Repurposing of the multiciliation gene regulatory network in fate specification of Cajal-Retzius neurons

Cajal-Retzius (CR) neurons are key players of cortical development that display a very unique transcriptomic identity. However, little is known about the mechanisms involved in their fate specification. Here we use scRNAseq to reconstruct the differentiation trajectory of hem-derived CR cells (CRs) and unravel the transient expression of a complete gene module previously known to control the cellular process of multiciliogenesis. However, we find that CRs do not undergo centriole amplification or multiciliation. We show that upon genetic disruption of Gmnc, the master regulator of the multiciliation cascade, CRs are initially produced but fail to reach their normal identity and lean towards an aberrant fate resulting in their massive apoptosis. We further dissect the contribution of multiciliation effector genes and identify Trp73 as a key determinant. Finally, we use in utero electroporation to demonstrate that the intrinsic competence of hem progenitors as well as the heterochronic expression of Gmnc prevent centriole amplification in the CR lineage. Our work exemplifies how the co-option of a complete gene module, repurposed to control a completely distinct process, may contribute to the emergence of novel cell identities. The hem region of four E11.5 PGKCre;Rosa26YFP and four E12.5 Wnt3aCre;Rosa26tdTomato mouse embryos were dissected and subjected to scRNAseq. Two libraries were generated from the initial single cell suspension. A third library was obtained from four E12.5 Gmnc-/- embryosCajal-Retzius (CR)神经元是皮层发育的关键参与者，具有极为独特的转录组特征，但目前对其命运特化的分子机制仍知之甚少。本研究利用单细胞RNA测序（scRNAseq）重构了脑半球来源的CR细胞（CRs）的分化轨迹，并揭示了一个此前被证实调控多纤毛发生（multiciliogenesis）过程的完整基因模块的瞬时表达。然而本研究发现，CR细胞并未发生中心粒扩增或多纤毛化。我们证实，当多纤毛发生级联反应的主调控因子Gmnc发生遗传破坏后，CR细胞虽可正常产生，但无法获得正常的细胞身份，反而倾向于异常分化命运，最终导致其大量凋亡。我们进一步解析了多纤毛发生效应基因的功能贡献，并鉴定出Trp73作为关键调控因子。最后，我们利用子宫内电穿孔（in utero electroporation）技术证实，脑半球祖细胞的内在感受性以及Gmnc的异时表达，共同阻止了CR细胞谱系中的中心粒扩增。本研究阐释了完整基因模块的共选择与功能重编程，如何通过调控完全迥异的细胞过程，助力新型细胞身份的形成。我们对4只E11.5龄PGKCre;Rosa26YFP小鼠胚胎及4只E12.5龄Wnt3aCre;Rosa26tdTomato小鼠胚胎的脑半球区域进行解剖，并开展scRNAseq测序；从初始单细胞悬液中构建了2个测序文库，另有1个测序文库来自4只E12.5龄Gmnc敲除（Gmnc-/-）小鼠胚胎。