Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.     

Non-conventional Frizzled ligands and Wnt receptors

The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of β-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate β-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.     

Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain

The Norrie disease gene (Ndp) codes for a secreted protein, Norrin, that activates canonical Wnt signaling by binding to its receptor, Frizzled-4. This signaling system is required for normal vascular development in the retina and for vascular survival in the cochlea. In mammals, the pattern of Ndp expression beyond the retina is poorly defined due to the low abundance of Norrin mRNA and protein. Here, we characterize Ndp expression during mouse development by studying a knock-in mouse that carries the coding sequence of human placental alkaline phosphatase (AP) inserted at the Ndp locus (Ndp(AP)). In the CNS, Ndp(AP) expression is apparent by E10.5 and is dynamic and complex. The anatomically delimited regions of Ndp(AP) expression observed prenatally in the CNS are replaced postnatally by widespread expression in astrocytes in the forebrain and midbrain, Bergman glia in the cerebellum, and Müller glia in the retina. In the developing and adult cochlea, Ndp(AP) expression is closely associated with two densely vascularized regions, the stria vascularis and a capillary plexus between the organ of Corti and the spiral ganglion. These observations suggest the possibility that Norrin may have developmental and/or homeostatic functions beyond the retina and cochlea.     

Norrin immunolocalization and its possible functions in rat endometrium during the estrus cycle and early pregnancy

Mutations in Norrie Disease Pseudoglioma (NDP) gene cause serious sight loss, deafness and mental retardation in Norrie disease patients via the impairment of angiogenesis. Since norrin is a Wnt pathway ligand, it could function in several tissues other than eye and nervous systems. Therefore, the aim of the present study was to determine the possible function of norrin in angiogenesis, cellular differentiation in stroma and in decidua and the survival of those cells using immunofluorescent labeling. While norrin had a uniform distribution in stroma and in blood vessels, it had a strong expression in luminal and glandular epithelia during the estrus cycle. Norrin had strong immunolocalization in the antimesometrial decidual reaction zone on day 7 of gestation, whereas it had a decreased expression in the mesometrial uterine luminal epithelium along with an increased localization in blood vessels and decidual cells of the same region on day 8 of gestation. As from day 9 of gestation, norrin demonstrated rather strong expression in the decidual cells and blood vessels of the mesometrial region in which the chorioallantoic placenta was going to develop. In all periods studied, norrin had rather weak expression in the primary decidual zone surrounding the embryo. Findings of the present study suggested that norrin might regulate the decidual reaction and the placental angiogenesis along with the survival and the differentiation of luminal and glandular epithelial and decidual cells in rats. In addition, it could play indirect important roles in the control of trophoblastic invasion and the programmed cell death.     

Maternal xNorrin, a canonical Wnt signaling agonist and TGF-β antagonist, controls early neuroectoderm specification in Xenopus

Dorsal-ventral specification in the amphibian embryo is controlled by β-catenin, whose activation in all dorsal cells is dependent on maternal Wnt11. However, it remains unknown whether other maternally secreted factors contribute to β-catenin activation in the dorsal ectoderm. Here, we show that maternal Xenopus Norrin (xNorrin) promotes anterior neural tissue formation in ventralized embryos. Conversely, when xNorrin function is inhibited, early canonical Wnt signaling in the dorsal ectoderm and the early expression of the zygotic neural inducers Chordin, Noggin, and Xnr3 are severely suppressed, causing the loss of anterior structures. In addition, xNorrin potently inhibits BMP- and Nodal/Activin-related functions through direct binding to the ligands. Moreover, a subset of Norrin mutants identified in humans with Norrie disease retain Wnt activation but show defective inhibition of Nodal/Activin-related signaling in mesoderm induction, suggesting that this disinhibition causes Norrie disease. Thus, xNorrin is an unusual molecule that acts on two major signaling pathways, Wnt and TGF-β, in opposite ways and is essential for early neuroectoderm specification.     

Close linkage between Norrie disease,a cloned DNA sequence from the proximal short arm,and the centromere of the X chromosome

Summary Norrie disease (ND) is an X-linked recessive disorder with congenital blindness (atrophia bulborum hereditaria, pseudoglioma). Six kindreds segregating for ND were studied for linkage with polymorphic markers of the human X chromosome. No recombination was observed between the ND-locus (NDP) and the DXS7 locus, the latter followed as a DNA-restriction fragment length polymorphism, detected by the recombinant DNA probe L1.28, and assigned to the region Xp11.2–Xp11.3. The maximum lod scores are at . Linkage data between NDP and the other genetic markers used in the present study are in keeping with this assignment of the mutation to the proximal Xp.     

Localisation of the gene for Norrie disease to between DXS7 and DXS426 on Xp

Summary A highly informative microsatellite marker, DXS426, which maps proximal to DXS7 in the interval Xp11.4–Xp11.23, has been used to refine further the localisation of the gene for Norrie disease (NDP). The results from a multiply informative crossover localize the NDP gene proximal to DXS7. In conjunction with information from 2 NDP patients who have a deletion for DXS7 but not for DSX426, our data indicate that the NDP gene lies between DXS7 and DXS426 on proximal Xp.     

Clinical and molecular findings in osteoporosis-pseudoglioma syndrome

Mutations in the low-density lipoprotein receptor-related protein 5 gene (LRP5) cause autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG). We sequenced the coding exons of LRP5 in 37 probands suspected of having OPPG on the basis of the co-occurrence of severe congenital or childhood-onset visual impairment with bone fragility or osteoporosis recognized by young adulthood. We found two putative mutant alleles in 26 probands, only one mutant allele in 4 probands, and no mutant alleles in 7 probands. Looking for digenic inheritance, we sequenced the genes encoding the functionally related receptor LRP6, an LRP5 coreceptor FZD4, and an LRP5 ligand, NDP, in the four probands with one mutant allele, and, looking for locus heterogeneity, we sequenced FZD4 and NDP in the seven probands with no mutations, but we found no additional mutations. When we compared clinical features between probands with and without LRP5 mutations, we found no difference in the severity of skeletal disease, prevalence of cognitive impairment, or family history of consanguinity. However, four of the seven probands without detectable mutations had eye pathology that differed from pathology previously described for OPPG. Since many LRP5 mutations are missense changes, to differentiate between a disease-causing mutation and a benign variant, we measured the ability of wild-type and mutant LRP5 to transduce Wnt and Norrin signal ex vivo. Each of the seven OPPG mutations tested, had reduced signal transduction compared with wild-type mutations. These results indicate that early bilateral vitreoretinal eye pathology coupled with skeletal fragility is a strong predictor of LRP5 mutation and that mutations in LRP5 cause OPPG by impairing Wnt and Norrin signal transduction.     

Mutational analysis of Norrin-Frizzled4 recognition

Norrin and Frizzled4 (Fz4) function as a ligand-receptor pair to control vascular development in the retina and inner ear. In mice and humans, mutations in either of the corresponding genes lead to defects in vascular development. The present work is aimed at defining the sequence determinants of binding specificity between Norrin and the Fz4 amino-terminal ligand-binding domain (the "cysteine-rich domain" (CRD)). The principal conclusions are as follows: 1) Norrin binds to the Fz4 CRD and does not detectably bind to the 14 other mammalian Frizzled and secreted Frizzled-related protein CRDs; 2) Norrin and Xenopus Wnt8 recognize largely overlapping regions of the Fz4 CRD; 3) surface determinants on the Fz4 and Fz8 CRDs that allow Norrin to distinguish between these two CRDs reside within several small regions on one face of the CRD; 4) Norrin function depends critically on three pairs of cysteines that form the highly conserved trio of disulfide bonds shared among all cystine knot proteins, but the remaining two putative disulfide bonds are less important; 5) Norrin-CRD binding depends on a largely contiguous group of amino acids in the extended beta-sheet domain of Norrin that are predicted to face away from the interface between the two monomers in the Norrin homodimer; 6) Norrin-CRD binding is strongly modulated by interactions involving charged amino acid side chains; and 7) Norrin-CRD binding is enhanced approximately 10-fold by the addition of heparin. These observations are discussed in the context of Frizzled signaling and the structure and function of other cystine knot proteins.     

Wnt signaling: Ig-norrin the dogma

Secreted Wnt proteins trigger the intracellular Wnt signaling cascade upon engagement of dedicated Frizzled-Lrp receptor complexes. Unexpectedly, a non-Wnt ligand for this receptor complex has now been discovered. This novel ligand, Norrin, is mutated in the hereditary ocular Norrie syndrome.     

Norrie disease gene is distinct from the monoamine oxidase genes

The genes for MAO-A and MAO-B appear to be very close to the Norrie disease gene, on the basis of loss and/or disruption of the MAO genes and activities in atypical Norrie disease patients deleted for the DXS7 locus; linkage among the MAO genes, the Norrie disease gene, and the DXS7 locus; and mapping of all these loci to the chromosomal region Xp11. The present study provides evidence that the MAO genes are not disrupted in "classic" Norrie disease patients. Genomic DNA from these "nondeletion" Norrie disease patients did not show rearrangements at the MAOA or DXS7 loci. Normal levels of MAO-A activities, as well as normal amounts and size of the MAO-A mRNA, were observed in cultured skin fibroblasts from these patients, and MAO-B activity in their platelets was normal. Catecholamine metabolites evaluated in plasma and urine were in the control range. Thus, although some atypical Norrie disease patients lack both MAO-A and MAO-B activities, MAO does not appear to be an etiologic factor in classic Norrie disease.     

An essential role of the cysteine-rich domain of FZD4 in Norrin/Wnt signaling and familial exudative vitreoretinopathy

The Wnt pathway plays important yet diverse roles in health and disease. Mutations in the Wnt receptor FZD4 gene have been confirmed to cause familial exudative vitreoretinopathy (FEVR). FEVR is characterized by incomplete vascularization of the peripheral retina, which can lead to vitreous bleeding, tractional retinal detachment, and blindness. We screened for mutations in the FZD4 gene in five families with FEVR and identified five mutations (C45Y, Y58C, W226X, C204R, and W496X), including three novel mutations (C45Y, Y58C, and W226X). In the retina, Norrin serves as a ligand and binds to FZD4 to activate the Wnt signaling pathway in normal angiogenesis and vascularization. The cysteine-rich domain (CRD) of FZD4 has been shown to play a critical role in Norrin-FZD4 binding. We investigated the effect of mutations in the FZD4 CRD in Norrin binding and signaling in vitro and in vivo. Wild-type and mutant FZD4 proteins were assayed for Norrin binding and Norrin-dependent activation of the canonical Wnt pathway by cell-surface and overlay binding assays and luciferase reporter assays. In HEK293 transfection studies, C45Y, Y58C, and C204R mutants did not bind to Norrin and failed to transduce FZD4-mediated Wnt/β-catenin signaling. In vivo studies using Xenopus embryos showed that these FZD4 mutations disrupt Norrin/β-catenin signaling as evidenced by decreased Siamois and Xnr3 expression. This study identified a new class of FZD4 gene mutations in human disease and demonstrates a critical role of the CRD in Norrin binding and activation of the β-catenin pathway.     

Bone morphogenetic proteins in development and homeostasis of kidney

Bone morphogenetic proteins play a key role in kidney development and postnatal function. The kidney has been identified as a major site of bone morphogenetic protein (BMP)-7 synthesis during embryonic and postnatal development, which mediates differentiation and maintenance of metanephric mesenchyme. Targeted disruption of BMP-7 gene expression in mice resulted in dysgenic kidneys with hydroureters, causing uremia within 24h after birth. Several experimental animal models of acute and chronic renal injury have all unequivocally shown beneficial effect of BMP-7 in ameliorating the severity of damage by preventing inflammation and fibrosis. Apart from the beneficial effect on kidney disease itself, BMP-7 improves important complications of chronic renal impairment such as renal osteodystrophy and vascular calcification.     

Regulation of Cellular Functions by Nucleoside Diphosphate Kinases in Mammals

The role of nucleoside diphosphate (NDP) kinases in cell growth, differentiation, and tumormetastasis in relation to signal transduction was investigated. The essential role of NDP kinasein cell growth was validated by coupling between reduced NDP kinase levels, induced byantisense oligonucleotides, and the suppression of proliferative activity of a cultured cell line.In addition, because NDP kinase levels are often enhanced with development and differentiation,as has been demonstrated in postmitotic cells and tissues, such as the heart and brain, wefurther examined this possibility using the bone tissue (osteoblasts) and a cultured cell linePC12D. The enhanced NDP kinase accumulation was demonstrated in the matured osteoblastsin vivo and in vitro by immunohistochemistry. In PC12D cells neurite outgrowth took placein NDP kinase -transfected clones without differentiation inducers, which was accompaniedby prolongation of doubling time. Neurite outgrowth, triggered by nerve growth factor and acyclic AMP analog, was down-regulated upon forced expression of inactive mutant NDPkinase by virtue of a dominant negative effect. NDP kinase -transfected rat mammaryadenocarcinoma cells (MTLn3) and nm23-H2-transfected human oral squamous cell carcinomacells (LMF4) manifested reduced metastatic potential and were associated with an alteredsensitivity to environmental factors, such as motility and growth factors. NDP kinase ,compared to NDP kinase , was involved in a wide variety of the cellular phenomena examined.Taken together, NDP kinase isoforms appear to elicit both their own respective and commoneffects. They may have an ability to lead cells to both proliferative and differentiated statesby modulating responsiveness to environmental factors, but their fate seems to depend on theirsurrounding milieu.     

Novel insights into the development and maintenance of the blood–brain barrier

The blood–brain barrier (BBB) is essential for maintaining homeostasis within the central nervous system (CNS) and is a prerequisite for proper neuronal function. The BBB is localized to microvascular endothelial cells that strictly control the passage of metabolites into and out of the CNS. Complex and continuous tight junctions and lack of fenestrae combined with low pinocytotic activity make the BBB endothelium a tight barrier for water soluble moleucles. In combination with its expression of specific enzymes and transport molecules, the BBB endothelium is unique and distinguishable from all other endothelial cells in the body. During embryonic development, the CNS is vascularized by angiogenic sprouting from vascular networks originating outside of the CNS in a precise spatio-temporal manner. The particular barrier characteristics of BBB endothelial cells are induced during CNS angiogenesis by cross-talk with cellular and acellular elements within the developing CNS. In this review, we summarize the currently known cellular and molecular mechanisms mediating brain angiogenesis and introduce more recently discovered CNS-specific pathways (Wnt/β?catenin, Norrin/Frizzled4 and hedgehog) and molecules (GPR124) that are crucial in BBB differentiation and maturation. Finally, based on observations that BBB dysfunction is associated with many human diseases such as multiple sclerosis, stroke and brain tumors, we discuss recent insights into the molecular mechanisms involved in maintaining barrier characteristics in the mature BBB endothelium.     

Expression of EGFL7 in primordial germ cells and in adult ovaries and testes

We have previously reported the isolation and characterization of a novel endothelial-restricted gene, Egfl7, that encodes a secreted protein of about 30-kDa. We and others demonstrated that Egfl7 is highly expressed by endothelial cells during embryonic development and becomes down-regulated in the adult vasculature. In the present paper, we show that during mouse embryonic development, Egfl7 is also expressed by primordial germ cells (PGC). Expression is down-regulated when PGCs differentiate into pro-spermatogonia and oogonia, and by 15.5 dpc Egfl7 can no longer be detected in the germ line of both sexes. Notably, Egfl7 is again transiently up-regulated in germ cells of the adult testis. In contrast, expression in the ovary remains limited to the vascular endothelium. Our results provide the first evidence of a non-endothelial expression of EGFL7 and suggest distinctive roles for Egfl7 in vascular development and germ cell differentiation.     

Moderate reduction of Norrin signaling activity associated with the causative missense mutations identified in patients with familial exudative vitreoretinopathy

Mutations in Norrin signaling genes (NDP, FZD4 and LRP5) have been found in patients with familial exudative vitreoretinopathy (FEVR) and the altered signaling is suspected to play a critical role in its pathogenesis. To better understand this relationship, we systematically performed functional analyses on previously identified single nucleotide variants of LRP5, FZD4 and NDP, utilizing the Norrin dependent Topflash reporter assay. Cell surface binding assays and protein electrophoresis analysis of Norrin were also performed. Seven causative mutations and five possibly causative but indecisive variants were examined. We found: (1) a nonsense mutation in FZD4 completely abolished its signaling activity, while single missense mutations in LRP5 and FZD4 caused a moderate level of reduction (ranging from 26 to 48, 36% on average) and a double missense mutation in both genes caused a severe reduction in activity (71%). These observations correlated roughly with clinical phenotypes. (2) A mutational effect is suggested in four of five indecisive variants by signaling reductions comparable to those of missense mutations. (3) Norrin mutants demonstrated variable effects on signal transduction, and no apparent correlation with clinical phenotypes was observed. (4) The Norrin mutants examined demonstrated impaired cell surface binding, and some may have partially lost their ability to form a complex with unknown high molecular weight material(s). Our results illustrate the nature of FEVR in relation to Norrin signaling and further suggest the complexity of its disease causing mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.