Tissue-specific and developmental regulation of rod opsin chimeric genes in transgenic mice

Neuron,

Vol. 6, 201-210, February, 1991, Copyright

0 1991 by Cell Press

Tissue-Specific and Developmental Regulation of Rod Opsin Chimeric Genes in Transgenic Mice janis Lem,* Meredithe 1. Applebury,+ Jeffrey D. Falk,+§ John C. Flannery,* and Melvin I. Simon* *Division of Biology California Institute of Technology Pasadena, California 91125 +The University of Chicago Eye Research Laboratories Chicago, Illinois 60637 *Jules Stein Eye Institute University of California, Los Angeles School of Medicine Los Angeles, California 90024

Summary Chimeric gene fusions between 4.4 kb of rod opsin 5’ flanking sequence fused to a diphtheria toxin gene and 4.4 kb or 500 bp of rod opsin 5’ flanking sequence fused to the E. coli /acZgene were used to generate transgenic mice for analysis of cell type-specific expression and temporal and spatial distribution of reporter gene product during retinal development. Opsin-diphtheria toxin transgene expression evoked photoreceptor-specific cell death. The 4.4 kb opsin-lacztransgene followed temporal and spatial gradients of expression that approximate opsin expression. The 500 bp opsin fragment targeted expression to photoreceptors, but expression was weaker and nonuniform, suggesting that elements located upstream may be required for enhanced and uniform spatial expression. introduction The development of the retina is remarkably conserved among vertebrate species (Ramon y Cajal, 1894, 1929; Griin, 1982). Elegant studies indicate that both neural and glial cells composing the retina are generated from proliferating precursor neuroepithelial cells that are determined near the time of the final mitotic division (Turner and Cepko, 1987; Turner et al., 1990; Holt et al., 1988; Wetts and Fraser, 1988). A complex developmental program must specify the characteristic stratification of retinal cells, patterns of cell distribution, and for photoreceptors in particular, cell type commitment with delayed morphogenesis (Polley et al., 1989; Zimmerman et al., 1988; Watanabe and Raff, 1990). Determining factors likely to be involved in controlling development include intrinsic genetic programming, response to cell-cell interactions, and factors from the microenvironment (Reh and Tully, 1986; Moscona, 1987; Watanabe and Raff, 1990). The molecular mechanisms directing gene exOPresent address: Department of Molecular Biology, institute of Scripps Clinic, La Jolla, California 92037.

Research

pression in the vertebrate retina are relatively unexplored. Promoter analysis of retinal genes would be invaluable in elucidating mechanisms of coordinate gene regulation. Such studies have recently become possible with the molecular cloning of a set of genes encoding the protein components of visual transduction in vertebrate rod and cone photoreceptor cells (Falk et al., 1987; Baehr et al., 1988; Nathans and Hogness, 1984; Nathans et al., 1986; Lochrie et al., 1985; Tanabe et al., 1985; Lerea et al., 1986; Raport et al., 1989; Lipkin et al., 1990; Kaupp et al., 1989; Lochrieand Simon, 1988). Thus, the analysis of genetic sequences controlling photoreceptor development as well as those responding to signaling factors from the environmentisnowpossible.Todescribethemechanisms of gene expression that define this program, the identityof thesesignals and howtheyare interpreted must be established. With this goal In mind, molecular genetic studiesof murine rod photoreceptor opsin gene expression in the retina have been initiated. Thevisual pigment protein encoded by this gene is expressed only in rod photoreceptors in mammals. To identify the controlling regions of the rod opsin gene that specify photoreceptor-specific and appropriate temporal expression, transgenic mouse lines have been generated with chimeric gene fusions of the rod opsin 5’ flanking regions with two different reporter genes. One study of a transgenic line was carried out with the diphtheria toxin A (DTA) subunit as a reporter gene. The DTA gene encodes an ADP ribosyltransferase, which ribosylates elongation factor 2, inhibiting protein synthesis and leading to cell death. As a single molecule of diphtheria toxin is sufficient to kill a cell, the toxin provides a very sensitive assay for the analysis of tissue-specific and developmental gene expression (Yamaizumi et al., 1978). Typically, DTA chimeric gene fusions are used to study cell lineage relationships between precursor cells and subsequently differentiating cell types (Palmiter et al., 1987). It is generally accepted that rod photoreceptor cells are the last cells to develop in the retina, and little information with regard to cell lineage is expected. However, the ablation of rods might affect development of neighboring cells and define the role that rod photoreceptor cells play in the generation or maintenance of other retinal cell types. A second study was carried out with two different opsin fragments, the 4.4 kb upstream region and a truncated 500 bp piece, fused with the E. coli /acZ structural gene. The latter reporter gene encodes the bacterial Bgalactosidase enzyme (LacZ), whose activity is readily detected with the chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-f%o-galactopyranoside) or by immunocytochemistry with the appropriate antibody. Histochemical staining of tissues produces an insoluble blue precipitate visible by light microscopy in cells expressing active LacZ enzyme.

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4 4 kb T”d ODSl”

DTA

\

sv40 POSY A

B 4 4 Lb rod “psm POSY A

C poly

Figure 1. Chimeric

Fusion

A

Gene Constructs

(A) The4.4 kb rod opsin Sflanking upstream sequences fused to the DTA reporter gene and SV40 small t antigen polyadenylation sequences. (B) The same 4.4 kb rod opsin fragment fused to the /acZ reporter gene and the SV40 VP1 polyadenylation sequence. (C) Rod opsin fragment truncated at the Bglll restriction site to generate a 500 bp 5’flanking sequence fused to the /acZ reporter gene.

Studies of opsin-/acZ chimeric genes will provide information about regions controlling cell-specific expression and developmental patterns of gene expression. In addition, both temporal and spatial aspects of gene expression can be examined in the developing and adult retina.

Expression of a Rod Opsin-Diphtheria Toxin Fusion Gene in Transgenic Mice The rod opsin-DTA fusion transgene plasmid, pRDT1, was created by the fusion of 4.4 kb of 5’ flanking rod opsin sequence to an 800 bp DTA subunit structural gene (Figure IA; see Experimental Procedures for details). The fusion genewas microinjected without plasmid sequences into one-cell mouse embryos. Six of 102 animals analyzed carried the pRDT1 transgene, as determined by slot blot hybridization analysis of genomic DNA isolated from tail samples (data not shown). Five transgenic mouse lines displayed no phenotypic evidence of diphtheria toxin expression in retinas of heterozygous animals examined. The sixth transgenic line, designated pRDT9-1, exhibited cellular ablation of photoreceptor cells in all adult heterozygotes examined carrying the transgene (Figure 2B). The outer nuclear layer, the outer plexiform

layer, and photoreceptor inner and outer segments were conspicuously absent, as compared with normal adult retina (Figure 2A). Very little cellular debris was observed adjacent to the pigment epithelium. Macrophages or proliferating pigment epithelial cells, however, wereobserved in theganglion and inner nuclear cell layers. The remainder of the retina, composed of the ganglion cell, inner plexiform, and inner nuclear layers, appeared normal and contained a normal complement of cells, as determined by electron microscopy. The transgene appeared to be integrated at l-5 copies, as determined by Southern blot analysis (data not shown). All results described in this study were from heterozygous animals. Therewas no phenotypic evidence of transgene expression in any other tissues of the body, including the pineal gland, which has been reported to exhibit immunoreactivities to opsin, S-antigen, a-transducin, and IRBP in a number of poikilothermic vertebrates (Vigh-Teichmann and Vigh, 1986; Korf et al., 1985a, 1985b, 1989; van Veen et al., 1986a, 1986b). The onset of diphtheria toxin expression was ascertained by backcrossing RDT9-1 heterozygous animals with wild-type animals. Retinal degeneration cosegregated with the transgene as a dominant allele. Retinas from age-matched heterozygous transgenic mice and their normal siblings were compared from postnatal days 5 (P5) to adulthood by histological staining of frozen serial sections. DTA expression was first noted in transgenic retinas at P7 (Figure 2E) as compared with an age-matched normal sibling (Figure 2D). The outer plexiform layer, readily apparent in the normal sibling, was disrupted in the retina of the transgenie sibling. In addition, the frequency of small pyknotic cells in the transgenic retina was noticeably greater than the numbers observed in the control retina due to normal histogenetic cell death (Young, 1984) and was confined to photoreceptor layers. Otherwise, development of photoreceptors appeared quite normal in thetransgenic mouse retina. Photoreceptor layers became increasingly disorganized in older transgenie animals. By P9, the inner segments of the retina were gone. By P13, all photoreceptor cell layers were ablated. Only the ganglion cell, inner plexiform, and inner nuclear cell layers remained at PI8 (Figure 2C). The thickness of the inner nuclear cell layer gradually decreased as the animal aged (compare with Figures 2B and 2C), but this is a normal consequence of photoreceptor loss, as it has also been reported in mouse models of spontaneous retinal degeneration (Blanks and Bok, 1977). Electron microscopic examination of adult degenerate retinas revealed the complete loss of both rod and cone photoreceptors at 3 months of age (data not shown). However, no attempt was made in these studies to look for persistence of the less abundant cone cells at earlier ages, as is apparent in spontaneous retinal degenerations of mouse, when cone cells have been reported to persist for a short time after rod photoreceptor degeneration.

Rod Opsin Fusion Gene Analysis in Transgenic 203

Mice

pe in1

ipl @l

w is OPl

in1

ipl gel

Figure 2. Retinal

Sections

from

RDT9-1

Transgenic

Mice

Eyecups were sequentially embedded in polyacrylamide and frozen in OCT compound for sectioning. (A) Normal adult retina. (6) Adult transgenic retina devoid of photoreceptor layers and showing a thinning inner nuclear layer. (C) PI8 transgenic retina devoid of photoreceptor cells. Remaining layers of the retina appear morphologically normal, and there is no obvious thinning of the inner nuclear layer. (D) Normal P7 retina. (E) P7 transgenic retina and sibling of mouse retina shown in (D). Note the disruption of the outer plexiform layer and the increased frequency of pyknotic cells in the transgenic retina. Sections were stained with hematoxylin and eosin. pe, pigment epithelium; OS, outer segment; is, inner segment; onl, outer nuclear layer; opl, outer plexiform layer; inl, inner nuclear layer; ipl, inner plexiform layer; gel, ganglion cell layer. Magnification, 144x.

Expression of a Rod Opsin Promoter-/acZ Gene in Transgenic Mice

Fusion

To verify cell-specific expression and study temporal and spatial patterns of developmental expression directed bythe rod opsin upstream flanking sequences, the same 4.4 kb rod opsin promoter region used in pRDT1 was fused to a 3.8 kb IacZ structural gene to create the plasmid pRG3 (Figure IB; see Experimental Procedures for details). The fusion gene was microinjetted after the removal of plasmid sequences. The pRG3 transgene was detected in 2 of 49 animals analyzed by slot blot hybridization analysis of genomic DNA isolated from tails and appeared to be present at l-5 copies in each line, as determined by Southern blot analysis (data not shown). The two transgenic mouse lines, designated RG2-2 and RG4-4, exhibited photoreceptor cell-specific LacZ expression in heterozygous adult animals, as determined by staining of lightly fixed histological sec-

tions with the chromogenic substrate X-Gal. LacZ staining was apparent in the myoid region of the photoreceptor inner segments, throughout the nuclear region, and in the outer plexiform layer of RG2-2 (Figure 3A) and RG4-4 (data not shown) transgenic mice. lmmunohistochemistry using a polyclonal antibody to the LacZ at the electron microscope level localized the enzyme to the inner segments and, to a lesser extent, to outer segments of rod photoreceptor cells of RG2-2 transgenic mice (Figure 4). No product was detected in the retinal pigment epithelium nor in other cells of the neural retina. These observations areconsistentwith X-Gal staining seen by light microscopy and confirm the localization of the transgene product to the rod cell. We have examined some cone cells for LacZ expression, but have not been able to confirm or reject expression in this cell type definitively. In adult animals aged 5 months and older, LacZ staining was also observed in photoreceptor outer

is

OPl

Figure 3. Transgenic

Mouse

Retinas Stained

for LacZ Activity

(A) Frozen retinal section from an RC2-2 transgenic mousewith the4.4 kb rod opsin-IacZtransgene, X-Gal staining of the section detects LacZ as an intense blue precipitate localized in the inner segments (is) and outer plexiform layer (opt) of the retina. Magnification, 124x (8) Retinal section from an RGA6-3 transgenic mouse with the truncated 500 bp rod opsin-lacZ transgene. Occasional photoreceptor inner segments and outer plexiform cells exhibited LacZ. Magnification, 124x. (C-E) Gradient of LacZ stai.ning in representative serial sections from RC2-2 transgenic mouse retinas at P13, P15, and 6 months ot age, respectively. Retinas werecut along a horizontal meridian, with the temporal aspect to the left and the nasal aspect to the right. Retina\ showed an expanding crescent of staining with a temporal bias, which persisted to adulthood. Magnification, 16x. (F-H) Whole mount retinas from RG2-2, RG4-4, and RGA6-3 transgenic animals, respectively. Retinas were stained with X-Cal to show LacZ staining patterns. Temporal aspect is to the left, nasal to the right, superior to the top, and inferior to the bottom. The RG2-2 retina (F) from a l-year-old animal shows a gradient of expression with uniform, even staining in the superior-temporal hemisphere of the eye and mottled staining in the inferior-nasal hemisphere. The RG4-4 transgenic retina from a l-year-old animal (C) shows uniform, even staining for LacZ expression across the entire retina. The RGA6-3 whole-mount retina (H) from a Cmonth-old animal stained for LacZ activity shows a diagonal band of staining just temporal of the optic nerve. Magnification, 16x.

segments by light microscopy. No expression was detected in adult pineal, brain, heart, kidney, spleen, lung, or testes by X-Gal staining of sectioned or whole tissues in either line of transgenic animals. Embryos taken at two stages of gestation were stained and examined for LacZ activity. At embryonic day 14 (E14) RG2-2 mice exhibited transient expression of LacZ in somites, limbs, and a spot in the brain (data not shown). This expression was no longer detectable in El7 embryos. In the RG4-4 line of animals no expression was observed in any tissue at E14.

Expression of a Truncated 500 bp Rod Opsin Promoter-/acZ Fusion Gene in Transgenic Animals In addition to the 4.4 kb rod opsin fusion constructs, a truncated 500 bp rod opsin-/acZfusion gene (Figure IC) was introduced into transgenic animals. From a total of 61 animals born, 4 transgenic animals were identified. We were only able to breed two of the lines. One of the two lines, RGA6-3, expressed the /acZ product as determined by reaction with X-Gal. No expression was detected in heart, spleen, kidney,

Rod Opsn 205

Fusion Gene Analysis in Transgenic

Mice

brain, liver, or testes of adult animals. No expression was detectable in the other line. Expression of LacZ in the RGA6-3 animal was observed specifically in photoreceptor cells of the retina (Figure 38). Enzyme activity was found primarily in the inner segments and, to a lesser extent, in the outer plexiform layer. However, expression was sporadic. It was confined to a few individual photoreceptor cells rather than uniformly expressed in a majority of photoreceptor cells across the surface of the retina, as was seen in transgenic animal lines made with the4.4 kb rod opsin promoter. The degree of enzyme activity observed based on staining was clearly less in these lines, Developmental and Spatial Expression in Rod Opsin-/acZ Transgenic Mice The developmental time course of rod opsin-/acZ expression in the RG2-2 and RG4-4 transgenic animal lines was studied by examining frozen serial tissue sections of the entire retina cut along the horizontal meridian of the eye. Retinas from P5 to adult animals were stained for LacZ. In these two lines of transgenic mice, temporal patterns of expression were similar and LacZ was first detectable at P13. Adetailed characterization of RG2-2 retinal development is described. Staining first appeared as a blue crescent just temporal to the center of the retina (Figure 3C). By P14-P15, the crescent of staining had expanded peripherally toward the margins of the retina, still displaying a temporal bias (Figure 3D). The crescent continued to expand until the entire breadth of the retina was covered by P18. The intensity of X-Gal staining continued to increase with age, with the gradient evident up to 6 months of age. In this animal line, the nasal portion of the retina showed mottled, punctate staining (Figure 3E). To determine further the spatial distribution of the LacZ, whole-mount retinas were stained with X-Gal. RG2-2 retinas revealed a uniform blue precipitate in the superior-temporal aspects of the retina (Figure 3F). In contrast, the inferior-nasal aspect showed a very uneven mottling. This gradient of expression was present in retinas of l-month-old animals and persisted up to a year later. While retinal sections of RG4-4 animals showed the same temporal and spatial patterns of staining during development as described for RG2-2 animals above, stained whole-mount retinas of mature RG4-4 animals showed relatively even, uniform staining across the retina (Figure 3G). There was no evidence of a spatial gradient in stained wholemount retinas of RC4-4 animals either at 1 month or at 1 year of age. Preliminary studies of developmental expression in the truncated rod opsin RGA6-3 animals clearly showed expression by Pll, as determined by X-Gal staining of whole-eye mountsof P7and PI1 transgenic mouse retinas. Stained whole-eye mounts of 1.5month-old adult transgenic animals exhibited a mottled band of LacZ expression that ran diagonally from

the temporal-inferior quadrant to the nasal-superior quadrant. This distinctive band of expression persisted up to 4 months of age (Figure 3H). Discussion The 5’ Regulatory Region of the Opsin Gene Targets Expression to Photoreceptors To define regions of the mouse opsin gene responsible for rod-specific expression, we have examined a 4.4 kb 5’flanking region of the murine rod opsin gene. The fragment was ligated to two different reporter genes, the DTA subunit and IacZ genes and used to generate transgenic mouse lines for study of expression. In addition to the longer 4.4 kb fragment, a truncated 500 bp opsin fragment was ligated to the lacZ gene and used to generate another line of mice. The information necessary for targeting expression of a structural gene to photoreceptor cells was found to lie within the 5’ flanking region. Both the longer and truncated fragments were capable of cell-specific targeting; however, distinct differences in spatial expression were noted. As judged by histochemical detection of LacZ activity or ablation of cells, transgenic lines carrying any of the rod opsin constructs expressed activity only in the photoreceptor layer of the retina (Figures 2-4). Expression of the diphtheria toxin reporter gene emphasizes the specificity imposed by the opsin gene regulatory region. Cell death was observed only in the photoreceptor cell layer of the retina (Figure 2). Clearly, little or no leakage of expression in other tissues occurred that was detrimental to the development or viability of the animal. In transgenic lines carrying the longer 4.4 kb rod opsin region, expression was distributed across the entire retinal surface of mature animals (Figure 3). The majority of photoreceptor cells expressed LacZ, consistent with mouse rod cell distribution (Figure 3A; Carter-Dawson and LaVail, 1979). LacZ expression was primarily evident in photoreceptor inner segments, but it also extended into the outer nuclear layer and into the outer plexiform layer. In the mature animal, histochemical staining was observed in the outer segments. Higher resolution immunoelectron microscopic studies using a polyclonal antibody raised against LacZappropriately localized LacZ in the rod inner segment and myoid region and, to a lesser extent, in outer segments and the synaptic processes (Figure 4). No product was detected in other retinal cell types or the pigment epithelium. The localization of LacZ to inner segments and the myoid region is consistent with the expression of rod opsin early in cell morphogenic development. Later, the majority of rod opsin protein is sequestered within rod outer segments of the mature photoreceptor, and only a small pool of newly synthesized opsin is found in the inner segment region (Nir et al., 1984; Hicks et al., 1989). As LacZ is not a transmembrane protein, it is not surprising that it is ineffi-

Neuron 206

gene, since RG4-4 animals did not exhibit embryonic staining at E14. For each of the other three transgenic lines lines studied, expression was restricted to photoreceptor cells and was not detectable in any other tissues of the body. In addition to cone cells, there is potential for expression in the pineal gland, which has cells with structures resembling photoreceptors in some species. We do not observe LacZ staining in the the pineal gland or anywhere in the brain.

Figure 4. Electron Micrograph of Photoreceptors from a IS-MonthOld RG2-2 Transgenic Mouse Expressing LacZ Polyclonal antibody labeling shows LacZ localized to the inner segment of the rod photoreceptor and, to a lesser extent, in the photoreceptor outer segment. Magnification, 25,000x.

ciently transported to the outer segments. Because of the low abundance of cone cells and the dense packing of photoreceptors, our current methods of examination neither confirm nor eliminate the possibility of expression in cone ceils. A more detailed study is underway. One transgenic line, RG2-2, also showed transient expression of LacZ activity at El4 in somites, limbs, and a region of the brain in the developing embryo. The activity ceased by E17, and further expression was detected only postnatally in photoreceptor cells. This transient expression may reflect the influence of neighboring sequences at the site of integration of the trans-

A 4.4 kb 5’ Regulatory Region of the Opsin Gene Directs Appropriate Temporal and Spatial Expression during Development The timing of developmental expression in the various transgenic mouse lineswas determined using animals between the ages of P5 and adulthood. The RDT9-1 transgenic mouse line first exhibited detectable phenotypic effects of diphtheria toxin at P7 (Figure 2). The outer plexiform layer was disorganized, and a high frequency of pyknotic cells were observed in photoreceptor cell layers compared with that seen in the retina of an age-matched normal sibling. The developmental onset of diphtheria toxin expression correlates with the time reported for rod opsin expression in mouse (Bowes et al., 1988; lshiguro et al., 1987). Thus the 4.4 kb rod opsin 5’ untranslated flanking sequences are capable of directing appropriate temporal expression. The RG2-2 and RG4-4 transgenic mouse lines containing the full-length rod opsin fusion gene showed initial expression of active LacZ at PI3 (Figure 3C), about the time of eye opening when rod opsin expression is at its peak. Expression of LacZ is much delayed compared with diphtheria toxin expression and rod opsin expression. Zack et al. (1991) similarly report delayed detection of LacZ activity at PI0 to PI2 in transgenic mice carrying a bovine rod opsin-/acZ chimeric gene fusion. The delay in developmental onset may simply reflect differences in assay sensitivity. A single molecule of diphtheria toxin is sufficient to induce cell death (Yamaizumi et al., 1978). However, ~1000 molecules of LacZ per cell are required for detection, and the LacZ molecule must tetramerize to be active (MacGregor et al., 1991). Thus levels of LacZ at P7 may be below the level of sensitivity of the X-Gal chromogenic assay used in these experiments. The initial expression of LacZ in RG2-2 and RG4-4 appeared centrally with a temporal bias (Figure 3C). This central crescent of LacZ staining spread peripherally, mirroring the central to peripheral pattern of rod cell birth dates observed during mouse retinal development (Young, 1985). Thus the spreading pattern of LacZ expression may reflect normal photoreceptor cell development in the mouse retina. Whole-mount retinal stains of the two transgenic mouse lines revealed two very different spatial patterns of expression in adult animals. RG2-2 clearly showed persistence of a gradient 1 year later that produced a very even stain in the superior-temporal quadrant of the retina (Figure 3F). Staining over the nasal-inferior

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Opsin

Fusion

Gene

Analysis

in Transgenic

Mice

quadrant of the retina was mottled. In contrast, RG4-4 showedaveryuniform,evenstainingacrosstheentire retina with no evidence of mottling (Figure 3G). Staining patterns in right and left eyes of any given transgenic animal were mirror images of one another, suggesting that the observed gradient of staining is genuine. The difference in spatial patterns of expression in the two mouse lines may reflect differences in levels of expression, since retinal sections of RG4-4 animals showed more intense staining of LacZ in the temporal aspect of the eye compared with the nasal aspect. Increased levels of LacZ expression may obscure our ability to detect a gradient of expression. However, we tried a variety of staining conditions and were unable to distinguish a gradient with shorter periodsof staining. Noobvious rearrangements in the integrated transgenes were detected by Southern blot analysis. The transient embryonic LacZ expression in RG2-2 animals but not in RG4-4 animals suggests that different sites of integration can affect expression and might account for differences in spatial patterns of expression in adult animals. Zack et al. (1991) also report a similar gradient of expression in some transgenie lines and uniform expression in others. Understanding the significance of these spatial patterns of expression will require further study.

Sequences Upstream of the Promoter Region of the Opsin Gene Are Important for Enhanced Levels and Uniform Expression across the Retina RGA6-3 transgenic animals with the truncated 500 bp rod opsin fusion transgene specifically expressed LacZ in photoreceptor inner segments and, at a lower level, in the outer plexiform layer (Figure 3B). However, levels of expression and numbers of expressing photoreceptors were significantly lower than those observed in RG2-2 and RG4-4 transgenic retinas. Preliminary studies of developmental onset show clear LacZ expression by Pll. Whole-mount retina staining revealed a gradient of LacZ staining that appeared as a band runningdiagonallyfrom the inferior-temporal quadrant to the superior-nasal quadrant (Figure 3H). In comparison with the spatial gradient of expression seen in the RG2-2 transgenic mouse retina, the orientation of this band of LacZ expression parallels the position of the border between the evenly staining and mottled regions of the RG2-2 retina. These observations suggest that 500 bp of 5’ flanking sequences are sufficient to confer cell-specific expression and that within the excised upstream fragment there may be enhancer sequences that increase the efficiency of rod opsin expression and direct appropriate spatial expression across the entire retina. Determining whether expression is confined to rods, cones, or both will require further study at the electron microscope level. Additional lines of transgenic animals are being investigated.

The Expression of DTA Induces Retinal Degeneration The expression

of the rod opsin-DTA

transgene

in the

mouse line RDT9-1 has established an animal model that displays early progressive retinal degeneration. Expression of diphtheria toxin was studied in heterozygous animals and led to the ablation of the entire photoreceptor layers. In mature animals, thinning of the inner nuclear layer was also observed. This is comparable to the spontaneous retinal degeneration observed in the rd mouse (Blanks and Bok, 1977) and reflects the importance of cell-cell contacts and/or the secretion of signal factors from photoreceptors for the viability of neighboring cells. The presence of the rd locus in the strain of mouse used in these studies has been ruled out; moreover, the rd locus is expressed recessively, whereas the opsin-DTA gene is dominant. The absence of the diphtheria toxin fi subunit, which facilitates toxin binding to cell surfaces and transport into the cytosol, makes it unlikely that adjacent cells are killed by leakage of the toxin. In addition, expression of the comparable 4.4 kb rod opsin-/acZ gene shows that product is absent in other retinal cells, arguing against spurious expression of the toxin in nonphotoreceptor cells. Rod cell degeneration proceeds much more rapidly than cone cell degeneration in the rd mouse line (Carter-Dawson et al., 1978). Whether cone cells persist in the RDT9-1 animals is under investigation. Although an apparently normal complement of cells is present in the inner nuclear layer of adult transgenic animals, a more detailed study may also indicate whether specific cell subtypes are affected and whether development or stability of mature neighboring cells is perturbed. In addition, comparative studies with other retinal degenerative animal models may give insight into the course of cell death induced by defects in other gene products.

Conclusions In conclusion, using chimeric gene fusions between mouse rod opsin 5’ upstream flanking sequences and diphtheria toxin or /acZ structural reporter genes, we have shown photoreceptor cell-specific expression. Full-length 4.4 kb rod opsin sequences were capable of appropriate temporal patterns of expression approximately mimicking normal opsin gene expression. Spatial patterns of expression were consistent with our limited knowledge of the spatial expression of opsin protein during mouse retinal development. Truncated 500 bp rod opsin flanking sequences, though still able to direct cell-specific expression, required sequences further upstream of the promoter region for enhanced levels and uniform expression across the retina. A detailed promoter truncation analysis is required to define more precisely tissue-specific and developmental regulatory sequences. In addition, characterization of the rod opsin upstream promoter region will provide a way to introduce other phototransduction genes or their mutants (using the rod opsin upstream region) into transgenic animals to study the role of these genes in normal development and retinal degenerative disease.

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Experimental

Procedures

Transgene Plasmid Construction The mouse rod opsin gene LMOPSI was obtained from an EMBL3 genomic library, prepared from the wild-type strain 610 as previously described (Baehr et al., 1988). An 11 kb BamHl fragment was excised, cloned into pUC13 as pMOPS5, and mapped; the 11 kb insert includes ~5 kb of 5’ upstream flanking sequences and the complete ~5 kb rod opsin gene. A 4.4 kb Kpnl-Xhol 5’ flanking fragment extending 15 bp upstream of the ATC start site to approximately 4.4 kb upstream was excised and cloned into the multiple cloning site of pBluescript KS(+), creating the 7.3 kb plasmid pRho4-I. In addition to the -4.4 kb upstream sequences, the fragment contains typical CCAAT and TATA sequences and ~80 bp of the opsin transcript 5’ untranslated region just preceding the opsin translation start site. The 800 bp Bglll fragment from the plasmid pDTA (Palmiter et al., 1987) encoding the diphtheria toxin structural gene and the SV4Cl small t antigen poly(A) sequence was ligated into BarnHI-linearized pRho4-I to generate pRDT1. The rod opsin-diphtheria gene chimera was excised as a 5.2 kb Kpnl-Xbal fragment for microinjection. The pRC3 transgene was made by subcloning the 4.4 kb Kpnl-Clal rod opsin 5’ regulatory region from pRho4-I into the multiple cloning site of pCEM-7Zf(+) to generate the 7.4 kb plasmid pRho4-5C. A vector, pGal5, was constructed by ligating the 3.8 kb Hindlll-BamHI fragment of pCH126 carrying lacZ (Hall et al., 1983) into the multiple cloning site of pBluescript KS(+). The 3.8 kb Sall-BamHI fragment of pGAL5, containing /acZstructural gene and the SV4OVPl poly(A) consensus sequence, was ligated into the corresponding restriction sites of pRho4-5C to generate pRG3. The 8.2 kb rod opsin-/acZ fusion gene was excised with Xbal and BamHl for microinjection. The truncated rod opsin promoter transgene was excised from pRG3 as a 4.3 kb BglllBamHl fragment and microinjected. The cloning vector pBluescript KS(+) was purchased from Stratagene (La Jolla, CA). pCEMJZf(+) was obtained from Promega (Madison, WI). pCH126 was kindly provided by Dr. F. Lee (DNAX, Palo Alto, CA), and pDTA was kindly provided by Dr. I. Maxwell (University of Colorado, Denver, CO). Transgenic Mouse Production DNA fragments for microinjection were isolated with Gene&an (Bio 101, La Jolla, CA). Chimeric fusion genes were microinjected in theabsenceof plasmid sequencesand injected into the pronuclei of fertilized one-cell eggs from superovulated C57BUbJ x DBA2 Fl mice (Jackson Laboratories, Bar Harbor, ME). Microinjetted eggs were incubated overnight at 37”C, 5% CO> in Ml6 media (Hogan et al., 1986) and allowed to develop to the two-cell stage. Surviving two-cell stage eggs were transplanted into the oviducts of pseudopregnant foster mothers. Transgenic animals were identified by slot blot hybridization or polymerase chain reaction (PCR) analysis of genomic DNA prepared from tail samples as described (Hogan et al., 1986). For slot blot hybridizations, 7 pg of each DNA sample was applied to Zetabind membrane (Cur-to, Meriden, CT) using a Minifold II slot blot apparatus (Schleicher and Schuell, Keene, NH). Seven micrograms of genomic DNA was digested to completion with EcoRl or Hindlll and run on a 0.5% agarose gel for Southern blot analysis. Slot blots were probed with oligo-labeled lad or DTA DNA fragments labeled to a specific activity of 8 x ICP to 1 x IO9 cpm/pg with [a-12P]dATP (Feinberg and Vogelstein, 1983,1984; Amersham Corporation,Arlington Heights, IL). ForSouthern blotanalyses, blots were probed with the rod opsin 4.4 kb fragment, which labeled both the endogenous opsin gene and the transgene, allowing assessment of copy number integration. For PCR analyses, a /acZ 5’ primer (S-CTCCCCTTACCCCGGTGATTT-3’) and 3’ primer (5’.TTGCCAACGCTTATTACCCAGC-3’) were used. The 5’ primer used to assay for diphtheria toxin was S-GATCCTCATGATGTTCTTGATTCT-3, and the 3’primer was S-TCGCCACClllTCCACGCCTT3’. PCR reactions were run for 30 cycles at 92°C for 1 min, 64OC for 30 s, and 72OC for 1 min, followed by a 10 min extension at 72OC in a Perkin-Elmer thermal cycler (Norwalk, CT). Amplified DNA fragments were approximately 500 bp in size.

Histological and Histochemical Analysis of Retinas Animals were sacrificed by cervical dislocation, and eye orientation was marked with awater-insoluble dye prior to enucleation. Enucleated eyes were punctured with a hypodermic needle and immediately fixed in 2% paraformaldehyde and 0.2% glutaraldehyde in IO m M PBS (pH 7.2). The cornea and lens were excised from fixed eyes and eye cups rinsed in 10 m M PBS (pH 7.0-7.4) and embedded in polyacrylamide by the method of Johnson and Blanks (1984). Polyacrylamide-embedded eyecups were mounted inOCTcompound,and4-8CLmsectionswerecutandtransferred to gelatin-subbed slides. Sections from RDT9-1 transgenic animals were refixed after sectioning with 2% paraformaldehyde, 0.2% glutaraldehyde and stained with hematoxylin and eosin. Sections from RG transgenic animals were stained with 2% X-Gal, 3 m M potassium ferricyanide, 3 m M potassium ferrocyanide in 0.01 M PBS containing 1 m M magnesium chloride and 1 m M calcium chloride (Goring et al., 1987). Sections were stained at 37OC for 2 hr to overnight, refixed, and stained with hematoxylin and eosin. Whole-mount retinas were prepared by fixation in 4% paraformaldehyde, 0.2% glutaraldehyde immediately after euthanization. The cornea and lens were removed, eyes fixed for 30 min, and rinsed extensively with ice-cold PBS every IO min for 1 hr. Whole-mount retinas, whole tissues, and embryos were stained overnight at 37OC in X-Gal staining buffer supplemented with 0.02% Nonidet P-40 and 0.01% sodium deoxycholate (Sanes et al., 1986). Retinas from multiple litters of RC2-2, RG4-4, and RDT9-1 animals were used in these studies. Three litters of RGA6-3 animals were examined. For embryonic temporal studies, EO was defined as the day of plugging. For postnatal developmental studies, pregnant mice were checked daily and the day of birth designated PO. Tissue Processing for Electron Microscopy Mouse eyes were enucleated immediately after euthanrration. Each eye was opened through the pars-plana and immediately processed for electron microscopy. Eyes were divided into two groups for further processing. Eyes for ultrastructural examination were postfixed in 4% OsOa, dehydrated in ethanol, infiltrated in propylene oxide, and embedded in Araldite 502 epoxy resin (Ciba Products Co., Summit, NH). Eyes for postembedding immunoelectron microscopy were processed according to the methods of Erickson etal. (1987Jand were embedded in LR White methacrylate resin (Polysciences, Inc., Warrington, PA). Thin sec.tions were placed on Formvar-coated nickel grids. Grids were incubated on drops of a blocking solution consisting of 0.8% bovine serum albumin, 0.1% IGSS gelatin, 5% normal serum, and 2 m M NaNl in PBS (pH 7.4) for 15 min. Grids were then floated on drops of a polyclonal antibody to B-galactosidase (5Prime-3Prime, West Chester, PA), diluted I:10 in PBS supplemented with 0.8% bovine serum albumin for 2 hr. Cuds were rinsed in PBS, bovine serum albumin and then incubated on drops of 1:20dilution of goat anti-rabbit immunoglobulin G complexed with 15 nM colloidal gold (Janssen Pharmaceutics, Olen, Belgium). lmmunocytochemistry controls consisted of replacement of the primary antibodies with preimmune immunoglobulin G. Thin sections for electron microscopy were stained with uranyl acetate and lead citrate and examined with a JEOL 100-C electron microscope. Acknowledgments Special thanks to J. Dausman for teaching transgenic microinlection technique; D. M. Gee for photographic consultation; C. Segal for histological preparation and sectioning; J. Ito for secretarial assistance; J. Blanks for teaching acrylamide embedding technique; and J. Pollock for teaching cryosectioning technique. Thisworkwas supported in part bya National Institutesof Health Program Project Grant AC 97687 (M. I. S.), National Institutes of Health Grant EY04801 (M. L. A.), and a grant from the George Cund!National Retinitis Pigmentosa Foundation (M. L. A.). M. L. A. is the recipient of a Jules and Doris Stein Research to Prevent Blindness Professorship. J. L. was supported by American Cancer

Rod Opsln Fusion Gene Analysis in Transgenic 209

Mice

Society, National Office, Grant #PF-3033 and a grant from the George GundlNational Retinitis Pigmentosa Foundation. 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

October

16, 1990; revised

November

21, 1990.

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