A Blueprint of Sex-Specific Neuronal Regulation in the C. elegans Nervous System at Single-Cell Resolution

Sex-specific behaviors within a species are often attributed to variances in neuronal wiring and molecular signatures. However, how the genetic sex shapes the molecular architecture of the nervous system at a single neuron level is still unknown. To address this gap, we used single-cell RNA-sequencing (scRNA-seq) to profile the transcriptome of the sex-shared nervous system of adult male and hermaphrodite Caenorhabditis elegans. By ranking neurons based on the degree of molecular dimorphism, we uncover novel sexually dimorphic neurons such as PLM neurons, where functional dimorphism was corroborated by diminished posterior touch responses in hermaphrodites. We identify sex-specific regulators of mechanosensory behavior and neuronal function by combining our dataset with reverse genetic screens. While most sex-shared neurons retain their neurotransmitter identity, the male neuropeptide connectome undergoes substantial remodeling, with most of the neuropeptides showing a male-bias expression. This reinforces the notion that neuropeptides are crucial for diversifying connectome outputs. Furthermore, by correlating gene expression with outgoing synaptic connectivity, we identified regulatory candidates for synaptic wiring, including both shared and sex-specific genes. This comprehensive resource provides foundational insights into the molecular drivers of sexual dimorphism, facilitating future exploration of regulatory mechanisms and their impact on sex-specific behaviors in higher organisms. Overall design: Single-cell RNA-sequencing profiles were generated from young adult C. elegans larvae of both sexes (hermaphrodites and males), utilizing strains with pan-neuronal labeling. Neurons were isolated using fluorescence-activated cell sorting (FACS).

物种内的性别特异性行为通常被归因于神经元连接模式与分子特征的差异。然而，遗传性别如何在单个神经元层面塑造神经系统的分子架构，目前仍不明朗。为填补这一研究空白，我们利用单细胞RNA测序（single-cell RNA-sequencing, scRNA-seq）技术，对成年雄性与雌雄同体秀丽隐杆线虫（Caenorhabditis elegans）的性别共享神经系统的转录组进行了图谱绘制。通过基于分子二态性程度对神经元进行排序，我们鉴定出了包括PLM神经元在内的新型性别二态性神经元；该神经元的功能二态性已通过雌雄同体个体的后触觉反应减弱得到佐证。我们将本数据集与反向遗传筛选相结合，成功识别出调控机械感觉行为与神经元功能的性别特异性因子。尽管多数性别共享神经元保留了其固有神经递质表型，但雄性个体的神经肽连接组发生了显著重塑，绝大多数神经肽呈现雄性偏向性表达。这一发现进一步印证了神经肽对于多样化连接组输出具有关键作用的论断。此外，通过将基因表达水平与神经元传出突触连接进行关联分析，我们鉴定出了一批调控突触连接形成的候选基因，其中既包含共享基因，也涵盖性别特异性基因。本综合性研究资源为性别二态性的分子驱动机制提供了基础性认知，将助力未来探索高等生物体内的调控通路及其对性别特异性行为的影响。实验设计概述：我们采用携带全神经元标记的秀丽隐杆线虫品系，分别获取了雌雄两性（雄性与雌雄同体）年轻成年个体的单细胞RNA测序转录组图谱。神经元通过荧光激活细胞分选（fluorescence-activated cell sorting, FACS）完成分离。