Abstract
Purpose :
The role of laminins, which are principal components of the extracellular matrix (ECM), has been well-studied in vertebrate retinal development. However, far less is known about their potential roles in neuronal regeneration, which is similar to development but is not a mere recapitulation. Therefore, we expect that laminins, which are critical for development, are also involved in regeneration. Here, we examine the role of β1b chain-containing laminins (CCLs) in the regenerative response of Müller glia (MG) in the zebrafish retina.
Methods :
Tg(gfap:eGFP) zebrafish, which express enhanced green fluorescent protein within the MG, were dark-adapted for two weeks and used for in vivo morpholino-mediated gene knockdown of Lamb1b protein expression in the retina. The fish were then exposed to intense light for up to 96 hours, resulting in photoreceptor death and initiating a regenerative response, which was assessed via immunohistochemistry in retinal cryosections. Cell-fate tracking studies were performed using intraperitoneal injections of EdU (5-Ethynyl-2′-deoxyuridine), a thymidine analogue that is incorporated into proliferating cells. Finally, the role of lamb1b in the expression of other laminins and cell-surface receptors was assessed via qRT-PCR.
Results :
Expression of lamb1b is upregulated in the regenerating retina after 48 hours of light damage (4-fold increase) and remains highly expressed throughout the regeneration process. Morpholino-mediated knockdown of Lamb1b expression results in a significant decrease in the number of proliferating MG and neuronal progenitor cells (NPC) during retinal regeneration at 36 (decreased by 60%; p≤0.01) and 72 hours (decreased by 30%; p≤0.0001) of light treatment. Lamb1b protein knockdown also results in significantly fewer EdU+ cells within the outer nuclear layer (decreased by 30%; p≤0.01). Moreover, the expression of other laminin chains and several integrin receptors is downregulated in the lamb1b morphant.
Conclusions :
These data illustrate that β1b CCLs regulate the regenerative response in the zebrafish retina. Specifically, MG and NPC proliferation, as well as the expression of other laminins and their receptors, are both disrupted in response to reduced β1b CCL expression. These results suggest that the ECM plays a crucial role in regeneration and begin to uncover the molecular mechanisms of these processes.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.