To test whether the all-cone outer retina influences organization of the inner retina, we analyzed expression of specific cell markers using immunofluorescence (
Fig. 2a). The M- and S-cone opsins (OPN1MW and OPN1SW) were detected almost exclusively in outer segments of
wt and
R91W;Nrl−/− mice. In
R91W, cone opsins were mislocalized to synaptic terminals, as reported previously.
8 Some rosettes in
Nrl−/− retinas were positive for both cone opsins, as reported before.
23 Calbindin (CALB), protein kinase C alpha (PKC-A) and visual system homeobox 2 (VSX2, also known as CHX10), synaptophysin (SYP), and calretinin (CR) were used as markers for horizontal, bipolar, and synaptic terminals and amacrine cells, respectively. They showed no overall difference between rod-dominated (
wt and
R91W) and the all-cone
R91W;Nrl−/− retinas. In contrast, protein localization in
Nrl−/− was irregular and followed the contours of rosettes (
Fig. 2a). The POU domain, class 4, transcription factor 1 (POU4F1, also known as BRN3A) localized to the ganglion cell layer in all strains. No clear signs of gliosis were visible in 6-week-old mice, as judged by GFAP immunofluorescence. However, increased GFAP immunoreactivity and higher
Gfap mRNA and protein levels were observed in older
R91W;Nrl−/− and
Nrl−/− mice (data not shown). Thus no overall differences were observed between
wt and
R91W;Nrl−/− mice in cell organization of the inner retina. This was corroborated by gene expression analysis of retina-specific cell markers in
wt,
Nrl−/− , and
R91W;Nrl−/− mice (
Fig. 2b; for validation of selected genes see
Supplementary Fig. S3). Overexpression of
Opn1sw was detected in both
Nrl−/− and
R91W;Nrl−/− mice. Expression of
Opn1mw was slightly increased but generally comparable between all-cone and
wt retinas (
Fig. 2b). These findings are consistent with reports showing that the lack of
Nrl leads to increased
Opn1Sw expression and enhanced S-cone but not the M-cone activity, as measured by ERG.
4,24,25 No difference among the three strains was found for
Vsx2 (
Chx10), a marker for bipolar cells. Markers for amacrine cells (
Myf2) and ganglion cells (
Pou4f1) were similarly expressed in
wt and
R91W;Nrl−/− , but were significantly reduced in
Nrl−/− mice at later time points (statistics are presented in
Supplementary Table S2). These data are consistent with a recent study reporting loss of cells in the ganglion and inner nuclear layer of
Nrl−/− mice.
23 Atrophic axon terminal arbors, reduced higher-order branchlets, and smaller dendritic fields were reported for horizontal cells in dysmorphic
Nrl−/− mice,
26 suggesting that horizontal cells may suffer directly from the irregular arrangement of cones. Notably, mitochondrial amidoxime reducing component 1 (
Marc1), a marker gene for horizontal cells, was significantly reduced in
Nrl−/− , whereas expression in
R91W;Nrl−/− was similar to
wt. These data suggest that the normal tissue lamination found in
R91W;Nrl−/− mice supports survival and normal physiology of second- and third-order neurons downstream of the all-cone nuclear layer.