Comparative transcriptomic analyses of developing melanocortin neurons reveal new regulators for the anorexigenic neuron identity

Despite their opposing actions on food intake, POMC and NPY/AgRP neurons in the arcuate nucleus of the hypothalamus (ARH) are derived from the same progenitors that give rise to ARH neurons. However, the mechanism whereby common neuronal precursors subsequently adopt either the anorexigenic (POMC) or the orexigenic (NPY/AgRP) identity remains elusive.We hypothesize that POMC and NPY/AgRP cell fates are specified and maintained by distinct intrinsic factors. In search of them, we profiled the transcriptomes of developing POMC and NPY/AgRP neurons in E15.5 mice embryos. Moreover, cell-type-specific transcriptomic analyses revealed transcription regulators that are selectively enriched in either population, but whose developmental functions are unknown in these neurons.Among them, we found the expression of the PR domain-containing factor 12 (Prdm12) was enriched in POMC neurons but absent in NPY/AgRP neurons. To study the role of Prdm12 in vivo, we developed and characterized a floxed Prdm12 allele. Selective ablation of Prdm12 in embryonic POMC neurons led to significantly reduced Pomc expression as well as early-onset obesity in mice of either sex that recapitulates symptoms of human POMC deficiency. Interestingly, however, specific deletion of Prdm12 in adult POMC neurons showed that it is no longer required for Pomc expression nor energy balance. Collectively, these findings establish a critical role for Prdm12 in the anorexigenic neuron identity and suggest that it acts developmentally to program body weight homeostasis. Finally, the combination of cell-type-specific genomic and genetic analyses provides a means to dissect cellular and functional diversity in the hypothalamus whose neurodevelopment remains poorly studied. Transcriptomic profiling of purified (Fluorescence activated cell sorting) hypothalamic E15.5 Pomc and NPY neurons using an Illumina HiSeq 2500.尽管下丘脑弓状核（arcuate nucleus of the hypothalamus, ARH）中的POMC神经元与NPY/AgRP神经元对食物摄取的调控作用截然相反，但二者均起源于产生ARH神经元的同一群神经祖细胞。然而，这类共同的神经前体细胞后续如何分化为厌食性（POMC）或促食性（NPY/AgRP）神经元的具体分子机制仍未阐明。我们提出假说，认为POMC与NPY/AgRP神经元的细胞命运由不同的内在因子决定并维持。为筛选这类关键因子，我们对胚胎期15.5天小鼠胚胎中发育中的POMC及NPY/AgRP神经元开展了转录组测序分析。此外，通过细胞类型特异性转录组分析，我们鉴定出了在两类神经元群体中选择性富集的转录调控因子，但目前尚未明确这些因子在这类神经元中的发育功能。其中，我们发现含PR结构域因子12（PR domain-containing factor 12, Prdm12）的表达在POMC神经元中显著富集，而在NPY/AgRP神经元中完全缺失。为在体内研究Prdm12的生物学功能，我们构建并鉴定了Prdm12的floxed等位基因。在胚胎期的POMC神经元中选择性敲除Prdm12，会导致雌雄小鼠均出现Pomc表达水平显著降低以及早发性肥胖，该表型重现了人类POMC缺乏症的临床症状。有趣的是，在成年POMC神经元中特异性敲除Prdm12则显示，此时Prdm12不再是维持Pomc表达及机体能量平衡所必需的因子。综上，本研究结果确立了Prdm12在厌食性神经元身份确立中的关键作用，并提示其在发育阶段通过调控体重稳态发挥功能。最后，细胞类型特异性基因组与遗传分析的结合，为解析下丘脑的细胞与功能多样性提供了全新的研究策略——目前下丘脑的神经发育过程仍鲜有深入研究。本研究通过荧光激活细胞分选（Fluorescence activated cell sorting, FACS）纯化下丘脑胚胎期15.5天的Pomc与NPY神经元，并使用Illumina HiSeq 2500测序平台完成了转录组测序分析。