Dataset Information

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

ABSTRACT: Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamtsl4. Homozygous Adamtsl4(tvrm267) mice recapitulate the EL phenotype observed in humans, and our analysis strongly suggests that ADAMTSL4 is required for stable anchorage of zonule fibers to the lens capsule. Unexpectedly, homozygous Adamtsl4(tvrm267) mice exhibit focal retinal pigment epithelium (RPE) defects primarily in the inferior eye. RPE dedifferentiation was indicated by reduced pigmentation, altered cellular morphology and a reduction in RPE-specific transcripts. Finally, as with a subset of patients with ADAMTSL4 mutations, increased axial length, relative to age-matched controls, was observed and was associated with the severity of the RPE phenotype. In summary, the Adamtsl4(tvrm267) model provides a valuable tool to further elucidate the molecular basis of zonule formation, the pathophysiology of EL and ADAMTSL4 function in the maintenance of the RPE.

SUBMITTER: Collin GB

PROVIDER: S-EPMC4654052 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Collin Gayle B GB Hubmacher Dirk D Charette Jeremy R JR Hicks Wanda L WL Stone Lisa L Yu Minzhong M Naggert Jürgen K JK Krebs Mark P MP Peachey Neal S NS Apte Suneel S SS Nishina Patsy M PM

Human molecular genetics 20150924 24

Human gene mutations have revealed that a significant number of ADAMTS (a disintegrin-like and metalloproteinase (reprolysin type) with thrombospondin type 1 motifs) proteins are necessary for normal ocular development and eye function. Mutations in human ADAMTSL4, encoding an ADAMTS-like protein which has been implicated in fibrillin microfibril biogenesis, cause ectopia lentis (EL) and EL et pupillae. Here, we report the first ADAMTSL4 mouse model, tvrm267, bearing a nonsense mutation in Adamt ...[more]

PMID: 26405179

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Project description:PurposeRetinal detachments (RDs), a separation of the light-sensitive tissue of the retina from its supporting layers in the posterior eye, isolate retinal cells from their normal supply of nourishment and can lead to their deterioration and death. We identified a new, spontaneous murine model of exudative retinal detachment, nm3342 (new mutant 3342, also referred to as rpea1: retinal pigment epithelium atrophy 1), which we characterize herein.MethodsThe chromosomal position for the recessive nm3342 mutation was determined by DNA pooling, and the causative mutation was discovered by comparison of whole exome sequences of mutant and wild-type controls. The effects of the mutation were examined in longitudinal studies by clinical evaluation, electroretinography (ERG), light microscopy, and marker and Western blot analyses.ResultsNew mutant 3342, nm3342, also referred to as rpea1, causes an early-onset, complete RD on the ABJ/LeJ strain background, and central exudative RD and late-onset RPE atrophy on the C57BL/6J background. The ERG responses were normal at 2 months of age but deteriorate as mice age, concomitant with progressive pan-retinal photoreceptor loss. Genetic analysis localized rpea1 to mouse chromosome 2. By high-throughput sequencing of a whole exome capture library of an rpea1/rpea1 mutant and subsequent sequence analysis, a splice donor site mutation in the Prkcq (protein kinase C, θ) gene, was identified, leading to a skipping of exon 6, frame shift and premature termination. Homozygotes with a Prkcq-targeted null allele (Prkcqtm1Litt) have similar retinal phenotypes as homozygous rpea1 mice. We determined that the PKCθ protein is abundant in the lateral surfaces of RPE cells and colocalizes with both tight and adherens junction proteins. Phalloidin-stained RPE whole mounts showed abnormal RPE cell morphology with aberrant actin ring formation.ConclusionsThe homozygous Prkcqrpea1 and the null Prkcqtm1Litt mutants are reliable novel mouse models of RD and can also be used to study the effects of the disruption of PRKCQ (PKCθ) signaling in RPE cells.

Project description:The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNA-Seq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd) were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67 ≥ FC ≥ 1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, and tight junctions were downregulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNA-Seq results. Thus, the RPE quickly downregulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space.

Project description:The retinal pigment epithelium (RPE) maintains photoreceptor viability and function, completes the visual cycle, and forms the outer blood-retinal barrier (oBRB). Loss of RPE function gives rise to several monogenic retinal dystrophies and contributes to age-related macular degeneration. Retinal detachment (RD) causes separation of the neurosensory retina from the underlying RPE, disrupting the functional and metabolic relationships between these layers. Although the retinal response to RD is highly studied, little is known about how the RPE responds to loss of this interaction. RNA sequencing (RNAseq) was used to compare normal and detached RPE in the C57BL6/J mouse. The naïve mouse RPE transcriptome was compared to previously published RPE signature gene lists and from the union of these 14 genes (Bmp4, Crim1, Degs1, Gja1, Itgav, Mfap3l, Pdpn, Ptgds, Rbp1, Rnf13, Rpe65, Slc4a2, Sulf1 and Ttr) representing a core signature gene set applicable across rodent and human RPE was derived. Gene ontology enrichment analysis (GOEA) of the mouse RPE transcriptome identified expected RPE features and functions, such as pigmentation, phagocytosis, lysosomal and proteasomal degradation of proteins, and barrier function. Differentially expressed genes (DEG) at 1 and 7 days post retinal detachment (dprd). were defined as mRNA with a significant (padj≤0.05) fold change (FC) of 0.67≥FC≥1.5 in detached versus naïve RPE. The RPE transcriptome exhibited dramatic changes at 1 dprd, with 2297 DEG identified. The KEGG pathways and biological process GO groups related to innate immune responses were significantly enriched. Lipocalin 2 (Lcn2) and several chemokines were upregulated, while numerous genes related to RPE functions, such as pigment synthesis, visual cycle, phagocytosis, andtight junctions were down regulated at 1 dprd. The response was largely transient, with only 18 significant DEG identified at 7 dprd, including upregulation of complement gene C4b. Validation studies confirmed RNAseq results. Thus, the RPE quickly down-regulates cell-specific functions and mounts an innate immune defense response following RD. Our data demonstrate that the RPE contributes to the inflammatory response to RD and may play a role in attraction of immune cells to the subretinal space and the opsonization of orphaned outer segments following RD.

Project description:MicroRNA expression in the mouse eye.MicroRNAs (miRNAs) are key regulators of biological processes. To define miRNA function in the eye, it is essential to determine a high-resolution profile of their spatial and temporal distribution. In this report, we present the first comprehensive survey of miRNA expression in ocular tissues, using both microarray and RNA in situ hybridization (ISH) procedures. We initially determined the expression profiles of miRNAs in the retina, lens, cornea and retinal pigment epithelium of the adult mouse eye by microarray. Each tissue exhibited notably distinct miRNA enrichment patterns and cluster analysis identified groups of miRNAs that showed predominant expression in specific ocular tissues or combinations of them. Next, we performed RNA ISH for over 220 miRNAs, including those showing the highest expression levels by microarray, and generated a high-resolution expression atlas of miRNAs in the developing and adult wild-type mouse eye, which is accessible in the form of a publicly available web database. We found that 122 miRNAs displayed restricted expression domains in the eye at different developmental stages, with the majority of them expressed in one or more cell layers of the neural retina . This analysis revealed miRNAs with differential expression in ocular tissues and provided a detailed atlas of their tissue-specific distribution during development of the murine eye. The combination of the two approaches offers a valuable resource to decipher the contributions of specific miRNAs and miRNA clusters to the development of distinct ocular structures. microRNA profiling of ocular tissues from mouse. In particular we analysed the cornea, lens, Retina Pigment Epithelium (RPE) and retina and compared them against RNA extracted from the entire eye. The purpose of this experiment was to understand which microRNAs are present nd/or show differential expression in the various structures of the eye (cornea, lens, RPE, retina). The samples numbered 1 & 2 (i.e. CORNEA1, CORNEA2 etc ) are biological replicates, prepared from tissues dissecyed from different groups of wild-type animals. RNA extracted from the entire eye (EYE) served as the unique reference sample. For each tissue to be analysed we performed the following hybridizations: - 2 slides for lens (LENS1, LENS2) vs entire eye (EYE) - 2 slides for RPE (RPE1, RPE2) vs entire eye (EYE) - 2 slides for retina (RETINA1, RETINA2) vs entire eye (EYE) - 2 slides for cornea (CORNEA1, CORNEA2) vs entire eye (EYE) - 1 slide for entire eye (EYE) vs entire eye (EYE)

Dataset Information

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

Publications

Disruption of murine Adamtsl4 results in zonular fiber detachment from the lens and in retinal pigment epithelium dedifferentiation.

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