Involvement of sonic hedgehog and notch signaling in regenerative neurogenesis in adult zebrafish optic tectum after stab injury

Abstract

Unlike humans and other mammals, adult zebrafish have the superior capability to recover from central nervous system (CNS) injury. We previously found that proliferation of radial glia (RG) is induced in response to stab injury in optic tectum and that new neurons are generated from RG after stab injury. However, molecular mechanisms which regulate proliferation and differentiation of RG are not well known. In the present study, we investigated Shh and Notch signaling as potential mechanisms regulating regeneration in the optic tectum of adult zebrafish. We used Shh reporter fish and confirmed that canonical Shh signaling is activated specifically in RG after stab injury. Moreover, we have shown that Shh signaling promotes RG proliferation and suppresses their differentiation into neurons after stab injury. In contrast, Notch signaling was down-regulated after stab injury, indicated by the decrease in the expression level of her4 and her6, a target gene of Notch signaling. We also found that inhibition of Notch signaling after stab injury induced more proliferative RG, but that inhibition of Notch signaling inhibited generation of newborn neurons from RG after stab injury. These results suggest that high level of Notch signaling keeps RG quiescent and that appropriate level of Notch signaling is required for generation of newborn neurons from RG. Under physiological condition, activation of Shh signaling or inhibition of Notch signaling also induced RG proliferation. In adult optic tectum of zebrafish, canonical Shh signaling and Notch signaling play important roles in proliferation and differentiation of RG in physiological and regenerative conditions.

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

“…No significant change of neuron production from injury-induced proliferative RG labeled during 1–3 dpi is observed (Figure 7H, J, K, O and P). Ueda et al recently showed that Notch inhibition during 4–7 dpi decreases the number of newborn neurons (Ueda et al, 2018), which is in contrast to our findings. This inconsistency is likely to be because the time window of EdU or BrdU treatment is different.…”

“…RO4929097, another Notch signaling inhibitor, resulted in a similar phenotype (Figure 5—figure supplement 1A–D). Consistent with the findings of a recent study (Ueda et al, 2018), Notch inhibition was also sufficient to trigger the proliferation of tectal RG even without any injury (DMSO-treated: 6.3 ± 0.48 cells, n = 4; LY411575-treated: 26.7 ± 2.4 cells, n = 5; mean ± SEM; p>0.05; Figure 5D, E, H, I, J, Figure 5—figure supplement 1C and D), which was reminiscent of the increase of constitutively proliferative RG in the zebrafish telencephalon by Notch inhibition (Chapouton et al, 2010).…”

“…Besides, long-term experiments unexpectedly showed that over-produced neurons disappeared by 25 dpi. Earlier studies also showed that manipulation of Notch signaling could drive more RG into the cell cycle in the injury context (Dias et al, 2012; Ueda et al, 2018; Wan et al, 2012). However, none of them investigated the long-term survival of those newborn cells.…”

“…Zebrafish optic tectum, the higher sensory integration center, possesses a large population of RG (Galant et al, 2016; Ito et al, 2010). Unlike other brain regions where RG present as both dormant and proliferative forms at physiological conditions, tectal RG has been reported to be dormant, and are reactivated by injury to give rise to newborn neurons via Wnt signaling as well as Notch signaling (Shimizu et al, 2018; Ueda et al, 2018). Interestingly, a recent study showed that tectal RG produced a significant number of glial cells (~25%) but not neurons (Lindsey et al, 2019).…”

Dopaminergic neurons regenerate following chemogenetic ablation in the olfactory bulb of adult Zebrafish (Danio rerio)

“…This ability to hold numerous cells in a proliferating stage allows for the recruitment of stem-progenitor cells to multiply and migrate following damage to brain tissue in order to allow cells to then remodel the brain environment in a regenerative fashion 8 , 9 . While much of our knowledge about the regenerative capacity of the adult zebrafish brain have come from invasive stab lesion studies 8 – 11 , few studies have investigated the zebrafish ability to regenerate a specific neuronal subtype through a conditional and specific ablation approach in the adult brain.…”