabolic control.

Neuropeptide S (NPS) neurons: Parabrachial identity and novel distributions.

Neuropeptide S (NPS) increases wakefulness. A small number of neurons in the brainstem express Nps. These neurons are located in or near the parabrachial nucleus (PB), but we know very little about their ontogeny, connectivity, and function. To identify Nps-expressing neurons within the molecular framework of the PB region, we used in situ hybridization, immunofluorescence, and Cre-reporter labeling in mice. The primary concentration of Nps-expressing neurons borders the lateral lemniscus at far-rostral levels of the lateral PB. Caudal to this main cluster, Nps-expressing neurons scatter through the PB and form a secondary concentration medial to the locus coeruleus (LC). Most Nps-expressing neurons in the PB region are Atoh1-derived, Foxp2-expressing, and mutually exclusive with neurons expressing Calca or Lmx1b. Among Foxp2-expressing PB neurons, those expressing Nps are distinct from intermingled subsets expressing Cck or Pdyn. Examining Nps Cre-reporter expression throughout the brain identified novel populations of neurons in the nucleus incertus, anterior hypothalamus, and lateral habenula. This information will help focus experimental questions about the connectivity and function of NPS neurons.

TFH cells depend on Tcf1-intrinsic HDAC activity to suppress CTLA4 and guard B-cell help function.

Precise regulation of coinhibitory receptors is essential for maintaining immune tolerance without interfering with protective immunity, yet the mechanism underlying such a balanced act remains poorly understood. In response to protein immunization, T follicular helper (TFH) cells lacking Tcf1 and Lef1 transcription factors were phenotypically normal but failed to promote germinal center formation and antibody production. Transcriptomic profiling revealed that Tcf1/Lef1-deficient TFH cells aberrantly up-regulated CTLA4 and LAG3 expression, and treatment with anti-CTLA4 alone or combined with anti-LAG3 substantially rectified B-cell help defects by Tcf1/Lef1-deficient TFH cells. Mechanistically, Tcf1 and Lef1 restrain chromatin accessibility at the Ctla4 and Lag3 loci. Groucho/Tle corepressors, which are known to cooperate with Tcf/Lef factors, were essential for TFH cell expansion but dispensable for repressing coinhibitory receptors. In contrast, mutating key amino acids in histone deacetylase (HDAC) domain in Tcf1 resulted in CTLA4 derepression in TFH cells. These findings demonstrate that Tcf1-instrinsic HDAC activity is necessary for preventing excessive CTLA4 induction in protein immunization-elicited TFH cells and hence guarding their B-cell help function.

Tcf1 and Lef1 are required for the immunosuppressive function of regulatory T cells.

Tcf1 and Lef1 have versatile functions in regulating T cell development and differentiation, but intrinsic requirements for these factors in regulatory T (T reg) cells remain to be unequivocally defined. Specific ablation of Tcf1 and Lef1 in T reg cells resulted in spontaneous multi-organ autoimmunity that became more evident with age. Tcf1/Lef1-deficient T regs showed reduced protection against experimentally induced colitis, indicative of diminished immuno-suppressive capacity. Transcriptomic analysis revealed that Tcf1 and Lef1 were responsible for positive regulation of a subset of T reg-overrepresented signature genes such as Ikzf4 and Izumo1r Unexpectedly, Tcf1 and Lef1 were necessary for restraining expression of cytotoxic CD8+ effector T cell-associated genes in T reg cells, including Prdm1 and Ifng Tcf1 ChIP-seq revealed substantial overlap between Tcf1 and Foxp3 binding peaks in the T reg cell genome, with Tcf1-Foxp3 cooccupancy observed at key T reg signature and cytotoxic effector genes. Our data collectively indicate that Tcf1 and Lef1 are critical for sustaining T reg suppressive functions and preventing loss of self-tolerance.

High-throughput screening of mouse knockout lines identifies true lean and obese phenotypes.

We developed a high-throughput approach to knockout (KO) and phenotype mouse orthologs of the 5,000 potential drug targets in the human genome. As part of the phenotypic screen, dual-energy X-ray absorptiometry (DXA) technology estimates body-fat stores in eight KO and four wild-type (WT) littermate chow-fed mice from each line. Normalized % body fat (nBF) (mean KO % body fat/mean WT littermate % body fat) values from the first 2322 lines with viable KO mice at 14 weeks of age showed a normal distribution. We chose to determine how well this screen identifies body-fat phenotypes by selecting 13 of these 2322 KO lines to serve as benchmarks based on their published lean or obese phenotype on a chow diet. The nBF values for the eight benchmark KO lines with a lean phenotype were > or =1 s.d. below the mean for seven (perilipin, SCD1, CB1, MCH1R, PTP1B, GPAT1, PIP5K2B) but close to the mean for NPY Y4R. The nBF values for the five benchmark KO lines with an obese phenotype were >2 s.d. above the mean for four (MC4R, MC3R, BRS3, translin) but close to the mean for 5HT2cR. This screen also identifies novel body-fat phenotypes as exemplified by the obese kinase suppressor of ras 2 (KSR2) KO mice. These body-fat phenotypes were confirmed upon studying additional cohorts of mice for KSR2 and all 13 benchmark KO lines. This simple and cost-effective screen appears capable of identifying genes with a role in regulating mammalian body fat.