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Transcriptome (RNA-seq) analysis of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.

ABSTRACT: The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity. When human CRB1 is mutated, it results in autosomal recessive Leber congenital amaurosis and retinitis pigmentosa, with no established genotype-phenotype correlation. Using the oko meduzym289/m289 (crb2a-/-) zebrafish model, we performed integrative transcriptomic and methylomic analysis to identify dysregulated genes and pathways. We reveal delayed retinal cell type specification confirmed in patient-derived retinal organoids, with disruption to cell cycle modulation and epigenetic transcriptional control. Hence, using reduced representation bisulphite sequencing (RRBS) we explored differential DNA methylation, identifying hypermethylated pathways involving biological adhesion, Hippo and transforming growth factor beta (TGFbeta) signalling. Functional epigenetic modules (FEM) were highlighted through the integration of RNA-seq and RRBS, confirming cell cycle involvement and disturbance of TGFbeta, BMP, Hippo and SMAD protein signal transduction. Taken together our work provides insights for epigenetic regulation in early retinal development and considerations for future therapeutic development.

ORGANISM(S): Danio rerio

PROVIDER: GSE178709 | GEO | 2023/01/25

REPOSITORIES: GEO

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Reduced representation bisulfite sequencing (RRBS) of the retina of crumbs 2a (crb2a m289/m289) zebrafish model of Leber congenital amaurosis and retinitis pigmentosa.

Project description:The crumbs cell polarity complex plays a crucial role in apical-basal epithelial polarity. When human CRB1 is mutated, it results in autosomal recessive Leber congenital amaurosis and retinitis pigmentosa, with no established genotype-phenotype correlation. Using the oko meduzym289/m289 (crb2a-/-) zebrafish model, we performed integrative transcriptomic and methylomic analysis to identify dysregulated genes and pathways. We reveal delayed retinal cell type specification confirmed in patient-derived retinal organoids, with disruption to cell cycle modulation and epigenetic transcriptional control. Hence, using reduced representation bisulphite sequencing (RRBS) we explored differential DNA methylation, identifying hypermethylated pathways involving biological adhesion, Hippo and transforming growth factor beta (TGFbeta) signalling. Functional epigenetic modules (FEM) were highlighted through the integration of RNA-seq and RRBS, confirming cell cycle involvement and disturbance of TGFbeta, BMP, Hippo and SMAD protein signal transduction. Taken together our work provides insights for epigenetic regulation in early retinal development and considerations for future therapeutic development.

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Histological and single cell transcriptomic analysis of human retinal organoid models of Leber Congenital Amaurosis identifies endoplasmic reticulum stress as a common degenerative pathway

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids

Project description:Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids

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Gene therapy of dominant CRX-Leber congenital amaurosis using patient retinal organoids I

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Human CRB1 and CRB2 form homo- and heteromeric protein complexes in the retina

Project description:Crumbs homolog 1 (CRB1) is one of the key genes linked to retinitis pigmentosa and Leber congenital amaurosis, which are characterized by a high clinical heterogeneity. The Crumbs family member CRB2 has a similar protein structure to CRB1, and in zebrafish, Crb2 has been shown to interact through the extracellular domain. Here, we show that CRB1 and CRB2 co-localize in the human retina and human iPSC-derived retinal organoids. In retina-specific pull-downs, CRB1 was enriched in CRB2 samples, supporting a CRB1–CRB2 interaction. Furthermore, novel interactors of the crumbs complex were identified, representing a retina-derived protein interaction network. Using co-immunoprecipitation, we further demonstrate that human canonical CRB1 interacts with CRB1 and CRB2, but not with CRB3, which lacks an extracellular domain. Next, we explored how missense mutations in the extracellular domain affect CRB1–CRB2 interactions. We observed no or a mild loss of CRB1–CRB2 interaction, when interrogating various CRB1 or CRB2 missense mutants in vitro. Taken together, our results show a stable interaction of human canonical CRB2 and CRB1 in the retina.

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