The spinal cord is a crucial component of the central nervous system that facilitates sensory processing and motor performance. Despite its importance, the spatiotemporal codes underlying human spinal cord development have remained elusive. In this study, we have introduced an image-based single-cell transcription factor (TF) expression decoding spatial transcriptome method (TF-seqFISH) to investigate the spatial expression and regulation of TFs during human spinal cord development. By combining spatial transcriptomic data from TF-seqFISH and single-cell RNA-sequencing data, we uncovered the spatial distribution of neural progenitor cells characterized by combinatorial TFs along the dorsoventral axis, as well as the molecular and spatial features governing neuronal generation, migration, and differentiation along the mediolateral axis. Notably, we observed a sandwich-like organization of excitatory and inhibitory interneurons transiently appearing in the dorsal horns of the developing human spinal cord. In addition, we integrated data from 10× Visium to identify early and late waves of neurogenesis in the dorsal horn, revealing the formation of laminas in the dorsal horns. Our study also illuminated the spatial differences and molecular cues underlying motor neuron (MN) diversification, and the enrichment of Amyotrophic Lateral Sclerosis (ALS) risk genes in MNs and microglia. Interestingly, we detected disease-associated microglia (DAM)-like microglia groups in the developing human spinal cord, which are predicted to be vulnerable to ALS and engaged in the TYROBP causal network and response to unfolded proteins. These findings provide spatiotemporal transcriptomic resources on the developing human spinal cord and potential strategies for spinal cord injury repair and ALS treatment.

脊髓是中枢神经系统的重要组成部分，有助于感觉处理和运动表现。尽管它很重要，但人类脊髓发育的时空密码仍然难以捉摸。在本研究中，我们引入了一种基于图像的单细胞转录因子（TF）表达解码空间转录组方法（TF-seqFISH）来研究人类脊髓发育过程中TF的空间表达和调控。通过结合 TF-seqFISH 的空间转录组数据和单细胞 RNA 测序数据，我们揭示了以沿背腹轴的组合 TF 为特征的神经祖细胞的空间分布，以及控制神经元生成、迁移、以及沿中外侧轴的分化。值得注意的是，我们观察到兴奋性和抑制性中间神经元的三明治状组织短暂地出现在发育中的人类脊髓的背角中。此外，我们整合了 10× Visium 的数据来识别背角神经发生的早期和晚期波，揭示了背角椎板的形成。我们的研究还阐明了运动神经元（MN）多样化背后的空间差异和分子线索，以及运动神经元和小胶质细胞中肌萎缩侧索硬化症（ALS）风险基因的富集。有趣的是，我们在发育中的人类脊髓中检测到了与疾病相关的小胶质细胞（DAM）样小胶质细胞群，预计它们容易受到 ALS 的影响，并参与 TYROBP 因果网络和对未折叠蛋白的反应。这些发现为发育中的人类脊髓提供了时空转录组资源以及脊髓损伤修复和 ALS 治疗的潜在策略。