Outline of Neurodevelopment Fertilization Embryonic morphogenesis Induction of Neuroectoderm Neurulation Differentiation: 1. Formation and placement of neuroblasts 2. Axonal outgrowth 3. Growth cones, selective migration 4. Selective fasciculation 5. Target selection 6. Synaptogenesis 7. Etc(cell shape, neurotransmitter, ionic channels, receptors) Adult neuronal plasticity (Activity-dependent?) Segmentation Physical forces of surface contacts largely determine cell shape: Drosophila cone cell morphology modeled by soap bubbles! (1 cell) (2 cells) (3 cells) (5 cells) (6 cells) Drosophila Rough eye (Roi) mutants (Hayashi and Carthew, 2004) Drosophila retina WT (4 cells) Soap bubbles Selective Adhesion Determines Specificity of Tissue and Cellular Associations Selective Aggregation of dissociated embryonic tissues (vertebrate and invertebrate) suggests ancient (surface) Adhesion Molecules (Townes and Holtfretter, 1955) 1. Sponges (Wilson, 1907) 2. Amphibians (Townes and Holtfretter, 1955) 3. Chick (Moscona, 1952) Epidermis + Mesoderm Experimental recreation of morphogenesis by mixing cells expressing low and high levels of one surface adhesion gene (N-cadherin) Green = high N-cadherin Red = low N-cadherin +4 hrs +24 hrs (Foty and Steinberg, 2004) V D N T D V N T (Friche,et al. 2001) Retinotectal Mapping Visualized by Dye Injection in Zebrafish Do Molecular Cues Determine the Retinotectal Spatial-topic Map? A (T) D V TN Retina P (N) M (D) L (V) Optic Tectum A (T) P (N) dorsalventral temporal nasal L (V) M (D) Optic tectum D V TN Retina Optic Tectum (T) (N) (D) (V) Retinotectal Map is Preserved Despite Experimental Rotation of the Eye: Chemaffinity Hypothesis (Sperry, 1956) Subjective up Rotate Eye 180 o N V D T (T) (N) (D) (V) Subjective down Early Embryonic Insect Neurons form a Repeated Segmental Scaffold: Favorable preparation for studying axonal guidance Grasshopper embryo Commissural Tracts Longitudinal Tracts Identified Neurons Q1 pCC aCC MP1 Q1 MP1 pCC aCC Q1 (Meyers and Bastiani, 1993) Pioneer Neurons Create the Early Scaffold of the Adult Nervous System pioneer neuron guidepost cells growth cone selective fasciculation Pioneer neuron and guidepost cells may die after pathway is pioneered, by apoptosis Pioneer Neurons and Guidepost Cells guide the initial path of peripheral nerve tracts in embryonic grasshopper limbs Pioneer Neurons Guidepost Cells (Bentley and Caudy, 1983) Growth Cone CT1 PhotoablatedControl Growth Cones are Dynamic Sensory Organelles that Guide the Growth of Embryonic Axons Sensing and Transducing: Diffusible Cues Contact-dependent Cues Trophic Factors Neurotransmitters (Play GFP-Actin Growth Cone Movie) Dr. Andrew Matus Friedrich Miescher Institute, Switzerland F-actin Tubulin lamellipodia filapodia Extracellular Cues Intracellular Signaling Pathways Cytoskeletal Rearrangment Ca +2 GTP cAMP 2nd Messengers (Forscher lab) Identification of Molecules Mediating Axonal Guidance using Model Systems 1. Biochemical approach: Friedrich Bonhoeffer, retinotectal culture assay. TemporalNasal Functional Assay Fractionate Native Factors Observe Neuronal Specificity Purify and Identify Factor (Ephrins...) Temporal Axons Nasal Axons 2. Molecular genetic approach: Corey Goodman, Drosophila screens for neurodevelopmental defects. Identification of Molecules Mediating Axonal Guidance using Model Systems Screen for Mutants of Neuronal Specificity Clone Mutant Genes Observe WT Neuronal Specificity Identify Factors (Semphorins, Slit, Robo, Commissureless...) Conserved Structural Classes of Axonal Guidance Molecules: Modular Construction and Multifunctionality 1. Laminin, fibronectin and extracellular matrix proteins. 2. Cadherins and catenins. (Ca +2 dependent) 3. Cell adhesion molecules (CAMs) (containing IgG domains). 4. Receptor tyrosine kinases and receptor phosphatases. Functional Classes of Axonal Guidance Molecules (Secreted) (Membrane Associated) (netrin) (sema, slit) (fas)(eph) Molecules may function for both: 1. Selective adhesion 2. Intracellular signaling Axonal Guidance Cues selective fasciculation diffusible attractant diffusible repellant Contact-dependent attractant Contact-dependent repellant (Timing is critical) Axonal Guidance 1. Pioneer neurons construct the earliest scaffold of the nervous system, following chemical cues. 2. Multiple chemical cues guide growth cones, including long-range diffusible cues (secreted molecules) and short-range contact mediated cues (membrane associated). 3. Chemical cues may be attractive or repulsive. 4. Chemical cues mediate both selective adhesion and intercellular signaling. 5. Axonal guidance molecules are ancient conserved molecules, including a large class with structural similarity to immunoglobulins. 6. Final axonal pathways likely specified by unique combinations of molecular cues expressed by growing neurons and targets (Sperrys Chemoaffinity Hypothesis). 7. Human mutations of axonal guidance genes may underlie many hereditary neurological conditions affecting complex cognitive functions. Zebrafish ROBO Mutant (astray) Disrupts Midline Retinotectal Axonal Projections (Fricke, et al. 2001) Drosophila robo disrupts longitudinal tract formation WT astWT astWT ast Robo acts as a receptor for a midline repulsive cue Human ROBO Mutation causes HGPPS (Horizontal Gaze Palsy with Progressive Scoliosis) HGPPS Normal (reduced hindbrain volume)(scoliosis) (horizontal gaze palsy) (Jen, et al., 2004) Drosophila robo disrupts longitudinal tract formation The Axon Guidance Receptor Gene ROBO1 Is a Candidate Gene for Developmental Dyslexia Katariina Hannula-Jouppi 1, Nina Kaminen-Ahola 1, Mikko Taipale 1,2, Ranja Eklund 1, Jaana Nopola Hemmi 1,3, Helena Kaariainen 4,5, Juha Kere 1,6 * 1 Department of Medical Genetics, University of Helsinki, Finland, 2 European Molecular Biology Laboratory, Gene Expression Programme, Heidelberg, Germany, 3 Department of Pediatrics, Jorvi Hospital, Espoo, Finland, 4 Department of Medical Genetics, The Family Federation of Finland, Helsinki, Finland, 5 Department of Medical Genetics, University of Turku, Turku, Finland, 6 Department of Biosciences at Novum and Clinical Research Centre, Karolinska Institutet, Stockholm, Sweden PLOS Genetics (2005) 1: 0467 Development Proceeds by Progressive Developmental Restrictions (pluripotent) (differentiated)