Abstract
Purpose :
Following damage to the human retina, the light-sensitive photoreceptor cells cannot be regenerated or replaced. However, the zebrafish (Danio rerio) retina can fully regenerate photoreceptors 14 days after sustaining an injury using intrinsic stem cells called Müller glia (MG). MicroRNAs (miRNAs) are highly conserved non-coding RNAs that target mature messenger RNA (mRNA) from other genes. The microRNA-155 (miR-155) is commonly expressed in cells of the immune system and is known to regulate inflammation, but its functions in the developing and injured/regenerating retina are unknown. This study will help to determine the role miR-155 plays during photoreceptor development and regeneration.
Methods :
In-situ hybridization was used to determine the temporal and spatial expression of miR-155 in the embryonic and adult regenerating retina. Immunolabeling was used to label proliferating and Müller glial cells. RT-qPCR was performed to quantify the expression of pre-miR-155 at 2-, 3-, 5-, and 7-days post lesion (dpl) and TaqMan RT-qPCR was used to quantify expression of the mature miR-155. Morpholino oligonucleotides (MO) were used to knock down miR-155 in the embryonic retina and adult retina, followed by immunolabeling of proliferating cells and mature adult photoreceptors. Histology was performed and retinal cross-sections from MO injected fish were compared to those of fish injected with standard control morpholinos.
Results :
In-situ hybridization and immunolabeling showed that miR-155 is expressed between 48-72 hours post fertilization (hpf) throughout the developing retina. During photoreceptor regeneration in adult retinas, miR-155 is expressed at 3 dpl in dividing Müller glia and progenitor cells. RT-qPCR results show an increase in expression of pre-miR-155 at 5 dpl compared with uninjured retinas. The miR-155 MO knockdowns show fewer proliferating cells in the embryonic retina at 48 hpf and fewer rod and cone photoreceptors at 72 hpf. In adult retinas, miR-155 knockdown reduces the number of proliferating cells in the inner nuclear layer (INL). CRISPR miR-155 mutant fish have been generated and are currently being characterized.
Conclusions :
In the developing retina, miR-155 is required for normal progenitor cell proliferation and photoreceptor development. During photoreceptor regeneration in adult retinas, miR-155 is required for the normal proliferation of MG-derived progenitors.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.