Abstract
Purpose :
Extracellular matrices (ECMs) are an important source of developmental and homeostatic molecular cues. Although much is known regarding the molecular mechanisms of retinal regeneration, our understanding of the ECM’s role in this context remains unclear. This study investigates the role of the ECM molecule nephronectin (Npnt) in regulating Müller glia (MG) and neural progenitor cell (NPC) proliferation and retinal neuron survival in the light-damaged adult zebrafish retina.
Methods :
We used in vivo morpholino-mediated gene knockdown to repress Npnt expression in the adult zebrafish dorsal retina. Anti-PCNA immunolabeling of Tg(gfap:egfp) zebrafish retinal sections was used to visualize proliferating MG and NPCs. TUNEL labeling was used to assess cell death. qRT-PCR was used to examine expression of target genes. Protein expression levels and subcellular distribution were assessed using protein immunoblotting of samples enriched for either cytosolic or nuclear proteins from dorsal retinas.
Results :
Npnt is present in the adult zebrafish retina and exhibits increased expression 48h after intense light treatment. Knockdown of Npnt results in decreased NPC proliferation and GDNF expression, leading to a significant increase in the death of inner retinal neurons. Protein immunoblotting revealed a novel pattern on Npnt processing and translocation. Under normal conditions, Npnt is observed as a 60 kDa protein, with a portion being cleaved into 20 and 40 kDa fragments. Upon light damage, the entire population is cleaved into the two fragments. Furthermore, our data suggest that one of these fragments is translocated into the nucleus, suggesting that it may directly regulate expression of target. Indeed, Npnt knockdown results in decreased expression of integrins α3b, α4, α11a, and β1, as well as ECM components, such as laminins α1 and α5. Knockdown of these genes also results in decreased MG and NPC proliferation.
Conclusions :
These findings demonstrate that the ECM provides regulatory cues guiding zebrafish retinal regeneration. Npnt appears to regulate retinal neuron survival and NPC proliferation. Interestingly, our data suggest that upon damage, Npnt is transported into the nucleus, where it may directly regulate expression of several ECM-related genes that govern the regeneration response. These findings suggest a novel role for Npnt, and expand our understanding of the role of the ECM in retinal regeneration.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.