Abstract
Most studies of c-Jun N-terminal Kinase (JNK) activation in retinal tissue were done in the context of neurodegeneration. In this study, we investigated the behavior of JNK during mitosis of progenitor cells in the retina of newborn rats. Retinal explants from newborn rats were kept in vitro for 3 hours and under distinct treatments. Sections of retinal explants or freshly fixed retinal tissue were used to detect JNK phosphorylation by immunohistochemistry, and were examined through both fluorescence and confocal microscopy. Mitotic cells were identified by chromatin morphology, histone-H3 phosphorylation, and location in the retinal tissue. The subcellular localization of proteins was analyzed by double staining with both a DNA marker and an antibody to each protein. Phosphorylation of JNK was also examined by western blot. The results showed that in the retina of newborn rats (P1), JNK is phosphorylated during mitosis of progenitor cells, mainly during the early stages of mitosis. JNK1 and/or JNK2 were preferentially phosphorylated in mitotic cells. Inhibition of JNK induced cell cycle arrest, specifically in mitosis. Treatment with the JNK inhibitor decreased the number of cells in anaphase, but did not alter the number of cells in either prophase/prometaphase or metaphase. Moreover, cells with aberrant chromatin morphology were found after treatment with the JNK inhibitor. The data show, for the first time, that JNK is activated in mitotic progenitor cells of developing retinal tissue, suggesting a new role of JNK in the control of progenitor cell proliferation in the retina.
Highlights
The retina is part of the central nervous system and is widely used as a model to study mechanisms of neurogenesis [1], due to knowledge of the spatio-temporal development of various retinal cell types
We first examined the phosphorylation of Jun N-terminal Kinase (JNK) in retinal explants of newborn rats maintained in vitro for 3 hours, and double stained with the phospho-JNK and phospho-histone-H3 antibodies
Because JNK phosphorylation can be induced by cellular stress, we examined the phosphorylation of JNK in freshly fixed retinal tissue from rats at embryonic day 14 (E14), 18 (E18), 21 (E21) and at postnatal day 1 (P1), by double staining with the phospho-JNK and phospho-histone-H3 antibodies
Summary
The retina is part of the central nervous system and is widely used as a model to study mechanisms of neurogenesis [1], due to knowledge of the spatio-temporal development of various retinal cell types. There is an immature inner nuclear layer (INLi), followed by the proliferative neuroblastic layer (NBL). The NBL of neonatal rats contains both proliferating progenitor and postmitotic cells, including early developing horizontal cells [4]. The cell cycle in the proliferative zone of the retina, similar to other parts of the CNS, is tightly controlled and proceeds in synchrony with interkinetic migration of the progenitor cell nuclei along the depth of the NBL [5]. The phases of the cell cycle are identified in retinal progenitor cells, due to interkinetic nuclear migration [6]. The spatial segregation of the phases of the cell cycle along the interkinetic migration pathway facilitates experimental studies of cell proliferation in the retina. The intracellular mechanisms that control phase transitions during the cell cycle are still poorly understood
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