Purchase this article with an account.
Dani Sarkis, Diane M. Cockburn, Jason P. Charish, Nardos G. Tassew, Philippe P. Monnier; Dissecting the biological functions of Pumilio 2 in the developing visual system. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1122.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The purpose of this study is to characterize the role of Pumilio2 (Pum2), a founding member of the Puf family of mRNA binding proteins and translational regulators, in the developing visual system. Particularly, the aim is to identify the effects of Pum2 on eye development and axonal outgrowth.
The experimental model used in this study is the chicken visual system. To examine the role of Pum2 in axonal outgrowth, retinal flat mount preparations were electroporated with cDNA constructs. For the purpose of these experiments, constructs over expressing, down regulating (Pum2 miRNA), and a dominant negative form of Pum 2 (dnPum2) were cloned. For in vivo studies, the optic vesicles of embryonic day 1.5 (E1.5) chicken embryos were electroporated with the above constructs and eye size was measured at later stages of development.
In situ hybridization confirmed the expression of Pum2 transcript in the retina, including the retinal ganglion cell (RGC) layer, of chicken embryos. Immunohistochemical staining revealed the expression of Pum2 in the optic fiber layer of the retina. Also, when retinal explant preparations were stained for Pum2, the protein was detected in the axons and growth cones of RGCs. When Pum2 is electroporated into dissociated neurons, the length of neurites is approximately 84.7 ± 2.7% (p < 0.0001) and 64.2 ± 8.2% (p = 0.001) shorter when compared to control GFP and dnPum2 transfected neurons, respectively. Furthermore, a small eye phenotype (micropthalmia) was observed at E9 following Pum2 overexpression in the developing eye at E1.5.
Pum2 overexpression significantly decreased the length of neurites in dissociated neurons and the eye size at E9. These preliminary data indicate a significant involvement of Pum2 in the developing eye and axonal outgrowth. More experiments are underway to investigate other functional aspects of Pum2 in eye development.
This PDF is available to Subscribers Only