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. 2014 Jul 14;9(7):e102510.
doi: 10.1371/journal.pone.0102510. eCollection 2014.

Exogenous Oct-4 inhibits lens transdifferentiation in the newt Notophthalmus viridescens

Rital B Bhavsar  1 , Panagiotis A Tsonis  1
Affiliations

Exogenous Oct-4 inhibits lens transdifferentiation in the newt Notophthalmus viridescens

Rital B Bhavsar et al. PLoS One. .

Abstract

From the cocktail of four factors that were able to induce pluripotent stem cells from differentiated cells, Oct-4, c-Myc, Sox-2 and Klf4, only Oct-4 was not expressed during regeneration in newts. To explore the possible action of this stemness factor we developed an assay where we introduced exogenous Oct-4 protein to an in vitro system for lens regeneration in newts. We found that exogenous Oct-4 inhibits differentiation of iris pigmented epithelial cells into lens cells and also regulates Sox-2 and Pax-6, both important players during lens development. Thus, presence of Oct-4 hinders transdifferentiation of iris cells.

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Conflict of interest statement

Competing Interests: P.A. Tsonis is a PLOS ONE Academic Editor. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Newt IPE cells treated with Oct-4 protein.
a) IPE cells after 7 hours of protein treatment (earliest time to visualize Oct-4 protein). Cells (arrow) show Oct-4 protein (green) transfer to nucleus (blue). b) Confocal image merged with bright field showing Oct-4 (green) localization in the cells at higher magnification. c) IPE cells not treated with oct-4 antibody show only nuclear stain (blue). The efficiency of oct-4 translocation increases at prolonged incubation time points (data not shown).
Figure 2
Figure 2. Live images of IPE cell aggregates placed on matrigel.
a) Dorsal control aggregate showing transdifferentiated transparent lentoid (arrow) on day 4 of matrigel treatment. b) Dorsal Oct-4 protein treated aggregate without lentoid formation at day 14 on matrigel. c) Ventral Oct-4 protein treated aggregate without lentoid formation at day 14 on matrigel. Scale bar = 50 µm.
Figure 3
Figure 3. Immunohistochemistry of aggregates placed on matrigel.
Control dorsal aggregate showing transdifferentiated transparent lentoid (arrow, a) and positive for crystallin expression (c). Dorsal Oct-4 treated aggregate negative for crystallin expression (d, f). Ventral Oct-4 treated aggregate negative for crystallin expression (g, i). Control dorsal aggregate image was captured using confocal microscope. The lentoid in the control aggregates was observed from day 4 to day 15 of matrigel treatment. Oct-4 treated aggregates were sectioned into 10 µm sections for staining. Nuclear staining of aggregates (b, e and h).
Figure 4
Figure 4. Dual staining of transfected IPE cells with Oct-4 (red) and Tunel (green).
a) Cells expressing Oct-4 (arrows) are Tunel-negative. b) Cells positive for Tunel (arrowheads) are Oct-4-negative.
Figure 5
Figure 5. mRNA expression analysis of a) Oct-4 b) Sox-2 c) c-Myc d) Pax-6 in newt dorsal and ventral IPE cells with or without Oct-4 protein treatment.
The control samples were cells not treated with Oct-4 protein. P value was obtained by anova analysis for Sox-2, Pax-6 and c-Myc. For oct-4 expression, p value was calculated by independent t-test for oct-4 treated groups. *Groups show significant difference between the control and oct-4 treated cells.
See this image and copyright information in PMC

References

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