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Objective: Proopiomelanocortin (POMC) neurons release potent anorexigenic neuropeptides, which suppress food intake and enhance energy expenditure via melanocortin receptors. Although the importance of central melanocortin in physiological regulation is well established, the underlying genetic mechanisms that define the functional identity of melanocortin neurons and maintain hypothalamic Pomc expression remain to be fully determined. In this study, we investigate the functional significance of Six3, a transcriptional regulator notably expressed in embryonic and adult mouse POMC neurons, in the regulation of hypothalamic Pomc expression and downstream physiological consequences., Methods: We first evaluated the expression of Six3 in the developing and adult hypothalamus by double fluorescence in situ hybridization. Next, we assessed POMC immunoreactivity in mutant mice selectively lacking Six3 from Pomc-expressing neurons and quantified Pomc mRNA levels in a tamoxifen-inducible Six3 knockout mouse model activated at embryonic E9.5 days. We also determined glucose and insulin sensitivity, daily food intake, body composition and body weight in adult male and female mice lacking Six3 specifically from POMC neurons. Lastly, we assessed the physiological consequences of ablating Six3 from POMC neurons in adult mice., Results: Six3 and Pomc were co-expressed in mouse hypothalamic neurons during development and adulthood. Mouse embryos deficient in Six3 showed reduced Pomc expression in the developing hypothalamus. Targeted deletion of Six3 specifically from POMC neurons resulted in decreased hypothalamic Pomc expression, increased daily food intake, enhanced glucose sensitivity and mild obesity in male but not in female mice. Finally, conditional removal of Six3 from POMC neurons in adult mice led to a reduction in hypothalamic POMC immunoreactivity with no significant effects in body weight or food intake., Conclusions: Altogether, our results demonstrate that Six3 plays an essential role in the early establishment of POMC neuron identity and the maintenance of physiological levels of hypothalamic Pomc expression. In addition, our study demonstrates that the functional significance of Six3 expression in POMC neurons is sexually dimorphic and age-dependent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Food intake and energy balance are tightly regulated by a group of hypothalamic arcuate neurons expressing the proopiomelanocortin ( POMC) gene. In mammals, arcuate-specific POMC expression is driven by two cis -acting transcriptional enhancers known as nPE1 and nPE2. Because mutant mice lacking these two enhancers still showed hypothalamic Pomc mRNA, we searched for additional elements contributing to arcuate Pomc expression. By combining molecular evolution with reporter gene expression in transgenic zebrafish and mice, here, we identified a mammalian arcuate-specific Pomc enhancer that we named nPE3, carrying several binding sites also present in nPE1 and nPE2 for transcription factors known to activate neuronal Pomc expression, such as ISL1, NKX2.1, and ERα. We found that nPE3 originated in the lineage leading to placental mammals and remained under purifying selection in all mammalian orders, although it was lost in Simiiformes (monkeys, apes, and humans) following a unique segmental deletion event. Interestingly, ablation of nPE3 from the mouse genome led to a drastic reduction (>70%) in hypothalamic Pomc mRNA during development and only moderate (<33%) in adult mice. Comparison between double (nPE1 and nPE2) and triple (nPE1, nPE2, and nPE3) enhancer mutants revealed the relative contribution of nPE3 to hypothalamic Pomc expression and its importance in the control of food intake and adiposity in male and female mice. Altogether, these results demonstrate that nPE3 integrates a tripartite cluster of partially redundant enhancers that originated upon a triple convergent evolutionary process in mammals and that is critical for hypothalamic Pomc expression and body weight homeostasis., Competing Interests: Competing interests statement:The authors declare no competing interest.