Data CitationsShuguang Yu, Jie He. (250K) DOI:?10.7554/eLife.48660.029 Data Availability StatementData has been deposited in Dryad (https://doi.org/10.5061/dryad.31t3425). The following dataset was generated: Shuguang Yu, Jie He. 2019. Data from: Stochastic cell-cycle entry and cell-state-dependent fate outputs of injury-reactivated tectal radial glia in zebrafish. Dryad Digital Repository. [CrossRef] Abstract Gliosis defined as reactive changes of resident glia is the primary response of the central nervous system (CNS) to trauma. The proliferation and fate controls of injury-reactivated glia are essential but remain largely unexplored. In zebrafish optic tectum, we found that stab injury drove a subset of radial ABH2 glia (RG) into the cell cycle, and surprisingly, proliferative RG responding to sequential injuries of the same site were distinct but overlapping, which was in agreement with stochastic cell-cycle entry. Single-cell RNA sequencing analysis and functional assays further revealed the involvement of Notch/Delta lateral inhibition in this stochastic cell-cycle entry. Furthermore, the long-term clonal analysis showed that proliferative RG were largely gliogenic. Notch inhibition of reactive RG, not dormant and proliferative RG, resulted in an increased production of neurons, which were short-lived. Our findings gain new insights into the proliferation and fate controls of injury-reactivated CNS glia in zebrafish. promoter. In Tg(drives the expression of the mCherry fluorescent protein and CreERT2 recombinase in tectal RG Cyclo (-RGDfK) (Figure 1H). By crossing this line with Tg((and and (in the tectal RG (Figure 3E). Thus, we excluded cluster 5 cells from further analysis. Cell cycle phases analysis (Figure 3J) and pseudo-time analysis (Figure 3K and Figure 3figure supplement 2J) were performed and suggested the temporal order of 4 remaining cell clusters, thereafter termed as the state of dormant RG (dRG), the state of reactive RG (reactive RG), the state of proliferative-S RG and the state of proliferative-G2 RG. Open in a separate window Figure 3. Single-cell RNAseq revealing cellular states underlying the cell-cycle entry of reactive RG.(A) Workflow for single-cell RNA-seq (scRNA-seq) of tectal RG after stab injury. Optic tecta are dissected from 3 dpi Tg((OCP1), (QCR1) and (QCR1) in the optic tecta after injury. The white arrowheads shown in (O and O1) indicate PCNA+ proliferative RG are (Q and Q1) or (S and T1) mRNA signals are located in processes of proliferative RG. White dashed lines represent the tectal Cyclo (-RGDfK) ventricle boundary. t-SNE, t-stochastic neighbor embedding; RG, radial glia; PGZ, periventricular gray zone, TS, torus semicircularis. Scale bars, 30 m. See also Figure 3figure supplements 1 and Cyclo (-RGDfK) ?and22 and Materials and methods. Figure 3figure supplement 1. Open in a separate window Glial and Non-glial cell clusters identification from the scRNA-seq data.(ACA2) Tg(mRNA is highly Cyclo (-RGDfK) expressed in RG from TPZ (open white arrows in (D)) and dormant RG (open white arrowheads in (E and F)) in central-dorsal region of optic tectum, whereas its expression is down-regulated in RG underneath the injury site ((F), white arrow). White dashed lines represent the tectal ventricle boundary. (GCI1) Representative images of Tg(was abundant in dormant RG (cluster 1), began to decrease in reactive RG (cluster 2) and became rapidly diminished in proliferative RG (cluster 3 and 4) (Figure 3L). Kruppel-like transcription factor 6a (was down-regulated in injured-induced PCNA+ proliferative RG at 3 dpi (Figure 3OCP1), whereas and mRNA expression increased in the 2-dpi (Figure 3QCR1) and 3-dpi (Figure 3SCT1) optic tecta, respectively. Interestingly, the signals of (Figure 3Q and Q1) and (Figure 3S and S1) were mainly distributed in the processes of RG. Notch/Delta expression pattern correlated with the cell-cycle entry of reactive RG Notably, during the transition of reactive (cluster 2) and proliferative states (cluster 3 and 4), the expression.