Single-cell genomics of the mouse olfactory cortex reveals contrasts with neocortex and ancestral signatures of cell type evolution

Understanding the molecular logic of cortical cell-type diversity can illuminate cortical circuit function and evolution. Here, we performed single-nucleus transcriptome and chromatin accessibility analyses to compare neurons across three- to six-layered cortical areas of adult mice and across tetrapod species. We found that, in contrast to the six-layered neocortex, glutamatergic neurons of the three-layered mouse olfactory (piriform) cortex displayed continuous rather than discrete variation in transcriptomic profiles. Subsets of piriform and neocortical glutamatergic cells with conserved transcriptomic profiles were distinguished by distinct, area-specific epigenetic states. Furthermore, we identified a prominent population of immature neurons in piriform cortex and observed that, in contrast to the neocortex, piriform cortex exhibited divergence between glutamatergic cells in lab versus wild-derived mice. Finally, we showed that piriform neurons displayed greater transcriptomic similarity to cortical neurons of turtles, lizards, and salamanders than to those of the neocortex. In summary, despite over 200 million years of co-evolution alongside the neocortex, olfactory cortex neurons retain molecular signatures of ancestral cortical identity. Nuclei from micro-dissected cortical areas were isolated in C57Bl/6 mice and wild-derived mice, and 10x multiome or 10x RNA sequencing 3.1 version (only for some wild-derived mice replicates) were performed. Areas collected were: A = anterior piriform cortex P = posterior piriform cortex T = agranular insular cortex N = primary somatosensory cortex W = data from wild-derived mice 1, 2, 3... = biological replicate ID解析大脑皮层细胞类型多样性的分子逻辑，可为皮层环路功能与演化研究提供关键洞见。本研究通过单细胞核转录组（single-nucleus transcriptome）与染色质可及性分析（chromatin accessibility analysis），对成年小鼠3至6层皮层脑区的神经元以及四足类（tetrapod）物种的神经元开展对比研究。研究发现，与6层新皮层（neocortex）不同，3层小鼠嗅觉（梨状皮层，piriform cortex）的谷氨酸能神经元（glutamatergic neurons）的转录组特征呈现连续变异，而非离散分化。具有保守转录组特征的梨状皮层与新皮层谷氨酸能神经元亚群，可通过独特的区域特异性表观遗传状态加以区分。此外，本研究在梨状皮层中鉴定出一类显著的未成熟神经元群体；同时观察到，与新皮层不同，梨状皮层的谷氨酸能神经元在实验室饲养小鼠与野生来源小鼠（wild-derived mice）间存在显著分化。最后，研究表明，相较于新皮层神经元，梨状皮层神经元与龟、蜥蜴、蝾螈的皮层神经元转录组相似性更高。综上，尽管与新皮层共同演化超过2亿年，嗅觉皮层神经元仍保留了祖先皮层身份的分子特征。本研究从C57Bl/6小鼠与野生来源小鼠的显微解剖皮层脑区中分离细胞核，并开展10x multiome或10x RNA sequencing 3.1版本（仅部分野生来源小鼠重复样本采用该方案）。采集的脑区如下：A = 前梨状皮层；P = 后梨状皮层；T = 无颗粒岛叶皮层；N = 初级躯体感觉皮层；W = 野生来源小鼠数据；1, 2, 3… = 生物学重复编号（biological replicate ID）。