The effect of EDN2 loss on mutant PR survival <i>in vivo</i> and in retinal explants.

<p>(A) At PN40, the histology and the thickness of the ONL (n = 5;p>0.05) was normal in toluidine-blue stained <i>Edn2^+/+</i> and <i>Edn2^−/−</i> retinas. (B,C) The loss of EDN2 in <i>Tg(RHO P347S)</i> retinas resulted in a mean 63% increase in ONL thickness at PN40 (n = 6; p<0.005) (C) and a mean 110% increase in ONL thickness in <i>Pde6b^rd1/rd1</i> retinas at PN15 (n = 6; p<0.005) (B). (D) ONL thickness in WT, <i>Pde6b^rd1/rd1</i> and <i>Tg(RHO P347S)</i> retinas in mice expressing or lacking EDN2 (^**p<0.005). (E) qRT−PCR assays of the <i>Edn2</i> mRNA, normalized to <i>Gapdh</i> mRNA, in <i>in vivo</i> WT, WT explants and <i>Pde6b^rd1/rd1</i> explants (n = 3;^*p<0.05). Values were compared to the mean <i>Edn2</i> mRNA levels in WT <i>in vivo</i> samples (arbitrarily given a value of 1). <i>Edn2</i> transcripts were significantly increased in WT as well as <i>Pde6b^rd1/rd1</i> explants at PN12 following retinal dissection at PN7, likely as a result of dissection-induced mechanical stress. (F) WT retinal explants cultured <i>ex vivo</i> from PN7-PN17 had an average of 7–8 rows of PR nuclei at PN17 (n = 10 retinas, one representative shown). Owing to artifacts in frozen sections, the number of nuclei, instead of ONL thickness, was assessed in retinal explants. The absence of EDN2 in <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> retinal explants did not increase PR survival. Both <i>Pde6b^rd1/rd1</i> and <i>Pde6b^rd1/rd1</i>; <i>Edn2^−/−</i> explants cultured from PN7-PN17 had an average of 3 rows of PR nuclei at PN17 (n = 4;p>0.05, one representative shown) (H&E staining). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (Black bar = 25 µm in A–C, and F). Error bars indicate SEM.