Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids [scATAC-seq]. Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids [scATAC-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 during a timeseries of gastruloid development using wildtype and genetic knockouts of CDX1/2/4 and MSGN1.

基因在时空维度上的精准表达水平，对于个体正常发育至关重要。基因组学领域的进展，使得我们能够推断发育过程中活跃的基因调控程序。然而，该方法无法捕捉胚胎发生过程中发生的复杂多细胞相互作用。与果蝇、斑马鱼等模式生物相比，哺乳动物胚胎在子宫内的发育过程，进一步复杂化了发育过程中细胞间通信的分析工作。不过，类原肠胚（gastruloid）等哺乳动物发育体外模型，能够克服这一局限。本研究借助时间分辨率单细胞染色质可及性分析，刻画了类原肠胚发育过程中的调控图谱，并由此确定了发育转变的关键驱动因子。我们观察到，类原肠胚由以转录因子（TF）二聚体OCT4-SOX2为驱动的多能细胞发育而来，并沿两个主要分支进行分化：一个为受转录因子MSGN1调控的中胚层分支，另一个为受CDX1、2、4（CDX）调控的脊髓分支。与我们的谱系重建结果一致，ΔCDX类原肠胚无法形成脊髓组织。反之，正如预期那般，敲除Msgn1会抑制轴旁中胚层的发育。但该操作同时也消除了脊髓细胞，这一结果令人意外——因为MSGN1并未被报道与该分支的分化相关。由此可见，轴旁中胚层的形成是脊髓发育的必要条件。为验证这一结论，我们利用ΔMSGN1细胞与野生型细胞构建了嵌合类原肠胚，这类嵌合体可同时形成脊髓与轴旁中胚层。值得注意的是，ΔMsgn1细胞特异性地参与了脊髓的形成，这表明轴旁中胚层与脊髓之间的细胞间通信，是脊髓形成不可或缺的条件。本研究对于发育过程中细胞间通信的研究，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桥梁，均具有重要的启示意义。实验整体设计：针对野生型及CDX1/2/4、MSGN1基因敲除的类原肠胚，在其发育的时间序列中开展染色质可及性分析。