Neuron,

Vol.

9, 815-830,

November,

1992,

Copyright

0

1992

by Cell

Press

Transgenic M ice with a Rhodopsin Mutation (Pro23His): A Mouse Model of Autosomal Dominant Retinitis Pigmentosa Jane E. OIsson,* Jon W. Gordon,+ Basil S. Pawlyk,* Dorothy Roof,* Annmarie Hayes,* Robert S. Molday,* Shizuo Mukai,* Glenn S. Cowley,* Eliot 1. Berson,* and Thaddeus P. Dryja* *Berman-Cund Laboratory for the Study of Retinal Degenerations and the Howe Laboratory of Ophthalmology Harvard Medical School Massachusetts Eye and Ear Infirmary Boston, Massachusetts 02114 +Department Obstetrics and Gynecology Mount Sinai School of Medicine New York, New York 10029 *Department of Biochemistry University of British Columbia Vancouver, British Columbia V6T 123 Canada

W e inserted into the germline of mice either a mutant or wild-type allele from a patient with retinitis pigmentosa and a missense mutation (P23H) in the rhodop sin gene. All three lines of transgenic mice with the mutant allele developed photoreceptor degeneration; the one with the least severe retinal photoreceptor degeneration had the lowest transgene expression, which was one-sixth the level of endogenous murine rod opsin. Of two lines of mice with the wild-type allele, one expressed approximately equal amounts of transgenic and murine opsin and maintained normal retinal function and structure. The other expressed approximately 5 times more transgenic than murine opsin and developed a retinal degeneration similar to that found in mice carrying a mutant allele, presumably due to the overexpression of this protein. Our findings help to establish the pathogenicity of mutant human P23H rod opsin and suggest that overexpression of wild-type human rod opsin leads to a remarkably similar photoreceptor degeneration. Introduction Retinitis pigmentosa is the term given to a group of hereditary progressive retinal degenerative diseases in humans. Patients with retinitis pigmentosatypically experience night blindness early in the disease, suggesting that rods are dysfunctional. As the condition progresses, patients gradually lose their peripheral vision and eventually their central vision as well. Many are blind by age 60. There is extensive genetic heterogeneity, with some families exhibiting an autosomal dominant pattern of inheritance, others an autosomal recessive pattern, and still others an X-linked recessive pattern. Even families with the same inheritance pattern may have nonallelic mutations. For example,

linkage studies have demonstrated at least two distinct loci on Xp that can cause X-linked retinitis pigmentosa (Wirth et al., 1988; Chen et al., 1989). Also, loci on chromosomes 3q (McWilliam et al., 1989), 6p (Kajiwara et al., 1991; Farrar et al., 1991), and 8 (Blanton et al., 1991) have been implicated in the autosomal dominant forms of the disease. Regardless of the genetic type of retinitis pigmentosa, the affected retina has a distinctive appearance late in the disease, characterized by attenuated retinal vessels, a pale optic disc, and clumps of intraretinal pigment distributed circumferentially around the midperipheral fundus. Early in the disease, even before these abnormalities are visible, patients show light-evoked electrical responses or electroretinograms (ERGS) that are reduced and delayed (Berson et al., 1968, 1969). Based on clinical findings, ERGS, and histopathology, it appears that photoreceptors are the retinal cells first affected by the disease (Verhoeff, 1931; Cogan, 1950; Berson et al., 1968; Kolb and Gouras, 1974). Recent studies have demonstrated that approximately25%-30% of patientswith autosomal dominant retinitis pigmentosa have a defect in the rod opsin (rhodopsin) gene on chromosome 3q (Dryja et al., 1990a, 1990b, 1991; Bhattacharya et al., 1991; Gal et al., 1991; Heckenlively et al., 1991; lnglehearn et al., 1991; Keen et al., 1991; Stone et al., 1991; Sung et al., 1991a). Over 31 distinct mutations that cosegregate with the disease have been reported so far. Each of these mutations alters the open reading frame and would be expected to result in an abnormality in the encoded amino acid sequence. None of these mutations has been found in individuals without retinitis pigmentosa. Finally, at least one example of a new germline mutation in the rhodopsin gene has been observed in a patient whose parents were unaffected (Dryja et al., 1991). These data indicate that mutations in the rhodopsin gene by themselves can cause autosomal dominant retinitis pigmentosa. W e began this study in order to substantiate this inference and to explore the mechanisms by which these mutant alleles lead to photoreceptor degeneration. Since there is no known animal model with retinal degeneration due to a dominant mutation of the rhodopsin gene, we constructed transgenic mice that carry either a normal or a mutant human allele. W e report here the funduscopic, electroretinographic, and histopathologic features of the retinal degeneration observed in these mice. Results Construction of Transgenic Mouse Lines W e chose as transgenes the normal and mutant alleles from patient AD191, a40-year-old man who is affected with autosomal dominant retinitis pigmentosa and

SCALE (kb)

Figure

-10 I

CONSENSUS

PR023HIS

I

I

I,

-5

I,,

RESTRICTION .y---

E ;

(P23H)

- 17.5

I,,

0

,

,

I,

5

MAP - HUMAN RHODOPSIN H

EB

l-m

I

I

I

III

I

QENE II

E i;

II

10

w

E

(t;

-

-3,

kb

1. Restriction

Map ot Transgenes

Both the mutant (P23H)and normal human rhodopsin (NHR) constructs include the entire transcriptional unit and introns as well as 4.2 or 4.8 kb of upstream sequence and 8.4 or 6.2 kb of downstream sequence, respectively. Upstream sequence was measured from the 5’ end of the transgene to the start codon, and the downstream sequence was measured from the termination codon to the 3’ end of the transgene.

I

NORMAL HUMAN RHODOPSIN (NHR) - 15.9

kb I

I Coding Region8

B

1 BarnHI

E , ECORI

carries heterozygously a missense mutation in the rhodopsin gene. The mutation, called Pro23His or P23H, is a C-to-A transversion in codon 23. It corresponds to a proline to histidine substitution near the amino terminus of rhodopsin in an intradiscal region of the protein. This is the mutant allele found most frequently among patients with autosomal dominant retinitis pigmentosa in North America (Berson et al., 1991a, 1991 b, 1991c; Dryja et al., 1991; Heckenlively et al., 1991; Stone et al., 1991; Sung et al., 1991a). We expected that cis-acting sequences would be necessary for the transgenes to be expressed specifically in rodsandatthelevelscommensuratewiththosefound in nature. Consequently, the transgenes used in this investigation were human genomicfragments that encompass the entire transcriptional unit as well as 4.8 kb of upstream and 6.2 kb of downstream DNAfor the wild-type allele and 4.2 kb and 8.4 kb, respectively, for the mutant allele (Figure 1). These constructs were isolated from a genomic library constructed from the patient’s leukocyte DNA and cloned in the bacteriophage vector EMBL3. The purified bacteriophage inserts were microinjetted into the male pronuclei of single-cell mouse embryos. Embryos were harvested from B6D2F, females that had previously been mated to B6D2F1 males. This strain of mice does not carry the rd allele. We assayed for the P23H or wild-type sequence in the founder animals by direct genomic sequencing of a portionofexonl. Inaddition,theentirecodingregion of the transgenes in the NHR-A and NHR-E lines was sequenced to ensure that no mutations were inadvertently acquired during the manipulation of the constructs. We traced the transmission of the transgenes by using human-specific primers designed to amplify exons 2 or 5 by the polymerase chain reaction. Four out of twelve founder animals carried the mutant transgene in their germline, while two out of six carried the wild-type human allele.The transgenic founders were bred with pigmented B6D2F1 mice to pro-

H , Hlndm

duce six lines of transgenic mice. The four lines carrying a mutant rhodopsin allele were designated P23H-B, P23H-D, P23H-E, and P23H-L, and the two lines with the wild-type human allele were designated NHR-A and NHR-E. In each line, the transgene segregated as an autosomal dominant allele. Mice from the P23H-B line reproduced poorly, preventing their detailed characterization; this line was excluded from subsequent analysis. Funduscopic Appearance of the Transgenic Mice The fundi of both the control B6D2F1 mice and the NHR-E mice had retinal vessels of normal caliber and homogenous background pigmentation (Figures 2a and 2b). Mice from all three P23H lines (-D, -E, and -L) aswell asoneNHRIine(NHR-A) hadattenuated retinal vessels and flat, hyper-, and hypopigmented areas. Figures2c-2earefundus photographsof P23H-D mice at 11 days, 20 days, and 5 months of age, respectively. These fundi are typical of those from the NHR-A, P23H-E, and P23H-L lines at comparable ages. Although the hyper- and hypopigmented patches gradually became more prominent over the first few months of life, they and the vascular attenuation were already present when the mice first opened their eyes at 11 days of age. The vascular attenuation mimicked that seen in humans with retinitis pigmentosa. The pigmentary abnormality differed in that the retinal hyperpigmentation in humans takes the form of discrete, dendritic clumps of pigment in the retina rather than thegeographic patchesof subretinal (retinal pigment epithelial) hyperand hypopigmentation observed in these mice. ERGS ERGS were recorded from members of each of five transgenic lines (Figure 3). NHR-E mice had b-waves (135-193 uV) and response times (66-80 that fell within the range of ERGS recorded from matched, nontransgenic B6D2F1 controls (134-618

the ERG ms) agepV;

Retinal Degeneration

in Mice with Pro23His Rhodopsin

817

Figure 2. Indirect

Ophthalmoscopy

Fundus photographs of retinas from (a) a wild-type B6D2F1 mo use at postnatal day 20, (b) an NHR-E mouse at postnatal day 20, I:c) a P23H-D mouse at postnatal day 11, (d) a P23H-D mouse at postn latal day 20, and (e) a P23H-D mouse at 5 months. The difference in the pigmentation of the fundi of animals photographed in (a) and (b) is within the range of normal variability among B6D2Fr mice and corresponds to the coat colors of these mice.

Neuron 018

(White

O.lHz)

45-99 ms) (Table 1). In contrast, ERGS recorded from the NHR-A mice and the P23H-D, P23H-E, and P23H-L mice showed reduced a- and b-wave amplitudes and delayed b-wave response times (see Table I). Some members of the P23H-L line displayed the largest amplitudes among all P23H lines (up to 25 uV) at 20 days, but they were clearly below normal amplitudes and had delayed response times (122-154 ms). The ERG abnormalities found in the P23H and NHR-A lines correspond to those found in patients with retinitis pigmentosa.

I

5O)lV

NHR-E

NHR-A

P23H-D 2OkiV I

P23H-E

P23H-L I

125

Time Figure

3. ERGS from

Transgenic

250

(msec) Mice (Postnatal

Day 20)

This figure illustrates representative dark-adapted, computeraveraged (sum = 4), full-field ERGS recorded intravitreally from postnatal day 20 mice in response to 0.1 Hz flashes of white light. The top trace is from a B6D2FI mouse and is similar to that recorded from an NHR-E mouse. Below the dotted line are ERGS from P23H-D, P23H-E, P23H-L, and NHR-A mice also at postnatal day 20 (note the different vertical scale). Arrows designate the b-wave response times.

Table 1. Ratio of Murine to Human Rod Opsin (Postnatal Day 20), As Monitored by the ERG

mRNA

in Transgenic

Line

Mouse: Human Rod Opsin mRNA in Retinas

Severity of Retinal Degeneration

P23H-D P23H-E P23H-L NHR-A NHR-E B6D2F,

1:3 I:1 6:l I:5 I:1 I:0

Severe Severe Moderate Severe None None

Retina-Specific Expression of the Transgenes Human and murine rod opsin transcripts have similar sizes and cannot be reliably distinguished by Northern blot analysis (Al-Ubaidi et al., 1990). We therefore synthesized a pair of 20 base oligomers that correspond to two coding regions of the rhodopsin gene that are identical in humans and mice and that are able to amplify the intervening mRNA. Since the amplified segments from the two species differ at 26 bases, the fragments amplified from the human transgene mRNA are unambiguously separable from those amplified from native murine rod opsin mRNA byelectrophoresis through a nondenaturing polyacrylamide gel. By this method we found that in every transgenic line, the transgene was expressed in the retina at notably higher levels than in any other tissue that was surveyed (see Figure 4 for representative results for an animal from the NHR-E line). This suggests that the sequences necessary for retina-specific expression of rod opsin in the retina are within the 15.5 kb shared by the mutant and wild-type transgenes. However, compared with the level of native, murine rod opsin mRNA, the levels of human mRNA in the retina at postnatal day20varied over a6-fold range in thedifferent lines of mice (Figure 5; Table 1). This latter finding may be due to variation in the transgene copy number

Retinas

Compared

with

the Severity

of Retinal

Degeneration

ERG b-Wave Amplitude (uV + SD)

ERG b-Wave Response Time (ms f SD)

11 + 4 ND 17 * 8

106 ND 132 147 74 67

a+5 160 f 24 286 f 146

i 14 k * f k

11 12 2 13

The ratio of mouse to human opsin mRNA was obtained by densitometry of bands on autoradiographs such as those shown in Figure 5. The severity of retinal degeneration was scored on the basis of remaining photoreceptors at 20 days of age. A “severe” rating indicates that only one row of photoreceptor cell nuclei remained in the outer nuclear layer, and no inner or outer segment material was observed. A “moderate” rating indicates that some animals in a line retained inner and outer segment material and had four to six rows of photoreceptor cell nuclei at 20 days of age. The number of animals tested by ERG at postnatal day 20 was as follows: P23H-D, 6; P23H-E, 2; P23H-L, 7; NHR-A, 5; NHR-E, 3; and B6D2F1, 38. ND, ERGS that could not be detected.

Retinal Degeneration

in Mice with ProZ3His Rhodopsin

a19

Figure 4. Retina-Specific man Rod Opsin mRNA

This autoradiograph shows the separation of amplified murine and human rod opsin mRNA sequences after electrophoresis through a nondenaturing polyacrylamide gel. Each lane shows the amplified cDNA fragments derived from the tissues, as indicated. Both homoduplexes and heteroduplexes are observed in the NHR-E retinas, whereas only homoduplexes are seen in human and wild-type mouse retinas. No amplified fragments are seen in the liver, lung, brain, or spleen of NHR-E mice.

r)Heteroduplexes -Human -Mouse

homoduplex homoduplex

or to factors specific to each integration site that modulate the level of mRNA expression in the retina. The level of retinal expression of the transgene appeared to be a factor that qualitatively correlated with the severity of the retinal degeneration. For example, the more mildly affected mice in the P23H-L line (as measured by the number of remaining photoreceptors at 20 days of age) had on average the lowest levels of transcript from the transgene at postnatal day 20, i.e., only approximately one-sixth of the endogenous murine rod opsin transcript. On the other hand, the transgenic opsin mRNA level was equal to or 3-fold greater than the native murine rod opsin mRNA in the severely degenerating P23H-E and P23H-D lines, respectively. Furthermore, the two transgenic lines with the wild-type human allele (NHR-A and NHR-E) differed in that mice from the NHR-A line expressed about 5 times more human than murine rod opsin mRNA, whereas mice from the NHR-E line expressed approximately equal amounts of human and murine mRNA. The retinal degeneration in the NHR-A line simulated that seen in the P23H lines, while the NHR-E line had no detectable retinal degeneration.

Expression of Huin NHR-E Mice

Retinal Histology Light Microscopy The retinas from mice of the NHR-E line were indistinguishablefromthoseof nontransgenic normal B6D2F1 mice(Figure6)witheighttoten rowsof photoreceptor cell nuclei and well-formed stacks of discs in theouter segments. Mice from transgenic lines NHR-A, P23H-D, P23H-E, and P23H-L had marked photoreceptor degeneration by postnatal day20. In the NHR-A, P23H-D, and P23H-E lines (see Figure 6 and Figure 7), the severity of this degeneration was similar. The outer nuclear layer was reduced to a single row of sparse photoreceptor nuclei. We could not determine whether the residual photoreceptors were rods or cones. The inner retinal layers (inner nuclear, inner plexiform, and ganglion cell layers) appeared normal. The retinal pigment epithelial cells exhibited variation in individual cell height as well as melanin content (Figure 7, top left). These variations appear to explain the areas of patchy hyper- and hypopigmentation observed by funduscopy. Among the 20-day-old retinas from the P23H-L line that were examined histologically, many had the se-

3

Heteroduplexes

-Human homoduplex - Mouse homoduplex Figure

5. Strain-Specific

Variation

in Levels of Transgenic

Rod Opsin

mRNA

(Postnatal

Day 20)

Each lane shows the retinal cDNA fragments amplified from either human, wild-type mouse, or the transgenic strain, as indicated above the autoradiograph. The amount of amplified cDNA differs between samples due to variation in the stage of photoreceptor degeneration and due to technical factors (such as amount of amplified product loaded). Within each lane, however, the relative intensities of human and murine fragments are a measure of the expression of the transgene compared with native, murine opsin gene. For example, the P23H-E and NHR-E lines have human and murine homoduplex expression of approximately equal intensity. The P23H-D and NHR-A lines have more intense human than mouse homoduplex bands. Most of the human opsin sequences in the P23H-L line are complexed with the more abundant murine sequences, thereby explaining the undetected human homoduplex band. The heteroduplex bands from this line are better seen in the right panel.

NHR-

NHR- -A

Figure 6. Light Micrographs of Retinas from Normal Mice and from Those Carrying the NHR Transgene (Postnatal Day 20) This figure shows light micrographs of photoreceptors from a control BbD2F, mouse (top), an NHR-E mouse (center), and an NHR-A mouse (bottom). The bar (bottom right) represents IO urn. INL, inner nuclear layer, consisting of bipolar, horizontal, amacrine, and Mtiller cell nuclei; ONL, outer nuclear layer, i.e., photoreceptor nuclei; RIS, rod inner segments; ROS, rod outer segments; RPE, retinal pigment epithelium.

vere photoreceptor degeneration (Figure 7, bottom left) similar to that seen in the P23H-D, P23H-E, and NHR-A animals. However, some age-matched P23H-L animals had a somewhat milder photoreceptor degeneration with four to six rows of photoreceptor cell nuclei, shortened inner segments, and shortened, haphazardly arranged outer segments (Figure 7, bottom right). The variation in the preservation of the photoreceptors at age 20 days was seen even among littermates. Electron Microscopy Photoreceptors from 20-day-old NHR-E mice (Figure 8A) had the same ultrastructural findings as control B6D2F1 mice. However, the ultrastructure of photoreceptors from the NHR-A, P23H-D, and P23H-E mice

was markedly abnormal at 20 days. These lines had a single, attenuated layer of photoreceptor cells, some with pyknotic nuclei (Figure 8B). The inner aspect of these cells abutted the synaptic region of the cells of the inner nuclear layer. The tight junctions between remaining photoreceptors, comprising the external limiting membrane, were apposed to the villous processes of the retinal pigment epithelium with no intervening inner or outer segment material. The remaining cytoplasm of the photoreceptors varied in density and contained mitochondria, free ribosomes, and rough and smooth endoplasmic reticulum. Figure 8C shows electron micrographs exemplifying the less severe retinal degeneration sometimes observed in members of the P23H-L line at postnatal day 20. Unlike the P23H-D, P23H-E, and NHR-A lines, inner and outer segments were present. However, the inner segments were irregularly dilated, and the density of mitochondria was reduced. There was no evident overabundance of rough endoplasmic reticulum. Golgi bodies were present and often contained aberrant, amorphous, osmophilic material of unknown origin. Photoreceptor connecting cilia, normally found at the distal end of the inner segment, were instead found at all levels of the inner segment, with some even seen near its proximal end. Correspondingly, outer segments were irregularly oriented and often found adjacent to inner segments rather than as distal extensions of them. The retinal pigment epithelium varied in both thickness and number of melanin granules, as previously observed by light microscopy. The apices of the retinal pigment epithelium cells had numerous microvillous projectionsand contained phagosomes encompassing fragments of disc material, indicating phagocytic function.

Localization of Human Rod Opsin in Transgenic Photoreceptors Two previously described monoclonal antibodies (Hodges et al., 1988) were used to study rod opsin expression in the transgenic mouse retinas. One of the antibodies, monoclonal rho 3A6, specifically recognizes an epitope that is present near the carboxyl terminus of human but not mouse rod opsin (Nathans and Hogness, 1984; Hodges et al., 1988; Al-Ubaidi et al., 1990). This antibody strongly stains unfixed cryostat sections of normal adult human retinas, but fails to stain above background levels on similar sections of adult mouse retinas (data not shown). This allows us to distinguish human from mouse rod opsin in unfixed retinal sections. A second monoclonal antibody, rho lD4, recognizes an epitope that is also near the carboxyl terminus and is present in both mouse and human rod opsin. Antibody rho ID4 strongly stains cryostat sections of both adult mouse and adult human retinas. We used this second antibody to assess total rod opsin expression (mouse plus human) within unfixed sections of the transgenic retinas. Retinas of 20-day-old mice from the nondegenerating NHR-E line showed staining with the human-

Retinal Degeneration

in Mice with Pro23His Rhodopsin

821

P23H-L Figure 7. Light Micrographs

of Retinas

P23H-L from

Transgenic

Mice

Carrying

the P23H Transgene

(Postnatal

Day 20)

This figure shows light micrographs of retinas from the three P23H lines studied: P23H-D (top left), P23H-E (top right), P23H-L (bottom left and right). The bar (bottom right) represents 10 pm. INL, inner nuclear layer, consisting of bipolar, horizontal, amacrine, and Miiller cell nuclei; ONL, outer nuclear layer, i.e., photoreceptor nuclei; RIS, rod inner segments; ROS, rod outer segments; RPE, retinal pigment epithelium.

specific antibody rho3A6 in the appropriate locations, primarily in the inner segment/outer segment layer with a small amount of fluorescence spillover into the outer nuclear layer (Figure 9). There was no staining in the outer plexiform layer. As expected, nontransgenic B6D2F1 retinas did not react with antibody rho 3A6 (Figure 9). As mentioned above, light and electron microscopy showed that photoreceptors in the P23H-D, P23H-E, and NHR-A lines were severely damaged by postnatal day 20 (see Figure 6, Figure 7, and Figure 8). Correspondingly, both human rod opsin and total rod opsin were undetectable in the central and midperipheral P23H-D and P23H-E transgenic retinas at that age. Only a small amount of residual rod opsin persisted at the extreme superior and inferior periphery of the retina and was localized to the area of the retina with the highest density of remaining photoreceptor nuclei (data not shown). Consequently, we studied these transgenic lines at an earlier stage of retinal development. At 10 days of age, retinas from the transgenic lines P23H-D, P23H-E, and NHR-A had an outer nuclear layerthatwas moderatelythinned comparedwith normal mouse retinas of that age. The overall pattern of

total rod opsin staining in these three lines, as detected with antibody rho lD4, was similar to that in normal mouse retinas (Figure 10; Figure 11). Human rod opsin staining, detected with rho 3A6 antibody, was present in the outer nuclear layer (especially prominent in the P23H-D line), as well as in the inner segments and in the rudimentary outer segments (P23H-D, P23H-E, and P23H-L lines). However, human rod opsin was also detected anomalously in the synaptic layer (i.e., outer plexiform layer) in the P23H-D and P23H-E mice (Figure IO). Comparing day 10 with day 20 animals, it appears that the P23H-L transgenic line has a different pattern of retinal degeneration and rod opsin expression from animals in the P23H-D, P23H-E, and NHR-A transgenic lines. At day 10, the overall retinal morphology in the P23H-L line was relatively normal, with an outer nuclear layer of normal thickness across the retina (Figure IO). The pattern of total rod opsin expression as seen with antibody rho ID4 was also comparable to age-matched controls (Figure IO). Human rod opsin was detected in the developing inner and outer segments in a pattern similar to the nondegenerating NHR-E line at day 20. Unlike the P23H-D, P23H-E, and NHR-A lines, there was no stain-

Neuron 822

NHR-E Figure 8. Electron

Micrographs

of Retinas

from

Transgenic

Mice

(Postnatal

Day 20)

(A) These electron micrographs compare the ultrastucture of retinas from NHR-E mice (left) with age-matched P23H-L mice (right) at the same magnification. In the P23H-L retina, the inner and outer segments are greatly shortened and are compressed into a single region designated with a bracket. Bar, 3 urn. (B) A micrograph from a P23H-D retina shows the photoreceptor degeneration also observed in P23H-E, NHR-A, and some P23H-L mice at age 20 days. No outer or inner segment material remains, and there is only one row of photoreceptor cell nuclei, some with pyknotic nuclei. Zonula adherens (ZA) remain between adjacent photoreceptors as a vestige of the external limiting membrane. The cells at the bottom of the figure are from the inner nuclear layer. Bar, 1 urn. (C)The left micrograph shows outer segments adjacent to inner segments in a P23H-L retina, as well as a Golgr body(G) with amorphous, osmophilic material. The right micrograph, also from a P23H-L mouse shows a phagosome (P) in the retinal pigment epithelium. Bar, 1 pm. Other abbreviations are as follows: ELM, external limiting membrane; ER, endoplasmic reticulum; IS, inner segment; M, mitochondria; MV, microvillous processes of the retinal pigment epithelium; ONL, outer nuclear layer; OS, outer segment; RER, rough endoplasmir reticulum; RPE, retinal pigment epithelium.

ing in the nuclear layer or the outer plexiform layer of P23H-L mice (Figure IO). We also examined rod opsin expression in four P23H-L transgenic individuals at postnatal day 15. At this age there was a noticeable decrease in the number of nuclei. The remaining photoreceptors had relatively long outer segments. As seen in Figure 12, these outer segments stained stronglywith the rho 3A6antibody specific for human rod opsin, suggesting that in murine photoreceptors,

P23H opsin can be translocated to the outer segment and be subsequently assembled into disc membranes. Discussion These studies were initiated because of the discovery that dominant mutations in the rhodopsin gene cosegregate with retinitis pigmentosa in some families.

Rettnal Degeneration 823

in Mice with ProZ3His Rhodopsin

Neuron 824

NOMARSKI

CONTROL

Figure 9. Human

Rod Opsin

Expression

within

NHR-E Transgenic

Mouse

Retinas

(Postnatal

Day 20)

Unfixed cryostat sections of retinas from NHR-E (top) and nontransgenic littermates (B6DZF,) (bottom) were stained with either monoclonal rho ID4 (left), which recognizes both mouse and human rod opsin, or rho 3A6 (right), which recognizes an epitope of human, but not mouse rod opsin. Nomarski images (center) are shown for reference. The nontransgenic retinas show intense staining of the outer segment layers with rho ID4 and no staining with rho 3A6. Retinas from NHR-E animals show morphology similar to the normal ones (Nomarski), but with high levels of human rod opsin expression in the inner segments (IS), outer segments (OS), and outer nuclear layers (ONL). OPL, outer plexiform layer. Bar, 21 urn.

The inference was that these mutations cause retinal degeneration. This conclusion, although compelling, did little to clarify the mechanisms responsible for the retinal degeneration. Consequently, we sought an experimental system that would allow detailed studies of the effects of these mutations. Since defects in the rhodopsin gene are not known to be responsible for retinal degeneration in any mammalian model of retinitis pigmentosa, we undertook to develop a murine model as described in this paper. In the process, we hoped to determine whether a contiguous segment of human genomic DNA encompassing the rhodopsin gene specifies photoreceptor-specific expression in transgenic mice,whether murine photoreceptors express and incorporate wild-type human rod opsin in photoreceptor outer segments, and whether abnormalities in the structure or function of murine

photoreceptors arise due to the expression of wildtype or mutant forms of human rod opsin. To develop these transgenic mice, we utilized human genomic DNA fragments corresponding to the wild-typeand mutantallelesfrom apatientwith retinitis pigmentosa who carries the P23H mutation. The choice of transgenes used in this experiment was based on the assumptions that the sequences directing photoreceptor-specific expression of rod opsin are within the transcriptional unit or the flanking upstream or downstream sequences and that there is sufficient conservation in these sequences and in the factors that recognize them to allow xenogenous expression. Both assumptions appear to be correct, since both transgenes are expressed specifically in murine photoreceptors. Experiments performed by two other groups (Zack et al., 1991; Lem et al., 1991)

NOMARSKI

104

P23H-E

P23H-L

CONTROL

Figure

IO. Human

Rod Opsin

Expression

within

P23H Transgenic

Mouse

Retinas

(Postnatal

Day IO)

P23H-D, P23H-E, P23H-L, and B6D2F1 (control) retinas are shown from top to bottom of this figure. Cryostat sections of each retina were stained with either monoclonal rho ID4 (left column), or rho 3A6 (right column). Nomarski images (center) are shown for reference. P23H-D retinas show more staining with rho 3A6 in the outer nuclear layer than P23H-E and P23H-L retinas. Note the specific staining in the outer plexiform layer of P23H-D and P23H-E retinas with the human-specific antibody. OS/IS, outer segmentiinner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Bar, 25 urn.

NHR-A

Figure Unfixed (center) 21 pm.

11. Human

Rod Opsin

Expression

within

NHR-A

Transgenic

Mouse

Retinas

(Postnatal

Day IO)

cryostat sections of an NHR-A retina were stained with either monoclonal rho ID4 (left) or rho 3A6 (right). Nomarski images are shown for reference. OS/IS, outer segment/inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer. Bar,

indicate that the 4.4 kb immediately upstream of the transcription start site of the rhodopsin gene are sufficient to direct photoreceptor-specific expression of a reporter gene. Of interest is that those investigators described a geographic variation in the expression of the reporter gene in the retina when 2 kb or fewer of upstream promoter sequence was used, with photoreceptors in some areas showing a higher level of expression than others. We did not observe a commensurate regional variation in the expression of the transgenic rod opsin, judged by the degree of bound rho 3A6 antibody in sections of eyes from the NHR-E and P23H-L lines of mice. The absence of any conspicuous geographic variation in the expression of a transgenic, human rhodopsin allele possibly indicates that some sequences necessary for panretinal expression of rod opsin may lie in regions included in our constructs but not included in some of the reporter gene constructs (i.e., DNA sequences lying more than 2 kb upstream of the cap site, within the transcriptional unit, or downstream of the last exon).

The observation of photoreceptor-specific expression of our constructs has relevance to the nature of dominant mutations in the rhodopsin gene that cause retinitis pigmentosa. One concern early in this project was whether photoreceptors express these mutant opsins. Although most of the rhodopsin gene mutations found in patients with dominant retinitis pigmentosa are missense, the possibility remained that they might be nullor inactive alleles and that photoreceptor degeneration was caused by a hypothetical reduction in the amount of functional rod opsin synthesized by photoreceptors with only one wild-type allele. Some support for this hypothesis comes from a collection of mutant alleles in the Drosophilia Rhl opsin gene (ninaE) that cause photoreceptor degeneration (Washburn and OTousa, 1989). Many of the ninaE alleles are missense mutations; nevertheless, they behave as nullalleles. If this were the situation in dominant retinitis pigmentosa, then adding a mutant human allele to the mouse genome with two preexisting, wild-type alleles should result in no discernible

RPE >os

>IS

ONL

Figure 12. Human Rod Opsin Expression within P23H-L Transgenic Mouse Retinas (Postnatal Day 15) Unfixed cryostat sections of P23H-L retinas were stained with monoclonal rho 3A6. Note the staining in the developing outer segments. IS, inner segment; OS, outer segment; ONL, outer nuclear layer; RPE, retinal pigment epithelium. Bar, 15 pm.

Retinal a27

Degeneration

in Mice

with

Pro23His

Rhodopsin

phenotype. Our observation of the expression of human rodopsin in theP23Hlinesof miceandthesubsequent retinal degeneration eliminates these concerns and substantiates the position that the mutations associated with dominant retinitis pigmentosa are either gain of function or dominant negative alleles. Having established that the P23H allele is not a null allele, one is faced with the question of what property of P23H rod opsin causes retinal degeneration. It is conceivable that it is the consequence of some toxic property shared by both wild-type and P23H human rod opsins when they are expressed in the murine photoreceptors. The NHR-E strain of mice effectively eliminates this possibility, since these mice express wild-type human rod opsin in amounts comparable with murine rod opsin (judging from mRNA levels), yet suffer no retinal degeneration.Twoviable explanations remain: the photoreceptorsdegenerate because of a property specific to P23H opsin and/or because they express a deleterious overabundance of opsin. To answer this question, it would be helpful to measure the quantity of transgenic opsin mRNA and translated protein that is produced per rod. Unfortunately this is problematic for all but the NHR-E strain of mice, since the rapidly progressive photoreceptor degeneration would confound such measurements. However, it is straightforward to measure the relative proportions of transgenic (human) versus murine rod opsin mRNA. Such measurements clearly distinguish the nondegenerating NHR-E line of mice from the degenerating NHR-A line. The former has approximately equal amounts of murine and human rod opsin mRNA, while the latter has a 6-fold excess of human rod opsin. Assuming that the retinal degeneration in the NHR-A line is not due to the fortuitous alteration of an unspecified “retinal degeneration” gene at the transgene insertion site, it appears that the photoreceptor degeneration in this line is related to an overproduction of transgenic (human) opsin. Supporting this reasoning are the previously documented associations of cell death due to cell-specificoverexpression of a transgene (Gilfillan et al., 1990; Turnley et al., 1991). For example, pancreatic overexpression of either calmodulin (Epstein et al., 1989), the H-ras oncoprotein (Efrat et al., 1990), class 1 histocompatibility molecules (Allison et al., 1988), or Bz microglobulin (Allison et al., 1991) causes diabetes by destroying insulin-producing B cells. It is intriguing to speculate that some patients with autosomal dominant retinitis pigmentosa might have a defect in cis-acting sequences, leading to overexpression of rod opsin and consequent retinal degeneration. On the other hand, it is possible that the deleterious effects of human opsin overproduction in mice might be species specific, in which case one would not expect elevated levels of human opsin in humans to lead to degeneration. Although all P23H lines of mice exhibit photoreceptor degeneration, the variation in the severity of the degeneration correlates with the level of transgenic opsin expression in comparison with the native, mu-

rine rod opsin expression. However, even the P23H-L line, with a transgene mRNA level considerably less than that seen in the NHR-E line, develops retinal degeneration that is strikingly apparent by age 20 days. Evidently a property of P23H opsin induces degeneration. The precise mechanism by which this occurs remains to be defined. It appears to be different from that responsible for hereditary retinal degeneration in the RCS rat (Mullens and LaVail, 1976), which has a defect in the ability of retinal pigment epithelial cells to phagocytose outer segments. P23H opsin does not seem to interfere in this process, since we observed phagosomes containing outer segment disc material in the retinal pigment epithelial cells of P23H-L mice. Sung et al. (1991b) expressed P23H rod opsin in vitro and found that unlike wild-type opsin, it was absent from the cell surface membrane. Instead, P23H rod opsin accumulated in the endoplasmic reticulum. Similarly ninaA mutations in Drosophilia lead to an accumulation of Rhl opsin in the cytoplasm of RI-R6 photoreceptors. In those flies, the opsin is presumably abnormally folded and/or poorly transported within the photoreceptors due to a defect in the ninaA gene product, a peptidyl-prolyl-cis-trans-isomerase (Colley et al., 1991). Based on these findings, one might speculate that structurally defective opsins, possibly including P23H opsin, aberrantly accumulate within the inner segment, thereby leading to cell death. The dilated inner segments found in the early stages of degeneration in the P23H-L line are consistent with such a mechanism. However, we did not detectthedramaticaccumulationsof roughendoplasmic reticulum as seen in ninaA mutants (Colley et al., 1991). Also, some P23H opsin was detected in photoreceptor outer segments. These results imply that alternative explanations for the photoreceptor degeneration are still tenable. For instance, P23H might be a dominant negative allele coding for an opsin that interferes with the molecular machinery responsible for the processing and transport of wild-type opsin that is concurrently being expressed. The frequent disruption of Golgi bodies by amorphous, osmophilic material suggests that this organelle might be involved in this hypothetical mechanism. Alternatively, the unexpected presence of P23H rod opsin at the synaptic end of the rods speaks for a possible mechanism involving a deranged intracellular transport system, and it is conceivable that photoreceptor death is a consequence of the rod opsin’s interference with the function of the proximal segment of the rod. Resolution of these issues will probably await the development of monoclonal antibodies that can distinguish human P23H opsin from murine opsin and that are suitable for ultrastructural studies. W e know of only one published report of postmortem ultrastructural findings from a patient with autosomal dominant retinitis pigmentosa due to a P23H allele (Kolb and Couras, 1974). That paper pertains to a 68-year-old woman who was an affected member of a family that we now know to carry this mutation

Neuron a28

(individual l-2 in family 1566 described in Berson et al. [1991a]). It is difficult to compare the histological abnormalities found in that case with those found in the transgenic mice reported here, because the patient had advanced disease with only fovea1 cones present and no remaining rods. Since no eyes from younger patients have yet been examined, it remains to be seen to what extent structural changes in these mice simulate those seen in humans with the early stage of degeneration. This study provides unequivocal support for the conclusion derived from clinical studies that mutant copies of the rhodopsin gene cause photoreceptor degeneration. Transgenic mice expressing a mutant human rhodopsin allele develop photoreceptor degeneration with funduscopic and electroretinographic abnormalities similar to those seen in patients with retinitis pigmentosa.These micewill permit studies aimed at better understanding the pathophysiologyof retinal degeneration and at evaluating thevalue of hypothetical therapeutic modalities that may alter its course.

Experimental

Procedures

Isolation of the Transgenes DNA was extracted from IO ml of peripheral blood from patient AD191. One hundred micrograms was partially digested with Mbol, and fragments between 12 kb and 20 kb were isolated after preparative agarose gel electrophoresis. These fragments were ligated into the bacteriophage vector EMBL3 (Frischauf et al., 1983) and packaged with Gigapack II Cold Packaging extract (Stratagene). Approximately 1.6 x IO6 independent recombinant phage were screened by plaque hybridization using a ‘*P-labeled 6.8 kb BamHI-Hindlll human genomic probe derived from the rhodopsin gene, kindly provided by J. Nathans (Nathans and Hogness, 1984). Positive plaques were purified and amplified using standard techniques. Purified phage DNA was analyzed by direct genomic sequencing to distinguish those phage with the P23H allele from those with the wild-type allele (Dryja et al., 1990a). The extent of the 5’ and 3’ flanking sequences was determined by comparing restriction digests of the phage DNA with the previously published map of the human rhodopsin locus (Nathans and Hogness, 1984). Construction of Transgenic Mice Inserts from two recombinant phage (one P23H allele and one wild-type allele) were selected for making transgenic mice. The genomic inserts were excised from the cloning vector with Sall, electroeluted from agarose gels, and dialyzed extensively against 10 m M Tris-HCI, 0.18 m M EDTA (pH 7.8), after dilution to a concentration of loo0 DNA molecules per pl. B6D2F1 female mice were superovulated and mated to males of the same strain. Zygotes were recovered and microinjected with either of the two DNA constructs as described by Gordon et al. (1980) and Gordon and Ruddle (1981). Screening of Mice for the Presence of Human Transgene in Tail DNA W e synthesized pairs of primers that amplified exons 2 or 5 specifically from the human rhodopsin gene. These exons were separately amplified using 100 ng of mouse tail DNA as a template for the polymerase chain reaction. (For details of primers and conditions see Dryja et al. [199Ob].) Since the primers were based on nonconserved intron sequences, they amplified only human rhodopsin gene sequences, and the presence of amplified fragments of 290 bp (exon 2) or 277 bp fexon 5) signified that a mouse carried the transgene.

Indirect Ophthalmoscopy and Fundus Photography Pupils were dilated with topically applied 5.0% phenylephrine hydrochloride, 0.8% tropicamide. Fundi were examined using a standardindirectophthalmoscope(KeelerFisionorMlRASchepens-Pomerantzeff) and a 90 diopter (Nikon) or a 60 diopter (Volk) condensing lens. Fundus photographs were taken, using the Kowa RC-2 hand-held fundus camera and a 90 diopter or a 60 diopter condensing lens, on Kodak Ektrachrome 100 Professional color reversal film. Electroretinographic Testing Mice were dark adapted for at least 12 hr prior to ERG testing. Under dim red illumination, pupils were dilated with one drop of 0.2% phenylephrine hydrochloride and 0.02% cyclopentolate hydrochloride, concentrations that in our experience sustained maximal dilation without cornea1 opacification in mice. Animals were anesthetized with 80 mglkg sodium pentobarbital injected intraperitoneally. An insulated platinum wire within a 30 gauge (approximately 0.25 m m diameter) stainless steel cannula was inserted through the pars plana into the vitreous to monitor the ERG; acotton wick saturated with saline was placed in the mouth as reference electrode. A subdermal electrode served as a ground. ERGS were elicited from one eye of each animal with 0.1 Hz, 10 us flashes of bright white light (Sandberg et al., 1986). Signals were differentially amplified, recorded on magnetic tape, digitized, filtered, and averaged by computer. Amplitudes were measured from baseline to the peak of the cornea-negative deflection for the a-wave and from the latter (or baseline if absent) to the highest cornea-positive peak for the b-wave. Response times were measured from the flash onset to the peak of the b-wave. Following the ERG testing, animals were immediately sacrificed, and the fellow, untested eye was enucleated and fixed for histological study. Quantitation of Mouse and Human Rod Opsin mRNA in Retinas Retinas were surgically removed from sacrificed 20-day-old mice and frozen in liquid nitrogen. Messenger RNA was isolated from between three and six pooled retinas from the same strain using the lnvitrogen Fast Track mRNA kit (version 3.0). Messenger RNA was similarly isolated from the normal retina recovered from an 8year-old boy without retinal degeneration who underwent therapeutic enucleation for an intraocular tumor. Oligomers were synthesized corresponding to regions of the rhodopsin gene coding sequence that were identical in humans and mice. Oligo 1125 was a sense sequence derived from exon 1 (S-TCAACTACATCCTCCTCAAC3’), and oligo 1126 was an antisense sequence derived from exon 2 (S-GGCTTCGCCTTCAACGAGTA-3’). These exons are separated by an intron that is 1782 bp in the human and 1487 bp in the mouse rhodopsin gene (Al-Ubaidi et al., 1990). The antisense oligomer 1126 was used to prime first-strand cDNA synthesis using the retinal mRNA as a template. Single-strand cDNA molecules were used as templates to amplify simultaneously the human and murine cDNA sequences between the two priming sites. The human and murine genomic sequences including intron 1 will not amplify from any possibly contaminating genomic DNA with these primers and polymerase chain reaction conditions (data not shown). [a-32P]dCTP was incorporated during the polymerase chain reaction, in which each target sequence was amplified for 35 cycles using anEricompProgrammableCyclicReactor.Annealingwasat520C for 30 s, polymerization was 71°C for 30 s, and denaturation was 94OC for 30 s. The last cycle was for 90 s at 52OC and 4 min at 94OC. Five microliters of the labeled amplification product was diluted in 5 or 10 ul of 10 m M EDTA, 0.1% SDS. Human and murine cDNA sequences amplified by primers 1125 and 1126 were separated by electrophoresis through 6 % polyacrylamide gels containing 10% glycerol. To quantitate the relative levels of human and murine mRNA, a microdensitometer was used to record the intensity of bands seen on X-ray film exposed to the acrylamide gels at room temperature for 24-48 hr. Fixation of Eyes for Histology Enucleated eyes were placed

in 1 % formaldehyde,

2.5% glutaral-

Retinal Degeneration 829

in Mice with Pro23His Rhodopsin

dehydeinO.l Mcacodylated buffer(pH7,5)at4”Cforaminimum of 2 hr. Eyes were postfixed in osmium tetroxide and prepared for both light and electron microscopy. For light microscopy, 1 pm thick sections were taken through the horizontal meridian at or near the level of the optic disc and through the superior and inferior retina in some eyes. These sections were stained with 50% azure II and 50% methylene blue. For electron microscopy, 0.1 gm sections were stained with lead citrate and uranyl acetate. lmmunofluorescence Monoclonal antibody rho 3A6 (Hodges et al., 1988) recognizes an epitope encompassing amino acids 337-341. The antibody binds strongly to bovine and human rod opsins but does not recognize the homologous mouse opsin. (Amino acid 337 is re sponsible for the inability of rho 3A6 to bind mouse opsin.) Antibody rho lD4 (Hodges et al., 1988) recognizes an epitope encompassing amino acids 341-348 and recognizes both human and mouse rod opsin. Secondary antibodies, Cy3-conjugated goat anti-mouse immunoglobulin G’s, were commercially obtained (Jackson ImmunoResearch, West Grove, PA). The sclera from eyes of sacrificed mice were marked to indicate ocular orientation. Eyes were then enucleated and frozen in OCT medium without prior aldehyde fixation. Cryostat sections (4-6 J.tm) taken in the superior midsagittal plane were mounted on silanized slides.Thesectionswerestainedas previouslydescribed(Roof et al., 1991) and mounted in Mowiol medium. Indirect fluorescence images were recorded using TriX film. Animal Procedures All animal procedures conformed to the Association for Research in Vision and Ophthalmology and National Institutes of Health statements on the use of animals in ophthalmic and vision research. Acknowledgments We gratefully acknowledge J. Morrow and M. Shokravi for assistance; M. Adamian, M. Sandberg, and C. Heth for useful discussions; R. York, D. Shen, and P. Kizelewicz for maintenance of the animal colony; and F. Delori for helpwith the microdensitometry measurements. This work was supported by grants from the National Institutes of Health (EY 08683, EY00169, EY06514, EY02422,and HD20484), the Australian Retinitis PigmentosaAssociation, the National Retinitis Pigmentosa Foundation Fighting Blindness (USA), the Retinitis Pigmentosa Research Foundation (Canada), the George Cund Foundation (Cleveland, Ohio), and a gift from the Smart Family Foundation. E. L. 6. is a Research to Prevent Blindness Senior Scientific Investigator. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

May 19, 1992; revised

July 21, 1992.

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