Abstract
Purpose:
The purpose of this project is to identify the signals that are required to initiate Müller glia dedifferentiation and proliferation in the damaged retina in order to regenerate the lost neurons.
Methods:
Dark-adapted adult albino zebrafish were exposed to constant intense light for 96 hours to kill rod and cone photoreceptors. Gene microarrays and two-dimensional gel electrophoresis identified candidate genes and proteins that increased in expression in the damaged retina. RNA and protein expression were examined by in situ hybridization and confocal microscopy, respectively. The function of candidate proteins during retinal regeneration was tested by electroporation of intravitreally injected morpholinos. The gamma-secretase inhibitor RO492 was injected intraperitoneally to block Notch signaling without causing retinal damage.
Results:
Two-dimensional gel electrophoresis suggested TNFα signaling proteins were more abundant in the light-damaged retina relative to the undamaged retina. Tissue in situ hybridization showed that tnfα mRNA expression increased in dying rod and cone photoreceptors. Morpholino-mediated knockdown of TNFα expression significantly reduced the number of Stat3-positive and proliferating Müller glia in the light-damaged retina. Inhibitors and morpholinos revealed that Jak1 is required for Stat3 phosphorylation/activation in the damaged retina. DNA microarray experiments revealed that genes encoding Notch signaling proteins are reduced in expression in light-damaged retinas. Intraperitoneal injection of the gamma-secretase inhibitor RO492 increased the number of Ascl1a-positive proliferating Müller glia in the undamaged retina. Morpholino-mediated knockdown of Ascl1a expression in the undamaged RO-492-stimulated retina significantly reduced the number of proliferating Müller glia, suggesting that Notch signaling suppresses Ascl1a expression to repress Müller glia proliferation.
Conclusions:
TNFα is expressed by dying neurons to induce Müller glia proliferation using the Stat3/Jak1 signaling pathway. Notch signaling, through repression of Ascl1a expression, maintains the Müller glia in a quiescent state in the undamaged retina. Thus, retinal damage increases TNFα expression and terminates Notch signaling to induce the expression of Stat3 and Ascl1a that results in Müller glia proliferation.
Keywords: 687 regeneration •
603 Muller cells •
721 stem cells