RESUMO
Dysfunction or death of photoreceptors is the primary cause of vision loss in retinal and macular degenerative diseases. As photoreceptors have an intimate relationship with the retinal pigment epithelium (RPE) for exchange of macromolecules, removal of shed membrane discs and retinoid recycling, an improved understanding of the development of the photoreceptor-RPE complex will allow better design of gene- and cell-based therapies. To explore the epigenetic contribution to retinal development we generated conditional knockout alleles of DNA methyltransferase 1 (Dnmt1) in mice. Conditional Dnmt1 knockdown in early eye development mediated by Rx-Cre did not produce lamination or cell fate defects, except in cones; however, the photoreceptors completely lacked outer segments despite near normal expression of phototransduction and cilia genes. We also identified disruption of RPE morphology and polarization as early as E15.5. Defects in outer segment biogenesis were evident with Dnmt1 exon excision only in RPE, but not when excision was directed exclusively to photoreceptors. We detected a reduction in DNA methylation of LINE1 elements (a measure of global DNA methylation) in developing mutant RPE as compared with neural retina, and of Tuba3a, which exhibited dramatically increased expression in mutant retina. These results demonstrate a unique function of DNMT1-mediated DNA methylation in controlling RPE apicobasal polarity and neural retina differentiation. We also establish a model to study the epigenetic mechanisms and signaling pathways that guide the modulation of photoreceptor outer segment morphogenesis by RPE during retinal development and disease.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , DNA (Citosina-5-)-Metiltransferases/genética , Morfogênese/genética , Segmento Externo das Células Fotorreceptoras da Retina/fisiologia , Epitélio Pigmentado da Retina/fisiologia , Animais , Permeabilidade da Membrana Celular/genética , Polaridade Celular/genética , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/genética , Embrião de Mamíferos , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Morfogênese/fisiologia , Especificidade de Órgãos/genética , Segmento Externo das Células Fotorreceptoras da Retina/metabolismo , Epitélio Pigmentado da Retina/embriologia , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/metabolismo , TranscriptomaRESUMO
Cerebral small vessel disease (SVD) affects the small vessels in the brain and is a leading cause of stroke and dementia. Emerging evidence supports a role of the extracellular matrix (ECM), at the interface between blood and brain, in the progression of SVD pathology, but this remains poorly characterized. To address ECM role in SVD, we developed a co-culture model of mural and endothelial cells using human induced pluripotent stem cells from patients with COL4A1/A2 SVD-related mutations. This model revealed that these mutations induce apoptosis, migration defects, ECM remodeling, and transcriptome changes in mural cells. Importantly, these mural cell defects exert a detrimental effect on endothelial cell tight junctions through paracrine actions. COL4A1/A2 models also express high levels of matrix metalloproteinases (MMPs), and inhibiting MMP activity partially rescues the ECM abnormalities and mural cell phenotypic changes. These data provide a basis for targeting MMP as a therapeutic opportunity in SVD.
Assuntos
Células-Tronco Pluripotentes Induzidas , Acidente Vascular Cerebral , Humanos , Células Endoteliais , Encéfalo/patologia , Acidente Vascular Cerebral/patologia , Matriz Extracelular , Metaloproteinases da Matriz/genética , Colágeno Tipo IV/genéticaRESUMO
Human induced pluripotent stem cells (hiPSC) offer a tractable system to model the blood-brain barrier (BBB). Here we detail the assembly of a triple co-culture hiPSC-BBB model, using hiPSC-derived brain microvascular endothelial cells (BMEC), astrocytes, and mural cells (MC). Transendothelial electrical resistance (TEER) and sodium fluorescein (NaFl) permeability can be used to test the barrier properties. The model has applications in studying BBB-related pathology and for drug screening.