Neural tube (NT) defects arise from abnormal neurulation and result in the most common birth defects worldwide. Yet, mechanisms of primate neurulation remain largely unknown due to prohibitions on human embryo research and limitations of available model systems. Here, we establish a three-dimensional (3D) prolonged in vitro culture (pIVC) system supporting cynomolgus monkey embryo development from 7 to 25 days post-fertilization. Through single-cell multi-omics analyses, we demonstrate that pIVC embryos form three germ layers, including primordial germ cells, and establish proper DNA methylation and chromatin accessibility through advanced gastrulation stages. In addition, pIVC embryo immunofluorescence confirms neural crest formation, NT closure, and neural progenitor regionalization. Finally, we demonstrate that the transcriptional profiles and morphogenetics of pIVC embryos resemble key features of similarly staged in vivo cynomolgus and human embryos. This work therefore describes a system to study non-human primate embryogenesis through advanced gastrulation and early neurulation.

神经管 (NT) 缺陷起因于神经发育异常，并导致全世界最常见的出生缺陷。然而，由于禁止人类胚胎研究和可用模型系统的限制，灵长类动物的神经形成机制在很大程度上仍然未知。在这里，我们建立了一个体外延长的三维 (3D)支持食蟹猴胚胎在受精后 7 至 25 天发育的培养 (pIVC) 系统。通过单细胞多组学分析，我们证明 pIVC 胚胎形成三个胚层，包括原始生殖细胞，并通过高级原肠胚形成阶段建立适当的 DNA 甲基化和染色质可及性。此外，pIVC 胚胎免疫荧光证实了神经嵴形成、NT 闭合和神经祖细胞区域化。最后，我们证明 pIVC 胚胎的转录谱和形态遗传学类似于体内阶段相似的食蟹猴和人类胚胎的关键特征。因此，这项工作描述了一个通过高级原肠胚形成和早期神经形成研究非人类灵长类动物胚胎发生的系统。