Deborah J. Good

The SIL gene is required for mouse embryonic axial development and left-right specification

The establishment of the main body axis and the determination of left–right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left–right anomalies. This association is also seen in a number of mutations in mouse and zebrafish,, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left–right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2 .

Reduced voluntary activity precedes adult-onset obesity in Nhlh2 knockout mice.

Targeted deletion of the neuronal basic helix-loop-helix transcription factor Nhlh2 results in adult-onset obesity in mice. Measurement of body weight and body composition in animals aged 3-25 weeks indicates that while male and female Nhlh2 knockout (N2KO) animals both show adult-onset obesity, the time frame for development of obesity is different, with females becoming obese by 7 weeks of age and males becoming obese by 10 weeks of age. Heterozygous (HET) animals also become obese but with a slower onset, indicating a dosage effect for the activity of the Nhlh2 transcription factor. Food intake, body temperature, and voluntary activity were measured in both preobese and obese N2KO, HET, and wild-type (WT) animals to determine which factors contributed to weight gain. While increased food intake and decreased body temperature were found in older obese N2KO animals, only reduced physical activity preceded the onset of obesity in N2KO mice. N2KO animals had no deficit in either circadian rhythm or balance and motor control, indicating that reduced voluntary activity is the result of a behavioral change. These data demonstrate a role for the Nhlh2 transcription factor in controlling genes important to energy expenditure, and more specifically voluntary physical activity of animals.

Nhlh2: A Basic Helix-Loop-Helix Transcription Factor Controlling Physical Activity

In mice, targeted deletion of the basic helix-loop-helix transcription factor, nescient helix-loop-helix 2 (Nhlh2), leads to adult-onset obesity and reduced physical activity. We propose the novel hypothesis that transcriptional activity by Nhlh2 (NHLH2 in humans) controls either the ability or the motivation for exercise.

Nescient Helix-Loop-Helix 2 Interacts with Signal Transducer and Activator of Transcription 3 to Regulate Transcription of Prohormone Convertase 1/3

Mechanisms controlling body weight involve gene regulation through the activation of signal transduction pathways. The Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway is the mechanism primarily used by leptin in the hypothalamus. The transcription factor nescient helix-loop-helix 2 (Nhlh2) is a downstream target of leptin signaling and is expressed in proopiomelanocortin arcuate neurons. Proopiomelanocortin is cleaved by prohormone convertase 1/3 (PC1/3) to produce peptides that regulate the bodys response to energy availability. Previous studies show that the PC1/3 promoter contains STAT3 sites mediating leptin-induced PC1/3 expression, and that Nhlh2 is required for hypothalamic PC1/3 expression because Nhlh2 knockout mice have reduced PC1/3 mRNA levels. Studies herein reveal that leptin-induced PC1/3 gene expression is abrogated in N2KO mice, and that in a hypothalamic cell line both STAT3 and Nhlh2 are required for the full transcriptional response of a PC1/3 reporter gene after leptin stimulation. Furthermore, it is shown that Nhlh2 binds to E-box motifs found adjacent to STAT3 sites in the PC1/3 promoter both in vitro and in chromatin immunoprecipitation assays. Finally, two different protein-protein interaction assays confirm the presence of a STAT3:Nhlh2 heterodimer on the PC1/3 promoter. The Nhlh2:STAT3 heterodimer may be an important transcriptional regulator of other hypothalamic genes in the leptin signaling pathway. These data confirm Nhlh2 as an integral element of the Janus kinase/STAT signaling pathway and are the first to demonstrate coordinated control of PC1/3 transcription by Nhlh2 and STAT3 after leptin stimulation.

Expression of the hypothalamic transcription factor Nhlh2 is dependent on energy availability

Mice with a deletion of the hypothalamic basic helix‐loop‐helix transcription factor Nhlh2 display adult onset obesity, implicating Nhlh2 in the neuronal circuits regulating energy availability. Nhlh2 colocalises with the hypothalamic thyrotrophin‐releasing hormone (TRH) neurones in the paraventricular nucleus (PVN) and pro‐opiomelanocortin (POMC) neurones in the arcuate nucleus. We show that Nhlh2 expression is significantly reduced in response to 24‐h food deprivation in the arcuate nucleus, PVN, lateral hypothalamus, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). Food intake for 2 h following deprivation stimulates Nhlh2 expression in the arcuate nucleus and the PVN, and leptin injection following deprivation results in increased Nhlh2 expression in the arcuate nucleus, PVN, lateral hypothalamus, VMH, and DMH. Hypothalamic Nhlh2 expression in response to leptin injection is maximal by 2 h. Following leptin injection, Nhlh2 mRNA colocalises in POMC neurones in the arcuate nucleus and TRH neurones in the PVN. Nhlh2 mRNA expression in POMC neurones in the arcuate nucleus and TRH neurones in the PVN is reduced with energy deprivation and is stimulated with food intake and leptin injection. Modulation of POMC expression in response to changes in energy availability is not affected in mice with a targeted deletion of Nhlh2. However, deletion of Nhlh2 does result in loss of normal TRH mRNA expression in mice exposed to food deprivation and leptin stimulation. These data implicate Nhlh2 as a regulatory target of the leptin‐mediated energy availability network of the hypothalamus, and TRH as a putative downstream target of Nhlh2.

Predisposition to Arrhythmia and Autonomic Dysfunction in Nhlh1-Deficient Mice

ABSTRACT Nhlh1 is a basic helix-loop-helix transcription factor whose expression is restricted to the nervous system and which may play a role in neuronal differentiation. To directly study Nhlh1 function, we generated null mice. Homozygous mutant mice were predisposed to premature, adult-onset, unexpected death. Electrocardiograms revealed decreased total heart rate variability, stress-induced arrhythmia, and impaired baroreceptor sensitivity. This predisposition to arrhythmia is a likely cause of the observed death in the mutant mice. Heterozygosity for the closely related transcription factor Nhlh2 increased the severity of the Nhlh1-null phenotype. No signs of primary cardiac structural or conduction abnormalities could be detected upon necropsy of the null mice. The pattern of altered heart rhythm observed in basal and experimental conditions (stress and pharmacologically induced) suggests that a deficient parasympathetic tone may contribute to the arrhythmia in the Nhlh1-null mouse. The expression of Nhlh1 in the developing brain stem and in the vagal nuclei in the wild-type mouse further supports this hypothesis. The Nhlh1 mutant mouse may thus provide a model to investigate the contribution of the autonomic nervous system to arrhythmogenesis.

Energy balance pathways converging on the Nhlh2 transcription factor

Multiple regulatory pathways exist to control the expression levels of neuropeptides in response to body weight and energy availability changes. Since many neuropeptides are first synthesized in a pro-neuropeptide form, the availability of processing enzymes in a neuron can control the amount of active mature neuropeptide produced at any given time. In this review, we will focus on the regulation of prohormone convertase 1 (PC1) and prohormone convertase 2 (PC2), as well as downstream neuropeptide genes. Evidence from our laboratory suggests that Nescient helix-loop-helix 2 (Nhlh2) regulates the transcription of PC1 and PC2, possibly in conjunction with the leptin-stimulated transcription factor, STAT3. Furthermore, Nhlh2 itself is a target of leptin and other energy availability signals, with high levels of expression during energy surplus, and low levels of expression in conditions of reduced energy availability such as food deprivation or cold exposure. Overall, coordinate regulation of Nhlh2, PC1, PC2 and downstream hypothalamic neuropeptides such as thyrotropin releasing hormone (TRH) and pro-opiomelanocortin (POMC) does lead to energy balance modulation and ensuing long-term changes in body weight.

Melanocortin 4 Receptor is a Transcriptional Target of Nescient Helix-Loop-Helix-2

Melanocortin 4 receptor (Mc4r/MC4R) is a G-Protein coupled receptor that is expressed in the hypothalamus and implicated in body weight control. Mutations in MC4R are the most frequent cause of monogenetic forms of human obesity. Despite its importance, the MC4R signaling pathways and transcriptional regulation underlying the melanocortin pathway are far from being fully understood. The transcription factor nescient helix-loop-helix 2 (Nhlh2) influences the melanocortin pathway through transcriptional regulation of prohormone convertase I, which influences the production of melanocortin peptides. In the present study, Nhlh2s role as a transcriptional regulator of Mc4r has been demonstrated. Nhlh2 knockout mice have reduced hypothalamic expression of Mc4r mRNA, suggesting that it could be a direct or indirect transcriptional regulator of the Mc4r promoter. To demonstrate direct transcriptional regulation, chromatin immunoprecipitation and electrophoretic gel shift assays show that Nhlh2 binds to the E-Boxes located at -551, -366 and +54 on the Mc4r promoter. Leptin-induced transactivation of the Mc4r promoter is significantly higher in the presence of exogenously added Nhlh2. siRNA knockdown of Nhlh2 leads to significantly reduced endogenous Mc4r mRNA expression levels in N29/2 cell line. Transactivation using promoters with mutations in each of the E-Boxes results in significantly reduced transactivation efficiency compared to the WT Mc4r promoter, suggesting that Nhlh2 regulates Mc4r transcription through these sites. Findings from these studies, combined with previous work implicating Nhlh2 as a transcriptional regulator of both the Mc4r gene and the melanocortin pathway, suggest that Nhlh2s transcriptional activity directly influences the human and rodent body weight control pathways.

Reduced activity without hyperphagia contributes to obesity in Tubby mutant mice.

The Tub gene was originally identified as a spontaneous mutation in C57Bl/6J mice, and associated with adult-onset obesity (Tub MUT mice). Although the original Tub MUT mouse was identified over 15 years ago, there have been few reports on the animals food intake, body fat percentage or energy expenditure. In this study, we report food intake, body weight from 5-20 weeks, body fat, body temperature and three different measures of physical activity behavior. Tub MUT mice display reduced food intake, uncharacteristic of many obese mouse models, and reduced voluntary wheel running with normal home cage ambulatory behavior. We conclude that motivation for food and exercise is an underlying defect in TUB MUT mice.

Deletion of Nhlh2 results in a defective torpor response and reduced beta adrenergic receptor expression in adipose tissue.

Background Mice with a targeted deletion of the basic helix-loop-helix transcription factor, Nescient Helix-Loop-Helix 2 (Nhlh2), display adult-onset obesity with significant increases in their fat depots, abnormal responses to cold exposure, and reduced spontaneous physical activity levels. These phenotypes, accompanied by the hypothalamic expression of Nhlh2, make the Nhlh2 knockout (N2KO) mouse a useful model to study the role of central nervous system (CNS) control on peripheral tissue such as adipose tissue. Methodology Differences in body temperature and serum analysis of leptin were performed in fasted and ad lib fed wild-type (WT) and N2KO mice. Histological analysis of white (WAT) and brown adipose tissue (BAT) was performed. Gene and protein level expression of inflammatory and metabolic markers were compared between the two genotypes. Principal Findings We report significant differences in serum leptin levels and body temperature in N2KO mice compared with WT mice exposed to a 24-hour fast, suggestive of a defect in both white (WAT) and brown adipose tissue (BAT) function. As compared to WT mice, N2KO mice showed increased serum IL-6 protein and WAT IL-6 mRNA levels. This was accompanied by slight elevations of mRNA for several macrophage markers, including expression of macrophage specific protein F4/80 in adipose, suggestive of macrophage infiltration of WAT in the mutant animals. The mRNAs for β3-adrenergic receptors (β3-AR), β2-AR and uncoupling proteins were significantly reduced in WAT and BAT from N2KO mice compared with WT mice. Conclusions These studies implicate Nhlh2 in the central control of WAT and BAT function, with lack of Nhlh2 leading to adipose inflammation and altered gene expression, impaired leptin response to fasting, all suggestive of a deficient torpor response in mutant animals.