Expression patterns of Tbx2, -3, and -5 genes were analyzed during chick embryonic heart development. Transcripts of these three cTbx genes were detected in overlapping patterns in the early cardiac crescent. cTbx2 and cTbx3 expression patterns closely overlapped with that of bmp2. cTbx5 expression diverged from cTbx2 and bmp2 during the elaboration and folding of the heart tube. In comparison, cTbx2 expression overlapped significantly with that of bmp2 and bmp4 during all stages of heart development and during later embryonic stages, suggestive of a specialized role for Tbx2 in septation. Coexpression of cTbx 2 and cTbx3 genes with bmp2 transcripts raised the possibility that these cTbx genes might be downstream bmp2 targets. Application of bmp2 selectively induced cTbx2 and cTbx3 expression in noncardiogenic embryonic tissue, and the bmp antagonist Noggin down-regulated cTbx2 gene activity. Moreover, the appearance of murine mTbx2 was blocked in bmp2 null mouse embryos. cTbx2 and to a lesser extent cTbx3 gene activity appears to be directed by BMPs during early cardiogenesis.

SummaryKinase suppressor of Ras 2 (KSR2) is an intracellular scaffolding protein involved in multiple signaling pathways. Targeted deletion of Ksr2 leads to obesity in mice, suggesting a role in energy homeostasis. We explored the role of KSR2 in humans by sequencing 2,101 individuals with severe early-onset obesity and 1,536 controls. We identified multiple rare variants in KSR2 that disrupt signaling through the Raf-MEK-ERK pathway and impair cellular fatty acid oxidation and glucose oxidation in transfected cells; effects that can be ameliorated by the commonly prescribed antidiabetic drug, metformin. Mutation carriers exhibit hyperphagia in childhood, low heart rate, reduced basal metabolic rate and severe insulin resistance. These data establish KSR2 as an important regulator of energy intake, energy expenditure, and substrate utilization in humans. Modulation of KSR2-mediated effects may represent a novel therapeutic strategy for obesity and type 2 diabetes.PaperFlick

The orphan nuclear receptor Ear2 (Nr2f6) is transiently expressed in the rostral part of the rhombic lip in which the locus coeruleus (LC) arises. LC development, regulated by a signaling cascade (Mash1 → Phox2b → Phox2a), is disrupted in Ear2 −/− embryos as revealed by an approximately threefold reduction in the number of Phox2a-and Phox2b-expressing LC progenitor cells. Mash1 expression in the rhombic lip, however, is unaffected, placing Ear2 in between Mash1 and Phox2a/b. Dopamine-␤-hydroxylase and tyrosine hydroxylase staining demonstrate that >70% of LC neurons are absent in the adult with agenesis affecting primarily the dorsal division of the LC. Normally, this division projects noradrenergic efferents to the cortex that appear to be diminished in Ear2 −/− since the cortical concentration of noradrenaline is four times lower in these mice. The rostral region of the cortex is known to contain a circadian pacemaker regulating adaptability to light-and restricted food-driven entrainment. In situ hybridization establishes that the circadian expression pattern of the clock gene Period1 is abolished in the Ear2 −/− forebrain. Behavioral experiments reveal that Ear2 mutants have a delayed entrainment to shifted light-dark cycles and adapt less efficiently to daytime feeding schedules. We propose that neurons in the dorsal division of LC contribute to the regulation of the forebrain clock, at least in part, through targeted release of noradrenaline into the cortical area.[Keywords: Circadian rhythm; Ear2; forebrain clock; locus coeruleus; nociception; nuclear orphan receptor] Supplemental material is available at http://www.genesdev.org.

The kinase suppressor of ras 2 (KSR2) gene resides at human chromosome 12q24, a region linked to obesity and type 2 diabetes (T2D). While knocking out and phenotypically screening mouse orthologs of thousands of druggable human genes, we found KSR2 knockout (KSR2−/−) mice to be more obese and glucose intolerant than melanocortin 4 receptor−/− (MC4R−/−) mice. The obesity and T2D of KSR2−/− mice resulted from hyperphagia which was unresponsive to leptin and did not originate downstream of MC4R. The kinases AMP‐activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) are each linked to food intake regulation, but only mTOR had increased activity in KSR2−/− mouse brain, and the ability of rapamycin to inhibit food intake in KSR2−/− mice further implicated mTOR in this process. The metabolic phenotype of KSR2 heterozygous (KSR2+/minus;) and KSR2−/− mice suggests that human KSR2 variants may contribute to a similar phenotype linked to human chromosome 12q24.

Targeted disruption of mouse ␤ 3 -adrenoceptor was generated by homologous recombination, and validated by an acute in vivo study showing a complete lack of effect of the ␤ 3 -adrenoceptor agonist CL 316,243 on the metabolic rate of homozygous null ( Ϫ / Ϫ ) mice. In brown adipose tissue, ␤ 3 -adrenoceptor disruption induced a 66% decrease ( P Ͻ 0.005) in ␤ 1 -adrenoceptor mRNA level, whereas leptin mRNA remained unchanged. Chronic energy balance studies in chow-fed mice showed that in Ϫ / Ϫ mice, body fat accumulation was favored ( ϩ 41%, P Ͻ 0.01), with a slight increase in food intake ( ϩ 6%, NS). These effects were accentuated by high fat feeding: Ϫ / Ϫ mice showed increased total body fat ( ϩ 56%, P Ͻ 0.025) and food intake ( ϩ 12%, P Ͻ 0.01), and a decrease in the fat-free dry mass ( Ϫ 10%, P Ͻ 0.05), which reflects a reduction in body protein content. Circulating leptin levels were not different in Ϫ / Ϫ and control mice regardless of diet. The significant shift to the right in the positive correlation between circulating leptin and percentage of body fat in high fat-fed Ϫ / Ϫ mice suggests that the threshold of body fat content inducing leptin secretion is higher in Ϫ / Ϫ than in control mice. Taken together, these studies demonstrate that ␤ 3 -adrenoceptor disruption creates conditions which predispose to the development of obesity. ( J. Clin. Invest. 1997. 100:1098-1106.)

Plasma kallikrein is a multifunctional serine protease involved in contact activation of coagulation. Deficiency in humans is characterised by prolonged activated partial thromboplastin time (aPTT); however, the balance between thrombosis and haemostasis is not fully understood. A study of plasma kallikrein-deficient mice revealed increased aPTT, without prolonged bleeding time. Prekallikrein antisense oligonucleotide (ASO) treatment in mice suggested potential for a positive therapeutic index. The current goal was to further define the role of plasma kallikrein in coagulation. Blood pressure and heart rate were normal in plasma kallikrein-deficient mice, and mice were completely protected from occlusion (100 ± 1.3% control flow) in 3.5% FeCl3 -induced arterial thrombosis versus heterozygotes (20 ± 11.4%) and wild-type littermates (8 ± 0%). Vessels occluded in 8/8 wild-type, 7/8 heterozygotes, and 0/8 knockouts. Anti-thrombotic protection was less pronounced in 5% FeCl3-induced arterial injury. Integrated blood flow was 8 ± 0% control in wild-type and heterozygotes, and significantly (p<0.01) improved to 43 ± 14.2% in knockouts. The number of vessels occluded was similar in all genotypes. Thrombus weight was significantly reduced in knockouts (-47%) and heterozygotes (-23%) versus wild-type in oxidative venous thrombosis. Average tail bleeding time increased modestly in knockout mice compared to wild-type. Average renal bleeding times were similar in all genotypes. These studies confirm and extend studies with prekallikrein ASO, and demonstrate that plasma kallikrein deletion prevents occlusive thrombus formation in mice with a minimal role in provoked bleeding. Additional support for the significance of the intrinsic pathway in the coagulation cascade is provided, as well as for a potential new anti-thrombotic approach.

Some blockers of beta1- and beta2-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta4-adrenoceptor) that resembles the beta3-adrenoceptor. It is likely but not proven that the putative beta4-adrenoceptor is genetically distinct from the beta3-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta3-adrenoceptor gene (beta3 KO). (-)-CGP 12177, a beta1- and beta2-adrenoceptor blocker that causes agonist effects through both beta3-adrenoceptors and cardiac putative beta4-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta3 KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 microM) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta3 KO. (-)-[3H]CGP 12177 labeled a similar density of the putative beta4-adrenoceptor in ventricular membranes from the hearts of both WT (Bmax = 52 fmol/mg protein) and beta3 KO (Bmax = 53 fmol/mg protein) mice. The affinity of (-)-[3H]CGP 12177 for the cardiac putative beta4-adrenoceptor was not different between WT (Kd = 46 nM) and beta3 KO (Kd= 40 nM). These results provide definitive evidence that the cardiac putative beta4-adrenoceptor is distinct from the beta3-adrenoceptor.

Capicua (CIC) regulates a transcriptional network downstream of the RAS/MAPK signaling cascade. In , CIC is important for many developmental processes, including embryonic patterning and specification of wing veins. In humans, CIC has been implicated in neurological diseases, including spinocerebellar ataxia type 1 (SCA1) and a neurodevelopmental syndrome. Additionally, we and others have reported mutations in in several cancers. However, whether CIC is a tumor suppressor remains to be formally tested. In this study, we found that deletion of in adult mice causes T cell acute lymphoblastic leukemia/lymphoma (T-ALL). Using hematopoietic-specific deletion and bone marrow transplantation studies, we show that loss of from hematopoietic cells is sufficient to drive T-ALL. -null tumors show up-regulation of the KRAS pathway as well as activation of the NOTCH1 and MYC transcriptional programs. In sum, we demonstrate that loss of CIC causes T-ALL, establishing it as a tumor suppressor for lymphoid malignancies. Moreover, we show that mouse models lacking CIC in the hematopoietic system are robust models for studying the role of RAS signaling as well as NOTCH1 and MYC transcriptional programs in T-ALL.