Transformation of cone precursors to functional rod photoreceptors by bZIP transcription factor NRL

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   Fig. 1.

   Expression of Nrl in cone precursors. (A–L) Toluidine blue stainings of WT (A), Crxp-Nrl/WT (B), Nrl ^−/− (C), and Crxp-Nrl/Nrl ^−/− (D) retinal sections demonstrate unique chromatin pattern in the photoreceptor layer for cones (indicated by arrowhead) and rods. Normal laminar structure is observed in both Crxp-Nrl/WT (B) and Crxp-Nrl/Nrl ^−/− (D) plastic sections. Immunohistochemical markers for rod photoreceptors (rhodopsin) can be detected in WT (E), Crxp-Nrl/WT (F), and Crxp-Nrl/Nrl ^−/− (H) retina but not in Nrl ^−/− (G). The pan cone photoreceptor marker, cone arrestin, is present only in WT (I) and Nrl ^−/− (K) retina, but is largely absent in the Crxp-Nrl/WT (J) and Crxp-Nrl/Nrl ^−/− (L). (M–P) ERG intensity series and responses were recorded from 2-mo-old WT, Nrl ^−/−, Crxp-Nrl/WT, and Crxp-Nrl/Nrl ^−/− mice under dark- (scotopic ERG; M and N) and light-adapted (photopic ERG; O and P) conditions. The x axes for M and O indicate time lapsed after flash. Stimulus energy is indicated (log cd-s/m²). OS, outer segments; IS, inner segments, ONL, outer nuclear layer; INL, inner nuclear layer. (Scale bars: A–D, 25 μm; E–L, 50 μm.)

   
   
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   Fig. 2.

   Synaptic organization of the inner retina in the absence of cones. (A) The glutamatergic receptor mGluR6 is clustered selectively at puncta (red) in the OPL, on the dendritic tips of ON bipolar cells, labeled by G_0α antibodies (green). (B) G_0α antibody labels the whole population of ON bipolar cells (green signal), whereas PKCα labels rod bipolar cells only (RBC; red). Rod bipolar neurons are therefore double-labeled by both antibodies and appear yellow. Green cells are ON cone bipolar cells (indicated as CBC). (C) mGluR6 receptors are labeled as red puncta located at the dendritic tips of rod bipolar cells, labeled green by PKCα antibodies. In addition, clusters of mGluR6 are visible in the OPL, but not in association with rod biolar cell dendrites. These clusters are likely to be associated to the dendrites of ON cone bipolar cells. (D) Rod bipolar cells (RBC), labeled by PKCα (red), are postsynaptic to photoreceptors in the OPL at ribbon synapses (indicated by R), as indicated by antibodies against kinesin, a synaptic ribbon marker (green). (E) High magnification of one type of cone bipolar cell (CBC), labeled with NK3-R antibody (red). Rod spherules (RS) are labeled with anti-PSD95 antibody (green). Few dendrites of cone bipolar cells reach the basal aspect of some spherules (arrows); however, many spherules do not appear apposed to CBC dendrites, although these belong to one of the most abundant types of retinal cone bipolar cell. (F) Calbindin staining (red) of the Crxp-Nrl/WT retina shows a normal distribution of intensely labeled horizontal cells and weakly fluorescent amacrine cells with their processes in the IPL. Occasionally, horizontal cell sprouts are observed (arrow). (G) AII amacrine cells (the most abundant population of mammalian amacrines) are specifically stained with DB3 antibodies (red). They exhibit a typical, bistratified morphology. The innermost dendrites terminate in apposition to the axonal endings of rod bipolar cells, stained green by PKCα antibodies. (H) Cholinergic amacrine cells are stained in the transgenic retina by ChAT antibodies (red). The cells form two mirror symmetric populations of neurons. Axonal complexes of horizontal cells are labeled with neurofilament antibodies (green). Axonal fascicles of ganglion cells are also intensely stained in the optic fiber layer. (I) Ethidium bromide nuclear staining (red) and ChAT immunostaining (green) demonstrate the normal layering and lamination of the transgenic retina. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; OPL, outer plexiform layer; IPL, inner plexiform layer.

   
   
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   Fig. 3.

   Ectopic expression of Nrl in S-opsin-expressing cone photoreceptors. (A and B) Quantification of S-cones in the inferior domain of flat-mounted retinas from WT and BPp-Nrl/WT mice with anti-S-opsin antibody (A) revealed a 40% decrease in S-cones. Light-adapted ERG photoresponses from WT and BPp-Nrl/WT mice are shown in B [photopic b-wave (Left) and photopic b-wave at maximum intensity (Right)]. In BPp-Nrl/WT mice, ≈50% reduction in the photopic b-wave amplitude is observed compared with the WT mice. (C–N) Immunostaining of cryosections from Nrl ^−/− retina show the lack of rhodopsin expression and higher S-opsin expression in the ONL (C–F). In the BPp-Nrl/Nrl ^−/− retina rhodopsin expression can be detected in the ONL and the OS (G and K). Hybrid photoreceptors expressing both S-opsin (H and L) and rhodopsin can be observed in the ONL, INL, and the GCL (G–N). OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer; BBZ, bisbenzamide. (Scale bar: C–N 50 μm.) Arrows refer to colocalization of S-opsin and rhodopsin in the INL and GCL; arrowheads refer to colocalization of S-opsin and rhodopsin in the ONL.

   
   
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   Fig. 4.

   Association of Nrl to cone-specific promoters. (A and B) EMSA. Radiolabeled double-stranded oligonucleotides from Thrb and S-opsin promoters were incubated with RNE, followed by nondenaturing PAGE. Lanes are as indicated. Arrows represent specific shifted bands. Competition experiments were performed with increasing concentration (1-, 5-, or 50-fold molar excess, respectively) of unlabeled specific oligonucleotide or 50-fold higher concentration of nonspecific (ns) oligonucleotide, to validate the specificity of band shift. Anti-NRL or normal rabbit IgG was added in some of the reactions, as indicated. Disappearance (see A) or increased mobility of the shifted band (B; shown by asterisk) was detected with anti-NRL antibody but not IgG. These experiments were performed three times, and similar results were obtained. (C) ChIP assay. WT or Nrl ^−/− mouse retina was used for ChIP with anti-NRL or rabbit IgG antibody. The positive and negative controls for ChIP assays are Pde6b and albumin, respectively. Lanes are as indicated. Input DNA served as positive control for PCR.

   
   
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   Fig. 5.

   A model of photoreceptor specification. Otx2 and Rb influence multipotent retinal neuroepithelial cells to exit cell cycle. We hypothesize that Crx is the competence factor in postmitotic photoreceptor precursors. The cells that express Nrl are committed to rod photoreceptor fate, with subsequent expression of Nr2e3. The cells expressing only Crx are cone precursors. We propose that a degree of plasticity exists in early retinal development, such that changes in Nrl and/or Nr2e3 expression can lead to alterations in final ratio of rod and cone photoreceptors. Additional transcription factors (14, 62) are required to guide the development to mature photoreceptors.