Figure 1.
Cre-mediated ablation of Ranbp2 selectively in cone photoreceptors.
(A) Schematic diagram for targeted Ranbp2 allele upon HGRP-driven Cre excision of exon 2 and production of the recombinant mRNA of Ranbp2. Pr1 and Pr2 are specific to fused exons 1 and 3, and exon 5, respectively, and were used to monitor the functional excision of Ranbp2Flox allele. (B) Schematic diagram of a constitutively targeted Ranbp2 allele upon insertion of a promoterless bicistronic (β-geo-PLAP) cassette with a splicing acceptor site (SA) between exons 1 and 2. (C) RT-PCR of recombinant Ranbp2 mRNA without exon 2 using Pr1 and Pr2 primers. Excision of exon 2 (ΔE2) was detectable in HRGP-cre:Ranbp2−/− (−/−) but not wild-type (+/+) mice at P7 of age, a day after expression of Cre recombinase. (D) Co-expression (a″″–c″″) of Arr4 (a′), M-opsin (b′), S-opsin (c′), Cre (a″–c″) and PNA (a′″–c′″) in cone photoreceptor neurons of the superior (dorsal, a–b″″) or inferior (ventral, c–c″″) regions of the retina of HRGP-cre:Ranbp2+/− mice at P15. Sections were counterstained with DAPI (a–c). Legends: HGRP, L/M opsin promoter; Arr4, cone arrestin 4; PNA, Peanut Agglutinin. Scale bars = 25 µm.
Figure 2.
Temporal and morphological profile of degeneration of cone photoreceptors in Ranbp2−/− mice.
(A–H′″) Immunohistochemistry of retinal sections of HRGP-cre:Ranbp2+/− (Ranbp2+/−) and HRGP-cre:Ranbp2−/− (Ranbp2−/−) mice at P9 (A–B′″), P13 (C–D′″) P20 (E–F′″) and P27 (G–H′″) of age with the antibodies against Cre recombinase and cone arrestin (Arr4) showing rapid loss of cone photoreceptors in RanBP2−/−. Cre co-localized with Arr4 only in DAPI-stained cell bodies of cone cells throughout all ages regardless of genotype. Arrowheads in indicate Cre+ nuclei retracted to the proximal ONL (outer nuclear layer) in Ranbp2−/− at P13, P20 and P27 of age, respectively. Arrows point to prominent swellings of synaptic pedicles of cone photoreceptors in Ranbp2−/− at P20 of age. Scale bars = 25 µm.
Figure 3.
Topographic degeneration of M-cone photoreceptors and their outer segments in HRGP-cre:Ranbp2−/−.
(A) Retinal flat mount images of HRGP-cre:Ranbp2−/− (−/−) and HRGP-cre:Ranbp2+/− (+/−) immunostained with an antibody against M-opsin showing progressive and severe M-cone cell loss in the retina of −/− mice. (a′–f′) Magnifications of the superior/central inset regions of a–f. (a″–f″) 3D-reconstruction images from a′–f′. (B–C) Quantitative and temporal analyses of the number (B) and length of outer segments (C) of M-cone photoreceptors in the superior (S)/central regions of retina of −/− and +/− mice. (D) Comparison of topographic distribution of Cre+ cells in the retina of −/− and +/− mice at P13 showing the proximal localization of a few displaced cone cells in −/− mice. White dashed line represents a virtual midline boundary between proximal and distal areas of the outer nuclear layer (ONL) used for tallying the topography of Cre+-cell bodies in (E). (E) Quantitative and morphometric analyses of the localization of Cre+ cells in the proximal versus distal ONL and central versus peripheral retina between −/− and +/− mice. About 20% of Cre+ cells are present in the proximal ONL region of −/− at P13 and there is greater number of Cre+-cell loss in the central retina of −/− mice at P20. Legend: Boxes in A (a–f) are ROIs used for quantitation of data shown in B and C. Data shown represent the mean ± SD, n = 3–5; **, p<0.01; ***, p<0.001. Scale bars = 150 µm (a–f), 20 µm (a′–f′), 50 µm (D).
Figure 4.
Spatiotemporal modalities of cell death between Cre+, Arr4+ and Arr4−-photoreceptor cell bodies in HRGP-cre:Ranbp2−/−.
(A) Comparison of the relative 3-D distribution between Cre+-cone and TUNEL+ cells in the outer nuclear layer of retinas of HRGP-cre:Ranbp2+/− (+/−, top) and HRGP-cre:Ranbp2−/− mice (−/−, bottom) at P20 shows that all TUNEL+ cells are Cre−. (B) Morphometric and topographic distribution of TUNEL+ cells in outer nuclear layer (ONL, proximal vs distal) and regions of retina (peripheral vs central) between HRGP-cre:Ranbp2+/− (+/−) and HRGP-cre:Ranbp2−/− mice (−/−) at P13, P20 and P27. TUNEL+ cells become prominent at P20 with most localizing at the distal ONL of central and peripheral retina. No TUNEL+ cells were identified by P27. Data shown represent the mean ± SD, n = 4–5; ***, p<0.001; o, p<0.0001. (C) Identification and localization of TUNEL+ (apoptotic) and EthD-III+ (necrotic) in Arr4−-photoreceptor cell bodies of P20 retinas of HRGP-cre:Ranbp2+/− mice (+/−; a–a′″) and HRGP-cre:Ranbp2−/− mice (−/−; b–b′″). Representative images of TUNEL+ (a′–b′) or EthDIII+ (a″–b″) cell bodies of Arr4−-photoreceptors (a′″–b′″) and co-localization of these (a″″–b″″) are shown. No TUNEL+ and EthD-III+-cell bodies in Arr4+-photoreceptor cell bodies were identified. Sections were counterstained with DAPI (a–b). White arrows in b′–b′″ indicate the localization of TUNEL+EthD-III+ cell bodies. (D) Quantification analysis of C. Among the Arr4−-photoreceptor cell bodies tallied, approximately 50, 30 and 20% were TUNEL−EthD-III+, TUNEL+EthD-III− and TUNEL+EthD-III+, respectively, in −/− mice, whereas they were negligible in +/− mice. Data shown represent the mean ± SD, n = 3; ***, p<0.001; **, p<0.02; *, p<0.005; Scale bars = 50 µm (A), 20 µm (C).
Figure 5.
Cell death in Nr2E3+-cell bodies of rod photoreceptors in HRGP-cre:Ranbp2−/−.
(A) Representative images of TUNEL+ (a′–d′), Cre+ (a″–d″) and Nr2E3+ (a′″–d′″) cell bodies and their colocalization (a″″–d″″) in rod photoreceptors of HRGP-cre:Ranbp2+/− (+/−; a–a″″) and HRGP-cre:Ranbp2−/− mice (−/−; b–d″″). d–d′″ are high magnifications of inset boxes of c–c″″. Sections were counterstained with DAPI (a–d). White arrowheads in a′–a″″ indicate Cre+Nr2E3− cells, white and green arrows in b′–b″″ point to TUNEL+Nr2E3+ and TUNEL+Nr2E3−, respectively. (B) Quantification analysis of A. Among the TUNEL+-cell bodies tallied, slightly over 50% were Nr2E3+ in −/− mice, whereas they were negligible in +/− mice. No TUNEL+Cre+-cell bodies were identified in −/− mice. % values are based on % of total DAPI+ cells (100%). Data shown represent the mean ± SD, n = 3; *; p<0.001. Scale bars = 20 µm (a–c″″), 5 µm (d–d″″).
Figure 6.
Spatiotemporal activation of caspases 3 and 7 and Parp1 in photoreceptors of HRGP-cre:Ranbp2−/− mice.
(A) Temporal profile of caspase 3/7 activation of retinal extracts of HRGP-cre:Ranbp2−/− mice (−/−) mice relative to age-matched +/− controls shows significant caspase3/7 activation at P13 and P20. The caspase 3/7 activities of +/− and +/+:Cre controls shown are at P20. (B) Quantitative, temporal and relative distributions between activated (cleaved) caspase 3 (c-casp3+), Cre+ and TUNEL+ in cell bodies of photoreceptors of HRGP-cre:Ranbp2+/− (+/−) and HRGP-cre:Ranbp2−/− mice (−/−) at P13 and P20. The majority of photoreceptor cell bodies are c-casp3+Cre−. The levels of any type of c-casp3+ cell bodies are negligible by P20. (C) Relative immunolocalizations of Cre+ and c-casp3+ (a–a′″, d–d′″), Cre+ and c-casp7+ (b–b′″, e–e′″), and Cre+ and activated Parp1 (c-Parp1+) (c–c′″, f–f′″) cell bodies of photoreceptors of HRGP-cre:Ranbp2−/− mice at P13 (a–c′″) and P20 (d–f′″). c-casp3+ develop at P13, c-casp7+ and c-Parp1+ at P20 and all c-Parp1+ cell bodies are Cre−, whereas c-casp7+ are Cre+. Legend: Data shown represent the mean ± SD, n = 4–5; *; p<0.05, **; p<0.01; scale bars = 25 µm; cleaved caspases 3 and 7, c-casp3 and c-casp7, respectively.
Figure 7.
Activation of matrix metalloproteinase 11 (MMP11) upon ablation of Ranbp2 in cone photoreceptors.
(A) Light microscopy images of methylene blue stained semi-thin retinal sections showing the presence of prominent interstitial spaces (black arrows) originating from cone cell bodies (white arrows) and between cell bodies of rod photoreceptors in HRGP-cre:Ranbp2−/− (−/−) compared to HRGP-cre:Ranbp2+/− (+/−) mice. * denote euchromatic nuclei of cones. (B) Electron micrographs depicting ultrastructural changes in the lower fiber of a −/− cone photoreceptor. Note the swelling of the lower fiber (arrows) traced to the cell body of a cone nucleus characterized by its prominent euchromatin (*). *, cone nuclei. (C) Temporal profile of MMP11 activity of retinal extracts of HRGP-cre:Ranbp2−/− mice (−/−) mice relative to age-matched +/− controls shows significant MMP11 activity at P13 and P20. The MMP11 activity of +/− and +/+:Cre controls shown are at P20. (D) Immunoblots of MMP11 from retinal homogenates of HRGP-cre:Ranbp2−/− (−/−) compared to +/− mice at P20 showing an increase of the active form of MMP11 in −/−. Hsc70 is cytosolic heat shock protein 70 used as loading control. (E) Quantitation analysis of the levels of active MMP11 in (D) of HRGP-cre:Ranbp2−/− (−/−) relative to age-matched +/− mice at P20. (F) Retinal sections immunostained with antibodies against cone arrestin (Arr4) and MMP11. (a–d) central retinal region of HRGP-cre:Ranbp2+/− (+/−); (e–f) central retinal region of HRGP-cre:Ranbp2−/− (−/−); (e′–f′) magnification of boxed regions shown in e–f; (i–l) peripheral retinal region of HRGP-cre:Ranbp2+/− (+/−); (m–p) peripheral retinal region of HRGP-cre:Ranbp2−/− (−/−); (m′–p′) magnification of lower fibers of cones of boxed regions shown in m–p. MMP11 predominantly localizes to cone photoreceptors, such as interstitial space around cell bodies, lower fibers and inner segments. Legend: Data shown represent the mean ± SD, n = 4; *, p<0.05; **, p<0.01; o, p<0.0001; scale bars = 25 µm (A, F), 5 µm (B).
Figure 8.
Ultrastructural changes of cone and rod photoreceptors upon ablation of Ranbp2 in cones at P20.
(A) Ultrastructural image of rod and cone photoreceptors outer and inner segments of HRGP-cre:Ranbp2+/− (+/−). All structures look unremarkable. (B–E) Representative ultrastructural images of multiple features rod and cone photoreceptors outer and inner segments of HRGP-cre:Ranbp2−/− (−/−). (B) shows the collapse of the cone outer segment and formation of a large electron lucent area, (C) shows extended discs and partial erosion of cone outer segment, (D) shows the accumulation of amorphous electron dense material at the connecting cilium of a cone photoreceptor, (E) shows the formation of electron lucent areas in the inner segment of a cone photoreceptor. White stars, euchromatic nuclei of cone photoreceptors. (A′–E′) are magnifications of boxed areas in A–E showing the aforementioned pathological features. (F) Synaptic pedicles of cones with normal morphology of HRGP-cre:Ranbp2+/− (+/−). (G) Multi-lamellar body with electrodense material in a cone synaptic pedicle of HRGP-cre:Ranbp2−/− (−/−). (H) Electron lucent area surrounding subcellular debris in a cone synaptic pedicle of HRGP-cre:Ranbp2−/− (−/−). F′–H′ are magnified images of boxed areas in F–H. (I) Synaptic spherule of a rod photoreceptor containing unremarkable mitochondria (black arrows) in HRGP-cre:Ranbp2+/− (+/−). (J) Synaptic spherule of a rod photoreceptor with abnormal mitochondria (black arrows) containing disorganized cristae and electron lucent areas in the matrix in HRGP-cre:Ranbp2−/− (−/−). Scale bars = 5 µm (A–E), 1 µm (A′–E′), 500 nm (F–H) and 200 nm (F′–H′).
Figure 9.
Temporal profiles of changes in gene expression by ablation of Ranbp2 in cone photoreceptors.
(A–G) qRT-PCR of cone-specific (A, B), pan-photoreceptor (C), cone survival (D) and cone transcription factors (E), col6α3 and col1α1 (F), and Gfap and Hif1α transcripts (G). There is a decrease of cone-specific transcripts beginning at P13 (A, B), whereas pan-photoreceptor (C) and cone survival genes (D) begin transient up-regulations at the same age. The expression of cone survival genes peaks at P20 (D). (E) The transcription factors, Crx and CoREST (Rcor1), are the first nuclear factors whose changes of their transcriptional levels coincides with the genetic ablation of Ranbp2 at P7. These events are followed by the up-regulations at P20 of the transcription factors, Nrp1, Trß2 and Otx2 (E). The up-regulations of transcripts encoding the substrate of MMP11, col6α3, begins at P9 and peaks at P20 (F), whereas those for the hypoxia marker, Hif1α, and inflammatory marker, Gfap, begin at P9 and P20, respectively (G). Legends: Data shown represent the mean ± SD, n = 3–4; *, p<0.05; **, p<0.01; ***, p<0.001; refer to table S1 for gene designations/symbols.
Figure 10.
Connectivity map of a gene network linked to Ranbp2 functions.
Ingenuity pathway analysis (IPA) of factors modulated by loss of Ranbp2 identified a top network implicated in “Cellular and Nervous System Development and Function, and Carbohydrate Metabolism” (score of 22). Dashed and solid blue arrows were added manually to the network to reflect the down-regulation and up-regulation of genes (shown in green and red), respectively, observed first upon ablation of Ranbp2 in cone photoreceptors as identified by this work. Note that Crx and Mpp11 expressions underwent first transient down-modulation at P7 followed by their sustained up-regulation. Legend: ARPP21, cAMP-regulated phosphoprotein, 21 kDa; BDNF, Brain derived neurotrophic factor; C5orf34, chromosome 5 open reading frame 34; cbp, CREB-binding protein; CCPG1, cell cycle progression 1; CEBPA, CCAAT/enhancer-binding protein alpha; CRX, Cone-rod homeobox protein; Cyp2d1/Cyp2d5, cytochrome P450, family 2, subfamily d, polypeptide 1/cytochrome P450, family 2, subfamily d, polypeptide 5; DNAJC21, DnaJ homolog subfamily C member 21; EP300, also called p300, E1A binding protein p300; FUT1, fucosyltransferase 1; HOXB2, homeobox B2; HTT or Hd, huntingtin; Kcnip2, Kv channel-interacting protein 2; KLF16, Kruppel-like factor 16; let-7, Let-7 microRNA precursor; LHX2, LIM/homeobox protein2; mir-124, microRNA 124; MMP11, Matrix Metalloproteinase 11; MNDA, myeloid cell nuclear differentiation antigen; NDUFA3, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 3; NEUN, Feminizing Locus on X-3, Fox-3, or Hexaribonucleotide Binding Protein-3; OPN1LW, long-wave-sensitive opsin (cone pigment); OTX2, orthodenticle homeobox 2; PCTP, Phosphatidylcholine transfer protein; PPP1R16B, protein phosphatase 1, regulatory subunit 16B; sn-glycero-3-phosphocholine, 2,3-dihydroxypropyl 2-(trimethylazaniumyl)ethyl phosphate; SP1, Sp1 transcription factor; THRB, thyroid hormone receptor, beta; Taf4, TAF4 RNA polymerase II, TATA box binding protein (TBP)-associated factor; Taf9b, TAF9B RNA polymerase II, TATA box binding protein (TBP)-associated factor; Tbp, TATA box binding protein; TR, Thyroid Hormone Receptor; RXR, Retinoic acid receptor; tretinoin, all-trans retinoic acid or ATRA; WNT2B, wingless-type MMTV integration site (WNT) family2B.
Figure 11.
Electrophysiological responses of cones and rod photoreceptors upon ablation of Ranbp2 in cones.
(A) Representative electroretinograms (ERGs) of control HRGP-cre:Ranbp2+/− (+/−, black) and HRGP-cre:Ranbp2−/− mice (−/−, red) mice at P13 and P29 to a 1.4 log cd s/m2 stimulus flash presented in darkness (left) or superimposed upon a steady rod-desensitizing adapting field (right). (B–F) Luminance-response functions for the major components of the dark-adapted rod (B, F) or light-adapted cone ERGs (C–E) at P13 (B, C), P22 (D) or P29 (E, F). Repeated measures analysis of variance was used to compare luminance-response functions of +/− and −/− mice for the amplitude of the major ERG components. Cone ERG amplitudes were significantly reduced in −/− mice at all ages examined (P13, P18, P22, P29, P150). Dark-adapted a-wave amplitudes were not different between +/− and −/− mice at any age examined. Dark-adapted b-wave luminance-response functions were also not different between +/− and −/− mice at any age examined. At P29 (F) and P150, dark-adapted ERG b-waves were significantly reduced (p<0.05, t-test, denoted by *) for stimuli above 0.0 log cd s/m2, but not for lower luminance stimuli. (G) Amplitude of −/− responses expressed relative to those of +/− littermates for the major ERG response components. Each measure reflects a single stimulus condition: ‘Light-adapted b-wave’ represents cone ERGs obtained to a 1.4 log cd s/m2 stimulus flash superimposed upon a steady adapting field; ‘Dark-adapted a-wave’ represents rod ERG a-waves obtained to a 1.4 log cd s/m2 stimulus flash presented in darkness; ‘Dark-adapted b-wave’ represents rod ERG b-waves obtained to a −1.2 log cd s/m2 stimulus flash presented in darkness. Data points indicate the average ± s.e.m. for n = 5–10 mice.