RNA-seq analysis of differences in gene expression between dorsal and ventral MEC

Neural circuits in the medial entorhinal cortex (MEC) encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations. Examination of dorsal and ventral regions from 4 replicate samples each containing pooled data from 3-4 mice内侧内嗅皮层（medial entorhinal cortex, MEC）中的神经环路可编码动物在空间中的位置与朝向。在内侧内嗅皮层内，包括网格放电野与方向放电野在内的空间表征具有分层与背腹侧组织模式，这与神经元连接特性及细胞属性的相似拓扑分布相对应。然而，部分由于难以整合皮层分层及边界分辨率下的解剖学数据，我们对该组织模式背后的分子机制仍知之甚少。为解决这一问题，我们开发了一套全新的计算流程，用于分层分辨率下的高通量分析与比较原位杂交（in situ hybridization, ISH）图像。我们将该流程应用于艾伦脑图谱（Allen Brain Atlas）中超过16000个基因的原位杂交数据，并通过成年小鼠内侧内嗅皮层组织的RNA测序验证了分析结果。研究发现，差异基因表达可勾勒出内侧内嗅皮层与邻近脑区的边界，并揭示其分层及背腹侧组织模式。我们的分析筛选出与离子通道、细胞黏附及突触相关的基因，作为内侧内嗅皮层分层功能分化，以及沿其背腹轴以不同尺度编码空间信息的候选靶点。研究结果支持这一假说：基因表达差异促成了内侧内嗅皮层表层的功能特化，以及空间表征尺度的背腹侧组织模式。本研究对4份重复样本的背侧与腹侧脑区进行了检测，每份样本均整合了3-4只小鼠的合并实验数据。