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Effect of Notch2 in chondrocytes

ABSTRACT: To investigate the role of Notch2 in the inflammatory response in chondrocytes, we cultured primary chondrocytes derived from wild type and Notch2 mutant mice and treated TNFalpha or vehicle. We then performed gene expression profiling analysis using data obtained from RNA-seq of wild type and Notch2 mutant chondrocytes.

ORGANISM(S): Mus musculus

PROVIDER: GSE224255 | GEO | 2023/10/16

REPOSITORIES: GEO

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Effect of Notch2 in chondrocytes

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NOTCH2 disrupts the synovial fibroblast identity and the inflammatory response of epiphyseal chondrocytes

Project description:Notch signaling plays a fundamental role in the inflammatory response and has been linked to the pathogenesis of osteoarthritis in murine models of the disease and in humans, but the mechanisms are poorly understood. We examined the effects of NOTCH2 in chondrocytes from Notch2tm1.1Ecan mice harboring a NOTCH2 gain-of-function mutation and from R26-NICD2 mice, a conditional gain-of-NOTCH2 function model, expressing the NOTCH2 intracellular domain (NICD2) from the Rosa26 locus. Bulk RNA-Sequencing (Seq) revealed increased expression of pathways associated with the phagosome, the role of osteoclasts in rheumatoid arthritis signaling and pulmonary fibrosis signaling in Notch2tm1.1Ecan and NICD2-expressing cultures. Collagen degradation was enhanced in Notch2tm1.1Ecan chondrocytes and the osteoarthritis pathway in NICD2-expressing cells. Single cell (sc)RNA-Seq analyzed 7,845 control and 7,141 Notch2tm1.1Ecan cells and used to define cell clusters. The more prevalent ones were constituted by limb mesenchyme, chondrogenic cells and fibroblasts including articular synovial fibroblasts. Pseudotime trajectory finding revealed a good association among clusters in control cultures, but this was disrupted in the articular/synovial cluster from Notch2tm1.1Ecan mice. scRNA-Seq of R26-NICD2 mice analyzed 9,957 cells in control and 9,924 in NICD2-expressing cultures. Neither the cluster distribution nor the pseudotime trajectory analysis revealed substantial differences between NIDC2-expressing and control cells except for an altered progression in an undefined cluster in NICD2-expressing cells. In conclusion, NOTCH2 enhances the activity of pathways associated with inflammation in epiphyseal chondrocytes and disrupts the transcriptome profile of articular/synovial fibroblasts.

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Conditional ablation of the Notch2 receptor in the ocular lens

Project description:Notch signaling is essential for proper lens development, however the specific requirements of individual Notch receptors has not been previously investigated. Here we report the lens phenotypes of Notch2 conditionally mutant mice, which exhibited severe microphthalmia, reduced pupillary openings, disrupted fiber cell morphology, eventual loss of the anterior epithelium, fiber cell dysgenesis, and cataracts. Notch2 mutants also had a persistent lens stalk phenotype at E11.5, and aberrant DNA synthesis in the fiber cell compartment by E14.5. Gene expression analyses showed elevated levels of the cell cycle regulators Cdkn1a (p21Cip1), Ccnd2 (CyclinD2) and Trp63 (p63) that negatively regulates Wnt signaling. Although removal of Notch2 phenocopied the increased proportion of fiber cells of Rbpj and Jag1 conditional mutant lenses, Notch2 is not required for AEL proliferation, suggesting that a different receptor regulates this process. Instead, we found that the Notch2 normally blocks progenitor cell death. Overall, we conclude that Notch2-mediated signaling regulates lens morphogenesis, apoptosis, cell cycle withdrawal, and secondary fiber cell differentiation. We have compared gene expression of ocular lenses of mice that are lens specific conditional mutants of Notch2 gene to that of littermate controls that had no ablation of Notch2 gene in the lens. Two lenses of each of the three conditional mutants and controls were pooled together and total RNA was harvested from embryonic day 19.5 (E19.5) lenses. Gene expression changes caused by absence of Notch2 gene in the lens were analyzed.

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