Injury-induced purinergic signalling molecules upregulate pluripotency gene expression and mitotic activity of progenitor cells in the zebrafish retina

Abstract

Damage in fish activates retina repair that restores sight. The purinergic signalling system serves multiple homeostatic functions and has been implicated in cell cycle control of progenitor cells in the developing retina. We examined whether changes in the expression of purinergic molecules were instrumental in the proliferative phase after injury of adult zebrafish retinas with ouabain. P2RY messenger RNA (mRNA) increased early after injury and showed maximal levels at the time of peak progenitor cell proliferation. Extracellular nucleotides, mainly ADP, regulate P2RY transcriptional and protein expression. The injury-induced upregulation of P2RY is mediated by an autoregulated mechanism. After injury, the transcriptional expression of ecto-nucleotidases and ecto-ATPases also increased and ecto-ATPase activity inhibitors decreased Müller glia-derived progenitor cell amplification. Inhibition of P2RY endogenous activation prevented progenitor cell proliferation at two intervals after injury: one in which progenitor Müller glia mitotically activates and the second one in which Müller glia-derived progenitor cells amplify. ADPβS induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. The expression of these genes, which regulate multipotent Müller glia reprogramming, was significantly inhibited by blocking the endogenous activation of P2RY early after injury. We consistently observed that the number of glial fibrillary acidic protein-BrdU-positive Müller cells after injury was larger in the absence than in the presence of the P2RY antagonist. Ecto-ATPase activity inhibitors or P2RY-specific antagonists did not modify apoptotic cell death at the time of peak progenitor cell proliferation. The results suggested that ouabain injury upregulates specific purinergic signals which stimulates multipotent progenitor cell response.

Characterization of proliferative, glial and angiogenic responses after a CoCl2‐induced injury of photoreceptor cells in the adult zebrafish retina

“…We observed that the injury following exposure to CoCl 2 , which targeted and killed virtually all photoreceptors, accelerated the induction and rate of progenitor proliferation activity, peaking between 72 and 96 hpl. In contrast, when the injury was caused with a single injection of ouabain (either at a low, medium or high dose), the Müller glia‐derived progenitor proliferative peak was observed at 5–7 dpl (Fimbel et al., ; Medrano et al., ; Sherpa et al., ).…”

“…A single intravitreal injection of ouabain at low concentrations damages inner retina cells (Fimbel et al., ), whereas higher doses of ouabain provoke cell death across all retina layers, including RPE and choroid cells (Medrano et al., ; Sherpa et al., ). In contrast, injury with intense light causes photoreceptor death as the original and principal effect (Rajaram et al., ; Weber et al., ; Wu et al., ).…”

“…Our results showed that TNFα transcriptional expression was increased by 72 hpl, supporting the idea of enhanced TNFα release and its involvement in the induction of multipotent cell proliferation, in addition to its known proinflammatory role. Nevertheless, the proliferative activity of retinal tissue might be activated by TNFα in specific zones, as well as by several other characterized extracellular signals provided by lytic photoreceptors (Battista et al., ; Lenkowski & Raymond, ; Medrano et al., ; Powell et al., ).…”

Neurodegeneration, Neuroprotection and Regeneration in the Zebrafish Retina

“…To date, there are various retinal neuronal injury paradigms used in zebrafish research. Undoubtedly, the most common of those paradigms is light-induced retinal degeneration [ 86 , 90 , 91 , 156 , 157 , 158 , 159 , 160 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 , 171 , 172 , 173 , 174 , 175 ], followed by chemically [ 95 , 103 , 155 , 176 , 177 , 178 , 179 , 180 , 181 , 182 , 183 , 184 , 185 , 186 ] or mechanically induced retinal damage [ 92 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 ]. The choice of paradigm depends on the desired outcome, for example, ouabain has a more global retinal degenerative effect (depending on concentration), while intense light exposure primarily affects the photoreceptors [ 156 , 169 ].…”

“…Interestingly, the extracellular nucleotide agonist, ADPβS, induced the expression of lin28a and ascl1a genes in mature regions of uninjured retinas. This gene expression was inhibited by blocking the early injury-induced activation of P2RY1, which also prevented progenitor cell proliferation [ 181 ]. Sonic hedgehog (shh) was also found to be upregulated after ouabain injury, and treating the retina with shh resulted in Müller cell hypertrophy followed by their conversion to retinal progenitor cells [ 169 ].…”