Abstract
Purpose :
Regeneration of the damaged adult zebrafish retina requires dynamic Notch signaling, including repressing Notch3 expression for Müller glia reprogramming and increasing Notch1a expression for neuronal progenitor cell (NPC) proliferation. Many hairy/enhancer of split-related (her) genes are direct targets of Notch signaling and transcriptionally repress pro-neural genes. To better understand the downstream effectors of Notch signaling in the adult zebrafish retina, we investigated her gene expression and function during regeneration of light-damaged retinas.
Methods :
The expression patterns of her genes were investigated using single-cell RNAseq data and quantitative RT-PCR. The heat shock-Gal4/UAS-NICD transgenic system was used to induce overexpression of Notch1a signaling and assess her gene expression via quantitative RT-PCR. Expression of specific Her proteins were knocked down during constant light damage with intravitreal morpholino injection and electroporation, and the regeneration response was assessed with immunohistochemistry.
Results :
Similar to notch1a, expression of her2, her4, her9, her12, and her15.1 increased during Müller glia activation and NPC proliferation. Also, like notch1a, expression of her2, her4.2, her9, and her12 were strongly localized to activated Müller glia, while her6 and her15.1 were expressed in both quiescent and activated Müller glia. Expression of her6, her9, and her15.1 were also expressed with notch1a in retinal vasculature cells. Induced expression of the Notch1a intracellular domain in undamaged retinas resulted in upregulation of her4, her9, and her12. Knockdown of Her4 expression during constant light damage resulted in a decreased number of PCNA-positive cells during photoreceptor regeneration.
Conclusions :
These results demonstrate that a subset of her genes are expressed in activated Müller glia and NPCs during regeneration of light-damaged retinas. Furthermore, expression of her4, her9, and her12 are responsive to Notch1a expression, and Her4 is required for Müller glia and NPC proliferation, while her6 and her15.1 are expressed in quiescent Müller glia. These results begin to elucidate the dynamic expression of her genes required to induce Müller glia reprogramming and for NPC proliferation during retinal regeneration.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.