Multi-omics analyses reveal DjTcf4 critical for proper timing of differentiation in planarian regeneration [scRNA_head]

The blastema is key to forming complete tissues in regenerating Dugesia japonica (D. japonica). However, the dynamic changes in cellular compositions and transcription landscapes in blastema during regeneration are understudied. Here, through genome reannotation, 3D spatial transcriptome construction, scRNA-seq and scATAC-seq analyses of changes in gene expression and chromatin structures, we delineated key transcription factors regulating the developmental trajectories of major cell clusters in the regenerating head. Importantly, we found that the T-cell factor 4 positive (DjTcf4+) cells highly accumulated at wound areas, and its gene network is critical for proper timing of development during regeneration in multiple progenitor cells. Depletion of DjTcf4 and its target genes led to singular eye and/or dull tail phenotypes and delayed regeneration. Taken together, we built multi-omics atlases in D. japonica and revealed noncanonical function of the DjTcf4 network in developmental pattern formation, laying a foundation for studies of regeneration in D. japonica. Examine single cell gene expression profiles of intact and 5 dpa animals by 10X scRNA-Seq platform. The experiment was performed in 2 samples.

芽基（blastema）是日本三角涡虫（Dugesia japonica）再生过程中形成完整组织的关键结构。然而，目前对再生过程中芽基内细胞组成与转录调控图谱的动态变化研究仍较为匮乏。本研究通过基因组重新注释、三维空间转录组构建、单细胞RNA测序（scRNA-seq）及单细胞转座酶可及性测序（scATAC-seq）分析基因表达与染色质结构的动态变化，阐明了调控再生头部主要细胞类群发育轨迹的关键转录因子。值得注意的是，本研究发现T细胞因子4阳性（DjTcf4+）细胞大量聚集于伤口区域，且其基因调控网络对多种祖细胞再生过程中的正常发育时序至关重要。敲低DjTcf4及其靶基因会导致单眼和/或尾部色泽暗淡的表型，并延缓再生进程。综上，本研究构建了日本三角涡虫的多组学图谱，揭示了DjTcf4调控网络在发育模式形成中的非经典功能，为日本三角涡虫的再生研究奠定了基础。本研究利用10X单细胞RNA测序平台检测了完整个体及截肢后5天（5 dpa）动物的单细胞基因表达谱，实验共设置2个生物学重复样本。