From ES cells to Neurons: A Road Map to Neurogenesis in the Embryo

1. 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

2. 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 cell-based replacement therapies of the nervous system

3. Embryonic stem cells & Neural Development ES cells Neural Progenitors Neurons Question: How do cells go from one stage to the other?

4. From ES cells to Neurons:In vitro Monolayer & Serum-free Protocol Rosettes neural progenitors Rosettes differentiating neurons ES cells Rosettes Neurons Time (days) (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)

5. Cluster of cells forming primitive epithelium and initiating neural commitment ZO1 Sox1:GFP ß-Catenin Sox1:GFP ZO1 Sox1:GFP

7. From ES cells to Neurons:In vitro Monolayer & Serum-free Protocol  Rapid & Reproducible process ES cells Rosettes Neurons 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

8. From ES cells to NeuronsMicroarray 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

9. Group I – ES cells ES cell Gene Signature (188 genes) 0 • “Stemness” character confirms the ES cell identity of the starting population

10. Group II – Primitive Ectoderm (PE) Primitive Ectoderm Gene Signature (66 genes) 1 • Known PE-like signature (FGF5+, Oct4+, Rex1-) • Calcium related genes

11. Group III – transient Neural Progenitors (tNPs) Transient NPs Gene Signature (61 genes) 3 • Genes important for neural progenitors specification that need to be switched off to allow progenitors to advance into the next stage

12. Group IV – neurogenic Neural Progenitors (nNPs) nNPs Gene Signature (763 genes) 3 • Genes important for the next stage of NP development, when competence to enter neurogenesis is acquired

13. Group V – Rosettes Rosettes Gene Signature (673 genes) 8 • Genes coupled to the final stages of NP development and commitment to neural differentiation • Notch pathway

14. From ES cells to NeuronsMicroarray analysis Time (days)

15. From ES cells to NeuronsMicroarray analysis • 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)

16. From ES cells to NeuronsNotch Pathway 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

17. From ES cells to NeuronsNotch 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

18. From ES cells to NeuronsNotch Pathway in vitro Tuj1 Sox1 Neural Progenitors in vitro Differentiating Neurons Tuj1 Sox1 Wild-type (Rosettes) Notch inhibition  Neural Progenitors Differentiating Neurons

19. From ES cells to NeuronsNotch Pathway Mouse Genome 430.2A Affymetrix 45101 ProbeSets Anova FDR <10-3(p-value < 1.8410-5) Notch-oriented criteria 701 Genes 4 Clusters AIM: Identify Notch Pathway components

20. From ES cells to NeuronsNotch Pathway Expression Level http://www.genepaint.org/Frameset.html FunGenES database: Different differentiation conditions Notch synexpression group Novel potential Notch components Dissect further the mechanisms underlying Notch activity during neural differentiation

21. From ES cells to NeuronsConclusions 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 resoursefor studies aimed at elucidating the genetic architecture underlying neural development Define more rational strategies to achieve controlled production of specific neuronal cell types

22. Collaborations: • Herbert Schulz, Oliver Hummel(MDC Berlin, Germany) • Raivo Kolde, Jaak Vilo (EGeen, Tartu, Estonia) • Laurent Pradier (Sanofi-Aventis, France) • Stem Cell Sciences (Edinburgh, UK) • Austin Smith lab (Cambridge, UK)