Sakae Ikeda

Corneal avascularity is due to soluble VEGF receptor-1.

Corneal avascularity—the absence of blood vessels in the cornea—is required for optical clarity and optimal vision, and has led to the cornea being widely used for validating pro- and anti-angiogenic therapeutic strategies for many disorders. But the molecular underpinnings of the avascular phenotype have until now remained obscure and are all the more remarkable given the presence in the cornea of vascular endothelial growth factor (VEGF)-A, a potent stimulator of angiogenesis, and the proximity of the cornea to vascularized tissues. Here we show that the cornea expresses soluble VEGF receptor-1 (sVEGFR-1; also known as sflt-1) and that suppression of this endogenous VEGF-A trap by neutralizing antibodies, RNA interference or Cre-lox-mediated gene disruption abolishes corneal avascularity in mice. The spontaneously vascularized corneas of corn1 and Pax6+/- mice and Pax6+/- patients with aniridia are deficient in sflt-1, and recombinant sflt-1 administration restores corneal avascularity in corn1 and Pax6+/- mice. Manatees, the only known creatures uniformly to have vascularized corneas, do not express sflt-1, whereas the avascular corneas of dugongs, also members of the order Sirenia, elephants, the closest extant terrestrial phylogenetic relatives of manatees, and other marine mammals (dolphins and whales) contain sflt-1, indicating that it has a crucial, evolutionarily conserved role. The recognition that sflt-1 is essential for preserving the avascular ambit of the cornea can rationally guide its use as a platform for angiogenic modulators, supports its use in treating neovascular diseases, and might provide insight into the immunological privilege of the cornea.

Aberrant actin cytoskeleton leads to accelerated proliferation of corneal epithelial cells in mice deficient for destrin (actin depolymerizing factor)

Corneal disease is the most common cause of bilateral blindness in the world. Visual loss in this condition is often due to changes in morphology and function of the corneal epithelial surface. Corneal disease-1 (corn1) and corn1(2J) are spontaneous mouse mutants that develop irregular thickening of the corneal epithelium, similar to that observed in human corneal surface disease. These autosomal-recessive mutations cause an increase in the rate of proliferation of the corneal epithelial cells. Here, we report that the phenotypes in both mutants are caused by mutations within the destrin gene (also known as actin-depolymerizing factor). By positional cloning, we identified a deletion encompassing the entire coding sequence of the destrin gene in corn1 mice, and a point mutation (Pro106Ser) in the coding sequence of destrin in corn1(2J) mice. In situ analysis showed that destrin is highly expressed in the corneal epithelium. Consistent with the cellular roles for destrin, an essential regulator of actin filament turnover that acts by severing and enhancing depolymerization of actin filament, we observed that the corn1 mutations increased the content of filamentous actin in corneal epithelial cells. Our results suggest an in vivo connection between remodeling of the actin cytoskeleton and the control of cell proliferation, and a new pathway through which an aberrant actin cytoskeleton can cause epithelial hyperproliferation.

Spontaneous Corneal Hem- and Lymphangiogenesis in Mice with Destrin-Mutation Depend on VEGFR3 Signaling

Lymphangiogenesis, the formation of new lymphatic vessels, is important for tumor metastasis and induction of immunity to peripheral antigens including organ transplants. We herein describe a novel mouse model of spontaneous, secondary lymphangiogenesis in the normally avascular cornea. corn1 mice, which suffer from a deletion in the gene encoding the cytoskeletal protein destrin, develop hemangiogenesis as well as spontaneous outgrowth of LYVE-1+++/CD31+ lymphatic vessels into the cornea starting at age 4 weeks. Corneal lymphangiogenesis is delayed in onset, is less intense, and regresses earlier compared with hemangiogenesis. Moreover, the lymphangiogenesis is preceded only by a mild recruitment of CD45+ inflammatory cells into the cornea. In contrast to mice with inflammation-induced hem- and lymphangiogenesis, corn1 mice do not develop breakdown of the blood-aqueous barrier. Finally, in this novel mouse model, a blocking anti-VEGFR3 antibody significantly inhibited not only lymph- but also hemangiogenesis. In summary, destrin deletion has differential effects on spontaneous hem- and lymphangiogenesis in the normally avascular cornea and represents a novel mouse model to study the mechanisms of lymphangiogenesis and to test the antihem- and antilymphangiogenic properties of known or new antiangiogenic agents.

GFP-tagged expression and immunohistochemical studies to determine the subcellular localization of the tubby gene family members.

The tubby gene family consists of four members, TUB, TULP1, TULP2 and TULP3, with unknown function. However, a splice junction mutation within the mouse tub gene leads to retinal and cochlear degeneration, as well as maturity onset obesity and insulin resistance. Mutations within human TULP1 have also been shown to co-segregate in several cases of autosomal recessive retinitis pigmentosa (RP) and TULP1 deficiency in mice leads to retinal degeneration. The primary amino acid sequences of the tubby family members do not predict a likely biochemical function. As a first step in defining their function, we present a detailed characterization of the cellular and subcellular localization of the human (TUB) and mouse (tub) homologous gene products. We report the isolation of TUB splice variants which have different subcellular localizations (nuclear versus cytoplasmic) and which define a nuclear localization signal. In addition, using green fluorescent protein (GFP) tags, we observe a nuclear localization for TULP1, similar to TUB splicing forms TUB 561 and TUB 506. Finally, we report tubby expression in mouse brain by in situ hybridization and by immunohistochemistry with polyclonal antibodies. Protein was found in both the hypothalamic satiety centers and in a variety of other CNS structures including the cortex, cerebellum, olfactory bulb and hippocampus. Both nuclear and cytoplasmic signals were detected with a series of independently generated polyclonal antibodies, consistent with the presence of multiple alternatively spliced isoforms within the CNS.

Effect of destrin mutations on the gene expression profile in vivo

Remodeling of the actin cytoskeleton through actin dynamics (assembly and disassembly of filamentous actin) is known to be essential for numerous basic biological processes. In addition, recent studies have provided evidence that actin dynamics participate in the control of gene expression. A spontaneous mouse mutant, corneal disease 1 (corn1), is deficient for a regulator of actin dynamics, destrin (DSTN, also known as ADF), which causes epithelial hyperproliferation and neovascularization in the cornea. Dstn(corn1) mice exhibit an actin dynamics defect in the corneal epithelial cells, offering an in vivo model to investigate cellular mechanisms affected by the Dstn mutation and resultant actin dynamics abnormalities. To examine the effect of the Dstn(corn1) mutation on the gene expression profile, we performed a microarray analysis using the cornea from Dstn(corn1) and wild-type mice. A dramatic alteration of the gene expression profile was observed in the Dstn(corn1) cornea, with 1,226 annotated genes differentially expressed. Functional annotation of these genes revealed that the most significantly enriched functional categories are associated with actin and/or cytoskeleton. Among genes that belong to these categories, a considerable number of serum response factor target genes were found, indicating the possible existence of an actin-SRF pathway of transcriptional regulation in vivo. A comparative study using an allelic mutant strain with milder corneal phenotypes suggested that the level of filamentous actin may correlate with the level of gene expression changes. Our study shows that Dstn mutations and resultant actin dynamics abnormalities have a strong impact on the gene expression profile in vivo.

Mutation in archain 1, a subunit of COPI coatomer complex, causes diluted coat color and Purkinje cell degeneration.

Intracellular trafficking is critical for delivering molecules and organelles to their proper destinations to carry out normal cellular functions. Disruption of intracellular trafficking has been implicated in the pathogenesis of various neurodegenerative disorders. In addition, a number of genes involved in vesicle/organelle trafficking are also essential for pigmentation, and loss of those genes is often associated with mouse coat-color dilution and human hypopigmentary disorders. Hence, we postulated that screening for mouse mutants with both neurological defects and coat-color dilution will help identify additional factors associated with intracellular trafficking in neuronal cells. In this study, we characterized a mouse mutant with a unique N-ethyl-N-nitrosourea (ENU)–induced mutation, named nur17. nur17 mutant mice exhibit both coat-color dilution and ataxia due to Purkinje cell degeneration in the cerebellum. By positional cloning, we identified that the nur17 mouse carries a T-to-C missense mutation in archain 1 (Arcn1) gene which encodes the δ subunit of the coat protein I (COPI) complex required for intracellular trafficking. Consistent with this function, we found that intracellular trafficking is disrupted in nur17 melanocytes. Moreover, the nur17 mutation leads to common characteristics of neurodegenerative disorders such as abnormal protein accumulation, ER stress, and neurofibrillary tangles. Our study documents for the first time the physiological consequences of the impairment of the ARCN1 function in the whole animal and demonstrates a direct association between ARCN1 and neurodegeneration.

Length regulation of mechanosensitive stereocilia depends on very slow actin dynamics and filament-severing proteins.

Auditory sensory hair cells depend on stereocilia with precisely regulated lengths to detect sound. Since stereocilia are primarily composed of cross-linked, parallel actin filaments, regulated actin dynamics are essential for controlling stereocilia length. Here, we assessed stereocilia actin turnover by monitoring incorporation of inducibly expressed β-actin-GFP in adult mouse hair cells in vivo and by directly measuring β-actin-GFP turnover in explants. Stereocilia actin incorporation is remarkably slow and restricted to filament barbed ends in a small tip compartment, with minimal accumulation in the rest of the actin core. Shorter rows of stereocilia, which have mechanically-gated ion channels, show more variable actin turnover than the tallest stereocilia, which lack channels. Finally, the proteins ADF and AIP1, which both mediate actin filament severing, contribute to stereocilia length maintenance. Together, the data support a model whereby stereocilia actin cores are largely static, with dynamic regulation at the tips to maintain a critical length.

Serum Response Factor is Essential for the Proper Development of Skin Epithelium

Mammalian epidermis is a stratified epithelium that serves as a barrier protecting the organism from mechanical stress and dehydration. Previous studies have demonstrated the importance of the actin cytoskeleton in the establishment of a functional skin epithelium. Despite what is known about the actin cytoskeleton in epithelial sheet formation, the molecules important for controlling the actin cytoskeleton during epidermal development have not been determined. Serum response factor (SRF) is a transcription factor that is considered to be an important regulator of the actin cytoskeleton. To examine the role of SRF in the developing mouse epidermis, we have employed gene targeting to ablate Srf in keratinocytes. Conditional inactivation of Srf during the embryonic timepoint leads to a defect in the organization of the epidermis. Immunohistochemical analyses demonstrated a marked loss of the filamentous actin cytoskeleton and E-cadherin localization in epidermis, as well as an aberration in the localization of tight junction proteins. Moreover, impairment of the “inside-out” epidermal barrier was shown. Srf conditional knockout keratinocytes are unable to establish proper intercellular connections or form an epithelial sheet as shown by histological examination and induced keratinocyte differentiation experiments. Our results demonstrate that Srf is essential for the actin-mediated sealing of epithelial cell-cell contacts and the development of functional stratified skin epithelium in vivo.

Reduced synaptic vesicle density and aberrant synaptic localization caused by a splice site mutation in the Rs1h gene.

X-linked retinoschisis (XLRS) is a common form of inherited macular degeneration caused by mutations in the RS1 gene. Whereas the role of RS1 has been implicated in the synaptic structure as well as layer organization in the retina, the pathological effect of a defective RS1 gene on the synaptic interaction between photoreceptor cells and second-order neurons has not been thoroughly investigated. In this study, we perform a detailed characterization of the retinal synaptic phenotypes caused by a splice site mutation in the murine RS1 homolog (Rs1h(tmgc1)). Electron microscopic analysis showed that presynaptic terminals of photoreceptor cells contain a lower areal density of synaptic vesicles in the Rs1h(tmgc1) retina. Examination of the synaptic interactions in the outer plexiform layer also revealed ectopic localization of photoreceptor cell presynaptic markers and elongation of neurites from postsynaptic neurons (bipolar and horizontal cells), which are observed in other mouse models with defective photoreceptor cell molecules. Consistent with these synaptic abnormalities, ERG analysis of young Rs1h(tmgc1) mice revealed attenuation of the b-wave with preservation of the a-wave. These results demonstrate that RS1H has functional significance in the morphology and function of the synapse between photoreceptors and second-order neurons. A developmental study from postnatal day (P) 15 through P19 showed that synaptic interactions form normally, and structural abnormalities occur after completion of synaptic formation suggesting that RS1H is important for the maintenance of this synaptic interaction. Thus, Rs1h(tmgc1) mice may serve as a new genetic model for human XLRS and other synaptic disorders.

An allele of microtubule-associated protein 1A (Mtap1a) reduces photoreceptor degeneration in Tulp1 and Tub Mutant Mice.

PURPOSE To identify genes that modify photoreceptor cell loss in the retinas of homozygous Tulp1(tm1Pjn) and Tub(tub) mice, which exhibit juvenile retinitis pigmentosa. METHODS Modifier loci were identified by genetic quantitative trait locus analysis. F2 Tulp1(tm1Pjn/tm1Pjn) mutant mice from a B6-Tulp1(tm1Pjn/tm1Pjn) × AKR/J intercross were genotyped with a panel of single nucleotide polymorphism markers and phenotyped by histology for photoreceptor nuclei remaining at 9 weeks of age. Genotype and phenotype data were correlated and examined with Pseudomarker 2.02 using 128 imputations to map modifier loci. Thresholds for the 63%, 10%, 5%, and 1% significance levels were obtained from 100 permutations. A significant, protective candidate modifier was identified by bioinformatic analysis and confirmed by crossing transgenic mice bearing a protective allele of this gene with Tulp1- and Tub-deficient mice. RESULTS A significant, protective modifier locus on chromosome 2 and a suggestive locus on chromosome 13 that increases photoreceptor loss were identified in a B6-Tulp1(tm1Pjn/tm1Pjn) × AKR/J intercross. The chromosome 2 locus mapped near Mtap1a, which encodes a protein associated with microtubule-based intracellular transport and synapse function. The protective Mtap1a(129P2/OlaHsd) allele was shown to reduce photoreceptor loss in both Tulp1(tm1Pjn/tm1Pjn) and Tub(tub/tub) mice. CONCLUSIONS It was demonstrated that the gene Mtap1a, which modifies hearing loss in Tub(tub/tub) mice, also modifies retinal degeneration in Tub(tub/tub) and Tulp1(tm1Pjn/tm1Pjn) mice. These results suggest that functionally nonredundant members of the TULP family (TUB and TULP1) share a common functional interaction with MTAP1A.