MYT1L deficiency impairs excitatory neuron trajectory during cortical development [P21]

MYT1L is a neuron-specific transcription factor routinely used in fibroblast-to-neuron transdifferentiation. Furthermore, mutations that reduce MYT1L function are associated with autism. Likewise, MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss, and the cumulative impact of haploinsufficiency on chromatin has been unclear. In this study, we profiled 277,698 nuclei from the forebrains of wild-type and MYT1L-deficient mice at two developmental stages: E14 at the peak of neurogenesis and P21, when neurogenesis is complete. We found that MYT1L deficiency had the strongest impact on deep layer excitatory neurons, specifically disrupting their trajectory of development and preventing arrival at a mature state. We also demonstrated that MYT1L acts primarily, but not exclusively, as a transcriptional repressor in these cell types, and most effects on gene expression are cell-autonomous. Furthermore, we developed and applied single-nucleus combinatorial indexing Calling Cards (sci-CC), an exponentially scalable method to simultaneously record gene expression and longitudinal enhancer activity in single nuclei over time. We find that the disruptions persist throughout neurodevelopment as excitatory neurons were disproportionately affected. These findings illuminate the intricate role of MYT1L in orchestrating gene expression dynamics during neuronal development, providing insights into the molecular underpinnings of MYT1L syndrome. For E21 samples, forebrains from mice were dissected and nuclei were harvested from 3 replicates each of MYT1L WT, Het, and KO mice. Males and females were collected and mixed for each genotype. Libraries were made using the Scale Biosciences Single Cell RNA Kit.MYT1L是一种神经元特异性转录因子（neuron-specific transcription factor），常规应用于成纤维细胞向神经元的转分化（fibroblast-to-neuron transdifferentiation）过程。此外，功能受损的MYT1L突变与自闭症存在显著关联。同样，学界此前曾假设MYT1L在神经元特化轨迹以及亚型特异性成熟过程中发挥作用，但该假说尚未得到直接验证；目前也尚不明确受MYT1L缺失影响最显著的神经元类型，以及单倍剂量不足（haploinsufficiency）对染色质的累积效应。本研究对两个发育阶段的野生型（wild-type）与MYT1L缺陷型小鼠前脑中的277698个细胞核进行了表征分析：分别是神经发生（neurogenesis）高峰期的胚胎期14天（E14），以及神经发生完成后的出生后21天（P21）。研究发现，MYT1L缺陷对深层兴奋性神经元（excitatory neurons）的影响最为显著，具体表现为破坏其发育轨迹，并阻碍其达到成熟状态。本研究还证实，在这类细胞中，MYT1L主要（但并非完全）作为转录抑制因子（transcriptional repressor）发挥作用，且多数基因表达调控效应具有细胞自主性（cell-autonomous）。此外，本研究开发并应用了单细胞核组合索引呼叫卡片技术（single-nucleus combinatorial indexing Calling Cards, sci-CC），这是一种可指数级扩展的实验方法，能够同时记录单个细胞核随时间变化的基因表达与增强子活性的纵向动态。研究发现，这种缺陷效应贯穿整个神经发育过程，兴奋性神经元受到的影响尤为显著。本研究结果阐明了MYT1L在神经元发育过程中调控基因表达动态的复杂作用，为解析MYT1L综合征的分子机制提供了全新见解。针对E21样本，研究人员解剖了小鼠前脑，分别从MYT1L野生型（WT, wild type）、杂合子（Het, heterozygous）与敲除型（KO, knockout）小鼠各3个生物学重复中提取细胞核；每个基因型的样本均混合了雌雄个体。测序文库构建采用Scale Biosciences公司的单细胞RNA试剂盒。