Ординатура / Офтальмология / Английские материалы / Eye, Retina, and Visual System of the Mouse_Chalupa, Williams_2008

and splice site mutations (Berger, van de Pol, et al., 1992; Meindl et al., 1992, 1995). Approximately 20% of patients have complete or partial gene deletions (see Berger, 1998, and references therein).

NDP mutations were also found in the DNA of patients with X-linked recessive familial exudative vitreoretinopathy (XLEVR), or Coats’ disease, and were associated with advanced stages of retinopathy of prematurity (Black et al., 1999; Chen et al., 1993a; Shastry et al., 1997). Thus, the clinical expressivity of mutations in the NDP gene is highly variable, even within a family, and both genetic and environmental modifiers are likely to influence the disease course (Zaremba et al., 1998). However, all these traits have the common feature of retinal blood vessel malformations. Therefore, a role of norrin in vascular development and homeostasis seems reasonable. It should be noted, however, that only classic Norrie disease is associated with deafness and mental retardation; the allelic traits do not show this pleiotropic effect.

Evolutionary conservation of the NDP gene and its expression pattern

Norrin shows high conservation in different species, as revealed by multiple protein sequence alignment (figure 43.1). The human sequence showed 95% identity on the amino acid level with rat, mouse, and dog, and 94% with cow. Lower levels of sequence identity of 87%, 60%, 57%, and 54% were observed with chicken, zebrafish, tetraodon, and fugu, respectively. This high degree of conservation implies an important function of norrin in vertebrates. In particular, the number and positions/spacing of 11 cysteine residues are highly conserved in all the species just mentioned. They form a cystine knot motif, which is a characteristic feature of a structural superfamily of growth factors that includes nerve growth factor (NGF), platelet-derived growth factor-β (PDGF-β), vascular endothelial growth factor (VEGF)-A and -B, placental growth factor (PlGF), and transforming growth factor-β (TGF-β) (McDonald and Hendrickson, 1993). The first cysteine residue occurs at position 39 in the human peptide sequence of norrin. The degree of conservation from this position toward the C-terminus of the protein is conspicuously higher in comparison to the N-ter- minal part, which contains the signal peptide and shows less conservation (see figure 43.1).

To date, tissueor cell type–specific expression of the NDP gene has been studied only at the transcriptional level. Protein expression by immunohistochemistry has not yet been reported. RNA in situ hybridizations (RISH) detected transcripts of the NDP gene in different tissues, including eye, ear, and brain. Thus, the gene is expressed in all tissues or organs that are affected in Norrie disease. In mouse, rabbit, and human retina, norrin mRNA is expressed in the inner

nuclear layer (INL) as well as the ganglion cell layer (GCL) (Chen et al., 1995; Berger et al., 1996; Hartzer et al., 1999), and might also be expressed in the outer nuclear layer (ONL) and the choroid (Hartzer et al., 1999). In mouse and rabbit brain, transcripts were detected by RISH in the olfactory bulbus and epithelium, cerebellum, cortex, and hippocampus (Chen et al., 1995; Berger et al., 1996; Hartzer et al., 1999). Transcriptional activity in the ear has been reported in neurons of the spiral ganglion cells and marginal cells of the stria vascularis of the cochlea (Chen et al., 1995; Hartzer et al., 1999). Furthermore, norrin has also been shown to be expressed in the endometrium of pregnant female mice (Luhmann et al., 2005b). In addition to these RISH studies, the more sensitive RT-PCR has also revealed norrin mRNA in other tissues, including several cell lines, as well as mouse endometrium and human placenta (Luhmann et al., 2005b), muscle, liver, and lung (Chen et al., 1995). The expression of norrin in mouse endometrium and human placenta is particularly interesting, because homozygous knockout mice show a characteristic infertility phenotype, as discussed later in the chapter.

Putative norrin function

Computational modeling of the gene product, norrin, revealed a three-dimensional structure very similar to TGF-β (Meitinger et al., 1993). The amino acid sequence contains a cystine knot motif that is present in several other proteins, including several growth factors, as well as in mucins, the Drosophila slit protein, von Willebrandt factor, and others. The common feature of the cystine knot in all these proteins is responsible for protein interaction and, most likely, dimerization of the respective molecules.

NDP mutations lead to exudative vitreoretinopathy, a disease characterized by an avascular retinal periphery, leakiness of retinal blood vessels, and vitreoretinal hemorrhages. Similar symptoms are observed in patients with a mutation in the Wnt receptor Frizzled-4 (FZD4) and its coreceptor LRP5 (low-density lipoprotein receptor–related protein 5) (Robitaille et al., 2002; Toomes et al., 2004). This observation and the fact that Frizzled-4 knockout mice show a similar phenotype prompted Xu, Wang, and co-workers to analyze norrin as a ligand for FZD4/LRP5 (Xu et al., 2004). The results of these experiments revealed that norrin is able to activate the canonical Wnt β-catenin pathway and to drive reporter gene transcription in a cell line (HEK293) coexpressing norrin, FZD4/LRP5, and a luciferase reporter gene under the control of transcription factor–binding sites (LEF/TCF). They concluded that norrin is a high-affinity ligand for FZD4/LRP5 and transcriptionally activates downstream target genes. Ligand binding depends on coexpression of both receptor molecules Lrp5 and Fzd4. No significant or very little activation of the Wnt pathway was

A

B

Figure 43.1 Multiple norrin protein sequence alignment from different species using the Clustal algorithm (Clustal W [1.83], www.ebi.ac.uk/clustalw). A, Alignment of the peptide sequences clearly shows a much higher conservation after the first of 11 cysteine residues (indicated by arrows above the mouse sequence of norrin). Asterisks in the bottom line indicate identical amino acids; dots designate similar properties of the residues. The putative signal

observed when cell lines were transfected with either of the two receptors. Mutations in LRP5 are also associated with autosomal recessive osteoporosis-pseudoglioma (OPPG) syndrome, which is characterized by congenital or early childhood onset visual loss and bone fragility (Gong et al., 2001; Ai et al., 2005).

peptide is highlighted in gray in the human sequence. B, The phylogenetic tree shows the evolutionary relationship of norrin protein sequences in different species. Sequence identity between human, dog, mouse, and rat is 95%. Identity with cow, chicken, zebrafish, tetraodon, and fugu is 94%, 87%, 60%, 57%, and 54%, respectively.

Generation of a knockout mouse model for Norrie disease

Given the high conservation between human and mouse and the tissue-specific transcription of the mouse norrin gene (gene symbol: Ndph, Norrie disease pseudoglioma homo-

logue) in eye, brain, and ear, the mouse may provide an adequate model for Norrie disease and allelic disorders. Reverse genetics is a powerful tool to establish models for human gene defects and use them to study the pathology at the phenotypic and molecular levels. The gene-targeting approach has been used to generate a knockout mouse model for Norrie disease (Berger et al., 1996). The targeting construct replaced the coding part of exon 2 with a neomycin cassette in opposite transcriptional orientation to Ndph. As a consequence of this replacement, the translation start codon, the splice donor site of exon 2 and 55 additional N-terminal amino acids, including the signal peptide for extracellular transport, are removed from the gene. After homologous recombination of this construct in embryonic stem cells, blastocysts were injected to produce chimeric embryos. Chimeric male offspring were crossed to wild-type females to obtain female carriers for the knockout allele. Approximately half of the male offspring were hemizygous for the knockout allele, which is consistent with Mendelian transmission of a viable mutation.

In an initial survey to characterize the corresponding phenotype, the retinal morphology of hemizygous knockout mice (Ndph−/y) was compared with that in wild-type (Ndph+/y) littermates (Berger et al., 1996). One of the most prominent findings in knockout mice was the presence of fibrous masses containing blood vessels in the vitreous of all nine mice analyzed. This is probably the anatomical correlate to the pre- cipitate-like structures that had been observed on slit lamp biomicroscopy. Likewise, an abnormal organization of nuclei in the GCL had been reported in all Ndph−/y mice examined. Occasionally, outer retinal layers, including the ONL and outer segments of photoreceptor cells, also showed a characteristic pattern of disorganization. Heterozygous female carriers (Ndph+/−) were inconspicuous. These initial data revealed a clear retinal phenotype in Ndph knockout mice. Histopathological data from human patients in whom the clinical diagnosis was confirmed by molecular testing are rare. There is one report in the literature in which retinal and vitreal tissues were examined after vitreoretinal surgery in a 6-month-old boy with a frameshift mutation in the NDP gene (Schroeder et al., 1997). In this case, a reduction in the number of ganglion cells was observed, along with a largely disarranged INL but otherwise normally differentiated retina. Massive fibrovascular proliferation was present in the vitreous.

Retinal physiology of knockout mice

To examine the consequences of the knockout mutation and the morphological changes on retinal physiology, electroretinography (ERG) was performed. This analysis allows direct measurement of the activity of photoreceptor cells and second-order neurons in the retina as a response to light stimuli. The two major ERG components are the a- and

b-waves, which reflect photoreceptor and second-order neuron (bipolar, amacrine, and horizontal cells) activities, respectively. Also, the two photoreceptor systems, rods and cones, can be discriminated by using dark adaptation and excitation with different light intensities. When the retina is dark adapted and the photoreceptors receive light stimuli of low to moderate intensity, the corresponding a-wave mainly represents the response of rod photoreceptors. In the lightadapted state, when bright flashes are utilized to excite photoreceptors, the corresponding a-wave primarily reflects cone photoreceptor activity. Therefore, the ERG is a suitable tool to characterize retinal function.

ERG measurements were performed in hemizygous knockout mice from 7 months of age up (Ruether et al., 1997). Data from hemizygous knockouts were compared with data from a group of heterozygous females and wildtype male litters. Both a- and b-waves were attenuated in hemizygous knockout mice but not in wild-type mice or female carriers. An interesting finding was that the a-waves of the three different genotypes (wild type, hemizygous knockout, heterozygous) were not much different at low intensities but became prominent at higher intensities of light stimuli. Conversely, the b-wave amplitudes were much lower in the group of hemizygous knockout mice at all intensities. Oscillatory potentials, which reflect the response of part of the second-order neurons in the retina, were severely reduced in male mice with the knockout allele. In summary, the ERG recordings revealed a more severe functional effect of the knockout mutation on the inner retina compared to photoreceptor cells. This is in agreement with molecular studies in which a late involvement of photoreceptor cells was found (Lenzner et al., 2002). In this study, the expression of photoreceptor-specific gene transcripts was normal until the age of 12 months in knockout mice and significantly reduced only after 2 years, indicating a rather slow progression of photoreceptor cell degeneration.

Abnormal retinal vascular development in norrin knockout mice

In normal mouse development, three vascular networks, superficial, deep, and intermediate, appear in the retina. The superficial retinal vasculature, located within the GCL/ nerve fiber layer, spreads from the entry point of the large artery and vein from the central retina (at the optic nerve head) toward the periphery between days 3 and 17 post partum (P3–P17). Around P7 or P8, the deep retinal vasculature starts forming by angiogenic sprouting from the superficial network. After completion, the deep vascular system will be present in the outer plexiform layer (OPL). From P12, an intermediate vascular system develops between the superficial and deep networks, close to the boundary between inner nuclear and plexiform layers.

Abnormalities and changes in the retinal vasculature in norrin knockout mice have been reported by several authors (Richter et al., 1998; Rehm et al., 2002; Luhmann et al., 2005a). Vascular development between P5 and P21 in knockout mice was studied in detail by Luhmann and co-work- ers and was correlated with molecular analyses of angiogenic signaling pathways. The outgrowth of the superficial vascular system was temporally retarded and never completed. In wild-type mice, the retina was entirely covered by superficial vessels at P21. The periphery of the retina in hemizygous knockout mice remained avascular, and only three-quarters of the retina was supplied with vessels. The development of the deep capillary network, which normally starts at P7, was completely abolished in norrin-deficient mice (figures 43.2 and 43.3). Although the vascular tubes of the superficial network branched out and tried to initiate angiogenic sprouting, this process was never completed but was entirely blocked at this point, leading to microaneurysmlike capillary lesions. Of note, the astrocytic network, which is important for vessel guidance, seemed to be normal in knockout mice in early disease phases. Rather, a primary effect on endothelial cells has been suggested by Luhmann and co-workers. This view is supported by some molecular findings. The transcript (mRNA) levels of Tie1, Tie2, and Pdgf b, which are involved in angiogenesis and vessel maturation and represent endothelial markers, were significantly lower in norrin-deficient mice at P5 and P10 than in wildtype controls.

The defect in vascular development in norrin knockout mice can be summarized as delayed and incomplete out-

growth of the superficial retinal blood vessels, leading to an avascular retinal periphery. Additionally, the deep and intermediate vessel networks fail to develop, owing to an arrest in sprouting angiogenesis.

Failure in sprouting angiogenesis leads to retinal hypoxia and leakiness of blood vessels, but not retinal neovascularization

As noted, the retinal vasculature in norrin-deficient mice does not develop properly. As a consequence, oxygen deficiency occurs in the inner retina. This hypoxic condition leads to an activation of angiogenic cascades, as shown by the transcriptional upregulation of Vegfa from P10 onward (Luhmann et al., 2005a). At P15, several other factors in the retina are transcriptionally activated: angiopoietin-2 (Agpt2), ephrin-B4 (EphB4), frizzled-4 (Fzd4), integrins α-V and β- III (Itga5, Itgb3), pdgf b and its receptor (pdgf rb), as well as Tie1. All of them also show higher transcript levels at P21 in addition to the Vegf receptors 1 and 2 (Vegfr1/Flt1 and Vegfr2/Flk1). This transcriptional activation of angiogenic factors indicates a response of retinal cells to hypoxia and provides an explanation for microaneurysms and the leakiness of blood vessels, which leads to retinal and vitreal hemorrhages and exudates. Another excellent indicator of hypoxia is the presence of hypoxia-inducible factor 1α (Hif1- α), which is known to be stabilized at the protein level under hypoxic conditions. Indeed, the Hif1-α protein level was increased in norrin knockout mice compared to wild-type litters at P15 and P21, but not at P5 and P10 (Luhmann

Figure 43.2 Sections of the central retina from wild-type and knockout mice at P21 after staining with a collagen IV antibody (red), which detects the extracellular matrix of endothelial cells, and diamidinophenylindol/DAPI (blue), which labels cell nuclei. Blood vessels (red) in wild-type mice are present in three layers, indicating completed development of the superficial (ganglion cell layer [GCL]), deep (outer plexiform layer [OPL]), and intermediate (border between the inner plexiform layer [IPL] and inner nuclear

layer [INL]) vessel networks. In contrast, age-matched knockout mice show only enlarged superficial vessels, while the deeper and intermediate networks failed to develop. In addition, the presence of nuclei in the IPL (white arrowheads) may reflect the characteristic disorganization in the GCL. See color plate 41. (Images acquired and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

Wild type P7

Knockout P7

Figure 43.3 Three-dimensional microscopy of retinal flat mounts stained with anticollagen IV from wild-type and knockout mice at P7. Top panels show an area of approximately 300 × 400 μm from wild-type and knockout mice. Vessels in the knockout are dilated, and the capillary network is less dense than in the wild-type control. Bottom panels (z-axis) show how blood vessels enter the deeper retinal

et al., 2005a). Particularly Hif1-α and Vegfa clearly indicate retinal hypoxia, and many aforementioned angiogenic factors are known to be regulated by them, including Pdgf b. Strikingly, reduced Pdgf b, as shown in norrin knockout mice at P10, results in the formation of microaneurysms, as reported previously (Enge et al., 2002).

In many other cases of retinal hypoxia, neovascularization occurs as a result of increased activity of pro-angiogenic signaling cascades. Obviously, this does not apply to norrindeficient mice. Retinal neovascularization has never been reported, not even in late stages of the disease. Thus, high VEGF-α alone is not sufficient to induce the formation of new blood vessels in the retina if norrin is lacking.

Regression of hyaloid vessels versus angiogenesis of retinal blood vessels

In addition to the characteristic abnormal pattern of retinal vasculature, norrin is required for normal regression of the hyaloid vessels, which is completed by P21 in control mice but heavily delayed in norrin knockout mice (figure 43.4) (Ohlmann et al., 2004; Luhmann et al., 2005a). Nevertheless, these hyaloid vessels are obliterated and most likely nonfunctional in knockout mice, as shown by angiography (Luhmann et al., 2005a). Between P14 and P21, the number and diameter of hyaloid vessels were reduced. This obliteration continued until the age of 6–8 weeks, when most of them became nonfunctional. Occasionally, hyaloid vessels

layers in wild-type mice by angiogenic sprouting. In contrast, this process is abolished in knockout mice, and the vessels do not invade the deeper retinal layers. See color plate 42. (Images acquired using the Zeiss ApoTome technology and provided courtesy of Nikolaus Schäfer, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

were found to grow into the peripheral retina of knockout mice (Ohlmann et al., 2004; Luhmann et al., 2005a).

There are two alternative hypotheses regarding the primary defect in norrin-deficient mice: (1) Norrin causes persistence of the hyaloid vessels that supply oxygen to the retina, and thus retinal vascular development is dispensable (Ohlmann et al., 2004). (2) Norrin deficiency in the retina results in an arrest of vascular development of the deep capillary networks, which leads to hypoxia and subsequent upregulation of VEGF. This excess of VEGF provides an antiapoptotic signal to the endothelial cells of the hyaloid vasculature and its persistence or extremely delayed regression (Luhmann et al., 2005a).

The second alternative is supported by the fact that hyaloid vessels are obliterated and most likely nonfunctional. Moreover, it has been shown in mice that, by blocking PGF, functional hyaloid vessels can persist without significantly affecting retinal vascular development (Feeney et al., 2003).

The two phases of Norrie disease in the eye

Examination of blood vessel development in norrin knockout versus wild-type mice has revealed a delayed outgrowth of the superficial retinal vasculature, an arrest in sprouting angiogenesis that forms the deep and intermediate retinal capillary networks, and a delay in hyaloid regression. According to Luhmann et al. (2005a), there are two phases of

Figure 43.4 Hematoxylin-eosin-stained sections of whole eyes from wild-type (wt) and norrin knockout (ko) mice at P5, P10, P15, and P21. The hyaloid vessel system (H) in the vitreous body (V) is clearly present in wild-type mice at P5 and P10, and remnants are evident at P15. In knockout mice, the hyaloid vasculature persists

pathology in knockout mice. The first phase is characterized by the abnormal vascular development and may last until P7 or P8. After this, hypoxia-driven activation of angiogenic signaling cascades lead to severe lesions in the vitreous and retina and the characteristic disease symptoms, which include vitreoretinal hemorrhages and exudates caused by leaky blood vessels in the vitreous and retina, an avascular retinal periphery, and retinal folding and detachment. Very similar symptoms were described in human patients. Thus, the Ndph knockout mouse may represent a suitable model not only for the classic form of Norrie disease but also for allelic traits such as exudative vitreoretinopathy or Coats’ disease, as well as more common vasoproliferative retinal diseases (retinopathy of prematurity, diabetic retinopathy, age-related macular dystrophy).

Hearing loss and abnormal cochlear vasculature in Ndph knockout mice

Because one of the clinical hallmarks of Norrie disease is progressive deafness in human patients, knockout mice were examined with respect to auditory performance. The auditory brainstem response (ABR) test is a useful diagnostic tool for measuring hearing performance. ABR records activities in the auditory centers of the brain in response to sound stimuli of different frequencies (e.g., 5–45 kHz). The test is considered reliable and objective, because it does not depend on subjective feedback as other, more conventional hearing tests do.

ABR test results were compared for hemizygous knockout mice at three ages (3–4, 6.5, and 15 months) and wild-type littermates (Rehm et al., 2002). ABR recordings were correlated with cochlear pathology that had been scored by histological approaches, namely, conventional histology of

through P21 and beyond, and regression of this vascular network is profoundly delayed. L, lens; O, optic nerve; R, retina. See color plate 43. (Images acquired and provided courtesy of Dr. Ulrich Luhmann, Division of Medical Molecular Genetics, Institute of Medical Genetics, University of Zurich, Switzerland.)

cochlear structures, including the stria vascularis, spiral ganglion, outer and inner hair cells, and cochlear vasculature. These analyses revealed a progressive hearing loss in hemizygous knockout mice across all frequencies (5–45 kHz). At younger ages (3–4 months), hearing impairment was more severe in the higher frequencies, but it progressed to a profound loss at all frequencies by the age of 15 months.

This functional decline in the auditory system in knockout mice was found to be consistent with morphological changes in the cochlea, which was completely normal at P12 (Rehm et al., 2002). Thus, cochlear structures progressively degenerate over time, while normal development is not affected by the knockout mutation. The most affected structure was the stria vascularis in all three age groups. Vessels were significantly enlarged, particularly in the apical cochlear turn. The loss of outer hair cells progressed consistently, with an increase in auditory threshold. With progression of hearing loss, neurons in the spiral ganglion degenerated. Obviously, the outer hair cells of knockout mice are more susceptible to premature cell death than the inner hair cells, which show significant cell death at 15 months but not in the two groups of animals aged 3–4 months and 6.5 months. In addition, characteristic changes were found in the cochlear vasculature. The average vessel size was larger in knockout mice than in wild-type mice, and the number of vessels decreased significantly over time. Differences in vessel quantity between control animal and knockout mice were marginal at 3–4 months. However, enlarged vessels in the stria vascularis and a sparse capillary network in the spiral ganglion were clearly visible at this age. At 15 months, two-thirds of vessels had disappeared.

To summarize these findings, the stria vascularis might be the primarily affected cochlear structure, followed by the spiral ganglion. Hearing loss is progressive in knockout mice,

with only mild symptoms or almost normal hearing present at younger ages, although the expressivity of hearing impairment shows a high degree of interindividual variability even in mice (Rehm et al., 2002). These observations suggest normal development of the auditory system but a defect in its homeostasis, probably resulting from degenerative processes in the cochlear vasculature. This is in contrast to the retina, where part of the vasculature fails to develop properly because of a defect in angiogenic sprouting. Thus, norrin might have different effects in eye and ear. Still, a major role in angiogenesis is reconcilable as the mechanism of angiogenic sprouting is necessary in both processes, development and homeostasis.

Generation and analysis of a transgenic mouse line with norrin overexpression in the lens

The role of norrin in vascular development has also been demonstrated in transgenic mice overexpressing norrin in the lens. The transgenic lines have been established by pronucleus injection of a transgene construct containing the chicken βB1-crystallin promoter in front of the coding region of the mouse norrin cDNA (Ohlmann et al., 2005). In addition, a polyadenylation signal was present downstream of the translation termination codon of the norrin cDNA. Four founder lines were obtained containing 3, 9, 10, and 11 copies of the transgene, respectively. At the transcript and protein level, norrin expression from the transgene construct approximately correlated with the copy number. The line with 3 copies of the transgene showed less norrin mRNA and protein than the transgenic lines with 9–11 copies but more than wild-type littermates. Expression studies also showed that norrin expression in transgenic animals was restricted to the lens; other ocular structures did not express the transgene.

At the morphological level, the lenses and eye bulbs of transgenic mice carrying between 9 and 11 copies were smaller than those of wild-type mice or the line with 3 transgene copies (approximately 30%). The tunica vasculosa lentis, an extensive blood vessel network of capillaries spreading over the posterior and lateral lens surface, in transgenic mice contained significantly more capillaries than in wildtype litters at P0–P2. Ultrastructurally, the capillaries were very similar in wild-type and transgenic mice. They were unfenestrated and surrounded by pericytes. Quantitatively, the number of capillaries was 1.5- to 2-fold higher in transgenic animals with 9–11 gene copies and increased by 20% in the transgenic line with 3 copies of the transgene.

Lenses with overexpression of norrin were also examined with respect to stimulation of growth and proliferation of endothelial cells. Indeed, there was a positive effect of lenses expressing norrin on human dermal microvascular endothelial cells (HDMECs). The presence of norrin resulted in a

66% or 40% increase in proliferation of HDMECs when the culture medium was preconditioned with lenses from transgenic mice at P1 or P7, respectively, which might also suggest a direct effect of norrin on endothelial cells.

Rescue of the knockout phenotype by ectopic norrin expression in the lens

Norrin-deficient mice (Berger et al., 1996) were crossed with transgenic mice carrying 11 copies of the transgene, showing norrin expression at the transcript and protein levels (Ohlmann et al., 2005). Offspring were examined morphologically and electrophysiologically (ERG). These tests showed a rescue of the vascular and ERG phenotype of norrin knockout mice.

The deep and intermediate vascular systems present in the OPL and IPL, respectively, were restored in knockout mice with ectopic norrin expression in the lens at P21 (Ohlmann et al., 2005). Also, disorganization of the GCL, which is seen in knockout animals, disappeared. The ERG pattern of knockout mice, characterized by a reduced b- wave and almost absent oscillatory potentials, was rescued completely, although the amplitudes of the responses were lower than in wild-type litters, probably due to the reduced size of the bulbus. These findings were surprising, since diffusion of norrin from the lens to the retina, a prerequisite for the observed rescue effects, might be limited by the high affinity of norrin for the extracellular matrix (ECM). However, this retarded spatial spreading had been reported for norrin expressed and secreted from COS-7 (African green monkey kidney fibroblasts) cells (Perez-Vilar and Hill, 1997). The differences in ECM composition between cultured cells and in the vitreous body might explain this observation.

Over and above the rescue effects of ectopic norrin, it might have a sort of neurotrophic or neuroprotective effect. Ohlmann and co-workers (2005) observed an increase in thickness of the ONL and INL, as well as an increase in the number of nuclei in the GCL. It is not yet clear whether these observations are mediated by Vegfa, which was shown to be more abundant in the retinas of transgenic mice. Compensation for a smaller eye size in transgenic mice may be another reason for this finding.

Fetal loss in homozygous knockout female mice

Almost complete infertility of female mice homozygous for the knockout allele (Ndph−/−) has been reported (Luhmann et al., 2005b). Breeding of Ndph−/− females revealed a mean litter size of 0.3, while the control litters had seven offspring. The number of implantation sites was not different in homozygous knockout females, indicating normal implantation. However, massive bleeding at the implantation sites was reported at all embryonic stages from E9 onward, and

occasionally at E7. Embryonic tissues were highly disorganized and surrounded by blood. A normal chorioallantoic placenta was never observed, but yolk sac and trophoblast derivatives were differentiated. However, the spatial pattern of the trophoblast was severely disrupted, and the number of spongiotrophoblast cells was reduced. The labyrinthine area failed to develop and was filled with blood. Implantation of blastocysts and decidual reaction still occurred in homozygous females, but decidualization was compromised, which led to severely reduced fertility. The observed bleeding, which occurred before normal development of the chorioallantois placenta, might be a result of defects in decidual angiogenesis and blood vessel remodeling due to impaired angiogenic processes similar to those observed in the retina. Homozygous Frizzled-4 knockout female mice were also reported to be infertile, but as a result of failure of corpora lutea formation and function, which causes a defect in implantation (Hsieh et al., 2005).

The expression of norrin has also been shown in human placenta at the RNA level (Luhmann et al., 2005b), implicating a role for norrin in human female reproduction. However, because of the low frequency of NDP gene mutations, homozygous females have never been described, and the function of this gene in human female reproductive tissues remains elusive. It might be noteworthy in this context that genotypic wild-type (for NDP) male and female offspring of NDP gene mutation carriers showed abnormal vascular patterns in the peripheral retina reminiscent of mild retinopathy of prematurity (Mintz-Hittner et al., 1996). This might be caused by the expression of norrin in the placenta of female mutation carriers from the mutant allele. The mildness of the phenotype can be explained by the presence of the wild-type allele in the embryos.

Summary

Taken together, the data that have emerged from these knockout and transgenic mouse models indicate that Norrie disease is caused by a primary defect in the inner retina and vitreous. In the retina, the failure of proper blood vessel development leads to oxygen deficiency (hypoxia), which activates angiogenic signaling cascades. Activation of these pathways causes subsequent leakiness of blood vessels, hemorrhages, and fibrovascular membranes. The vitreal phenotype may be caused primarily by a delayed regression of the hyaloid vasculature and leaky blood vessels. The data obtained in mice can explain the leading ocular symptoms in patients with Norrie disease, exudative vitreoretinopathy, Coats’ disease, and retinopathy of prematurity, and indicate a role for norrin in vascular development in the eye. In contrast, its function in the auditory system might be vascular maintenance in the stria vascularis and spiral ganglion rather than vascular development in these structures, as

shown in the knockout mouse. This is also consistent with the human condition, where auditory function is normal in the beginning and the onset of sensorineural hearing loss or impairment occurs in adolescence.

acknowledgments I thank Dr. Ulrich Luhmann and Nikolaus Schäfer for critical reading of the manuscript and helpful comments and suggestions, as well as for providing the images in figures 43.2, 43.3, and 43.4.

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