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From ES cells to Neurons: A Road Map to Neurogenesis in the Embryo Elsa Abranches, Domingos Henrique, Evguenia Bekman Unidade de Biologia do Desenvolvimento Instituto de Medicina Molecular Encontro Nacional de Ciência Lisboa, 29-07-2009 Embryonic stem cells & Neural Development in vitro generation of Neurons from ES cells Promising approach to: Gain a better knowledge of the cellular and molecular events that are involved in neural development Produce cells suitable for neural tissue repair and cellbased replacement therapies of the nervous system Embryonic stem cells & Neural Development ES cells Neural Progenitors Neurons Question: How do cells go from one stage to the other? From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol ES cells Time -1 (days) 0 1 2 Rosettes 3 4 5 6 7 Neurons 8 Rosettes neural progenitors High density Monolayer Replating Rosettes differentiating neurons (i) Have proper apico-basal polarity (Divide apically & Produce neurons at the basal side) (ii) Notch pathway is active (iii) The timing of production of neurons and glia is correct (iv) Cells show interkinetic nuclear movement in vitro model mimicks in vivo commitment to neural fate Rosettes are Neural tube-like in vitro structures Abranches et al. PlosOne (2009) Cluster of cells forming primitive epithelium and initiating neural commitment ZO1 Sox1:GFP ZO1 Sox1:GFP ß-Catenin Sox1:GFP From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol Rapid & ES cells Rosettes Neurons Reproducible process Large amounts of cells Homogeneous populations Define the transcriptional profile of different neural progenitors populations (Microarray analysis) Gain a better knowledge of the cellular and molecular events that are involved in neural development From ES cells to Neurons Microarray analysis Mouse Genome 430.2A Affymetrix 45101 ProbeSets Anova FDR <10-3 (p-value < 2.10-4) 6563 Differentially Expressed Genes Specific embryo-oriented criteria 1750 Genes 5 Clusters AIM: Identify different progenitor populations Group I – ES cells ES cells Frequency ES cell Gene Signature (188 genes) 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 1 Tim 3 ep oin t ES cells 3' 8 0 “Stemness” character confirms the ES cell identity of the starting population Group II – Primitive Ectoderm (PE) PE Frequency Primitive Ectoderm Gene Signature (66 genes) 0.60 0.50 0.40 0.30 0.20 0.10 0.00 0 1 Tim 3 ep oin t PE 3' 8 Known PE-like signature (FGF5+, Oct4+, Rex1-) Calcium related genes 1 Group III – transient Neural Progenitors (tNPs) tNPs Transient NPs Gene Signature (61 genes) Frequency 0.40 0.30 0.20 0.10 0.00 0 1 Tim 3 ep oin t tNPs 3' 8 3 Genes important for neural progenitors specification that need to be switched off to allow progenitors to advance into the next stage Group IV – neurogenic Neural Progenitors (nNPs) nNPs nNPs Gene Signature (763 genes) Frequency 0.40 0.30 0.20 0.10 0.00 0 1 Tim 3 ep oin t nNPs 3' 8 3 Genes important for the next stage of NP development, when competence to enter neurogenesis is acquired Group V – Rosettes 0.40 Frequency Rosettes Gene Signature (673 genes) Rosettes 0.30 0.20 0.10 0.00 0 1 Genes coupled to the final stages of NP development and commitment to neural differentiation Notch pathway Tim 3 ep oin t Rosettes 3' 8 8 From ES cells to Neurons Time (days) Microarray analysis From ES cells to Neurons Microarray analysis 1. Delineate transient cellular states that occur during neural development ( ES cells Primitive Ectoderm Neuroepithelial Progenitor populations) 2. Reveal signalling pathways associated with these transitions (e.g. Ca2+ signalling; Notch pathway) From ES cells to Neurons in vivo Wild-type Equilibrium between progenitors and differentiating neurons Activated Notch Excess of progenitors at the expense of neurons Notch inhibition Excess of neurons at the expense of progenitors Notch Pathway From ES cells to Neurons Notch Pathway A comprehensive analysis of the Notch pathway in mammalian neural development has never been done Problems • Pleiotropic effects of the pathway in the embryo; • Heterogeneity of embryonic cell stages and types that respond differently to Notch activity Explore the simplicity of the rosette culture system to address in detail how Notch operates to regulate neural development in vitro Tuj1 Sox1 Neural Progenitors Differentiating Neurons Notch Pathway Notch inhibition Wild-type (Rosettes) From ES cells to Neurons in vitro Tuj1 Sox1 Neural Progenitors Differentiating Neurons From ES cells to Neurons Notch Pathway Mouse Genome 430.2A Affymetrix 45101 ProbeSets Anova FDR <10-3 K-Group 0 Down regulate after 12 h LY treatment (p-value < 1.8410-5) Notch-oriented criteria K-Group 1 Up regulated after 6 h LY treatment 701 Genes K-Group 2 Up regulated b LY stronger effect after 12 h treatment 4 Clusters K-Group3 Down regulate after LY treatm AIM: Identify Notch Pathway components From ES cells to Neurons Notch Pathway Expression Level Notch synexpression group http://www.genepaint.org/Frameset.html FunGenES database: Different differentiation conditions Novel potential Notch components Dissect further the mechanisms underlying Notch activity during neural differentiation From ES cells to Neurons Conclusions in vitro model mimicks in vivo commitment to neural fate delineate transient cellular states that occur during neural development reveal signalling pathways associated with these transitions Comprehensive resourse for studies aimed at elucidating the genetic architecture underlying neural development Define more rational strategies to achieve controlled production of specific neuronal cell types Collaborations: • Herbert Schulz, Oliver Hummel (MDC Berlin, Germany) • Stem Cell Sciences (Edinburgh, UK) • Raivo Kolde, Jaak Vilo (EGeen, Tartu, Estonia) • Austin Smith lab (Cambridge, UK) • Laurent Pradier (Sanofi-Aventis, France)