Analyzing human knockouts to validate GPR151 as a therapeutic target for reduction of body mass index.

Novel drug targets for sustained reduction in body mass index (BMI) are needed to curb the epidemic of obesity, which affects 650 million individuals worldwide and is a causal driver of cardiovascular and metabolic disease and mortality. Previous studies reported that the Arg95Ter nonsense variant of GPR151, an orphan G protein-coupled receptor, is associated with reduced BMI and reduced risk of Type 2 Diabetes (T2D). Here, we further investigate GPR151 with the Pakistan Genome Resource (PGR), which is one of the largest exome biobanks of human homozygous loss-of-function carriers (knockouts) in the world. Among PGR participants, we identify eleven GPR151 putative loss-of-function (plof) variants, three of which are present at homozygosity (Arg95Ter, Tyr99Ter, and Phe175LeufsTer7), with a cumulative allele frequency of 2.2%. We confirm these alleles in vitro as loss-of-function. We test if GPR151 plof is associated with BMI, T2D, or other metabolic traits and find that GPR151 deficiency in complete human knockouts is not associated with clinically significant differences in these traits. Relative to Gpr151+/+ mice, Gpr151-/- animals exhibit no difference in body weight on normal chow and higher body weight on a high-fat diet. Together, our findings indicate that GPR151 antagonism is not a compelling therapeutic approach to treatment of obesity.

Discovery of a small molecule RXFP3/4 agonist that increases food intake in rats upon acute central administration.

The relaxin family peptide receptors have been implicated in numerous physiological processes including energy homeostasis, cardiac function, wound healing, and reproductive function. Two family members, RXFP3 and RXFP4, are class A GPCRs with endogenous peptide ligands (relaxin-3 and insulin-like peptide 5 (INSL5), respectively). Polymorphisms in relaxin-3 and RXFP3 have been associated with obesity, diabetes, and hypercholesterolemia. Moreover, central administration of relaxin-3 in rats has been shown to increase food intake, leading to body weight gain. Reported RXFP3 and RXFP4 ligands have been restricted to peptides (both endogenous and synthetic) as well as a low molecular weight positive allosteric modulator requiring a non-endogenous orthosteric ligand. Described here is the discovery of the first potent low molecular weight dual agonists of RXFP3/4. The scaffold identified is competitive with a chimeric relaxin-3/INSL5 peptide for RXFP3 binding, elicits similar downstream signaling as relaxin-3, and increases food intake in rats following acute central administration. This is the first report of small molecule RXFP3/4 agonism.

Identification of spinal circuits involved in touch-evoked dynamic mechanical pain.

Mechanical hypersensitivity is a debilitating symptom for millions of chronic pain patients. It exists in distinct forms, including brush-evoked dynamic and filament-evoked punctate hypersensitivities. We reduced dynamic mechanical hypersensitivity induced by nerve injury or inflammation in mice by ablating a group of adult spinal neurons defined by developmental co-expression of VGLUT3 and Lbx1 (VT3Lbx1 neurons): the mice lost brush-evoked nocifensive responses and conditional place aversion. Electrophysiological recordings show that VT3Lbx1 neurons form morphine-resistant polysynaptic pathways relaying inputs from low-threshold Aß mechanoreceptors to lamina I output neurons. The subset of somatostatin-lineage neurons preserved in VT3Lbx1-neuron-ablated mice is largely sufficient to mediate morphine-sensitive and morphine-resistant forms of von Frey filament-evoked punctate mechanical hypersensitivity. Furthermore, acute silencing of VT3Lbx1 neurons attenuated pre-established dynamic mechanical hypersensitivity induced by nerve injury, suggesting that these neurons may be a cellular target for treating this form of neuropathic pain.

MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus.

Activation of melanocortin-4 receptors (MC4Rs) restrains feeding and prevents obesity; however, the identity, location, and axonal projections of the neurons bearing MC4Rs that control feeding remain unknown. Reexpression of MC4Rs on single-minded 1 (SIM1)(+) neurons in mice otherwise lacking MC4Rs is sufficient to abolish hyperphagia. Thus, MC4Rs on SIM1(+) neurons, possibly in the paraventricular hypothalamus (PVH) and/or amygdala, regulate food intake. It is unknown, however, whether they are also necessary, a distinction required for excluding redundant sites of action. Hence, the location and nature of obesity-preventing MC4R-expressing neurons are unknown. Here, by deleting and reexpressing MC4Rs from cre-expressing neurons, establishing both necessity and sufficiency, we demonstrate that the MC4R-expressing neurons regulating feeding are SIM1(+), located in the PVH, glutamatergic and not GABAergic, and do not express oxytocin, corticotropin-releasing hormone, vasopressin, or prodynorphin. Importantly, these excitatory MC4R-expressing PVH neurons are synaptically connected to neurons in the parabrachial nucleus, which relays visceral information to the forebrain. This suggests a basis for the feeding-regulating effects of MC4Rs.

Melanocortin 4 receptors in autonomic neurons regulate thermogenesis and glycemia.

Whether melanocortin 4 receptors (MC4Rs) in extra-hypothalamic neurons, including cholinergic autonomic pre-ganglionic neurons, are required to control energy and glucose homeostasis is unclear. We found that MC4Rs in sympathetic, but not parasympathetic, pre-ganglionic neurons were required to regulate energy expenditure and body weight, including thermogenic responses to diet and cold exposure and 'beiging' of white adipose tissue. Deletion of Mc4r genes in both sympathetic and parasympathetic cholinergic neurons impaired glucose homeostasis.

Leptin-responsive GABAergic neurons regulate fertility through pathways that result in reduced kisspeptinergic tone.

The adipocyte-derived hormone leptin plays a critical role in the central transmission of energy balance to modulate reproductive function. However, the neurocircuitry underlying this interaction remains elusive, in part due to incomplete knowledge of first-order leptin-responsive neurons. To address this gap, we explored the contribution of predominantly inhibitory (GABAergic) neurons versus excitatory (glutamatergic) neurons in the female mouse by selective ablation of the leptin receptor in each neuronal population: Vgat-Cre;Lepr(lox/lox) and Vglut2-Cre;Lepr(lox/lox) mice, respectively. Female Vgat-Cre;Lepr(lox/lox) but not Vglut2-Cre;Lepr(lox/lox) mice were obese. Vgat-Cre;Lepr(lox/lox) mice had delayed or absent vaginal opening, persistent diestrus, and atrophic reproductive tracts with absent corpora lutea. In contrast, Vglut2-Cre;Lepr(lox/lox) females exhibited reproductive maturation and function comparable to Lepr(lox/lox) control mice. Intracerebroventricular administration of kisspeptin-10 to Vgat-Cre;Lepr(lox/lox) female mice elicited robust gonadotropin responses, suggesting normal gonadotropin-releasing hormone neuronal and gonadotrope function. However, adult ovariectomized Vgat-Cre;Lepr(lox/lox) mice displayed significantly reduced levels of Kiss1 (but not Tac2) mRNA in the arcuate nucleus, and a reduced compensatory luteinizing hormone increase compared with control animals. Estradiol replacement after ovariectomy inhibited gonadotropin release to a similar extent in both groups. These animals also exhibited a compromised positive feedback response to sex steroids, as shown by significantly lower Kiss1 mRNA levels in the AVPV, compared with Lepr(lox/lox) mice. We conclude that leptin-responsive GABAergic neurons, but not glutamatergic neurons, act as metabolic sensors to regulate fertility, at least in part through modulatory effects on kisspeptin neurons.

An excitatory paraventricular nucleus to AgRP neuron circuit that drives hunger.

Hunger is a hard-wired motivational state essential for survival. Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus (ARC) at the base of the hypothalamus are crucial to the control of hunger. They are activated by caloric deficiency and, when naturally or artificially stimulated, they potently induce intense hunger and subsequent food intake. Consistent with their obligatory role in regulating appetite, genetic ablation or chemogenetic inhibition of AgRP neurons decreases feeding. Excitatory input to AgRP neurons is important in caloric-deficiency-induced activation, and is notable for its remarkable degree of caloric-state-dependent synaptic plasticity. Despite the important role of excitatory input, its source(s) has been unknown. Here, through the use of Cre-recombinase-enabled, cell-specific neuron mapping techniques in mice, we have discovered strong excitatory drive that, unexpectedly, emanates from the hypothalamic paraventricular nucleus, specifically from subsets of neurons expressing thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP, also known as ADCYAP1). Chemogenetic stimulation of these afferent neurons in sated mice markedly activates AgRP neurons and induces intense feeding. Conversely, acute inhibition in mice with caloric-deficiency-induced hunger decreases feeding. Discovery of these afferent neurons capable of triggering hunger advances understanding of how this intense motivational state is regulated.

Leptin engages a hypothalamic neurocircuitry to permit survival in the absence of insulin.

The dogma that life without insulin is incompatible has recently been challenged by results showing the viability of insulin-deficient rodents undergoing leptin monotherapy. Yet, the mechanisms underlying these actions of leptin are unknown. Here, the metabolic outcomes of intracerebroventricular (i.c.v.) administration of leptin in mice devoid of insulin and lacking or re-expressing leptin receptors (LEPRs) only in selected neuronal groups were assessed. Our results demonstrate that concomitant re-expression of LEPRs only in hypothalamic γ-aminobutyric acid (GABA) and pro-opiomelanocortin (POMC) neurons is sufficient to fully mediate the lifesaving and antidiabetic actions of leptin in insulin deficiency. Our analyses indicate that enhanced glucose uptake by brown adipose tissue and soleus muscle, as well as improved hepatic metabolism, underlies these effects of leptin. Collectively, our data elucidate a hypothalamic-dependent pathway enabling life without insulin and hence pave the way for developing better treatments for diseases of insulin deficiency.

Double deletion of melanocortin 4 receptors and SAPAP3 corrects compulsive behavior and obesity in mice.

Compulsive behavior is a debilitating clinical feature of many forms of neuropsychiatric disease, including Tourette syndrome, obsessive-compulsive spectrum disorders, eating disorders, and autism. Although several studies link striatal dysfunction to compulsivity, the pathophysiology remains poorly understood. Here, we show that both constitutive and induced genetic deletion of the gene encoding the melanocortin 4 receptor (MC4R), as well as pharmacologic inhibition of MC4R signaling, normalize compulsive grooming and striatal electrophysiologic impairments in synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3)-null mice, a model of human obsessive-compulsive disorder. Unexpectedly, genetic deletion of SAPAP3 restores normal weight and metabolic features of MC4R-null mice, a model of human obesity. Our findings offer insights into the pathophysiology and treatment of both compulsive behavior and eating disorders.

Melanocortin 4 receptors reciprocally regulate sympathetic and parasympathetic preganglionic neurons.

Melanocortin 4 receptors (MC4Rs) in the central nervous system are key regulators of energy and glucose homeostasis. Notably, obese patients with MC4R mutations are hyperinsulinemic and resistant to obesity-induced hypertension. Although these effects are probably dependent upon the activity of the autonomic nervous system, the cellular effects of MC4Rs on parasympathetic and sympathetic neurons remain undefined. Here, we show that MC4R agonists inhibit parasympathetic preganglionic neurons in the brainstem. In contrast, MC4R agonists activate sympathetic preganglionic neurons in the spinal cord. Deletion of MC4Rs in cholinergic neurons resulted in elevated levels of insulin. Furthermore, re-expression of MC4Rs specifically in cholinergic neurons (including sympathetic preganglionic neurons) restores obesity-associated hypertension in MC4R null mice. These findings provide a cellular correlate of the autonomic side effects associated with MC4R agonists and demonstrate a role for MC4Rs expressed in cholinergic neurons in the regulation of insulin levels and in the development of obesity-induced hypertension.

Runx1 controls terminal morphology and mechanosensitivity of VGLUT3-expressing C-mechanoreceptors.

VGLUT3-expressing unmyelinated low-threshold mechanoreceptors (C-LTMRs) are proposed to mediate pleasant touch and/or pain, but the molecular programs controlling C-LTMR development are unknown. Here, we performed genetic fate mapping, showing that VGLUT3 lineage sensory neurons are divided into two groups, based on transient or persistent VGLUT3 expression. VGLUT3-transient neurons are large- or medium-diameter myelinated mechanoreceptors that form the Merkel cell-neurite complex. VGLUT3-persistent neurons are small-diameter unmyelinated neurons that are further divided into two subtypes: (1) tyrosine hydroxylase (TH)-positive C-LTMRs that form the longitudinal lanceolate endings around hairs, and (2) TH-negative neurons that form epidermal-free nerve endings. We then found that VGLUT3-persistent neurons express the runt domain transcription factor Runx1. Analyses of mice with a conditional knock-out of Runx1 in VGLUT3 lineage neurons demonstrate that Runx1 is pivotal to the development of VGLUT3-persistent neurons, such as the expression of VGLUT3 and TH and the formation of the longitudinal lanceolate endings. Furthermore, Runx1 is required to establish mechanosensitivity in C-LTMRs, by controlling the expression of the mechanically gated ion channel Piezo2. Surprisingly, both acute and chronic mechanical pain was largely unaffected in these Runx1 mutants. These findings appear to argue against the recently proposed role of VGLUT3 in C-LTMRs in mediating mechanical hypersensitivity induced by nerve injury or inflammation. Thus, our studies provide new insight into the genetic program controlling C-LTMR development and call for a revisit for the physiological functions of C-LTMRs.

Identification and characterization of a sleep-active cell group in the rostral medullary brainstem.

Early transection and stimulation studies suggested the existence of sleep-promoting circuitry in the medullary brainstem, yet the location and identity of the neurons comprising this putative hypnogenic circuitry remains unresolved. In the present study, we sought to uncover the location and identity of medullary neurons that might contribute to the regulation of sleep. Here we show the following in rats: (1) a delimited node of medullary neurons located lateral and dorsal to the facial nerve-a region we termed the parafacial zone (PZ)-project to the wake-promoting medial parabrachial nucleus; (2) PZ neurons express c-Fos after sleep but not after wakefulness and hence are sleep active; and (3) cell-body-specific lesions of the PZ result in large and sustained increases (50%) in daily wakefulness at the expense of slow-wave sleep (SWS). Using transgenic reporter mice [vesicular GABA/glycine transporter (Vgat)-GFP], we then show that >50% of PZ sleep-active neurons are inhibitory (GABAergic/glycinergic, VGAT-positive) in nature. Finally, we used a Cre-expressing adeno-associated viral vector and conditional Vgat(lox/lox) mice to selectively and genetically disrupt GABA/glycinergic neurotransmission from PZ neurons. Disruption of PZ GABAergic/glycinergic neurotransmission resulted in sustained increases (40%) in daily wakefulness at the expense of both SWS and rapid eye movement sleep. These results together reveal the location and neurochemical identity of a delimited node of sleep-active neurons within the rostral medullary brainstem.

GABAergic RIP-Cre neurons in the arcuate nucleus selectively regulate energy expenditure.

Neural regulation of energy expenditure is incompletely understood. By genetically disrupting GABAergic transmission in a cell-specific fashion, and by combining this with selective pharmacogenetic activation and optogenetic mapping techniques, we have uncovered an arcuate-based circuit that selectively drives energy expenditure. Specifically, mice lacking synaptic GABA release from RIP-Cre neurons have reduced energy expenditure, become obese and are extremely sensitive to high-fat diet-induced obesity, the latter due to defective diet-induced thermogenesis. Leptin's ability to stimulate thermogenesis, but not to reduce feeding, is markedly attenuated. Acute, selective activation of arcuate GABAergic RIP-Cre neurons, which monosynaptically innervate PVH neurons projecting to the NTS, rapidly stimulates brown fat and increases energy expenditure but does not affect feeding. Importantly, this response is dependent upon GABA release from RIP-Cre neurons. Thus, GABAergic RIP-Cre neurons in the arcuate selectively drive energy expenditure, contribute to leptin's stimulatory effect on thermogenesis, and protect against diet-induced obesity.

MEF2C ablation in endothelial cells reduces retinal vessel loss and suppresses pathologic retinal neovascularization in oxygen-induced retinopathy.

Ischemic retinopathies, including retinopathy of prematurity and diabetic retinopathy, are major causes of blindness. Both have two phases, vessel loss and consequent hypoxia-driven pathologic retinal neovascularization, yet relatively little is known about the transcription factors regulating these processes. Myocyte enhancer factor 2 (MEF2) C, a member of the MEF2 family of transcription factors that plays an important role in multiple developmental programs, including the cardiovascular system, seems to have a significant functional role in the vasculature. We, therefore, generated endothelial cell (EC)-specific MEF2C-deficient mice and explored the role of MEF2C in retinal vascularization during normal development and in a mouse model of oxygen-induced retinopathy. Ablation of MEF2C did not cause appreciable defects in normal retinal vascular development. However, MEF2C ablation in ECs suppressed vessel loss in oxygen-induced retinopathy and strongly promoted vascular regrowth, consequently reducing retinal avascularity. This finding was associated with suppression of pathologic retinal angiogenesis and blood-retinal barrier dysfunction. MEF2C knockdown in cultured retinal ECs using small-interfering RNAs rescued ECs from death and stimulated tube formation under stress conditions, confirming the endothelial-autonomous and antiangiogenic roles of MEF2C. HO-1 was induced by MEF2C knockdown in vitro and may play a role in the proangiogenic effect of MEF2C knockdown on retinal EC tube formation. Thus, MEF2C may play an antiangiogenic role in retinal ECs under stress conditions, and modulation of MEF2C may prevent pathologic retinal neovascularization.

Neuron-type-specific signals for reward and punishment in the ventral tegmental area.

Dopamine has a central role in motivation and reward. Dopaminergic neurons in the ventral tegmental area (VTA) signal the discrepancy between expected and actual rewards (that is, reward prediction error), but how they compute such signals is unknown. We recorded the activity of VTA neurons while mice associated different odour cues with appetitive and aversive outcomes. We found three types of neuron based on responses to odours and outcomes: approximately half of the neurons (type I, 52%) showed phasic excitation after reward-predicting odours and rewards in a manner consistent with reward prediction error coding; the other half of neurons showed persistent activity during the delay between odour and outcome that was modulated positively (type II, 31%) or negatively (type III, 18%) by the value of outcomes. Whereas the activity of type I neurons was sensitive to actual outcomes (that is, when the reward was delivered as expected compared to when it was unexpectedly omitted), the activity of type II and type III neurons was determined predominantly by reward-predicting odours. We 'tagged' dopaminergic and GABAergic neurons with the light-sensitive protein channelrhodopsin-2 and identified them based on their responses to optical stimulation while recording. All identified dopaminergic neurons were of type I and all GABAergic neurons were of type II. These results show that VTA GABAergic neurons signal expected reward, a key variable for dopaminergic neurons to calculate reward prediction error.

Presynaptic inhibition of gamma-aminobutyric acid release in the bed nucleus of the stria terminalis by kappa opioid receptor signaling.

BACKGROUND: The kappa opioid receptor (KOR) and its endogenous agonist, the neuropeptide dynorphin, are a critical component of the central stress system. Both dynorphin and KOR are expressed in the bed nucleus of the stria terminalis (BNST), a brain region associated with anxiety and stress. This suggests that KOR activation in this region may play a role in the regulation of emotional behaviors. To date, however, there has been no investigation of the ability of KOR to modulate synaptic transmission in the BNST. METHODS: We used whole-cell patch-clamp recordings from acutely prepared mouse brain slices to examine the actions of KOR on inhibitory transmission in the BNST. Additionally, we used neurochemical and pathway-specific optogenetic manipulations to selectively stimulate gamma-aminobutyric acid (GABA)ergic fibers from the central nucleus of the amygdala (CeA) to the BNST. RESULTS: We found that activation of KOR reduced GABAergic transmission through a presynaptic mechanism. Furthermore, we examined the signal transduction pathways that mediate this inhibition and provide the first functional information implicating extracellular signal-regulated kinase in KOR-mediated presynaptic modulation. Moreover, we found that at KOR signaling robustly reduced inhibitory synaptic transmission in the CeA to BNST pathway. CONCLUSIONS: Together, these results demonstrate that KOR provides important inhibitory control over presynaptic GABAergic signaling within the BNST and provides the first direct functional demonstration of KOR-sensitive long-range GABAergic connections between the CeA and the BNST.

Brainstem and spinal cord circuitry regulating REM sleep and muscle atonia.

BACKGROUND: Previous work has suggested, but not demonstrated directly, a critical role for both glutamatergic and GABAergic neurons of the pontine tegmentum in the regulation of rapid eye movement (REM) sleep. METHODOLOGY/PRINCIPAL FINDINGS: To determine the in vivo roles of these fast-acting neurotransmitters in putative REM pontine circuits, we injected an adeno-associated viral vector expressing Cre recombinase (AAV-Cre) into mice harboring lox-P modified alleles of either the vesicular glutamate transporter 2 (VGLUT2) or vesicular GABA-glycine transporter (VGAT) genes. Our results show that glutamatergic neurons of the sublaterodorsal nucleus (SLD) and glycinergic/GABAergic interneurons of the spinal ventral horn contribute to REM atonia, whereas a separate population of glutamatergic neurons in the caudal laterodorsal tegmental nucleus (cLDT) and SLD are important for REM sleep generation. Our results further suggest that presynaptic GABA release in the cLDT-SLD, ventrolateral periaqueductal gray matter (vlPAG) and lateral pontine tegmentum (LPT) are not critically involved in REM sleep control. CONCLUSIONS/SIGNIFICANCE: These findings reveal the critical and divergent in vivo role of pontine glutamate and spinal cord GABA/glycine in the regulation of REM sleep and atonia and suggest a possible etiological basis for REM sleep behavior disorder (RBD).

Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons.

Leptin acts in the brain to prevent obesity. The underlying neurocircuitry responsible for this is poorly understood, in part because of incomplete knowledge regarding first-order, leptin-responsive neurons. To address this, we and others have been removing leptin receptors from candidate first-order neurons. While functionally relevant neurons have been identified, the observed effects have been small, suggesting that most first-order neurons remain unidentified. Here we take an alternative approach and test whether first-order neurons are inhibitory (GABAergic, VGAT⁺) or excitatory (glutamatergic, VGLUT2⁺). Remarkably, the vast majority of leptin's antiobesity effects are mediated by GABAergic neurons; glutamatergic neurons play only a minor role. Leptin, working directly on presynaptic GABAergic neurons, many of which appear not to express AgRP, reduces inhibitory tone to postsynaptic POMC neurons. As POMC neurons prevent obesity, their disinhibition by leptin action on presynaptic GABAergic neurons probably mediates, at least in part, leptin's antiobesity effects.

Glucose stimulation of hypothalamic MCH neurons involves K(ATP) channels, is modulated by UCP2, and regulates peripheral glucose homeostasis.

Blood glucose levels are tightly controlled, a process thought to be orchestrated primarily by peripheral mechanisms (insulin secretion by ß cells, and insulin action on muscle, fat, and liver). The brain also plays an important, albeit less well-defined role. Subsets of neurons in the brain are excited by glucose; in many cases this involves ATP-mediated closure of K(ATP) channels. To understand the relevance of this, we are manipulating glucose sensing within glucose-excited neurons. In the present study, we demonstrate that glucose excitation of MCH-expressing neurons in the lateral hypothalamus is mediated by K(ATP) channels and is negatively regulated by UCP2 (a mitochondrial protein that reduces ATP production), and that glucose sensing by MCH neurons plays an important role in regulating glucose homeostasis. Combined, the glucose-excited neurons are likely to play key, previously unexpected roles in regulating blood glucose.

MEF2C is required for the normal allocation of cells between the ventricular and sinoatrial precursors of the primary heart field.

Targeted deletion of the mef2c gene results in a small left ventricle and complete loss of the right ventricle (Lin et al. [1997] Science 276:1404-1407). Absence of the right ventricle is from defective differentiation of cells from the secondary heart field. Our studies of the dysmorphogenesis of the left ventricle uncovered morphological and transcriptional abnormalities at the transition from the cardiac crescent to the linear-tube stage heart. Use of the cgata6LacZ transgene demonstrated that lacZ-positive cells, which normally mark the precursors to the atrioventricular canal and adjacent regions of the left ventricle and atria, remain in the sinoatrial region of the mutant. This, along with the absence of a morphologically distinct atrioventricular canal, indicates a misapportioning of cells between the inflow and outflow segments. The underlying genetic program was also affected with altered expression of mlc2a, mlc2v, and irx4 in outflow segment precursors of the primary heart field. In addition, the sinoatrial-enriched transcription factor, tbx5, was ectopically expressed in the primitive ventricle and ventricle-specific splicing of mef2b was lost, suggesting that the mutant ventricle had acquired atrial-specific characteristics. Collectively, these results suggest a fundamental role of MEF2C in ventricular cardiomyocyte differentiation and apportioning of cells between inflow and outflow precursors in the primary heart field.