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,

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

Embryonic stem cells & Neural Development Question: How do cells go from one stage to the other? ES cellsNeural ProgenitorsNeurons

(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 From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol (iv) Cells show interkinetic nuclear movement Abranches et al. PlosOne (2009) ES cellsRosettesNeurons in vitro model mimicks in vivo commitment to neural fate  Rosettes are Neural tube-like in vitro structures Time (days) Rosettes neural progenitors Rosettes differentiating neurons

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

Define the transcriptional profile of different neural progenitors populations (Microarray analysis) ES cellsRosettesNeurons Gain a better knowledge of the cellular and molecular events that are involved in neural development  Rapid & Reproducible process  Homogeneous populations  Large amounts of cells From ES cells to Neurons: In vitro Monolayer & Serum-free Protocol

Mouse Genome 430.2A Affymetrix ProbeSets Anova FDR <10 -3 (p-value < ) From ES cells to NeuronsMicroarray analysis 6563 Differentially Expressed Genes 1750 Genes Specific embryo-oriented criteria 5 Clusters AIM: Identify different progenitor populations

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

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

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

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

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

From ES cells to NeuronsMicroarray analysis Time (days)

1. Delineate transient cellular states that occur during neural development ( ES cells  Primitive Ectoderm  Neuroepithelial Progenitor populations) From ES cells to NeuronsMicroarray analysis 2. Reveal signalling pathways associated with these transitions (e.g. Ca2+ signalling; Notch pathway)

Wild-type Activated Notch Notch inhibition in vivo Equilibrium between progenitors and differentiating neurons Excess of progenitors at the expense of neurons Excess of neurons at the expense of progenitors 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

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

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

Novel potential Notch components From ES cells to Neurons Notch Pathway Notch synexpression group Expression Level FunGenES database: Different differentiation conditions Dissect further the mechanisms underlying Notch activity during neural differentiation

Comprehensive resourse for studies aimed at elucidating the genetic architecture underlying neural development  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 Define more rational strategies to achieve controlled production of specific neuronal cell types From ES cells to Neurons Conclusions

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)