A recent study from Dr. HE Jie’s lab at the Center for Excellence in Brain Science and Intelligence Technology, Institute of Neuroscience, State Key Laboratory of Neuroscience, Chinese Academy of Sciences, investigated two essential questions of the mechanisms underlying the responses of glia in the injured brain. How do the injury-induced reactivated glia enter the cell cycle?

　　How do the injury-induced reactivated glia choose to generate newborn glia or newborn neurons?

　　Traumatic brain injury (TBI) is one of the clinically principal type of central nervous system (CNS) insults. TBI causes glia (e.g., mammalian astrocytes) to undergo gliosis, which in turn hinders neuronal regeneration and tissue restoration. In mammals, gliosis undergoes three stages: Glia initially become reactive, hypertrophic; subsequently, a subset of reactivated glia re-enter the cell cycle and become proliferative; finally, proliferative glia undergo gliogenesis, the process of glial cell production, and form structures known as glial scars.

　　In contrast to the mammalian CNS, zebrafish exhibit a superior neural regeneration after TBI, which makes zebrafish an important model system to study the mechanisms underlying neuronal regeneration of injured CNS. The researchers found that the radial glia (RG) in adult zebrafish optic tectum are dormant under physiological conditions; physical injury resulted in their proliferation and gliosis. By taking advantage of stochastic modeling analysis, single-cell RNA-seq  and in-vivo cell lineage tracing, they found:

　　Surprisingly, only a subset of tectal RG were driven into the cell cycle and become proliferative by stab injury. Sequential injuries at the same injury site could induce two populations of proliferative RG, which were partially overlapping. The overlapping proportion could be well explained by a stochastic model. The analysis indicated reactive RG entered cell cycle at the fixed probability of ~ 25 % and become proliferative.

　　Single-cell RNA-seq analysis revealed different status of RG including dormant, reactive and proliferative. Furthermore, the analysis also revealed the stochastic cell-cycle entry was dependent on Notch/Delta lateral inhibition.

　　Injury-induced proliferative tectal RG mainly underwent gliogenesis to generate ~90% newborn glia and only ~5% newborn neurons, the newborn cells could survive up to at least 300 days. Interestingly, post-injury notch inhibition during a proper time window (4-5 day post-injury (dpi)) resulted in a significant increased neurogenesis (~20%). Further analysis suggested that the over-produced newborn neurons were likely derived from the injury-induced reactive RG which were driven into the cell cycle to become proliferative by post-injury Notch inhibition. However, the majority of over-produced newborn neurons diminished by approximately 25 dpi, which required further investigation.

　　This work entitled “Stochastic cell cycle-entry and cell state-dependent fate outputs of injury-reactivated tectal radial glia in Zebrafish” was published in eLife on August 23. This work was carried out by Graduate Student YU Shuguang, under the supervision of Dr. He Jie. It was supported by the grants from Shanghai Municipal Science and Technology Major Project , Strategic Priority Research Program of Chinese Academy of Science , State Key Laboratory of Neuroscience, Shanghai basic research field Project, National Natural Science Foundation of China . 

　　Figure legend: (A) Schematic representation of stab injury assay. (B) Sparse labeling of tectal RG. (C) The representative images of Tg(gfap:GFP) (green) and PCNA (red) immunofluorescences showing that injury induces the proliferation of RG (GFP+/PCNA+, yellow cells) underneath the injury site at 3 days post-injury (dpi). (D) A t-SNE plot of 1174 single tectal RG at 3 dpi revealing 5 cell clusters. Dormant RG (dRG, cluster 1) in orange; Reactive RG (rRG, cluster 2) RG in dark cyan; Proliferative-S RG (pRG-S, cluster 3) in Indian red; Proliferative-G2 RG (pRG-G2, cluster 4) in purple; Unidentified RG (cluster 5) in dark green. (E) Schematic summary of the working model. Injury induces all RG underneath the injury site to become reactive. Only ~25% of reactive RG enter the cell cycle and become proliferative. The cell-cycle entry of reactive RG is regulated by Notch/Delta lateral inhibition. In the injury condition, proliferative RG largely undergo gliogenesis (~3-5% newborn neurons). The resulting newborn cells could survive up to 300 dpi. In the Notch inhibition condition, dormant RG can become proliferative but only generate ~1% of newborn neurons. However, Notch inhibition during 4-5 dpi drives reactive RG into the cell cycle, giving rise to significant more neurons (~12-20%). Interestingly, these over-produced neurons are largely diminished by 25 dpi. RG, radial glia; TeO, tectum opticum; PGZ, periventricular gray zone; TS, torus semicircularis; Val, valvula cerebelli. Scale bars, 50 μm.