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Ariadna G. Battista, Maria P. Faillace; Injury of the Adult Zebrafish Retina Induces Expression of Purinergic Receptors and Ecto-nucleotidases that Control In Vivo Cell Proliferation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5926.
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© ARVO (1962-2015); The Authors (2016-present)
Regeneration and growth that occurs in the adult teleost retina by neurogenesis have been helpful in identifying molecular and cellular mechanisms underlying cell proliferation and differentiation. We have previously reported that cell proliferation, after injury of inner and ganglion cell layers, is precisely regulated by purinergic signals.We aimed to examine purinergic signal participation in controlling retinal progenitor cell proliferation following a high intravitreal ouabain concentration treatment, which provokes cell death in all retinal layers. Moreover, we intended to get insight into injury-associated mechanisms, by examining whether injury or the damaged environment was able to modify the expression pattern of purinergic signaling molecules.
Zebrafish were lesioned by one intravitreal injection of 10µM ouabain. Then, apyrase (enzyme that hydrolyses extracellular nucleotides), ARL67156 (inhibitor of ecto-ATPase activity) or antagonists of purinergic receptors were injected daily for 6 days in the ouabain-treated eyes, and cell proliferation was determined by BrdU incorporation. Purinergic receptor and ecto-nucleotidase mRNA levels were measured at different days post injury by RT-qPCR. Finally, P2Y1 receptor protein was assessed by western blot.
Lesion increased cell proliferation in mature retinal layers, as well as in the ciliary marginal zone, with a maximum at day 7. Treatment with MRS2179, an antagonist of the ADP-activated P2Y1 receptor, completely blocked the injury-induced increase in cell division. In contrast, cell proliferation was not affected by blocking P2Y13 receptor, or by the injection of antagonists of adenosine-activated P1 receptors or for ATP-activated P2Y11, P2X1, P2X2, P2X3 receptors. ARL67156, which has been shown to inhibit NTPDase activity, significantly decreased BrdU-positive nuclei number observed in ouabain-treated retinas. Lesioned retinas showed a significant increase of P2Y1 receptor mRNA levels in the proliferative phase, whereas P2Y12 and P2X7 receptor mRNA levels did not change. NTPDase 1, 2, and 3 mRNA levels also enhanced significantly following damage. Additionally, P2Y1 receptor protein exhibited a lesion-induced change in its expression pattern.
Our results showed that purinergic signals, particularly ADP binding to P2Y1 receptors, as well as the extracellular activity of NTPDases are key regulators of cell cycle timing during the regeneration process of the globally-damaged adult zebrafish retina.
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