Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids [ATAC-Seq]. Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids [ATAC-Seq]

Correct gene expression levels in space and time are crucial for normal development. Advances in genomics enable the inference of gene regulatory programs that are active during development. However, this approach cannot capture the complex multicellular interactions that occur in embryogenesis. Compared to model organisms such as fruit flies and zebrafish, the growth of mammalian embryos in utero further complicates the analysis of cell-cell communication during development. However, in vitro models of mammalian development such as gastruloids allow to overcome this limitation. Using time-resolved single-cell chromatin accessibility analysis, we have delineated the regulatory landscape during gastruloid development and thereby identified the critical drivers of developmental transitions. We observed that gastruloids develop from pluripotent cells driven by the transcription factor (TF) dimer OCT4-SOX2 and differentiate along two main branches. A mesoderm branch driven by the TF MSGN1 and a spinal cord branch driven by CDX1, 2, 4 (CDX). Consistent with our lineage reconstruction, ΔCDX gastruloids fail to form spinal cord. Conversely, Msgn1 ablation inhibits the development of paraxial mesoderm, as expected. However, this also abolished spinal cord cells, which is surprising given that MSGN1 is not associated with differentiation along this branch. Therefore, formation of paraxial mesoderm is required for spinal cord development. To validate this, we generated chimeric gastruloids using ΔMSGN1 and wildtype cells, which formed both spinal cord and paraxial mesoderm. Strikingly, ΔMsgn1 cells specifically contributed to spinal cord, suggesting that cell-cell interactions between paraxial mesoderm and spinal cord are necessary for the formation the latter. Our work has important implications for the study of cell-cell communication in development and how the bridge can be made between gene regulatory programs and complex multicellular developmental structures. Overall design: Analysis of accessible chromatin in wildtype mouse embryonic stem cells.

基因在时空维度上的精准表达对于个体正常发育不可或缺。基因组学技术的迭代进步，使得研究者能够推断发育进程中活跃的基因调控程序。但该类方法无法捕捉胚胎发生过程中存在的复杂多细胞互作网络。相较于果蝇、斑马鱼等经典模式生物，哺乳动物胚胎在子宫内的发育过程，进一步增加了发育阶段细胞间通讯分析的复杂度。而类原肠胚(gastruloids)等哺乳动物发育体外模型，则可有效突破这一研究局限。

本研究借助时间分辨率单细胞染色质开放性分析技术，绘制了类原肠胚发育全过程的调控图谱，并据此鉴定出发育转变过程中的关键调控因子。研究发现，类原肠胚由转录因子(TF)二聚体OCT4-SOX2调控的多能干细胞分化而来，并沿两大主要分支进行发育：其一为受转录因子MSGN1调控的中胚层分支，其二为受CDX1、2、4（统称CDX家族）调控的脊髓分化分支。

与本研究的谱系重构结果一致，CDX基因敲除（ΔCDX）类原肠胚无法形成脊髓组织。反之，MSGN1基因敲除会如预期般抑制轴旁中胚层的发育。但该操作同时也意外地消除了脊髓细胞——而MSGN1通常并不参与该谱系的分化调控。由此可见，轴旁中胚层的形成是脊髓发育的必要前提。

为验证这一结论，本研究利用MSGN1敲除（ΔMSGN1）细胞与野生型细胞构建了嵌合类原肠胚，此类嵌合体可同时形成脊髓与轴旁中胚层。值得注意的是，MSGN1敲除细胞仅特异性地整合至脊髓组织中，这表明轴旁中胚层与脊髓之间的细胞互作是脊髓形成的必要条件。

本研究对于发育过程中细胞间通讯的相关研究，以及如何搭建基因调控程序与复杂多细胞发育结构之间的研究桥梁，均具有重要的参考意义。

**实验设计概述**：针对野生型小鼠胚胎干细胞的开放性染色质进行分析。