Inhibition of muller glial cell division blocks regeneration of the light-damaged zebrafish retina

The adult zebrafish retina possesses a robust regenerative response. In the light-damaged retina, Muller glial cell divisions precede regeneration of rod and cone photoreceptors. Neuronal progenitors, which arise from the Muller glia, continue to divide and use the Muller glial cell processes to migrate to the outer nuclear layer and replace the lost photoreceptors. We tested the necessity of Muller glial cell division for photoreceptor regeneration. As knockdown tools were unavailable for use in the adult zebrafish retina, we developed a method to conditionally inhibit the expression of specific proteins by in vivo electroporation of morpholinos. We determined that two separate morpholinos targeted against the proliferating cell nuclear antigen (PCNA) mRNA reduced PCNA protein levels. Furthermore, injection and in vivo electroporation of PCNA morpholinos immediately prior to starting intense light exposure inhibited both Muller glial cell proliferation and neuronal progenitor marker Pax6 expression. PCNA knockdown additionally resulted in decreased expression of glutamine synthetase in Muller glia and Muller glial cell death, while amacrine and ganglion cells were unaffected. Finally, histological and immunological methods showed that long-term effects of PCNA knockdown resulted in decreased numbers of Muller glia and the failure to regenerate rod photoreceptors, short single cones, and long single cones. These data suggest that Muller glial cell division is necessary for proper photoreceptor regeneration in the light-damaged zebrafish retina and are consistent with the Muller glia serving as the source of neuronal progenitor cells in regenerating teleost retinas. (C) 2007 Wiley Periodicals, Inc.