Dax1 modulates ERα-dependent hypothalamic estrogen sensing in female mice.

Coupling the release of pituitary hormones to the developmental stage of the oocyte is essential for female fertility. It requires estrogen to restrain kisspeptin (KISS1)-neuron pulsatility in the arcuate hypothalamic nucleus, while also exerting a surge-like effect on KISS1-neuron activity in the AVPV hypothalamic nucleus. However, a mechanistic basis for this region-specific effect has remained elusive. Our genomic analysis in female mice demonstrate that some processes, such as restraint of KISS1-neuron activity in the arcuate nucleus, may be explained by region-specific estrogen receptor alpha (ERα) DNA binding at gene regulatory regions. Furthermore, we find that the Kiss1-locus is uniquely regulated in these hypothalamic nuclei, and that the nuclear receptor co-repressor NR0B1 (DAX1) restrains its transcription specifically in the arcuate nucleus. These studies provide mechanistic insight into how ERα may control the KISS1-neuron, and Kiss1 gene expression, to couple gonadotropin release to the developmental stage of the oocyte.

HypoMap-a unified single-cell gene expression atlas of the murine hypothalamus.

The hypothalamus plays a key role in coordinating fundamental body functions. Despite recent progress in single-cell technologies, a unified catalog and molecular characterization of the heterogeneous cell types and, specifically, neuronal subtypes in this brain region are still lacking. Here, we present an integrated reference atlas, 'HypoMap,' of the murine hypothalamus, consisting of 384,925 cells, with the ability to incorporate new additional experiments. We validate HypoMap by comparing data collected from Smart-Seq+Fluidigm C1 and bulk RNA sequencing of selected neuronal cell types with different degrees of cellular heterogeneity. Finally, via HypoMap, we identify classes of neurons expressing glucagon-like peptide-1 receptor (Glp1r) and prepronociceptin (Pnoc), and validate them using single-molecule in situ hybridization. Collectively, HypoMap provides a unified framework for the systematic functional annotation of murine hypothalamic cell types, and it can serve as an important platform to unravel the functional organization of hypothalamic neurocircuits and to identify druggable targets for treating metabolic disorders.

Relaxin/insulin-like family peptide receptor 4 (Rxfp4) expressing hypothalamic neurons modulate food intake and preference in mice.

OBJECTIVE: Insulin-like peptide 5 (INSL5) signalling, through its cognate receptor relaxin/insulin-like family peptide receptor 4 (RXFP4), has been reported to be orexigenic, and the high fat diet (HFD) preference observed in wildtype mice is altered in Rxfp4 knock-out mice. In this study, we used a new Rxfp4-Cre mouse model to investigate the mechanisms underlying these observations. METHODS: We generated transgenic Rxfp4-Cre mice and investigated central expression of Rxfp4 by RT-qPCR, RNAscope and intraparenchymal infusion of INSL5. Rxfp4-expressing cells were chemogenetically manipulated in global Cre-reporter mice using designer receptors exclusively activated by designer drugs (DREADDs) or after stereotactic injection of a Cre-dependent AAV-DIO-Dq-DREADD targeting a population located in the ventromedial hypothalamus (RXFP4VMH). Food intake and feeding motivation were assessed in the presence and absence of a DREADD agonist. Rxfp4-expressing cells in the hypothalamus were characterised by single-cell RNA-sequencing (scRNAseq) and the connectivity of RXFP4VMH cells was investigated using viral tracing. RESULTS: Rxfp4-Cre mice displayed Cre-reporter expression in the hypothalamus. Active expression of Rxfp4 in the adult mouse brain was confirmed by RT-qPCR and RNAscope. Functional receptor expression was supported by cyclic AMP-responses to INSL5 application in ex vivo brain slices and increased HFD and highly palatable liquid meal (HPM), but not chow, intake after intra-VMH INSL5 infusion. scRNAseq of hypothalamic RXFP4 neurons defined a cluster expressing VMH markers, alongside known appetite-modulating neuropeptide receptors (Mc4r, Cckar and Nmur2). Viral tracing demonstrated RXFP4VMH neural projections to nuclei implicated in hedonic feeding behaviour. Whole body chemogenetic inhibition (Di-DREADD) of Rxfp4-expressing cells, mimicking physiological INSL5-RXFP4 Gi-signalling, increased intake of the HFD and HPM, but not chow, whilst activation (Dq-DREADD), either at whole body level or specifically within the VMH, reduced HFD and HPM intake and motivation to work for the HPM. CONCLUSION: These findings identify RXFP4VMH neurons as regulators of food intake and preference, and hypothalamic RXFP4 signalling as a target for feeding behaviour manipulation.

Glucose-Dependent Insulinotropic Polypeptide Receptor-Expressing Cells in the Hypothalamus Regulate Food Intake.

Ambiguity regarding the role of glucose-dependent insulinotropic polypeptide (GIP) in obesity arises from conflicting reports asserting that both GIP receptor (GIPR) agonism and antagonism are effective strategies for inhibiting weight gain. To enable identification and manipulation of Gipr-expressing (Gipr) cells, we created Gipr-Cre knockin mice. As GIPR-agonists have recently been reported to suppress food intake, we aimed to identify central mediators of this effect. Gipr cells were identified in the arcuate, dorsomedial, and paraventricular nuclei of the hypothalamus, as confirmed by RNAscope in mouse and human. Single-cell RNA-seq identified clusters of hypothalamic Gipr cells exhibiting transcriptomic signatures for vascular, glial, and neuronal cells, the latter expressing somatostatin but little pro-opiomelanocortin or agouti-related peptide. Activation of Gq-DREADDs in hypothalamic Gipr cells suppressed food intake in vivo, which was not obviously additive with concomitant GLP1R activation. These data identify hypothalamic GIPR as a target for the regulation of energy balance.

Hypothalamic loss of Snord116 recapitulates the hyperphagia of Prader-Willi syndrome.

Profound hyperphagia is a major disabling feature of Prader-Willi syndrome (PWS). Characterization of the mechanisms that underlie PWS-associated hyperphagia has been slowed by the paucity of animal models with increased food intake or obesity. Mice with a microdeletion encompassing the Snord116 cluster of noncoding RNAs encoded within the Prader-Willi minimal deletion critical region have previously been reported to show growth retardation and hyperphagia. Here, consistent with previous reports, we observed growth retardation in Snord116+/-P mice with a congenital paternal Snord116 deletion. However, these mice neither displayed increased food intake nor had reduced hypothalamic expression of the proprotein convertase 1 gene PCSK1 or its upstream regulator NHLH2, which have recently been suggested to be key mediators of PWS pathogenesis. Specifically, we disrupted Snord116 expression in the mediobasal hypothalamus in Snord116fl mice via bilateral stereotaxic injections of a Cre-expressing adeno-associated virus (AAV). While the Cre-injected mice had no change in measured energy expenditure, they became hyperphagic between 9 and 10 weeks after injection, with a subset of animals developing marked obesity. In conclusion, we show that selective disruption of Snord116 expression in the mediobasal hypothalamus models the hyperphagia of PWS.