BETA2/NeuroD1 null mice: a new model for transcription factor-dependent photoreceptor degeneration

Abstract

BETA2/NeuroD1 is a basic helix-loop-helix transcription factor that is expressed widely throughout the developing nervous system. Previous studies have shown that BETA2/NeuroD1 influences the fate of retinal cells in culture. To analyze the effect of BETA2/NeuroD1 on the structure and function of the retina, we examined a line of BETA2/NeuroD1 knock-out mice that survives until adulthood. At 2-3 months of age, homozygous null mice showed a 50% reduction in rod-driven electroretinograms (ERGs) and a 65% reduction in cone-driven ERGs. ERGs measured from knock-out mice that were >9 months of age were undetectable. At 2-3 months, the number of photoreceptors in the outer nuclear layer was reduced by 50%. In addition, electron microscopy showed that the surviving photoreceptors had shortened outer segments. The number of cones labeled by peanut agglutinin was decreased 50-60%. By 18 months, retinas from null mice were completely devoid of photoreceptors. There appeared to be few changes in the inner retina, although BETA2/NeuroD1 is expressed in this area. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling staining revealed a dramatic increase in cell death, peaking at approximately postnatal day 3 and continuing into adulthood. No defects in cell birth were detected using bromodeoxyuridine staining. Our results reveal that BETA2/NeuroD1 not only plays an important role in terminal differentiation of photoreceptors but also serves as a potential survival factor. Loss of BETA2/NeuroD1 results in an age-related degeneration of both rods and cones.

Fig. 1.

Expression of BETA2/NeuroD1 in the developing and adult retina. A–F, X-gal staining from +/− mice at E10.5, E13, E16.5, E18.5, P3, and P30. G–L, X-gal staining from −/− mice at the same time points. The pattern of expression is similar for +/− and −/− mice all time points. However, null mice display a higher level of expression because of the presence of two copies of the β-gal gene. At earlier time points (A, B and G, H) BETA2/NeuroD1 is expressed central to the peripheral pattern in both the inner and outer retina. High levels of expression are seen in the outer layers of the retina, and punctate staining is seen in the ganglion cell layer.

Fig. 2.

ERGs from BETA2/NeuroD1 mice. Representative ERGs from 1- to 3-month-old (first three columns) and 10-month-old (fourth column) BETA2/NeuroD1 mice.A–D, Measurement of scotopic b waves. Traces were obtained using a series of 500 nm flashes with intensities ranging from −3.86 to −1.76 log scotopic cd-sec/m² (estimated to produce between 0.13 and 16.1 photoisomerizations per rod in photoreceptors with normal outer segments). Each trace represents the average of at least five flashes. E–H, ERG response to an intense flash (2.97 log scotopic cd-sec/m² or an estimated 800,000 photoisomerizations per rod). The initial negative deflection is the saturated a wave. I–L, Close-up of multiple a-wave responses to flashes varying in intensity from −0.51 to 2.97 log scotopic cd-sec/m². The traces have been fitted using the Lamb–Pugh model (dotted lines) as described in Materials and Methods. M–P, Cone-isolated ERGs using the double-flash method. Only the response to the second flash is displayed.

Fig. 3.

Light microscopy and EM of the retina in BETA2/NeuroD1 mice. Light microscopy of retinal slices shows an area taken from the posterior retina of 2-month-old +/+ mice (A), 2-month-old −/− mice (B), and 18-month-old −/− mice (C). In 2-month-old null mice, the ONL thickness is reduced to four to five cells. Null mice also demonstrate decreased thickness of the outer segments (OS), inner segments (IS), and OPL. In 18-month-old −/− mice, the ONL has completely degenerated. Scale bar, D (forA–C), 20 μm. EM for 2-month-old +/+ (D) and −/− (E) mice focusing on the OS is shown. In null mice, both outer and inner segments are shorter. Scale bar, F (for Dand E), 10 μm. F, EM of a rod synapse in a +/+ mouse. Note the presence of a synaptic ribbon (arrow), two postsynaptic horizontal cell processes (h), and a bipolar cell dendrite (b) forming a classic synaptic triad.G, OPL taken from a −/− mouse. Note the presence of multiple ribbons in rod terminals (arrows).H, Higher magnification taken from a −/− mouse showing multiple synaptic ribbons. RPE, Retinal pigment epithelial. Scale bar, 0.5 μm forF–H.

Fig. 4.

Analysis of cone photoreceptors and synaptic ribbons. A, B, Flat-mount retinas from +/+ and −/− mice stained with PNA to label cone photoreceptors. Labeled cones are reduced ∼60% in null mice. C–E, Retinal slices from +/+, +/−, and −/− mice double labeled with PNA (green) and anti-kinesin II (red). Null mice have decreased numbers of cone synapses (arrowheads), decreased thickness of anti-kinesin-labeled ribbons in the OPL (arrow), and aberrant ribbons present in the ONL (asterisks).F, G, Stacked images from flat-mount retinas taken at the level of the OPL and stained with anti-kinesin II. Labeled synaptic ribbons are significantly decreased in null mice. Some blood vessels (bv) are also labeled. Drawings of the retina are modified from Dowling and Boycott (1966). CB, Cone cell body; RB, rod cell body.

Fig. 5.

Detection of apoptosis in BETA2/NeuroD1 mice.A–F, TUNEL staining from (+/−) mice at E16.5, E18, P3, P5, P10, and P30. G–L, TUNEL staining from −/− mice at the same time points. In the control mice, some TUNEL-positive cells are observed from E18 to P3. In adult mice, few if any TUNEL-positive cells are observed. In contrast, null mice show a higher number of TUNEL-positive cells at E18, and the count increases at P3. The number of dying cells seems to decrease after P3, but cell death is present even in the adult retina. Most of the cell death seems to be occurring in the ONL, the location of developing photoreceptors.

Fig. 6.

Gliosis in BETA2/NeuroD1 null mice.A–C, Immunohistochemical staining with anti-glutamine synthetase. There is little difference in the number Müller glia in the different lines of mice. D–F, Immunohistochemical staining with anti-GFAP in +/+, +/−, and −/− mice. In wild-type and heterozygous mice, anti-GFAP stains only astrocytic processes near the inner limiting membrane. In null mice, anti-GFAP staining demonstrates a reactive gliosis in Müller glia.

Fig. 7.

Analysis of bipolar cells in BETA2/NeuroD mice.A–C, Immunohistochemical staining using anti-G_oα in +/+, +/−, and −/− mice. Anti-G_oα stains rod bipolar and ON-cone bipolar cells. There is little difference between wild-type, heterozygous, and null mice. D–F, Immunohistochemical staining using anti-PKC, which labels rod bipolar cells. G, H, Cross sections taken from anti-PKC-stained flat-mount retinas. There is no significant difference between the number of labeled cells. OS, Outer segment.