Choroid plexus derived Otx2 role during adult neurogenesis PLANQUES Anabelle, DI NARDO Ariel, PROCHIANTZ Alain Centre Interdisciplinaire de Recherche en Biologie, Collège de France [email protected] 5) Mouse adult neurogenesis study 1) Introduction / Keywords Experimental paradigm Premiminary data: Decrease Otx2 expression in the choroid plexus leads to decreased DCX staining Nature review / Ben Brahim Mohammed, Fès Faculty / InTechOpen 2) Otx2 is highly expressed by the mouse choroid plexus and is secreted in the CSF Otx2 is highly expressed by the choroid plexus In situ hydridization Otx2 is secreted in the CSF Immunolabelling of choroid plexus (coronal view) DCX stained area (% WT mean) 250 200 150 100 50 DCX 0 Vehicle Cre 6) Otx2 transcriptional targets Concept: (Non?) cell autonomous target Experimental paradigm RNAseq data from WT choroid plexus Otx2 / Dapi 3) Otx2 protein is non cell autonomous in SVZ Otx2 is not expressed in SVZ In situ hydridization Preliminary data: constitutive knock down model at P100 Otx2 protein is present in SVZ Immunoprecipitation Corpus callosum SVZ 247 genes are differentially expressed between WT and Otx2 188/247 genes analyzed by GSEA For p<0,001: 42 enriched in GO database Otx2 protein is present in migrating chains Wholemount +/GFP with p<0,001 240/247 genes analyzed by webgestalt: GO classification Otx2 is in the RMS astrocytes Coronal slice Striatum Otx2 / DCX Otx2 / DCX/ GFAP Otx2 / DCX/ GFAP 240/247 genes analyzed by webgestalt For p<0,001: 16 microRNA target enrichment found 240/247 genes analyzed by webgestalt For p<0,001 24 KEGG pathways are enriched Sugar and retinol synthesis metabolism, signaling, detoxification pathway 4) Models for studying Otx2 transfer from choroid plexus to SVZ A) Otx2 lox: knock down model Selected targets Choroid plexus specific recombination: Rosa mice, ß-gal staining Vehicle Cre Decreased Otx2 expression by choroid plexus in Otx2 lox model Vehicle Cre 7) Perspectives B) AAV5: gain of function model and Otx2 tracing Otx2 / Dapi Otx2 / Dapi Choroid plexus specific infection Immunolabeling Non injected AAV5 Cre-GFP Otx2 intensity in SVZ after Cre injection in Otx2 lox model Descriptive : - Confirm astrocyte localization of Otx2 - Perform AAV5 Otx2-HA infection to confirm Otx2 choroid plexus origin Functional : - Perform adult neurogenesis study - Confirm decrease of Otx2 in astrocytes after Otx2 choroid plexus KD Molecular : - Perform RNAseq on Otx2 lox model (choroid plexus and SVZ) - Perform ChIP on selected potential Otx2 target genes - Use secreted single chain antibody against Otx2 to differentiate between cell and non cell autonomous targets Single chain antibody model GFP / Dapi GFP / Dapi References: Spatazza J. et al., Choroid-Plexus-Derived Otx2 Homeoprotein Constrains Adult Cortical Plasticity. 2013 Projet soutenu par la région Ile-de-France dans le cadre du DIM Cerveau et Pensée
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