Doppler echocardiographic assessment of left ventricular diastolic function in myotonic dystrophy

To determine the increased production and release of vascular endothelial growth factor (VEGF) from the retina in the eye with non-angiogenic retinal detachment in which relative blood supply disturbance may be present, the concentration of VEGF in subretinal fluid and vitreous fluid collected from the eyes was investigated by enzyme linked immunospecific assay. The average concentration of VEGF was 0.5 +/- 1.1 ng/ml (mean +/- standard deviation) in nine samples of vitreous fluid collected from proliferative retinal detachment, and was 1.2 +/- 1.2 ng/ml in eleven samples of subretinal fluid from rhegmatogenous retinal detachment. The concentration of VEGF in six samples of vitreous fluid from angiogenic diabetic eyes (5.0 +/- 2.8 ng/ml) was significantly higher than in the samples from eyes with retinal detachment. The results suggest that the relative ischemic insult to the detached retina increases the release of VEGF into the vitreous cavity and subretinal space. The increased concentration of VEGF does not induce remarkable angiogenesis since the concentration is lower than the biological threshold, or the effect is modulated by inhibitors.     

Characterisation of the infarct-limiting effect of delayed preconditioning: timecourse and dose-dependency studies in rabbit myocardium

BACKGROUND: Although the choroidal neovascularization (CNV) is a common pathologic feature of a number of different eye diseases, its pathological mechanisms have not been fully elucidated. We investigated the expression of vascular endothelial growth factor (VEGF) in CNV using an experimental primate model. METHOD: CNV was induced by intense laser photocoagulation in four monkey eyes. Single eyes were enucleated at 1, 3, 7 or 14 days after photocoagulation and examined immunohistochemically for VEGF, macrophage antigen, von Willebrand factor and glial fibrillary acidic protein (GFAP). Expression of VEGF mRNA was examined by in situ hybridization. RESULTS: One day after photocoagulation, the normal structure of the outer portion of the retina and the inner portion of the choroid was destroyed. Three days after photocoagulation, choroidal vascular endothelial cells migrated into the subretinal space through the defect in Bruch's membrane. Increased expression of VEGF was detected in the accumulating macrophages, migrating retinal pigment epithelial (RPE) cells and Müller cells. Maximal expression of VEGF was observed between 3 and 7 days after wounding, and many newly formed vessels extended into the subretinal space 7-14 days after photocoagulation. CONCLUSION: VEGF derived from RPE cells, macrophages and Müller cells may play a role in the formation of CNV.     

The 67-kd laminin receptor is preferentially expressed by proliferating retinal vessels in a murine model of ischemic retinopathy

Endothelial cell association with vascular basement membranes is complex and plays a critical role in regulation of cell adhesion and proliferation. The interaction between the membrane-associated 67-kd receptor (67LR) and the basement membrane protein laminin has been studied in several cell systems where it was shown to be crucial for adhesion and attachment during angiogenesis. As angiogenesis in the pathological setting of proliferative retinopathy is a major cause of blindness in the Western world we examined the expression of 67LR in a murine model of hyperoxia-induced retinopathy that exhibits retinal neovascularization. Mice exposed to hyperoxia for 5 days starting at postnatal day 7 (P7) and returned to room air (at P12) showed closure of the central retinal vasculature. In response to the ensuing retinal ischemia, there was consistent preretinal neovascularization starting around P17, which persisted until P21, after which the new vessels regressed. Immunohistochemistry was performed on these retinas using an antibody specific for 67LR. At P12, immunoreactivity for 67LR was absent in the retina, but by P17 it was observed in preretinal proliferating vessels and also within the adjacent intraretinal vasculature. Intraretinal 67LR immunoreactivity diminished beyond P17 until by P21 immunoreactivity was almost completely absent, although it persisted in the preretinal vasculature. Control P17 mice (not exposed to hyperoxia) failed to demonstrate any 67LR immunoreactivity in their retinas. Parallel in situ hybridization studies demonstrated 67LR gene expression in the retinal ganglion cells of control and hyperoxia-exposed mice. In addition, the neovascular intra- and preretinal vessels of hyperoxia-treated P17 and P21 mice labeled strongly for 67LR mRNA. This study has characterized 67LR immunolocalization and gene expression in a murine model of ischemic retinopathy. Results suggest that, although the 67LR gene is expressed at high levels in the retinal ganglion cells, the mature receptor protein is preferentially localized to the proliferating retinal vasculature and is almost completely absent from quiescent vessels. The differential expression of 67LR between proliferating and quiescent retinal vessels suggests that this laminin receptor is an important and novel target for future chemotherapeutic intervention during proliferative vasculopathies.     

Basic fibroblast growth factor is neither necessary nor sufficient for the development of retinal neovascularization

Basic fibroblast growth factor (FGF2) is constitutively expressed in the retina and its expression is increased by a number of insults, but its role in the retina is still uncertain. This study was designed to test the hypothesis that altered expression of FGF2 in the retina affects the development of retinal neovascularization. Mice with targeted disruption of the Fgf2 gene had no detectable expression of FGF2 in the retina by Western blot, but retinal vessels were not different in appearance or total area from wild-type mice. When FGF2-deficient mice were compared with wild-type mice in a murine model of oxygen-induced ischemic retinopathy, they developed the same amount of retinal neovascularization. Transgenic mice with a rhodopsin promoter/Fgf2 gene fusion expressed high levels of FGF2 in retinal photoreceptors but developed no retinal neovascularization or other abnormalities of retinal vessels; in the ischemic retinopathy model, they showed no significant difference in the amount of retinal neovascularization compared with wild-type mice. These data indicate that FGF2 expression is not necessary nor sufficient for the development of retinal neovascularization. This suggests that agents that specifically antagonize FGF2 are not likely to be useful adjuncts in the treatment of retinal neovascularization and therapies designed to increase FGF2 expression are not likely to be complicated by retinal neovascularization.     

Anxiety and cognitive performance in adolescent women with disruptive behavior disorders

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and permeability-inducing factor that has been implicated in the pathogenesis of diabetic retinopathy. In the present study, the localization and magnitude of VEGF, VEGF receptor-1 (VEGFR-1), and VEGF receptor-2 (VEGFR-2) gene expression were examined in the eye of streptozotocin-induced diabetic rats using quantitative in situ hybridization. VEGF protein was also examined by immunohistochemistry. Abundant VEGF mRNA and protein were present in the retinae of control rats. In the retinae of diabetic rats, VEGF gene expression was increased compared with control animals (p = 0.001). The increase in VEGF mRNA was noted in the ganglion cell layer and inner nuclear layer but not in the pigment epithelium of the retina. VEGF was also detected in blood vessels, ciliary body, and lens epithelium in both control and diabetic rats. The distributions of VEGFR-1 and VEGFR-2 were similar in both control and diabetic rats. VEGFR-1 mRNA was present beneath the inner limiting membrane and in the ganglion cell layer, inner nuclear layer, outer plexiform layer, and outer limiting membrane of the retina; it was also detected in blood vessels, the ciliary body, and the cornea. The magnitude and distribution of ocular VEGFR-1 mRNA were not affected by experimental diabetes. Expression of VEGFR-2 mRNA was noted in the inner nuclear layer and pigment epithelium of the retina and in blood vessels. An increase in VEGFR-2 mRNA in the diabetic retina was restricted to the inner nuclear layer. The presence of VEGF and its receptors in the control retina suggests a physiologic role for VEGF within the eye. The changes in retinal expression of VEGF and VEGFR-2 in association with diabetes suggest a role for this pathway in diabetic retinopathy.     

Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate

PURPOSE: The purpose of the study is to determine the effect of exogenous vascular endothelial growth factor (VEGF) on the primate retina and its vasculature. METHODS: Ten eyes of five animals were studied. Physiologically relevant amounts of the 165 amino acid isoform of human recombinant VEGF were injected into the vitreous of six healthy cynomolgus monkey eyes. Inactivated human recombinant VEGF or vehicle was injected into four contralateral control subject eyes. Eyes were assessed by slit-lamp biomicroscopy, tonometry, fundus color photography, fundus fluorescein angiography, light microscopy, and immunostaining with antibodies against proliferating cell nuclear antigen and factor VIII antigen. RESULTS: All six bioactive VEGF-injected eyes developed dilated, tortuous retinal vessels that leaked fluorescein. Eyes receiving multiple injections of VEGF developed progressively dilated and tortuous vessels, venous beading, edema, microaneurysms, intraretinal hemorrhages and capillary closure with ischemia. The severity of the retinopathy correlated with the number of VEGF injections. None of the four control eyes exhibited any abnormal retinal vascular changes. The endothelial cells of retinal blood vessels were proliferating cell nuclear antigen positive only in the bioactive VEGF-injected eyes. CONCLUSION: Vascular endothelial growth factor is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies, such as hemorrhage, edema, venous beading, capillary occlusion with ischemia, microaneurysm formation, and intraretinal vascular proliferation.     

Retinal vascular changes in congenital hypertrophy of the retinal pigment epithelium

The overlying retinal blood vessels were abnormal in five cases of congenital hypertrophy of the retinal pigment epithelium. This illustrated the well-recognized association between outer retinal degeneration and obliteration of the overlying retinal vasculature. The proposed pathophysiological mechanisms, however, seem inadequate to explain completely the morphological changes of the retinal blood vessels in the presence of atrophy of the outer retina.     

Patterns of cell proliferation in the developing retina of the clawed frog in relation to blood supply and position of the choroidal fissure

The pattern of retinal vasculative is described and the position at which cell proliferation at the ventral retinal margin is maximal was shown to be at the point of entry of the ventral blood vessels. To test whether there is a causal relation between retinal blood supply and retinal cell production, surgical inversion of the eye, transplantations and excisions of retina were done to change the pattern of retinal vasculature. The growth pattern of inverted eyes was normal with respect to the internal axes of the eyes. After excision of part of the retina or after fusion of retinal fragments to form compound eyes, the pattern of retinal cell proliferation was not correlated with the distribution of retinal blood vessels, but was correlated with the position(s) of the choroidal fissure(s).     

An aldose reductase inhibitor and aminoguanidine prevent vascular endothelial growth factor expression in rats with long-term galactosemia

OBJECTIVE: To study the effects of an aldose reductase inhibitor (ARI-509, Wyeth-Ayerst, Princeton, NJ) and aminoguanidine (AMG), agents that have been reported to prevent or delay diabetic retinopathy, on retinal vascular abnormalities and the immunocytochemical expression in the retina of vascular endothelial growth factor (VEGF) in rats maintained for up to 2 years on a 50% galactose diet. METHODS: Albino rats were placed on a control diet, a diet containing 50% galactose, or the 50% galactose diet containing either ARI-509 or AMG. Treatment with ARI-509 or AMG was initiated at the beginning of the experiment or after 12 months of galactose feeding. After 22 to 24 months, the rats were killed and the retinal vasculature from half of one eye was isolated by trypsin-elastase digestion for semiquantitative evaluation of retinal vascular lesions. The other half of the retina was prepared for immunocytochemistry and stained for the presence of VEGF, factor VIII, vimentin, and glial fibrillary acidic protein. Red blood cells, sciatic nerves, and a portion of the retina from the second eye were assayed for glucose, galactose, fructose, sorbitol, galactitol, and myo-inositol. Red blood cells were also assayed for galactosylated hemoglobin. RESULTS: Galactose-fed animals developed a vascular retinopathy characterized by severe cellular loss in the retinal capillaries and intensification of periodic acid-Schiff staining of the vascular basement membranes. Some animals also displayed dilation and hypercellularity of vessels in the posterior retina. These changes were substantially reduced in animals receiving ARI-509 from the beginning of the galactose diet, but were unaffected in all of the other treatment groups. None of the rats receiving ARI-509 or AMG treatment, whether initiated from the onset or after 12 months of galactosemia, demonstrated VEGF immunoreactivity. With the exception of the animals receiving ARI-509 from the beginning of the experiment, all of the galactose-fed animals developed dense cataracts within 6 weeks of the beginning of the galactose diet. Galactitol levels in animals receiving ARI-509 were 86% to 93% lower in red blood cells, retina, and sciatic nerve than those in the other galactose-fed groups. CONCLUSIONS: Although ARI-509 and AMG have different abilities to delay or prevent the diabetic-like retinopathy in galactosemic rats, even when substantial retinal microvascular acellularity occurs, both drugs prevent the immunocytochemical expression of VEGF. These results suggest that factors other than hypoxia may be responsible for VEGF expression in the retina, and that aldose reductase inhibitors and AMG have potential roles in preventing such expression and, thus, perhaps preventing retinal neovascularization.     

Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity

PURPOSE: To assess the role of vascular endothelial growth factor (VEGF) in the feline model of retinopathy of prematurity (ROP). METHODS: Retinopathy of prematurity was induced in neonatal cats by raising them in an oxygen-enriched (70% to 80%) atmosphere for 4 days to suppress vessel formation and then returning them to room air for 3 to 27 days. In situ hybridization was used to detect the expression of VEGF and its high-affinity receptor, flk-1, in the retina of neonatal cats, and glial fibrillary acidic protein immunocytochemistry was used to assess astrocyte status. RESULTS: The expression of VEGF in the innermost layers of retina fell in hyperoxia and increased on return to room air. Vascular endothelial growth factor expression was transient; it was maximal where vessels were about to form, and it rapidly downregulated after vessels had formed. During the proliferative vasculopathy of ROP, VEGF expression was stronger than in the normally developing retina, and the astrocytes that normally express VEGF degenerated. After the degeneration of astrocytes, VEGF was expressed by neurones of the ganglion cell layer. flk-1 was expressed by intraretinal and preretinal vessels. Supplemental oxygen therapy reduced or eliminated the overexpression of VEGF expression, astrocyte degeneration, and formation of preretinal vessels. CONCLUSIONS: Regulation of VEGF by tissue oxygen mediates the inhibition of vessel growth during hyperoxia and the subsequent proliferative vasculopathy. Degeneration of retinal astrocytes creates conditions for the growth of preretinal vessels.     

Localization of vascular endothelial growth factor in human retina and choroid

OBJECTIVE: To examine the distribution and relative levels of vascular endothelial growth factor (VEGF) in the nondiabetic and preproliferative diabetic human retina and choroid. METHODS: Immunohistochemical localization was performed on frozen sections from cryopreserved postmortem human tissue using a polyclonal antibody against VEGF and a streptavidin peroxidase system. Eyes from 5 subjects without diabetes and 8 subjects with diabetes were examined and analyzed using a 7-point immunohistochemical grading system. RESULTS: In subjects without diabetes, weak or no VEGF immunoreactivity was associated with retinal blood vessels. In subjects with diabetes, we found significantly increased immunoreactivity in the retinal vascular endothelium and blood vessel walls. Vascular endothelial growth factor immunoreactivity was also associated with intravascular leukocytes in subjects with and without diabetes. In the choroid of subjects without diabetes, immunoreactivity was almost exclusively associated with intravascular leukocytes, whereas in diabetic subjects, immunoreactivity was localized within choriocapillaris endothelium, choroidal neovascular endothelium, and migrating retinal pigment epithelium cells. CONCLUSIONS: The observed increase in VEGF immunoreactivity in the diabetic retina and choroid suggests that VEGF may contribute to 2 well-documented events during retinopathy: increased vascular permeability and angiogenesis.     

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

A new method for noninvasive optical sectioning of the chorioretinal vasculature

PURPOSE: To report a new method for optical sectioning of the chorioretinal vasculature to improve the visualization of vascular abnormalities due to chorioretinal eye diseases. METHODS: An imaging system was developed for optical sectioning of the vasculature called chorioretinal optical sectioning (CROS). CROS consists of projecting a laser beam at an angle on the retina after injection of a fluorescent dye and viewing the fluorescence. On the fluorescence optical section (FOS) image, the vasculature of the retina and choroid appear laterally displaced according to their depth location. The laser beam is scanned over a 2 X 2-mm area to generate 40 FOS images, each spatially separated by 50 microm on the retina. Optical section images of the vascular layers are constructed from the series of FOS images. RESULTS: CROS permitted optical separation of vascular layers in living eyes. Optical section images of normal and laser-photocoagulated retinas had higher contrast than conventional angiography because of the separation of the fluorescence from the overlapping layers and allowed enhanced visualization of vascular abnormalities. CONCLUSIONS: CROS enhances the visualization of the retinal and subretinal vasculature and promises to be a beneficial tool for evaluation of chorioretinal diseases.     

Infectious simian varicella virus expressing the green fluorescent protein

PURPOSE: To determine if vascular occlusion and nonperfusion is associated with the outer retinal atrophy, retinopathy, and choroidopathy (chorioretinopathy) that occurs in the alpha H beta S[beta MDD] and alpha H beta S [alpha MD beta MDD] transgenic mouse models of sickle cell disease. METHODS: Mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] transgenic mouse lines that express high levels of human beta S globin were anesthetized and administered horseradish peroxidase (HRP) intracardially. After 1 min, the animals were sacrificed, and the retina from one eye was excised, fixed, and developed in diaminobenzidine (DAB). The contralateral eye was fixed, embedded whole in glycol methacrylate, and HRP developed in 2.5 microns sections. RESULTS: HRP reaction product (HRP-RP) and stained erythrocytes (RBCs) (due to endogenous peroxidase) were diffusely distributed within all vascular lumens in flatmount retinas from control animals (littermates homozygous for the mouse Beta Major deletion not expressing the beta S transgene). In 42.5% of the transgenic mice expressing beta S without any proliferative retinopathy, many blood vessels contained RBC plugs and lacked lumenal HRP-RP. In addition to packed RBCs, fibrin was sometimes present at sites of occlusion. In sections from whole eyes of the same animals, foci of photoreceptor degeneration were associated with areas of choriocapillaris nonperfusion (lumen that lacked HRP-PR). In areas with normal photoreceptors, the choriocapillaris appeared perfused (HRP-RP was present). In animals with proliferative chorioretinopathy, some neovascular formations lacked luminal HRP-RP, suggesting autoinfarction. CONCLUSIONS: Nonperfused retinal and choroidal vessels were observed in mice from the alpha H beta S[beta MDD] and alpha H beta S[alpha MD beta MDD] lines without retinal and choroidal neovascularization, whereas, all mice with neovascularization had nonperfused areas. Furthermore, small foci of PR loss were associated with areas of nonperfused choriocapillaris. These results suggest that sickle cell-mediated vaso-occlusions are an initial event in the chorioretinopathy and outer retinal atrophy that occurs in these models.     

ANCA testing. New developments and clinical implications

Upregulation of keratinocyte-derived VEGF-A expression has recently been established in non-neoplastic processes of skin such as wound healing, blistering diseases and psoriasis, as well as in skin neoplasia. To further characterize the effects of VEGF-A in skin in vivo, we have developed transgenic mice expressing the mouse VEGF120 under the control of a 2.4 kb 5' fragment of keratin K6 gene regulatory sequences that confers transgene inducibility upon hyperproliferative stimuli. As expected from the inducible nature of the transgene, two of the three founder mice obtained (V27 and V208), showed no apparent phenotype. However, one founder (V2), mosaic for transgene integration, developed scattered red spots throughout the skin at birth. The transgenic offspring derived from this founder developed a striking phenotype characterized by swelling and erythema, resulting in early postnatal lethality. Histological examination of the skin of these transgenics demonstrated highly increased vascularization and edema leading to disruption of skin architecture. Expression of the transgene was silent in adult animals of lines derived from founders V27 and V208. Phorbol ester-induced hyperplasia resulted in transgene induction and increased cutaneous vascularization in adult transgenic mice of these lines. Skin carcinogenesis experiments performed on hemizygous crosses of V208 mice with activated H-ras-carrying transgenic mice (TG.AC) resulted in accelerated papilloma development and increased tumor burden. Previous results from our laboratory showed that VEGF upregulation is a major angiogenic stimulus in mouse epidermal carcinogenesis. By overexpressing VEGF in the skin of transgenic mice we now move a step further toward showing that VEGF-mediated angiogenesis is a rate-limiting step in the genesis of premalignant lesions, such as mouse skin papilloma. Our transgenic mice constitute an interesting model system for in vivo study of the cutaneous angiogenic process and its relevance in tumorigenesis and other skin diseases.     

Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model

In vivo selection of phage display libraries was used to isolate peptides that home specifically to tumor blood vessels. When coupled to the anticancer drug doxorubicin, two of these peptides-one containing an alphav integrin-binding Arg-Gly-Asp motif and the other an Asn-Gly-Arg motif-enhanced the efficacy of the drug against human breast cancer xenografts in nude mice and also reduced its toxicity. These results indicate that it may be possible to develop targeted chemotherapy strategies that are based on selective expression of receptors in tumor vasculature.     

Retinal vaso-occlusion in sickling hemoglobinopathies

Sickle cell retinopathy, in all of its manifestations, represents the effects of arteriolar and capillary occlusions. Increased viscosity of circulating whole blood plus the microembolic action of individual sickled erythrocytes contribute to vasoocclusion. Decreased oxygenation and increased acidosis develop and lead to further sickling -- and further vaso-occlusion. The cycle of erythrostasis that characterizes sickling throughout the body is also applicable to the retina. The transparent media of the eye permit direct visualization of vaso-occlusions which occur preferentially in and about the macula and in the far periphery of the retina. Many of the occlusive episodes are transient. There dynamic events are simultaneously occurring elsewhere in the body but can only be visualized in the eye. The net effect in the retina is a remodeling of its vasculature, as some vessels close and others reopen. After the onset of arteriolar closure in the retina, affected blood vessels embark on a spontaneous, naturally evolving course of events leading to arteriolarvenular anastomoses, neovascular proliferations, vitreous hemorrhages, and retinal detachment. The advanced stages of proliferative sickle retinopathy are most commonly observed in SC disease and in Sthal, possibly because these two forms of sickling have significantly higher than normal whole blood viscosity. Retinal vaso-occlusions can also lead to blow-out hemorrhages which may evolve into salmon patches, iridescent spots, schisis cavities, and black sunbursts. In some respects sickle retinopathy is unique, but many of its manifestations are similar to those of retinopathies found in diabetes mellitus, AC hemoglobinopathy, Takayasu pulseless disease, sarcoidosis, chronic myelogenous leukemia, branch retinal vein occlusion, retrolental fibroplasia, and Eales disease.     

Ret 1, a cis-acting element of the rat opsin promoter, can direct gene expression in rod photoreceptors

The Ret 1 element, located at -136 to -110 in the rat opsin promoter, binds developmentally regulated retinal nuclear proteins. A similar sequence is found up-stream of opsin genes, from humans to Drosophila, as well as many other photoreceptor-specific genes. The function of the Ret 1 element was tested both in vitro and in two sets of transgenic mice. A mutated Ret 1 element did not bind retinal nuclear proteins in vitro. The same mutations in an otherwise normal 1.9-kb rat opsin promoter failed to drive expression of a lacZ reporter gene in nine of 12 lines. In the three other lines, expression in photoreceptors was very faint. Four tandem copies of the Ret 1 element maintained the Ret 1 binding specificity in vitro and were able to direct expression of a lacZ transgene in photoreceptors of all nine mouse lines obtained. In two lines, expression was also detected in the ganglion cell layer and the ciliary epithelium. In three lines, a characteristic pattern of expression was found in the nervous system in addition to the normal retinal expression. These results indicate that Ret 1 can and is necessary to drive gene expression in rod photoreceptors. Furthermore, our results suggest that Ret 1-like elements may also be important in the developing nervous system.