Stochastic cell-cycle entry and cell-state-dependent fate outputs of injury-reactivated tectal radial glia in zebrafish

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 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.

Single cell sequencing of radial glia progeny reveals the diversity of newborn neurons in the adult zebrafish brain

“…5A,B). Similarly, recent sc-RNAseq studies in the adult zebrafish brain with higher cell throughput, only identified cells with transcriptomic profiles of radial glia as proliferating neurogenic precursors (Cosacak et al, 2019;Yu and He, 2019). Our findingsthat a subset of zebrafish RG corresponds to intermediate neurogenic progenitors, the major proliferating and amplifying population from the postnatal and adult murine hippocampus, while a NBN.1 subset corresponds to neuroblast, their direct progenysupport this view.…”

Transcriptome Analyses Reveal IL6/Stat3 Signaling Involvement in Radial Glia Proliferation After Stab Wound Injury in the Adult Zebrafish Optic Tectum

“…To examine macrophage responses after injury, we performed immunostaining using an IB4 antibody conjugated with Dylight 594 (Lai et al, 2017) in the intact and injured optic tectum at 6, 12, and 24 hpi (Figures 2A-D and Supplementary Figure 2). Few IB4-positive cells were observed in the intact optic tectum (Figures 2A,E), suggesting that IB4-positive cells were not microglia as there were fewer of these cells in the intact optic tectum than GFP-positive cells in Tg(mpeg1:GFP) (Lindsey et al, 2019;Yu and He, 2019). Interestingly, we found that there were significantly more IB4-positive cells in the injured hemisphere than in the uninjured hemisphere at 6, 12, and 24 hpi.…”

“…At 6 hpi, the number of proliferative RG cells in the two hemispheres of the optic tectum did not significantly differ (Figure 1E); however, significant RG proliferation was observed on the injured side at 12 hpi in comparison to the contralateral uninjured hemisphere. Furthermore, the number of BLBP-PCNA+ cells, which likely include immune cells such as microglia and macrophages (Lindsey et al, 2019;Yu and He, 2019), also significantly increased from 12 to 24 hpi (Figure 1F). To examine macrophage responses after injury, we performed immunostaining using an IB4 antibody conjugated with Dylight 594 (Lai et al, 2017) in the intact and injured optic tectum at 6, 12, and 24 hpi (Figures 2A-D and Supplementary Figure 2).…”

“…Previously, we found that RG proliferation was induced by stab wound injury in the adult optic tectum (Shimizu et al, 2018) while other studies have shown that immune responses during the early stages after stab wound injury are required for RG proliferation in the adult telencephalon and optic tectum (Kyritsis et al, 2012;Ueda et al, 2018). Although molecular pathways that work in RG after the tectum injury have been well investigated by pharmacological experiments, transgenic lines and single cell RNA-seq (Shimizu et al, 2018;Ueda et al, 2018;Lindsey et al, 2019;Yu and He, 2019;Kiyooka et al, 2020), the global changes in gene expression related to immune activation and RG proliferation during the early stages of optic tectum regeneration had not yet been investigated. In this study, we first determined the time points for RNAseq by evaluating RG and macrophage responses at 6, 12, and 24 hpi.…”

“…On the other hand, most of RG are quiescent, while neuroepitheliallike stem cells contribute to adult neurogenesis in the adult optic tectum (Ito et al, 2010). Previous studies have shown that stab wound injury in the optic tectum induces RG proliferation and differentiation into neurons in young adult zebrafish (2-4 months old) under regenerative conditions (Shimizu et al, 2018;Ueda et al, 2018;Yu and He, 2019), but induces RG proliferation without neurogenesis in elderly adult zebrafish (6-12 months old) (Lindsey et al, 2019). Moreover, Yu and He found that only a few neurons were generated from RG using long-term EdU labeling and Cre-loxP-based clonal analysis.…”