Persistent Hyperplastic Primary Vitreous in Transgenic Mice Expressing IE180 of the Pseudorabies Virus

Abstract

purpose. Pseudorabies virus (PRV), a representative member of the α-herpesvirus family, causes nervous symptoms and ocular lesions, such as keratoconjunctivitis and retinal degeneration in piglets. The immediate-early protein IE180 of the PRV is known to be essential, not only in viral gene expression, but also in the cellular gene expression in host cells. The purpose of this study was to examine the effect of IE180 on the development of the mouse eye, by using transgenic technology.

methods. Transgenic mice expressing IE180 were generated and their eyes analyzed by histology, immunocytochemistry, and the bromodeoxyuridine cell proliferation assay.

results. A fibrovascular retrolental tissue analogous to persistent hyperplastic primary vitreous (PHPV) in humans was observed in a transgenic mouse line expressing IE180. The gross anatomy of the eye showed white pupils. Analysis of hematoxylin and eosin–stained sections revealed that the retrolental tissue adhered to the neuroretina, the inner nuclear and ganglion cell layers were disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were present. Bromodeoxyuridine-positive cells were detected in the retrolental tissues of postnatal day (P)1, P7, and P14 mice. The retrolental mass in the P7 transgenic mouse was composed of melanocytes and endothelial cells, which were detected by a cocktail of antibodies against endoglin, CD31, and VEGF receptor-2.

conclusions. The observation that the eye disease in transgenic mice is similar to that in PHPV in humans raises the possibility that expression of the immediate-early gene of α-herpesviruses may contribute to PHPV.

Persistent hyperplastic primary vitreous (PHPV) is thought to be a congenital anomaly in which the normal regression of the primary vitreous body and hyaloid vasculature does not occur. ¹ The primary vitreous is a part of the embryonic vasculature of the eye and supplies nutrients to the developing lens and retina during early gestation. ² It is composed of the hyaloid artery, the vasa hyaloidea propria, and the tunica vasculosa lentis. ³ PHPV, first identified by Reese, ¹ is a congenital malformation of the primary vitreous that is characterized by a retrolental white plaque of fibrovascular tissue. Subsequently, Pruett and Schepens ⁴ classified malformations involving a retrolental mass as anterior PHPV, as described by Reese, ¹ and malformations involving a congenital retinal fold or ablatio falciformis congenita as posterior PHPV. A variety of clinical findings are associated with PHPV, including microphthalmos, glaucoma due to closure of the chamber angle, shallowing of the anterior chamber, corneal opacity, cataract, uveal coloboma, and retinal degeneration. Most cases of PHPV have no known cause, are unilateral, and are not associated with diseases in other tissues of the body. They are therefore best considered to be idiopathic isolated sporadic congenital malformation syndromes localized to the eye. ⁵  

Pseudorabies virus (PRV) is classified into the genus Varicellovirus of the subfamily Alphaherpesvirinae. ⁶ PRV causes not only nervous symptoms, such as unbalanced walking, trembling, staggering, and convulsions, but also ocular lesions, such as keratoconjunctivitis ^{7 8} and retinal degeneration ⁹ in piglets. PRV invades and spreads within the trigeminal pathway (the nasal mucosa, the trigeminal ganglion, the pons/medulla, and the cerebellum/thalamus) of neonatal pigs. ^{10 11}  

PRV expresses a single IE protein (IE180), with a molecular weight of 180 kDa, for continuous transcription of late genes and shutting off the synthesis of its own RNA. ^{12 13 14} In addition, IE180 is known to be a strong transactivator of several promoters, including other viral and cellular genes, ^{12 14 15 16} and binds to specific sites on class II gene promoters ¹⁷ and single-stranded DNA. ¹⁸ Taken together, these findings suggest the possibility that IE180 is not only essential in viral gene expression, but also participates in gene expression in the host cells. To investigate whether IE180 affects the murine’s normal development, we have generated transgenic mouse lines expressing IE180 and have shown that IE180 affects the cascade of gene expression for development of the murine cerebellum. ¹⁹ IE180 is a multifunctional transcription factor that activates transcription from a variety of promoters and represses transcription from its own promoter. ^{12 13 14 15 16} We have reported that transcription factors were involved in development of the eye. ^{20 21 22 23 24}  

In the current study, a transgenic mouse line expressing IE180 showed an abnormality analogous to PHPV in humans in their eyes. The transgenic mice showing PHPV described in this study are the first demonstration that PHPV can be caused by expression of the immediate-early gene of α-herpesviruses. 

Materials and Methods

Generation of Transgenic Mice

Transgenic mice were generated as described previously. ¹⁹ Briefly, approximately 500 copies of the 7.2-kb HindIII transgene fragment and the 3.3-kb XhoI-NotI fragment containing the Tet promoter, the tetracycline transactivator (tTA) gene, and the SV40 intron/polyA signal from pTet-tTAk (Invitrogen, Carlsbad, CA) were comicroinjected into the pronuclei of fertilized B6C3F1 (C57BL/6 X C3H/He) mouse embryos. Genomic DNA was isolated from mouse tail. ²⁵ Both transgenes were detected by PCR analysis using specific primers, as described previously. ¹⁹ The transgene copy number was estimated by comparing the band intensity of transgenic mouse DNA with that of control DNA by Southern blot analysis. The DNA samples (10 μg) were digested with EcoRI, fractionated on 0.8% agarose gels, and transferred to membranes (Hybond N⁺; Amersham Pharmacia Biotech, Piscataway, NJ) by capillary blotting. Digoxigenin (DIG)-labeled DNA probes for detection of the transgene were derived from pTet/IE180 using the specific primers and a PCR DIG probe synthesis kit (Roche Diagnostics, Indianapolis, IN). Hybridization and detection of the transgene were performed as described previously. ²⁶ All animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Briefly, all mice were maintained in the animal facility at our institute and treated according to the Laboratory Animal Control Guidelines of our institute, which conform to those of the National Institutes of Health-American Association of Laboratory Animal Control. 

Analysis of the Transgene Expression

Transgenic mice (3 weeks old) were killed by decapitation, and tissue samples were immediately removed and frozen in liquid nitrogen. Total cellular RNA was isolated from various tissues of the transgenic mice and RT-PCR analyses were performed as described previously. ¹⁹ To analyze expression level of IE180 gene in transgenic eyes at postnatal day (P)1, P7 and P14, we performed quantitative RT-PCR assays, as described previously. ¹⁹  

Treadmill Test

A treadmill apparatus (Rotarod; Ugo Basile, Camerio, Italy) was used for measuring the mice’s fore- and hindlimb motor coordination and balance. ²⁷ Mice (8 weeks old) were placed on the treadmill at a constant speed (20 rpm/min) for a maximum of 300 seconds, and the time until they fell off the treadmill within this period was recorded. Mice underwent three trials per day, by which time a steady baseline level of performance was attained. 

Histological Procedures

Various tissue samples including eyes of decapitated animals (12 weeks old and P1, P7, and P14) were excised and immersion-fixed in 4% paraformaldehyde (PFA) solution for 24 hours at room temperature, embedded in paraffin, and cut into 4-μm-thick slices with a microtome. For bromodeoxyuridine (BrdU) labeling, mice were injected with BrdU peritoneally at a dosage of 30 mg/kg body weight 30 minutes before sampling. Anti-BrdU staining was performed as described previously. ^{28 29 30 31} Briefly, the sections were immersed in pepsin solution (0.4 mg/mL) in 0.1 M HCl at 30°C for 1 minute and then in 2 M HCl at 40°C for 1 hour. After a wash in PBS, the slides were incubated with normal goat serum and then with the anti-BrdU antibody (dilution, 1:1000; BD Biosciences, Franklin Lakes, NJ). Binding of the primary antibody was localized by fluorescence microscopy using FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a dilution of 1:200. The slides were examined by laser scanning confocal microscopy (MRC-1024; Bio-Rad, Hercules, CA; and LSM 510; Carl Zeiss Meditec, Dublin, CA). 

Tissue samples from mice at P7, after fixation, were immersed in 20% sucrose in PBS at 4°C, embedded in optimal temperature cutting compound (Sakura Finetek, Inc., Torrance, CA), and snap frozen. Frozen histologic sections (20 μm) were cut with a cryostat (Reichert, Inc., Buffalo, NY). Endothelial cells were identified by a cocktail of antibodies against endoglin (CD105), CD 31, and VEGF receptor-2 (flk-1; BD-PharMingen, San Diego, CA), a strategy that has been used to maximize labeling of all vascular endothelial cells, both immature and mature. ^{32 33}  

Results

Characterization of Transgenic Mouse Lines

To assess neuropathogenic potentials of pseudorabies virus IE180, transgenic mice expressing the immediate-early gene were generated. In the course of breeding of transgenic mice, we observed that one of the transgenic mouse lines (TgIE110) showed abnormalities in their eyes. White pupils were observed in 11 of 12 transgenic mice, but not in 12 of their nontransgenic littermates (Fig. 1) . TgIE110 mice gave rise to offspring in crosses with C57BL/6 mice and transmitted the introduced gene in Mendelian fashion. Southern blot analysis performed on genomic DNA from the transgenic mouse showed that 35 copies of the transgene were integrated (Fig. 2a) . The transgene expression in various tissues at 3 weeks of age was assessed by RT-PCR. The expected 296-bp PCR product of IE180 mRNA was detected in all the tested tissues, including the eye (Fig. 2b) . The expression level of the transgene varied among the tested tissues, with expression consistently high in cerebrum and cerebellum. However, the expression levels in cerebella of TgIE110 mice were rather low compared with TgIE96 mice showing severe cerebellar symptoms (approximately 5% of that of TgIE96 mice), although there was no difference between both lines in the tissue specificity of the transgene expression and in the expression levels in eyes (data not shown). In the tTA gene expression, a similar expression pattern was observed (data not shown). To examine whether IE180 expression level changed in early postnatal stages, quantitative RT-PCR analysis using transgenic eyes was performed. There was no difference in the levels of IE mRNA accumulation among the eyes at postnatal day (P)1, P7, and P14 (Fig. 2c) . Because we have reported that four transgenic mouse lines expressing IE180 showed motor discoordination, ¹⁹ motor coordination and balance of TgIE110 mice were measured on a treadmill (Rotarod; Ugo Basile). TgIE110 mice maintained constant balance on the treadmill, similar to their nontransgenic littermates (data not shown). Histopathological analyses of the cerebella of several TgIE110 mice were performed. Failure of layer formation and/or reduction in size, as reported previously, ¹⁹ was not observed in their cerebella (data not shown). 

Histopathological Analyses of Transgenic Eyes

In control mice at 12 week of age, there was no evidence of hyaloid vasculature and primary vitreous (data not shown). In contrast to the vitreous in littermates, fibrovascular retrolental tissue was detected in TgIE110 mice. Many melanocytes were present in the mesenchymal tissue on the vitreous (Figs. 3e 3f) . The manifestation of retrolental tissue in TgIE110 mice was variable. Some animals had only a thin, tapering stalk of persistent hyaloid artery (Figs. 3a 3c 3e) , which just reached the lens where it spread into a funnel-shaped retrolental mass. However, in more than half of the adult animals, a folding of the retina adjacent to the hyaloid artery was observed (Fig. 3b) . In these areas, the retrolental tissue adhered to the neuroretina (Figs. 3b 3d , arrowhead), the inner nuclear layer (INL) and the ganglion cell layer (GCL) were disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were observed (Fig. 3c) . 

On P1, hyaloid vessels were present in the vitreous of nontransgenic littermates (Figs. 4c 4e , arrowhead), as described previously. ³⁴ In contrast, a retrolental mass was present in the vitreous of TgIE110 eyes on P1 (Figs. 4b 4d 4f) . Retrolental tissue was observed in both eyes of all TgIE110 mice (3/3), but not in nontransgenic littermates (0/4). In the retrolental tissue on the vitreous of TgIE110 mice, melanocytes were present (Fig. 4f , arrows). Whereas retinal folds were observed in TgIE110 eyes, the rest of the neuroretinas of TgIE110 mice were morphologically similar to those of nontransgenic littermates. We examined the incorporation of BrdU, which was incorporated only into cells that were in the S-phase, and a portion of these reflected proliferative activity. When TgIE110 mice and nontransgenic littermates were injected with BrdU 30 minutes before death, BrdU-positive cells were detected in the retrolental tissue of the TgIE110 (Fig. 4h) , but not in the hyaloid vessels in the nontransgenic littermates (Fig. 4g) . The BrdU-positive retrolental mass was also present in the TgIE110 mice at P7 and P14, as shown in Figure 5 . Careful examination revealed that the retrolental mass was composed of melanocytes and endothelial cells, detected by a cocktail of antibodies against endoglin (CD105), CD 31, and VEGF receptor-2 (flk-1; Fig. 6 ). 

Discussion

A retrolental mass presented in the vitreous of TgIE110 eyes on postnatal day 1 (P1; Figs. 4b 4d 4f ). Retrolental tissue was observed in both eyes of all TgIE110 mice (3/3), but not in nontransgenic littermates (0/4). The phenotype was completely penetrant in the TgIE110 line. More than half of the adult animals showed a folding of the retina adjacent to the hyaloid artery. In these areas, the retrolental tissue adhered to the neuroretina (Figs. 3b 3d 3f) , the INL and GCL were disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were observed. The remainder of the neuroretina of TgIE110 mice was morphologically similar to that of nontransgenic littermates, suggesting that the progressive nature of the neuroretina was secondary to the progressive attachment of the retrolental tissue to these structures. 

Ocular abnormalities found in transgenic mice expressing PRV IE180 were similar to those in patients with PHPV, with the following pathologic characteristics: (1) the presence of retrolental tissue, which can contain melanocytes; (2) the attachment of the retrolental tissue to the inner neuroretina; (3) retrolental tissue-induced traction on the neuroretina, which causes neuroretinal detachment from the retina pigment epithelium; and (4) cellular disorganization and other dysplastic changes in the neuroretina. ^{5 35}  

The vessels in the retrolental mass of TgIE110 mice included endothelial cells that were detected by a cocktail of antibodies against endoglin (CD105), CD 31, and VEGF receptor-2 (flk-1; Fig. 6 ). When the transgenic mice were injected with BrdU 30 minutes before death, BrdU-positive cells were detected in the retrolental tissue of TgIE110 mice but were not detected in the hyaloid vessels in the wild-type mice. These findings suggest that IE180 is involved in cell proliferation during the development of the vitreous. The mesenchymal tissue of the vitreous of TgIE110 mice contained scattered melanocytes. It has been reported that the primary vitreous develops from melanocytes. ³⁶ The pathologic observation in TgIE110 mice indicates that the mesenchymal retrolental tissues were caused, at least in part, by the abnormal migration of excessive cells derived from the neural crest cells, because melanocytes and pericytes in the craniofacial structures including eye and brain have been shown to be of neural crest origin. ^{37 38 39 40}  

IE180 is a multifunctional transcription factor that activates transcription from a variety of promoters and represses transcription from its own promoter. It is therefore thought that IE180 affects the serially and precisely regulated gene expression in murine eye development, resulting in disruption of ocular morphogenesis. The observation that the eye disease in transgenic mice is similar to that in PHPV in humans raises the possibility that expression of the immediate early gene of α-herpesviruses may contribute to this disease, which is usually unilateral and sporadic. ^{5 35}  

³  Contributed equally to the work and therefore should be considered equivalent authors.

 Supported by Grants-in-Aid for Scientific Research (B)(2) and (C)(2), and Encouragement of Young Scientists (A) from The Ministry of Education, Culture, Sports, Science and Technology, Japan.

 Submitted for publication June 24, 2004; revised December 2, 2004; accepted January 10, 2005.

 Disclosure: S. Taharaguchi, None; K. Yoshida, None; Y. Tomioka, None; S. Yoshino, Gene Techno Science (E); T. Uede, None; E. Ono, None

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Eyes of TgIE110 and littermate mice showing the white pupil observed in the transgenic eye.

Figure 2.

 

(a) Southern blot analysis of the introduced transgene. Copy numbers of control DNA derived from pTet/IE180 are shown at the top of the four right lanes. Arrow: position of the 4.8-kb EcoRI fragment containing the IE180 gene. The copy number of the transgene integrated in the genomic DNA is shown below. (b) Expression of the IE180 gene in various tissues of TgIE110. Total RNAs prepared from the tissues shown were analyzed by RT-PCR. Shown is a representative result of Southern blot analysis using RT-PCR products from the various tissues. β-Actin was the control. +, Positive control product synthesized from the transgene DNA fragment by PCR. (c) Quantitative RT-PCR analysis of the transgene expression in the transgenic eyes at P1, P7, and P14.

Figure 3.

 

Abnormal retrolental tissue observed in the eyes of TgIE110 mice. Photomicrographs of sections of eyes from TgIE110 mice at 12 weeks of age. (c, e) Detail of (a); (d, f) detail of (b). Hyaloid arteries were present in the tissue (a–c, e, f, arrows). The retrolental tissue adhered to the neuroretina (b, d, arrowhead), (e, ) lens. Lens sections were stained with hematoxylin and eosin. Scale bar: (a, b) 330 μm; (c, d) 60 μm; (e, f) 20 μm.

Figure 4.

 

Abnormal retrolental tissues observed in the eyes of neonatal TgIE110 mice. Hematoxylin and eosin staining (a–f) and immunodetection of BrdU (g, h) of the nontransgenic (a, c, e, g) and TgIE110 (b, d, f, h) mice at postnatal day 1. The retrolental tissue was present in the TgIE110 mouse (b, arrow and arrowhead). Melanocytes were present in the tissue (f, arrows). BrdU-positive cells were detected in the retrolental tissue of the TgIE110 mouse (h), but not in the hyaloid vessels in the nontransgenic mouse (c, e, arrowhead). Scale bar: (a, b) 400 μm; (c, d) 100 μm; (e–h) 40 μm.

Figure 5.

 

Immunodetection of BrdU-positive cells in the retrolental tissue of the TgIE110 mice. Hematoxylin and eosin staining (a, c, e, g) and immunodetection of BrdU (b, d, f, h) of the nontransgenic (a, b, e, f) and TgIE110 (c, d, g, h) mice of P7 (a–d) and P14 (e–h). Scale bar: 100 μm.

Figure 6.

 

Immunodetection of endothelium cells in the retrolental tissue of the TgIE110 mice. (a) Detail in the retrolental tissues in the eye 7 days after birth. (b) Immunodetection of endothelium cells in the retrolental tissue. A serial section was processed by immunohistochemistry to identify the vascular endothelium (combined CD31+CD105+flk-1). Scale bars: 20 μm.