Molecular Vision 1998; 4:5 <>
Received 4 August 1997 | Accepted 16 February 1998 | Published 2 March 1998

Apoptosis in the Murine rd1 Retinal Degeneration is Predominantly p53-Independent

R. M. P. Hopp,1 N. Ransom,2 S. G. Hilsenbeck,3 D. S. Papermaster,2 and J. J. Windle4

1Department of Cellular and Structural Biology, 2Department of Pathology, 3Department of Medicine, The University of Texas Health Science Center at San Antonio, TX; 4Cancer Therapy and Research Center, San Antonio, TX

Correspondence to: Jolene J. Windle, Ph.D., Department of Cancer Biology, Institute for Drug Development, Cancer Therapy and Research Center, 8122 Datapoint Dr., Suite 700, San Antonio, TX, 78229-3219, email:
Dr. Papermaster's present address is: Department of Pharmacology, School of Medicine, University of Connecticut Health Center, Farmington, CT, 06030.


Purpose: To determine if p53 mediates apoptosis in photoreceptors of retinal degeneration, rd1, mice.

Methods: The rd1/rd1 mice were interbred with p53 null mice to generate p53-/- rd1/rd1 and p53+/+ rd1/rd1 mice. Rates of loss and incidence of apoptosis in rod photoreceptors were analyzed at appropriate ages (postnatal days 12, 14 and 16).

Results: The extent and kinetics of photoreceptor cell loss in rd1 mice were nearly indistinguishable in the p53+/+ and p53 null mice.

Conclusions: Photoreceptor cell apoptosis in the rd1 mouse model occurs by a predominantly p53-independent molecular pathway.


Several genes have recently been identified that either positively or negatively regulate apoptosis [1]. One such gene, p53, encoding the p53 tumor suppressor protein, mediates apoptosis in response to DNA damage and certain cell cycle perturbations. However, a variety of forms of p53-independent apoptosis have also been identified [2]. Importantly, nearly all normal developmental apoptosis must be p53-independent, since most p53 null mice develop essentially normally [3,4]. Although the ability of p53 to regulate apoptosis in response to DNA damage or cell cycle perturbation as well as during development has been extensively examined, the role of p53 in post-mitotic cells, such as photoreceptors, has not been well addressed. If p53 does play a role in the regulation of programmed cell death in photoreceptors, then this could be a potential target for the treatment of retinal degeneration.

Inherited retinal degenerations are characterized by a progressive deterioration of vision resulting from the loss of function and viability of photoreceptors. Animal models of retinal degeneration in which the phenotype and genetic mutation correspond to the human disease have been widely studied. One such model, the rd1 retinal degeneration mouse, exhibits a rapid retinal degeneration initiated by a recessive mutation of the gene encoding the rod ß-subunit of the cyclic GMP phosphodiesterase [5,6]. These mice develop fully differentiated photoreceptors during the second postnatal week, but then suffer nearly complete loss of the rod photoreceptors in the next week, followed by a slower loss of cone photoreceptors [7]. The photoreceptor death in this model exhibits the morphologic features and DNA fragmentation characteristic of apoptosis [8]. However, the regulation of retinal apoptosis in the rd1 mouse model is still poorly understood.

To determine if the presence of p53 influences apoptosis in the rods of rd1 mouse retinas, we interbred rd1 mice with mice deficient for p53. We show that the rapid apoptotic loss of photoreceptors observed in this model is largely p53-independent.


Experimental Animals

FVB/NJ (rd1/rd1) mice were obtained from the Jackson Laboratory, ME. TSG-p53 (p53-deficient) mice [3], obtained from GenPharm International, CA, are maintained in our laboratory in a C57Bl/6 X Balb/c genetic background. rd1/rd1 and p53-/- mice were interbred to generate p53+/- rd1/+ double heterozygous F1 offspring, and these were subsequently interbred to obtain mice with the following genotypes of interest: p53+/+ rd1/rd1, p53-/- rd1/rd1, p53+/+ +/+, and p53-/- +/+. Litters of mice were sacrificed at postnatal day 12, 14, or 16 (P12, P14, or P16; day of birth is defined as P0).

Identification of Genetic Mutations

Tail tissue was taken for DNA extraction and prepared as described [9]. Tail DNA was used to determine the presence of the p53 and rd1 mutations by PCR-based strategies. The genetic status of p53, mutated or wild-type, was determined by using a three primer-based strategy previously described [10]. The rd1 nonsense mutation within the ß-PDE gene was identified by PCR amplification of the mutated region, followed by digestion with a restriction enzyme that distinguishes between the wild type and mutant allele, as described by Pittler and Baehr [6].

Detection of Apoptotic Cells

Eyes were enucleated with the optic nerve left intact to be used for orientation of anterior-posterior sections. Eyes were fixed overnight at room temperature in phosphate buffered 4% paraformaldehyde fixative and then embedded in Poly-Fin paraffin (Triangle Biomedical Sciences). Apoptotic cells were detected using the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) procedure [11]. Incorporated biotin was detected using a streptavidin-horseradish peroxidase conjugate (DAKO) and visualized with a DAB (diaminobenzidine) chromagen (Sigma). Sections were counterstained with methyl green.

Quantitation of Apoptosis

Retinas were aligned along the maximal diameter of the 40x objective field (610 µm length of retina) and numbers of TUNEL labeled nuclei in the outer nuclear layer (ONL) per high power field (hpf) were counted. Only those cells in which label was confined to the nucleus were counted. Both totally stained nuclei as well as those with punctate or peripheral staining were scored as TUNEL positive. The number of photoreceptor nuclei per row in the ONL were also counted, with a maximum index of 10 (the normal ONL thickness). Four hpfs were counted for each retina examined, two central hpfs (averaged to generate a single datapoint) and two peripheral hpfs (also averaged). The central hpf was aligned with the optic nerve at one edge of the field. The peripheral hpf was aligned distal to the outer border of the central hpf and proximal to the tapering of the retina at the ciliary body. The second eye from each animal was not evaluated, unless the first eye was damaged due to handling and could not be assayed. Because the sample sizes were relatively small, data were summarized by plotting individual data points and medians, and compared using the Wilcoxon Rank Sum test, a nonparametric analogue of the two sample t-test that does not require the normality assumption.


Qualitative Analyses of Retinal Morphology

TUNEL assay results for p53+/+ rd1/rd1, p53-/- rd1/rd1, and p53+/+ +/+ (wild-type) retinas at P12 and P16 are displayed in Figure 1. These micrographs illustrate qualitatively that at the onset of degeneration (P12), central retinal cross sections from all three genotypes appear virtually the same with regard to ONL thickness and morphology, although a slight increase in TUNEL label in rd1 retinas compared to wild-type is detectable. Only four days later, however, the p53+/+ rd1/rd1 and p53-/- rd1/rd1 mice have lost nearly all rod photoreceptors. Moreover, the p53-/- rd1/rd1 mice are nearly indistinguishable from the p53+/+ rd1/rd1 mice at P16. At all time points examined, p53-/- +/+ retinas are indistinguishable from p53+/+ +/+ retinas (data not shown).

Quantitative Analyses of Photoreceptor Loss

The qualitative results clearly demonstrate that the retinal degeneration in rd1 mice is largely p53-independent. However, to determine whether p53 status might have a subtle effect on the kinetics of the process, the apoptotic index and ONL thickness were determined at P12, P14 and P16 for rd1 mice in the presence and absence of p53. We detected a slight decrease in TUNEL labeling in the central retina of p53-/- rd1/rd1 mice at P14, and in the peripheral retina at P16, relative to the p53+/+ rd1/rd1 mice (Figure 2A,B). A slight statistically significant difference was also identified in the ONL thickness of the central retina at P16 (Figure 2C). However, no statistically significant differences were identified at the other time points for the parameters measured.


These results indicate that the retinal degeneration observed in rd1 mice is predominantly p53-independent (Figure 1). While there may be a slight delay in the apoptosis observed in p53-/- rd1/rd1 retinas, reflected in small differences in apoptotic cell indices at P14 and P16, this effect is subtle and does not affect the end result caused by the rd1 mutation, that is, complete loss of rod photoreceptors.

The variability in both TUNEL positive cells and ONL thickness, most apparent at P14, may indicate that the rate of photoreceptor loss varies somewhat between individuals. It may also suggest that at this critical midway point in the degeneration, small differences in age of the mice could lead to large differences in the extent of degeneration. The age of mice from different P14 litters could have differed by nearly as much as 24 hrs, since cages were checked for newborn pups once each day. Nevertheless, by P16, most of the photoreceptors in all mice have degenerated.

The time course of degeneration in this study, regardless of p53 genotype, appears to be slightly accelerated relative to that reported in previous studies of the rd1 phenotype [8,12]. While the explanation for this difference in kinetics is not clear, one possibility is that the genetic background of the mice influences the rate of degeneration, since a different strain of mice carrying the rd1 mutation was characterized in each study.

We have previously demonstrated that mice expressing HPV-E7 (which binds and inactivates the retinoblastoma gene product, pRb) in photoreceptor precursors undergo profound retinal apoptosis, but that when bred into a p53-deficient background, these mice develop retinal tumors [10]. However, examination of E7/p53-/- retinas at early postnatal ages revealed that apoptosis was slightly delayed, but not completely abrogated [13].

A variety of studies have demonstrated p53-independent apoptosis of neurons in response to withdrawal of neurotrophins [14,15]. In addition, developmental apoptosis in the nervous system is generally p53-independent, since most p53-deficient mice develop normally [3]. Interestingly, a fraction of p53-deficient embryos exhibit exencephaly, but it is not clear whether a defect in apoptosis is responsible for the condition [4]. In contrast, cerebellar granule cells have been shown to exhibit p53-dependent apoptosis in response to gamma-irradiation [16]. Together, these studies and ours indicate that p53 may be involved in neuronal apoptosis in some situations, including cell cycle perturbation or DNA damage, but is not generally involved in mediating developmental or other forms of apoptosis in neurons.

Why a particular cell type, neuronal or otherwise, exhibits p53-dependent or -independent apoptosis, or both, remains poorly understood. There are clearly multiple molecular pathways regulating this process. In addition, a variety of factors, including cell type, stage of differentiation, cell-cell interactions, extracellular matrix interactions, availability of growth factors, the stimulus used to provoke the apoptosis, etc., can influence the cellular susceptibility to apoptosis. Understanding the mechanisms involved in the regulation of apoptosis of post-mitotic cells like the photoreceptors of rd1 mice may lead to therapies directed at preventing these devastating degenerations.


This work was supported by Grant EY10992 (JJW and DSP) from The National Eye Institute, National Institutes of Health, Bethesda, MD and Grant P30 CA54174 (San Antonio Cancer Institute) from the National Cancer Institute, National Institutes of Health, Bethesda, MD.


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