Abstract
Purpose::
Rho GTPases are important proteins in signaling pathways that regulate gene transcription, cell cycle entry and cell survival. We have previously shown that these proteins may participate in processes during retinal development, Müller cells morphology, and apoptosis (Santos-Bredariol et al., 2003, 2006; Belmonte et al., 2006). These proteins are activated by LPA, a lipid mediator with a wide variety of biological actions, particularly in proliferation, migration, differentiation and cell survival. The aim of the present study was to characterize the presence of these GTPases in retinal progenitor ciliary body (CB) cells of mice and the effects of their activation by LPA on cell proliferation and differentiation in the CB and central retina.
Methods::
Adult Balb/c mice were anesthetized and LPA (1µM) or PBS (control) with BrdU (1µg) were injected into the eyes. After 6, 12 and 24 hours, they were sacrificed and the retinas were processed for immunofluorescence with the antibodies against RhoA, RhoB and Rac1, and markers for microglia (Ox42), proliferation (BrdU and Ki67) and progenitor cells (Pax6, Chx10 and Nestin), and analyzed in a confocal microscope (Nikon PCM2000).
Results::
In control eyes, RhoA was observed in photoreceptors and inner nuclear layer in the retina, and it was weakly expressed in the CB. RhoB was predominantly expressed by Müller cells in the retina and pars plana of the CB, whereas Rac1 was mostly observed in photoreceptor segments and plexiform layers, but it was diffusely distributed in the CB. After LPA injections, RhoA and RhoB expression was increased in the retina and CB, while Rac1 was detected in photoreceptor cell bodies. In addition, Ki67 and BrdU-positive cells were observed in the plexiform and ganglion cell layers, and CB. Cell counts did not show statistical differences between the PBS and LPA injections; the Ox42 marker suggested that they were microglial cells. However, nestin-positive cells were seen in the CB 6h after LPA injection, disappearing after 12 and 24h, when Pax6 and Chx10 co-expression was observed.
Conclusions::
Although all investigated GTPases changed their expression after LPA activation, LPA did not induce proliferation of progenitor retinal cells. On the other hand, an increased number of progenitors in the CB, as shown by nestin expression, as well as Pax6 and Chx10 co-expression, indicated that LPA might induce undifferentiated patterns.
Keywords: retina • development • ciliary body