Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina

Abstract

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.

Figure 1. Normal expression of rod and cone molecular markers in early development in erd.

Sections from 4 wk normal (A) and 4.3 wk mutant (B) stained with H&E (A1, B1), or labeled with antibodies against rod opsin (A2, B2; green), L/M-cone opsin (COS-1, green)/hCAR (red) (A3, B3), and S-cone opsin (OS-2, green)/hCAR (red) (A4, B4) with a DAPI (blue) nuclear stain. With the exception of rod opsin which shows slight delocalization of label into the outer nuclear (*) and plexiform layers, opsin labeling is restricted to the outer segments. hCAR labeling of cones is present throughout the cell. RPE = retinal pigment epithelium, ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer. Scale bar 40 µm.

Figure 2. Early rod abnormalities in erd.

Immunofluorescence labeling with anti-rod opsin antibody (green) shown with (A1, B1, C1) and without (A2, B2, C2) the blue DAPI nuclear stain in normal (A1, A2) and erd-mutant (B1, B2, C1, C2) retinas of different ages (weeks = w). (A1, B1, C1) Images of H&E stained adjacent cryosections are included for illustration. The boxed areas in the first row are presented at higher magnification in the panels below. (A1, A2) In normals, rod opsin labeling is restricted to the well oriented outer segments. (B1, B2, C1, C2) Rod outer segments are variable in length and irregular in erd, and opsin delocalizes to the inner segments, ONL (*) and OPL synaptic terminals (oblique arrows). The rod opsin delocalization is visualized best without DAPI. ONL = outer nuclear layer, OPL = outer plexiform layer, INL = inner nuclear layer. Scale bar 40 µm for principal panels.

Figure 3. Photoreceptor cell death and proliferation in erd.

(A, C) TUNEL and PCNA labeling, respectively, in erd is sustained between 7.7–14.1 wks with many labeled cells in the ONL of the superior (Sup.) and inferior (Inf.) meridians (data from both quadrants combined for normal; data points expressed as mean ±1 SD). Color insets illustrate the labeled cells (green) from a 7.7 wk old mutant animal for the corresponding assay in sections with hCAR antibody that labels all cones (red). (B) The number of photoreceptors in the outer nuclear layer (ONL), expressed as the mean number of rows of nuclei, remains relatively constant until 14.1 wks, and then decreases. Sup.A1/Inf.A1 = superior/inferior area 1, 2000±500 µm from the optic disc; Sup.A2/Inf.A2 = superior/inferior area 2, mid point ±500 µm between optic disc and ora serrata. For the ONL, the mean nuclei counts are presented. Between 4.3–14.1 wks of age, all the SD for ONL were ≤15% of the mean, with greater than 60% of the values being less than 10% of the mean.

Figure 4. Phospho-histone H3 (PHH3) labeling of mitotic cells.

Sections examined by epifluorescence (A–E) or confocal (F–G) microscopy using single (PHH3; A, D1, D2) or double labeling (B, C -TUNEL; E, F, G -rod opsin), and DAPI (blue) nuclear staining. (A) Cell division occurs in the normal retinal periphery of young dogs [1 and 1.6 (inset) weeks of age] as the outer neuroblastic layer (ONbL) begins to separate (*), and the outer and inner nuclear (ONL, INL) and outer plexiform (OPL) layers form. Labeled nuclei (arrows) are large, ovoid and located at the external edge of the retina. (B, C, D1, D2). In erd, PHH3 labeled nuclei are small, round (arrows), located at all levels of the ONL, and labeling does not co-localize with TUNEL labeled (green) nuclei (B,C and D1,D2 are different dogs at 11.6 wk of age). (E,F) Epifluorescence (E, 11.6 wks of age) and maximally projected confocal image (F, 7.7 wks of age) shows individual or clustered (arrows or oval, respectively) PHH3 labeled nuclei (orange or purple nuclei) in the ONL, and rod opsin (green) delocalization into the ONL and synaptic terminals (E, arrowheads). (G, G1–G4) Single confocal sections from a different region of the same retina as (F) using rod opsin (green), PHH3 (red-purple), and DAPI (blue) labeling. (G1–G4) Higher magnification of the boxed region showing colocalization of rod opsin labeling surrounding PHH3 (purple) labeled nucleus (G2), and the individual channels for rod opsin (G1), PHH3 (G3) and DAPI (G4). Scale bars: a–e = 20 µm, f–g, g1–4 = 10 µm.

Figure 5. L/M-, S- cones, and hybrid rod/S-cones in the erd retina.

Double immunofluorescence (A: L/M-opsin = green, hCAR = red; B: S-opsin = green, hCAR = red; C: S-opsin = green, rod opsin = red; D: NRL = green, PNA = red; E: NR2E3 = green, PNA = red; DAPI nuclear stain = blue) in normal and erd retinas of different ages (weeks = w). The boxed areas in A and B are presented at higher magnification in the panels below, and vertical arrows identify the same cells. (A, B) Control cones label distinctly but variably with hCAR; L/M-cones labeled with COS-1are more numerous, and only few S-cones are present. Other than cone outer segment disorientation in the older erd retina, L/M-cone opsin labeling is normal and restricted to the outer segments of these cells. In mutants, the OS-2 antibody distinctly labels S-cone outer segments, and also there is more diffuse but less intense labeling over the rod outer segments. (C) Colocalization of S-opsin and rod opsin labeling in hybrid rod/S-cone photoreceptors is demonstrated in single and merged images. Because the S-opsin (green) labeling is weaker than rod opsin (red) in rods, the green signal has been enhanced in the merged image to illustrate colocalization. Closed (A, B) or open (C) arrows identify the same cells. (D, E) Double labeling with PNA and NRL or NR2E3, respectively, in normal and mutant retinas. In the mutant, NRL labels the outer segments of presumable hybrid rod/S-cones (vertical arrows) that are not invested by a PNA positive insoluble cone extracellular matrix; oblique arrows point to cone nuclei which are not labeled and * identifies an NRL labeled cone nucleus in normal retina. The mutant retina shows absence of NR2E3 labeling. ONL = outer nuclear layer, INL = inner nuclear layer. Scale bar 40 µm for principal panels in A, B, and 20 µm for C–E.

Figure 6. Retinal gene expression changes during normal development and disease.

Expression was analyzed for genes that are (A) causally associated with the mutation or are part of the same gene family, or (B) involved with the cell cycle. Statistical significance between different groups in comparison to the 16 wks old normal control retinas (n = 3), depicted by horizontal line at 1.0 Fold Change, was assessed with an unpaired t-test, and expressed as statistically significant (** = p≤0.05) or towards statistical significant (* = 0.05≤p≤0.1). For STK38L, one probe was located within the exonic deletion [STK38L (exon 4)], and the second in exon 6, 3′ to the splicing defect [STK38L (exon 6)]. Noted below each time point are the number of different samples used (n = ) at each time point.

Figure 7. Protein expression in erd retina.

Expression of (A) CNGA3, L/M-opsin, S-opsin, RDS, and (B) NRL, NR2E3, CRX proteins in cleared whole retinal lysates. (C) Net intensity densitometric values are expressed as corrected net intensity of the sample normalized to corrected net intensity of actin loading controls. Actin loading control is the same for S-opsin and RDS, and for NRL, NR2E3 and CRX, respectively. Results are the mean ± SD of four independent densitometer scans of two individual westerns. Confidence levels: * = 95%, ** = 99%.

Figure 8. Abnormal outer segment renewal in erd rods.

(A1–3) Rod outer segment renewal examined 4 days following intravitreal injection of ³H-fucose in 10.9 week old normal and (A4–6) erd-affected dogs. The images (A1,A2 and A4,A5) are serial sections taken from the posterior pole of the superior retinal quadrant. The transmitted bright-field autoradiogram (A2) shows in the normal a band of labeling whose leading edge extends to 2/3 of the rod OS length (A2, horizontal arrow) with a trailing tail of radioactivity. This is better visualized in the combined transmitted/epipolarizing image (A3, arrow) that also shows diffuse label in the cone OS (oblique arrows). The mutant retina (A4–6) shows diffuse label at all levels of the OS layer (A5,A6; brackets) that is similar in rods and cones. RPE = retinal pigment epithelium; OS = outer segments; IS = inner segments; ELM = external limiting membrane; ONL = outer nuclear layer. (B) Relative incorporation of ³H-fucose/¹⁴C-leucine into opsin at different time points following a single intravitreal injection. All data are normalized to incorporation of label at day 1. The erd outer segments initially take up both labels (day 1), but label intensity decreases at later post injection times, suggesting that label is diffusely distributed through the rod outer segment membranes after discs are formed, and outer segment tips are lost to the RPE by daily shedding/phagocytosis events.