|Molecular Vision 2000;
Received 20 April 2000 | Accepted 28 August 2000 | Published 8 September 2000
Ultrastructural and ERG findings in mice with adenomatous polyposis coli gene disruption
Dennis M. Marcus,1 Anil K.
Rustgi,2 Dennis Defoe,3 Raju Kucherlapati,4 Winfried
Edelmann,4 Duco Hamasaki,5 Gregory I. Liou,1,6 Sylvia
The Departments of 1Ophthalmology and 6Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA; 2Gastroenterology Division, Cancer Center, Department of Genetics, University of Pennsylvania, Philadelphia, PA; 3Department of Anatomy and Cell Biology, East Tennessee State University College of Medicine, Johnson City, TN; 4Department of Molecular Genetics, Albert Einstein College of Medicine, Bronx, NY; 5The Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami, Miami, FL
Correspondence to: Dennis M. Marcus, Department of Ophthalmology, Medical College of Georgia, 1120 15th Street, Augusta, GA; Phone: (706) 721-4804; FAX (706) 721-8328; email: firstname.lastname@example.org
Purpose: In order to continue the previous morphological studies of eyes from mice with adenomatous polyposis coli (APC) gene mutation at codon 1638, we determined the ultrastructural and electrophysiologic characteristics of these eyes.
Methods: Thirty-eight eyes from 20 mice heterozygous for APC gene mutation and 22 eyes from 11 wild-type mice were examined by light microscopy. Six APC-modified eyes without light microscopic abnormalities, four APC-modified eyes with focal light microscopic abnormalities, and four wild-type eyes were examined by electron microscopy. Electroretinograms were recorded from four APC-modified and three wild-type mice.
Results: Four of 38 APC-modified eyes demonstrated ultrastructural evidence of focal RPE cells with increased melanosome production and atrophy. Other areas of the RPE in these four eyes demonstrated no ultrastructural abnormalities. Three APC-modified eyes demonstrated electron and light microscopic evidence of RPE hyperplasia. Electron microscopic examination of APC-modified eyes without light microscopic evidence of abnormalities demonstrated no ultrastructural differences from age-matched controls. Electroretinography demonstrated no differences in the b-wave or c-wave amplitudes between APC-modified and wild-type mice.
Conclusions: While light microscopic RPE alterations are observed in these APC-modified mice, the absence of a generalized, ultrastructural murine RPE defect is in contradistinction to observations in electron microscopic investigations of humans with colonic polyposis, pigmented ocular fundus lesions, and APC gene mutations between codons 463 and 1444. Our results in mice with APC mutation at codon 1638, however, are consistent with a previously identified association between the expression of pigmented ocular fundus lesions and region-specific mutation in the human APC gene. The APC protein may possess a physiologic function for both retinal and RPE development.
Familial adenomatous polyposis (FAP) is an autosomal dominantly inherited disease that is characterized by numerous adenomatous colonic polyps that develop into adenocarcinoma of the colon . FAP may exhibit various extracolonic manifestations . Patients with FAP may present with ocular manifestations in the form of bilateral and multiple pigmented lesions of the retinal pigment epithelium (RPE) that represent a clinical marker for disease inheritance [2-7]. While the clinical appearance of these pigmented ocular fundus lesions (POFL) are similar to congenital hypertrophy of the RPE (CHRPE; sometimes found as unilateral and unifocal lesions in patients without polyposis) [8,9], postmortem studies in FAP patients demonstrate RPE hyperplasia and RPE hamartomatous growth in addition to CHRPE [5,6].
Mutations of the adenomatous polyposis coli (APC) gene, a tumor supressor gene mapped to chromosome 5q21, lead to the development and inheritance of FAP [2,10]. APC gene mutations have also been demonstrated to cause POFL in patients with FAP [11-13].
A murine model of colonic polyposis was generated by Fodde and coworkers by introducing a chain-termination mutation in codon 1638 of the exon 15 of the murine APC gene . While homozygosity is lethal, mice that are chimeric or heterozygous develop colonic polyposis and small intestinal tumors that result in gastrointestinal carcinoma. In order to better understand the effect of APC mutation on RPE proliferation and development, we studied the eyes from these APC-modified mice. We previously reported the light microscopic ocular findings and found unilateral or bilateral focal abnormalities of the RPE and outer nuclear layer (ONL) of the retina in 45% of eyes and in 75% of APC-modified animals . Abnormalities found were RPE coloboma, RPE hypertrophy, RPE hyperplasia, and RPE duplication with invasion of the outer and inner segments . We demonstrated that these mice not only develop intestinal polyposis and cancer in a similar fashion to humans, but also develop ocular manifestations resembling their human counterpart. Our light microscopic studies also confirmed clinical, molecular genetic, and histopathologic human investigations suggesting that the APC gene is critical in the regulation of RPE proliferation and development . In the present study, we performed electron microscopy and electrophysiologic studies in order to gain further insight into the role of APC codon 1638 mutation in retinal and RPE development.
A murine model with modification of the APC gene at position 1638 (exon 15) has been previously described . Polymerase chain reaction and Southern blot analysis identified mice heterozygous for the APC 1638 mutation . All animals used in this study were treated according to the Association for Research in Vision and Ophthalmology statement for use of animals in ophthalmic and vision research.
Light and electron microscopy
Thirty-eight eyes from 20 heterozygous APC 1638 mice of various ages (embryonic day 18, 1, 2, 3 and 4 months) were enucleated and fixed by immersion in paraformaldehyde/glutaraldehyde and 0.1 M Cacodylate buffer. The animals and eyes examined were different from our light microscopic study previously reported . Eyes were sectioned and examined by light microscopy. Tissues were fixed overnight in 0.2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2. Tissues were post-fixed in osmium tetroxide, rinsed, dehydrated through serial ethanol and processed through propylene oxide/Embed 812 overnight. Tissues were embedded in Embed 812. Thick (1 mm) and thin (90-150 nm) sections were prepared. Thick sections were stained by Hematoxylin/Eosin and examined using a Zeiss Axiophot microscope (Carl Zeiss, Inc. Thornwood, NY). Thin sections were stained in uranyl acetate/lead citrate and were examined and photographed using a Philips 400 transmission electron microscope. Four APC eyes with light microscopic abnormalities of the RPE were prepared for transmission electron microscopy with attention to both the focal abnormal and surrounding normal regions. Six APC eyes that did not demonstrate light microscopic abnormalities were also prepared for transmission electron microscopy. Eight C57BL wild-type mice (16 eyes) were matched for age with the APC 1638 mice and were also examined by light microscopy. Four age-matched wild-type eyes were prepared for transmission electron microscopy.
Electroretinography (ERG) was recorded at approximately 5,12, 18, and 26 weeks of age from three wild-type and four APC-modified mice. Responses were recorded from only the right eyes except at 26 weeks of age when responses were recorded from both eyes. After the ERG recordings at 26 weeks, the animals were sacrificed and seven APC-modified and six wild-type eyes were processed for light microscopy as described above.
A detailed description of the recording of ERGs from mice has been previously published . In brief, mice were anesthetized with a mixture of ketamine:xylazine:urethane (ketamine 0.011 mg/g: xylazine, 0.014 mg/g: urethane 0.5 mg/g body weight), and the pupils were dilated with 2% phenylephrine and 0.5% atropine. The mice were placed on a square (2 X 2 inch), aluminum stock that was placed on a heating pad. The right eye of the mouse was held open and slightly proptosed with masking tape. The animals were not restrained in any other way. The animals on the stock were placed in a Faraday cage whose temperature was warmed to approximately 28 °C with a separate heating pad. The ERGs were recorded between a wick-Ag:AgCl electrode placed on the cornea and a reference electrode placed subcutaneously on the head. The animal was grounded by an electrode placed subcutaneously in the neck region. The responses were fed to a Tektronix A39 preamplifier (Tektronix, Portland, OR) with the half-amplitude bandpass set at DC to 10 kHz (DC recordings; for c wave analysis only) or at the 0.1 Hz to 10 kHz (AC recordings). The output of the preamplifier was displayed on an oscilloscope and fed to the Biopac MP100 (Goleta, CA) signal averaging program.
The light for the stimulus was obtained from a quartz halogen lamp bulb. The filament of the lamp bulb was brought into focus in the plane of a Uniblitz shutter (Vincent Associates, Rochester, NY), and another lens focused the filament onto the tip of a fiber optic bundle. The other end of the fiber optic bundle was brought into the Faraday cage and the tip was placed 1-2 mm from the cornea.
The stimulus intensity was measured with a photometer (UDT Instruments, Orlando, FL) with the detector placed at the position of the cornea. The maximum stimulus luminance was 1.59 x 102 cd/m 2, and neutral density (ND) filters were used to attenuate the full intensity stimulus. The stimulus intensity was increased in the 0.5 log unit steps, and two responses were averaged at the lower stimulus intensities (ND = 6.0 to 3.5). Only one response was recorded at the higher stimulus intensities. The stimulus duration was 4.0 s, and the time between the recordings was increased from 1.0 min at the lowest stimulus intensity to 8 min at the highest intensity in 30 to 60 min steps.
Light and electron microscopy
The light microscopic findings and summary of eyes processed for electron microscopy and electroretinography are summarized in Table 1. Four of thirty-eight APC 1638 eyes demonstrated focal RPE atrophy and increased melanosone production. One embryonic eye, one 1-month old eye, and one 6-month old eye from APC 1638 mice demonstrated focal RPE hyperplasia (Table 1). Abnormalities were not observed in wild-type eyes. Electron microscopic examination of the embryonic APC 1638 eye with focal light microscopic RPE duplication and hyperplasia demonstrated RPE hyperplastic proliferation with increased melanosomes (Figure 1A). In eyes with focal RPE abnormalities, electron microscopic examination of the RPE and retina from normal-appearing light microscopic regions demonstrated no ultrastructural abnormalities (Figure 1B,C). There was no evidence of a diffuse ultrastructural abnormality in RPE melanogenesis as that described in human studies. Electron microscopy of four APC 1638 eyes demonstrated RPE atrophy with increased melanosome production (Figure 1D). Adjacent RPE in these four eyes did not demonstrate evidence of a diffuse ultrastructural abnormality (Figure 1E). Electron microscopic examination of APC 1638 eyes without light microscopic evidence of abnormalities demonstrated no ultrastructural differences from age matched controls (Figure 1F-G).
ERGs from wild-type mice
The ERGs elicited from the right eye of a wild-type mouse (mouse 9) by increasing stimulus intensities are shown in Figure 2A. The responses recorded at five weeks of age are shown in the left column and those recorded at 26 weeks of age are shown in the right column. The number between the ERGs represents the value of the neutral density filter used to attenuate the full intensity stimulus (at ND = 1.0, luminance = 1.59 x 102 cd/m2).
At five weeks, a small b-wave of 23.1 mV was elicited with the 6.0 ND filter, and increasing the stimulus led to an increase in the b-wave amplitude. The maximum b-wave amplitude was 608.3 mV and was elicited with the ND = 1.0 filter. The maximum b-wave amplitudes for the other two wild-type mice were 707.6 and 846.2 mV. The recordings at this age were DC-coupled, and demonstrated large c-waves beginning at a stimulus intensity of ND = 4.0. The a-wave was first recorded with an ND = 3.0 and increased with further increases of the stimulus intensity. The amplitude and shape of these ERGs are comparable to those recorded from other strains (Balb/c, C57B1/B6 CD-1).
At 26 weeks, the b-wave amplitudes were slightly larger than those recorded at five weeks. The maximum b-wave amplitude was 753.6 mV for wild-type mouse number 9 (Figure 2A), and it was 661.3 mV for the other wild-type mice tested at this age. A c-wave was present although it was not as large as that at five weeks because the recordings were AC-coupled.
From these wild-type ERGs, the amplitude of the b-waves of the right eye at 5 and 26 weeks were measured, and the amplitudes are plotted in Figure 2B. The b-wave amplitude of the left eye recorded at 26 weeks are also plotted. These intensity:response curves (V:logI curves) demonstrated that the b-wave amplitudes recorded from the right eye at 26 weeks were larger than at five weeks but were still within the range of normal eyes. The b-wave amplitudes for the left eye at 26 weeks were also comparable to those of normal eyes. These data demonstrated that the b-wave amplitudes do not change significantly during this recording period.
ERGs from APC-modified mice
The ERGs recorded from the right eye of APC-modified mouse 18 are shown in Figure 3A. The responses recorded at five weeks of age are shown in the left column and those recorded at 26 weeks are shown in the right column. The number between the ERGs represents the value of the neutral density filters.
As with the ERGs recorded from the wild-type mouse, a small b-wave of 53.8 mV was elicited by the ND = 6.0 stimulus intensity. Increasing the stimulus intensity increased the b-wave and the maximum b-wave amplitude was 800.0 mV. At 26 weeks of age, the b-wave amplitude elicited with the 6.0 ND filter was 38.5 mV and the maximum b-wave amplitude was 653.8 mV. The ERGs on both the fifth and 26th weeks were recorded with DC-coupling, and large amplitude c-waves were recorded.
The V:log I curves for these ERGs and for the left eye at 26 weeks are plotted in Figure 3B. Two findings are significant in the ERGs from APC-modified mouse 18. First, the amplitude of both the right and left eyes at 26 weeks did not differ from those at five weeks, and second, the b-wave amplitudes did not differ significantly from that of the wild-type mice at five and 26 weeks of age.
Similar findings were made from two of the other APC-modified mice (mice 19 and 20). However, the fourth APC-modified mouse (mouse 17) showed very different amplitude ERGs (Figure 4A). As opposed to the other APC-modified eyes, the b-wave amplitudes for APC-modified mouse 17 were significantly smaller at all intensities at five weeks and even smaller at 26 weeks. At both five and 26 weeks, the ERGs demonstrated large c-waves. The V:log I curves for these ERGs are plotted in Figure 4B along with the data for the left eye at 26 weeks. Also plotted are the data for the wild-type mouse at 26 weeks. By 26 weeks, a further decrease in the b-wave exists. The b-wave amplitudes for the left eye were large and comparable to those of the wild-type mice. Light microscopic findings in the right eye of this APC-modified mouse 17 with reduced ERG demonstrates a corneal scar, ruptured lens capsule, and disorganized retina most consistent with a traumatic rather than genetic etiology.
We have described the ultrastructural features of eyes from mice carrying a heterozygous mutation at codon 1638 of the murine APC gene. The present murine study has confirmed that, similar to human observations, focal lesions in a murine model are congenital occurring as early as embryonic day 18. Our present results reveal a lower incidence of murine RPE lesions than our previous light microscopic study . Electron microscopy confirms that the focal abnormalities of the murine RPE are confined to regions recognized by light microscopy and are not diffuse. The absence of a generalized RPE defect in the murine model distinguishes it from histopathologic findings in two humans with FAP and GS [5,6]. Traboulsi and coworkers  studied the eyes of a polyposis patient and found evidence of focal RPE hypertrophy, hyperplasia and hamartomatous proliferation. These investigators sampled several areas of normal RPE and demonstrated a generalized abnormality of the melanin pigment granules . Although the height and diameter of the RPE were normal, large spherical melanin granules were present . Kasner and coworkers  studied the eyes of a patient with FAP and identified focal RPE hypertrophy and hyperplasia. Focal lesions included a monolayer of hypertrophied RPE, a mound of pigmented cells interposed between Bruch's membrane and the RPE basement membrane, and a small mound of hyperplastic RPE . Abnormal RPE pigment granules were present within focal lesions and from areas of grossly normal RPE . While both human studies propose that a generalized disturbance of RPE melanogenesis exists [5,6], our murine findings indicate that a spectrum of RPE alteration may occur.
The results of our electrophysiologic studies demonstrated that the amplitude and shapes of the ERGs of the wild-type mice were comparable to those of other strains (Balb/c, C57B1/B6, CD-1) of mice. In addition, the amplitude and shape of the ERGs did not change significantly over the 26 week testing period. With the exception of one eye the was traumatized, b-wave amplitudes from APC-modified eyes at five and 26 weeks were comparable to those recorded from wild-type mice. The normal appearing c-waves in these APC-modified eyes demonstrated that the RPE giving rise to these waves were functional. This agrees with the morphological findings as we did not find evidence of a diffuse RPE or retinal disturbance nor did we find evidence of RPE basement membrane or pigment granule abnormalities in APC 1638 mice. The ultrastructural findings, thus, are supported by our electrophysiologic studies. Even in the APC-modified mice with the reduced b-wave, the c-wave was relatively large and comparable to the size of the b-wave (Figure 3A). Murine APC-modified eyes with focal RPE changes demonstrate normal ERGs. These electrophysiologic murine findings are consistent with a human study in a polyposis patient demonstrating normal electro-oculograms despite focal RPE abnormalities .
Discussion of the APC gene and its function are necessary to analyze the phenotypic variability and focal nature of POFL in the APC 1638 mouse (we found a lower incidence of abnormalities in the present study as compared to our previously reported study ). The APC gene is localized to human chromosome 5q21-22 [10-18] and is mutated in FAP patients and in sporadic colorectal tumors [19-23]. Various mechanisms about how APC mutations result in tumor formation have been proposed [24-26]. FAP patients have germline APC mutations in one allele and have one normal APC allele. Colorectal tumors show inactivation of the wild-type allele suggesting that APC is a tumor suppressor gene . The timing of the second mutation may well be subject to a wide range of external factors, so variation in the phenotypic manifestations between mice with the same mutation may be expected. In another hypothesis, chain terminating mutations in APC result in truncated protein which may inhibit the normal function of the wild-type APC gene product in a dominant negative effect . Tissue specificity of such mutant proteins may influence this dominant negative effect and account for variable phenotypic expression. Phenotypic variability may also be due to other factors such as modifier genes or environmental influences .
Phenotypic variation in FAP also depends, in part, on the location of the APC mutation. The APC gene consists of 15 exons that span 8.5 kilobases. The first 14 exons are relatively short, whereas exon 15 spans over 6.5 kilobases. POFL are often seen in humans with mutations between codons 463 (exon 9) and 1444 (exon 15) [11,12,29,30], or between codons 541 (exon 9) and 1309 (exon 150) [13,29-31], whereas ocular lesions are usually not observed with mutations proximal or distal to these codons. However, this association of POFL to specific regional mutation of the APC gene is not absolute, since humans with mutations at codons 215, 1546 and 1641 are reported to be associated with POFL [29,31,32]. The murine model investigated in the present study resembles the particularly variable phenotypic expression of POFL seen in FAP patients within the same family who inherit APC mutations located in codons distal to 1444 [31-34].
The APC protein may possess a physiologic function in retinal and RPE development. The ability of different regions of the APC protein to bind several proteins such as microtubules , beta catenin (which associates with E cadherin) [35-39], hDLG (human homologue of the Drosophila discs large tumor suppressor) and glycogen synthase kinase 3b[35,40-43] may provide additional molecular explanations for the resulting RPE and/or neural retinal abnormalities. The present study supports the hypothesis that APC mutation may lead to a primary disturbance in RPE development. The precise role and function of APC in RPE proliferation development remain to be elucidated.
The authors would like to thank Penny Roon and Diane Leibach for their technical assistance.
Supported by a grant from Fight for Sight Research Division of Prevent Blindness America and Research to Prevent Blindness (DMM) and by the Leonard and Madyla Abramson Family Cancer Research Fund (AKR).
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