Abstract
Purpose :
Extracellular matrix (ECM) is an important source of developmental and homeostatic molecular cues. Although much is known regarding the molecular mechanisms of retinal regeneration, ECM’s role in regeneration is 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 light-damaged adult zebrafish retinas.
Methods :
Constant light treatment induced photoreceptor cell death and MG-dependent neuronal regeneration. In vivo morpholino-mediated gene knockdowns were used to repress Npnt expression in adult zebrafish retinas. Anti-PCNA immunolabeling of Tg(gfap:egfp) zebrafish retinal sections visualized proliferating MG and NPCs. TUNEL labeling was used to assess cell death. qRT-PCR and FISH were used to examine target gene expression. Protein expression levels and subcellular distribution were assessed using protein immunoblotting of either cytosolic or nuclear proteins. Fate tracking was performed using EdU to label proliferating NPCs during regeneration, with cell-type-specific antibodies used to counterstain in recovered retinas.
Results :
npnt is expressed by HuC/D+ amacrine and ganglion cells in adult zebrafish retinas and is upregulated after light treatment. Knockdown of npnt results in decreased NPC proliferation and a significant increase in inner retinal neuron death via the GDNF pathway, as well as altered ratios of retinal cell types produced during regeneration. Protein immunoblotting revealed a novel pattern of Npnt processing and subcellular translocation. Upon light damage, Npnt is cleaved into two fragments. Our data suggest that one of these fragments is translocated into the nucleus by a mechanism involving itga11a-containing receptor, suggesting it may directly regulate expression of target genes.
Conclusions :
These findings reveal that the ECM provides regulatory cues guiding zebrafish retinal regeneration. Npnt regulates both 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 genes that govern the regeneration response and neuron survival. These findings suggest a novel mechanism for an ECM molecule and expand our understanding of the role of the microenvironment in retinal regeneration.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.