Notch3 and DeltaB maintain Müller glia quiescence and act as negative regulators of regeneration in the light‐damaged zebrafish retina

Abstract

Damage to the zebrafish retina stimulates resident Müller glia to reprogram, reenter the cell cycle, divide asymmetrically, and produce neuronal progenitor cells that amplify and differentiate into the lost neurons. The transition from quiescent to proliferative Müller glia involves both positive and negative regulators. We previously demonstrated that the Notch signaling pathway represses retinal regeneration by maintaining Müller glia quiescence in zebrafish. Here we examine which Notch receptor is necessary to maintain quiescence. Quantitative RT‐PCR and RNA‐Seq analyses reveal that notch3 is expressed in the undamaged retina and is downregulated in response to light damage. Additionally, Notch3 protein is expressed in quiescent Müller glia of the undamaged retina, is downregulated as Müller glia proliferate, and is reestablished in the Müller glia. Knockdown of Notch3 is sufficient to induce Müller glia proliferation in undamaged retinas and enhances proliferation during light damage. Alternatively, knockdown of Notch1a, Notch1b, or Notch2 decreases the number of proliferating cells during light damage, suggesting that Notch signaling is also required for proliferation during retinal regeneration. We also knockdown the zebrafish Delta and Delta‐like proteins, ligands for the Notch receptors, and find that the deltaB morphant possesses an increased number of proliferating cells in the light‐damaged retina. As with Notch3, knockdown of DeltaB is sufficient to induce Müller glia proliferation in the absence of light damage. Taken together, the negative regulation of Müller glia proliferation in zebrafish retinal regeneration is mediated by Notch3 and DeltaB.

Notch signaling via Hey1 and Id2b regulates Müller glia’s regenerative response to retinal injury

“…CRISPR-mediated dll4 gene editing had no significant effect on retinal cell death (Figure 5i). Finally, forced expression of Dll4 with heat shock in hsp70:dll4-P2A-GFP transgenic fish inhibited injury-dependent proliferation of MG-derived progenitors without affecting (Campbell et al 2021). Consistent with this result, we found that Notch3 knockdown with a translation-blocking MO that successfully suppressed chimeric notch3-GFP RNA expression increased the number of MG recruited to a regenerative response as indicated by an expanded zone of injuryresponsive MG (Figure 5-figure supplement 6a-e).…”

“…Like that observed for radial glial stem cells, Notch signaling must be suppressed for MG proliferation and retina regeneration (Campbell et al 2021, Conner et al 2014, Elsaeidi et al 2018, Wan & Goldman 2017, Wan et al 2012). Furthermore, pan-retinal Notch inhibition increases the number of MG that engage in a regenerative response in light and needle poke injury models(Campbell et al 2021, Wan & Goldman 2017, Wan et al 2012), and Notch inhibition enhances spontaneous MG proliferation in uninjured retinas treated with growth factors, cytokines, or forced expression of Ascl1a and Lin28a(Conner et al 2014, Elsaeidi et al 2018, Wan & Goldman 2017).…”

“…The correlation of dll4 expression with that of hey1 suggested Dll4 is a quiescence promoting factor. However, Dll4 was recently reported to stimulate proliferation of MG-derived progenitors in the injured retina (Campbell et al 2021). This inconsistency prompted us to reevaluate Notch signaling component gene expression in uninjured and injured retina.…”

“…MG's injury response threshold is reflected by the zone of proliferating MG responding to a needle poke injury (Wan et al 2012). A previous study suggested Dlb may be involved in regulating this injury response threshold since Dlb knockdown increased the number of MG that engage in a regenerative response in the light damaged retina (Campbell et al 2021). We obtained a similar result in the needle poke injured retina using a translation blocking MO that was validated in zebrafish embryos injected with a dlb-GFP chimeric transcript (Figure 5-figure supplement 5a-d).…”

“…Because Notch signaling controls MG’s responsiveness to injury-derived signals, understanding its mechanism of action may not only inform us of the underlying mechanism, but also may suggest strategies for enhancing MG’s regenerative potential in mammals. One recent study investigating Notch ligand and receptor encoding genes concluded Dlb, acting via the Notch3 receptor, maintains MG quiescence and suppresses MG proliferation in the light damaged retina(Campbell et al 2021). However, the mechanism by which these factors act to regulate MG’s injury response remains unknown.…”