Overexpression of Six3OS in developing retina inhibits changes in cell fate and photoreceptor morphology observed following Six3 overexpression. (A) Electroporation of CAG-Six3 led to an increase in the fraction of GFP-positive cells expressing the amacrine cell marker syntaxin and a decrease in cells expressing the rod bipolar marker PKCα, CAG-Six3OS led to a decrease in syntaxin-positive cells, and co-expression led to a cell composition that was indistinguishable from CAG-GFP controls. *P < 0.05. (B-E) Section immunohistochemistry of retinas electroporated with CAG-GFP, CAG-Six3OS, CAG-Six3 or both CAG-Six3OS and CAG-Six3. White dashed lines define the outer third of the outer nuclear layer (OONL). Syntaxin (red) is co-immunostained with GFP (green). (C) No obvious difference is observed in either amacrine cell number or morphology (white arrowheads) in retinas electroporated with CAG-Six3OS relative to CAG-GFP controls or in the fraction of the cells in the OONL. (D) An increase in amacrine cell number and in the number of cells in the OONL and a decrease in outer segment length is observed in the case of Six3. (E) An increase in the number of cells in the OONL is observed, but no difference in amacrine cell number or outer segment length is observed from controls following co-electroporation of CAG-Six3 and CAG-Six3OS. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OS, outer segment.. (F) Laminar position of cells within the OONL. Electroporation of CAG-Six3 leads to a shift of rod photoreceptor cell bodies to the OONL, and this effect is not affected by co-electroporation of CAG-Six3OS (white dashed lines in B-E). (G) Rod photoreceptors electroporated with CAG-Six3 show substantially shorter outer segments and this effect is reversed by co-electroporation with CAG-Six3OS. Cell type specific markers used: rhodopsin (Rho4D2), rod photoreceptors; glutamine synthetase (GS), Muller glia; protein kinase C alpha (PKCα), rod bipolar cells; syntaxin (Syn), amacrine cells.