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Familial exudative vitreoretinopathy (FEVR) is a human disease characterized by defective retinal angiogenesis and associated complications that can result in vision loss. Defective Wnt/β-catenin signaling is an established cause of FEVR, whereas other molecular alterations contributing to the disease remain insufficiently understood. Here, we show that integrin-linked kinase (ILK), a mediator of cell-matrix interactions, is indispensable for retinal angiogenesis. Inactivation of the murine Ilk gene in postnatal endothelial cells results in sprouting defects, reduced endothelial proliferation and disruption of the blood-retina barrier, resembling phenotypes seen in established mouse models of FEVR. Retinal vascularization defects are phenocopied by inducible inactivation of the gene for α-parvin ( Parva ), an interactor of ILK. Screening genomic DNA samples from exudative vitreoretinopathy patients identifies three distinct mutations in human ILK , which compromise the function of the gene product in vitro. Together, our data suggest that defective cell-matrix interactions are linked to Wnt signaling and FEVR. Integrin-linked kinase (ILK) is an important mediator of integrin signaling. Here Park et al. show that mice with endothelial-specific deletion of Ilk develop vascular defects that resemble familial exudative vitreoretinopathy, and identify mutations in ILK in patients with exudative vitreoretinopathy suggesting a potential role in human pathogenesis.

Proper connectivity of the nervous system requires temporal and spatial control of axon guidance signaling. As commissural axons navigate across the CNS midline, ROBO-mediated repulsion has traditionally been thought to be repressed before crossing, and then to become upregulated after crossing. The regulation of the ROBO receptors involves multiple mechanisms that control protein expression, trafficking, and activity. Here, we report that mammalian ROBO1 and ROBO2 are not uniformly inhibited precrossing and are instead subject to additional temporal control via alternative splicing at a conserved microexon. The NOVA splicing factors regulate the developmental expression of ROBO1 and ROBO2 variants with small sequence differences and distinct guidance activities. As a result, ROBO-mediated axonal repulsion is activated early in development to prevent premature crossing and becomes inhibited later to allow crossing. Postcrossing, the ROBO1 and ROBO2 isoforms are disinhibited to prevent midline reentry and to guide postcrossing commissural axons to distinct mediolateral positions.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to βcatenin‐dependent formation of the blood–retina–barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger βcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12 −/− mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen‐induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction. SYNOPSIS This study reports a FZD4:LRP5 antibody agonist (F4L5.13) that activates βcatenin signaling in endothelial cells. F4L5.13 shows efficacy in animal models by normalizing defective retinal angiogenesis and barrier function, providing a novel therapeutic strategy for eye diseases. βcatenin signaling was activated by F4L5.13, which functions as a Norrin surrogate in endothelial cells. Endothelial barrier function was promoted, and VEGF‐induced endothelial permeability was blocked by F4L5.13. Retinal barrier function was restored by F4L5.13 in Tspan12 −/− mice. Pathological neovascularization was reduced by F4L5.13 in an OIR model. Graphical Abstract This study reports a FZD4:LRP5 antibody agonist (F4L5.13) that activates βcatenin signaling in endothelial cells. F4L5.13 shows efficacy in animal models by normalizing defective retinal angiogenesis and barrier function, providing a novel therapeutic strategy for eye diseases.

Angiogenesis and blood–brain barrier formation are required for normal central nervous system (CNS) function. Both processes are controlled by Wnt or Norrin (NDP) ligands, Frizzled (FZD) receptors, and β-catenin-dependent signalling in vascular endothelial cells. In the retina, FZD4 and the ligand NDP are critical mediators of signalling and are mutated in familial exudative vitreoretinopathy. Here, we report that NDP is a potent trigger of FZD4 ubiquitination and induces internalization of the NDP receptor complex into the endo-lysosomal compartment. Inhibition of ubiquitinated cargo transport through the multivesicular body (MVB) pathway using a dominant negative ESCRT (endosomal sorting complexes required for transport) component VPS4 EQ strongly impairs NDP/FZD4 signalling in vitro and recapitulates CNS angiogenesis and blood-CNS-barrier defects caused by impaired vascular β-catenin signalling in mice. These findings provide evidence for an important role of FZD4 endocytosis in NDP/FZD4 signalling and in CNS vascular biology and disease. Multiple mechanisms regulate Wnt/ß-catenin signalling. Zhang et al . describe a novel regulatory pathway and show that the activator of canonical Wnt signalling, Norrin, triggers endocytosis of its receptor Frizzled4 by promoting Frizzled4 ubiquitination.

The neuron specific RNA-binding proteins NOVA1 and NOVA2 are highly homologous alternative splicing regulators. NOVA proteins regulate at least 700 alternative splicing events in vivo, yet relatively little is known about the biologic consequences of NOVA action and in particular about functional differences between NOVA1 and NOVA2. Transcriptome-wide searches for isoform-specific functions, using NOVA1 and NOVA2 specific HITS-CLIP and RNA-seq data from mouse cortex lacking either NOVA isoform, reveals that NOVA2 uniquely regulates alternative splicing events of a series of axon guidance related genes during cortical development. Corresponding axonal pathfinding defects were specific to NOVA2 deficiency: Nova2-/- but not Nova1-/- mice had agenesis of the corpus callosum, and axonal outgrowth defects specific to ventral motoneuron axons and efferent innervation of the cochlea. Thus we have discovered that NOVA2 uniquely regulates alternative splicing of a coordinate set of transcripts encoding key components in cortical, brainstem and spinal axon guidance/outgrowth pathways during neural differentiation, with severe functional consequences in vivo. The first step of producing a protein involves the DNA of a gene being copied to form a molecule of RNA. This RNA molecule can often be processed to create several different “messenger” RNAs (mRNAs), each of which are used to produce a different protein by a process known as alternative splicing. A class of proteins that bind to RNA molecules controls alternative splicing. These “splicing factors” ensure that the right protein variant is produced at the right time and in the right place to carry out the appropriate activity. Many genes that play important roles in the nervous system have been reported to undergo alternative splicing to generate different protein variants. However, it is unclear whether alternative splicing is important for controlling how the nervous system develops, during which time the neurons connect to the cells that they will communicate with. Forming these connections involves part of the neuron, called the axon, growing along a precise path through the nervous system to reach its destination. Two RNA-binding proteins called NOVA1 and NOVA2 are produced exclusively in the central nervous system, where they regulate a number of actions including alternative splicing. So far, no differences in the roles of NOVA1 and NOVA2 have been identified, and relatively little is known about their actions in the brain. Saito et al. have addressed these missing puzzle pieces by combining RNA analysis methods with an analysis of the structure of the nervous system of mice that lack either NOVA1 or NOVA2. This approach identified where NOVA1 and NOVA2 bind on mRNAs, and showed that the mRNAs are processed in different ways in the developing mouse brain depending on which form of the NOVA protein is bound to it. Further analysis of the data revealed that NOVA2, and not NOVA1, regulates splicing in a series of RNA molecules that help to guide axons to the correct locations in the developing mouse brain. A related study by Leggere et al. also reported on the role that NOVA proteins play in the alternative splicing of one of these genes, called Dcc. Saito et al. also found defects in the nervous systems of the mice that lacked NOVA2 that only occurred in these mice and resulted from certain axons being unable to follow the correct path to their target cells. These led to major defects, such as agenesis of the corpus callosum (a complete lack of connection between the right and left sides of the brain). Further defects affected how specific subsets of motor neurons connect to muscles and how cochlear neurons in the brainstem connect to the inner ear. The next steps are to explore how the processing of RNA molecules by NOVA2 causes these defects, and to assess whether these actions relate to developmental brain disorders in humans.

RNA-binding proteins (RBPs) control multiple aspects of post-transcriptional gene regulation and function during various biological processes in the nervous system. To further reveal the functional significance of RBPs during neural development, we carried out an in vivo RNAi screen in the dorsal spinal cord interneurons, including the commissural neurons. We found that the NOVA family of RBPs play a key role in neuronal migration, axon outgrowth, and axon guidance. Interestingly, Nova mutants display similar defects as the knockout of the Dcc transmembrane receptor. We show here that Nova deficiency disrupts the alternative splicing of Dcc, and that restoring Dcc splicing in Nova knockouts is able to rescue the defects. Together, our results demonstrate that the production of DCC splice variants controlled by NOVA has a crucial function during many stages of commissural neuron development. The first step of producing a protein involves the DNA of a gene being copied to form a molecule of RNA. This RNA molecule can often be processed to create several different “messenger” RNAs (mRNAs), each of which are used to produce a different protein by a process known as alternative splicing. A class of proteins that bind to RNA molecules controls alternative splicing. These “splicing factors” ensure that the right protein variant is produced at the right time and in the right place to carry out the appropriate activity. Many genes that play important roles in the nervous system have been reported to undergo alternative splicing to generate different protein variants. However, it is unclear whether alternative splicing is important for controlling how the nervous system develops, during which time the neurons connect to the cells that they will communicate with. Forming these connections involves part of the neuron, called the axon, growing along a precise path through the nervous system to reach its destination. If alternative splicing is important for this process, it is also important to ask: which splicing factors are relevant and which genes do these splicing factors regulate? Through genetic and molecular studies using mouse embryos, Leggere et al. found that the NOVA family of splicing factors are essential for the development of the nervous system. In particular, the NOVA splicing factors control the alternative splicing of a gene called Dcc. This gene produces proteins that play a number of roles, including helping axons to grow and guiding the axons to the correct location in the developing nervous system. A related study by Saito et al. showed that two forms of NOVA splicing factors – called NOVA1 and NOVA2 – have different roles in the nervous system, and describes the role of NOVA2 in more detail. Leggere et al. will now carry out additional studies to determine the unique role of each protein variant produced from the Dcc gene. Future research will also investigate how NOVA proteins help generate these variants at the right time and in the right place.

In the retina blood vessels are required to support a high metabolic rate, however, uncontrolled vascular growth can lead to impaired vision and blindness. Subretinal vascularization (SRV), one type of pathological vessel growth, occurs in retinal angiomatous proliferation and proliferative macular telangiectasia. In these diseases SRV originates from blood vessels within the retina. We use mice with a targeted disruption in the Vldl-receptor (Vldlr) gene as a model to study SRV with retinal origin. We find that Vldlr mRNA is strongly expressed in the neuroretina, and we observe both vascular and neuronal phenotypes in Vldlr-/- mice. Unexpectedly, horizontal cell (HC) neurites are mistargeted prior to SRV in this model, and the majority of vascular lesions are associated with mistargeted neurites. In Foxn4-/- mice, which lack HCs and display reduced amacrine cell (AC) numbers, we find severe defects in intraretinal capillary development. However, SRV is not suppressed in Foxn4-/-;Vldlr-/- mice, which reveals that mistargeted HC neurites are not required for vascular lesion formation. In the absence of VLDLR, the intraretinal capillary plexuses form in an inverse order compared to normal development, and subsequent to this early defect, vascular proliferation is increased. We conclude that SRV in the Vldlr-/- model is associated with mistargeted neurites and that SRV is preceded by altered retinal vascular development.

β-Catenin signaling controls the development and maintenance of the blood–brain barrier (BBB) and the blood–retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand–receptor systems—the Norrin and Wnt7a/Wnt7b systems—are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.

Background Newborn neurons often migrate before undergoing final differentiation, extending neurites, and forming synaptic connections. Therefore, neuronal migration is crucial for establishing neural circuitry during development. In the developing spinal cord, neuroprogenitors first undergo radial migration within the ventricular zone. Differentiated neurons continue to migrate tangentially before reaching the final positions. The molecular pathways that regulate these migration processes remain largely unknown. Our previous study suggests that the DCC receptor is important for the migration of the dorsal spinal cord progenitors and interneurons. In this study, we determined the involvement of the Netrin1 ligand and the ROBO3 coreceptor in the migration. Results By pulse labeling neuroprogenitors with electroporation, we examined their radial migration in Netrin1 (Ntn1), Dcc, and Robo3 knockout mice. We found that all three mutants exhibit delayed migration. Furthermore, using immunohistochemistry of the BARHL2 interneuron marker, we found that the mediolateral and dorsoventral migration of differentiated dorsal interneurons is also delayed. Together, our results suggest that Netrin1/DCC signaling induce neuronal migration in the dorsal spinal cord. Conclusions Netrin1, DCC, and ROBO3 have been extensively studied for their functions in regulating axon guidance in the spinal commissural interneurons. We reveal that during earlier development of dorsal interneurons including commissural neurons, these molecules play an important role in promoting cell migration.

Macular edema (ME) can cause profound vision impairment and occurs in several prevalent retinal diseases, including diabetic retinopathy, choroidal neovascularization, retinal vein occlusion, and uveitis. Retinal edema typically results from dysfunction of the blood-retina barrier (BRB), which is associated with increased retinal expression of complement components. It is unclear whether the classical complement pathway has detrimental or protective roles in the context of BRB dysfunction. Here, we characterized Tspan12-KODBM (disrupted BRB maintenance) mice, a mouse model of cystoid edema generated by genetically and pharmacologically manipulating β-catenin-dependent norrin/frizzled-4 (FZD4) signaling. We assessed BRB function, cystoid edema, electroretinogram, and microglia activation outcomes in an aging study with WT, C1qa-KO, Tspan12-KODBM, and Tspan12-KODBM; C1qa-KO compound mutant mice. Phenotypic analyses and cell-based experiments indicated that C1QA contributes to maintaining basal β-catenin-dependent signaling and that the absence of C1QA exacerbates BRB dysfunction, cystoid edema, and neuroinflammation in Tspan12-KODBM; C1qa-KO compound mutant mice. Activation of β-catenin-dependent signaling by an anti-FZD4 and anti-LRP5 agonistic antibody modality achieved complete resolution of cystoid edema. This study shows that reducing or enhancing norrin/FZD4 signaling can increase or decrease cystoid edema, respectively, underscoring its potential as a therapeutic target in ME. Furthermore, this study provides insights into the contribution of C1QA to BRB maintenance.