Neocortical temporal patterning by a two-layered regulatory structure

In the developing neocortex, a diverse array of neurons with defined types and abundances are systematically generated by a limited population of radial glial progenitors (RGPs) as they undergo successive fate changes. How this temporal patterning is regulated at the molecular level remains largely unknown. Here we present an in-depth single-cell multi-omics assay for deep characterization of regulatory programs, along with an analytical framework that integrates joint dynamics in single-cell transcription and chromatin structure to interrogate the regulation of temporal identities and fate progression of RGPs. We found that RGP temporal patterning is governed by a two-layered regulatory structure consisting of global regulators acting upon a cascade of transcription factor (TF) hubs. This cascade proceeds not by confining cascaded TF expression to specific temporal windows, but through synergistic transcriptional and epigenetic dynamics that temporally modulate TF regulatory activity. Furthermore, global regulators exhibit a progressively increasing expression pattern, and our computational modeling analysis found that the gradient of this pattern specifies the duration of each cascading stage and, subsequently, the abundance of specific progeny output. Together, this two-layered regulatory structure offers a mechanistic control of temporal patterning and emphasizes the importance of global regulators in managing temporal programs. Hi-C experiment in mouse cortical development (E10-E11, E12-E13, E14-E15, E12-E15). H3K27me3 and H3K27ac ChIP-seq in mouse cortical development (E10, E12). H3K27me3 and H3K27ac CUT&Tag in mouse cortical development (E12-E13, E14-E15).

在发育中的新皮层内，有限群体的放射状胶质祖细胞（radial glial progenitors, RGPs）在经历连续的命运转变时，会系统性地产生种类丰富、类型与丰度均已明确的各类神经元。目前，这种时序模式调控的分子机制仍在很大程度上未被阐明。
本研究开发了一种深度单细胞多组学检测方法，用于对调控程序进行深入表征，并构建了一套整合单细胞转录组与染色质结构联合动态变化的分析框架，以此探究放射状胶质祖细胞的时序身份调控与命运进展机制。
本研究发现，放射状胶质祖细胞的时序模式调控由双层调控结构介导：全局调控因子作用于一系列转录因子（transcription factor, TF）枢纽模块。该调控级联并非通过将级联转录因子的表达限定于特定时间窗口来实现，而是通过协同的转录与表观遗传动态变化，对转录因子的调控活性进行时间维度上的精准调控。
此外，全局调控因子的表达呈现逐步升高的模式；本研究的计算建模分析显示，该表达梯度决定了每个级联阶段的持续时长，进而调控特定子代细胞的产生丰度。综上，这种双层调控结构为时序模式调控提供了机制层面的解释，并凸显了全局调控因子在调控时序程序中的重要作用。
小鼠皮层发育各阶段（E10-E11、E12-E13、E14-E15、E12-E15）的Hi-C实验；小鼠皮层发育阶段（E10、E12）的H3K27me3与H3K27ac染色质免疫共沉淀测序（Chromatin Immunoprecipitation sequencing, ChIP-seq）实验；小鼠皮层发育阶段（E12-E13、E14-E15）的H3K27me3与H3K27ac CUT&Tag（Cleavage Under Targets and Tagmentation）实验。