Lecture VI. Making Connections

1. Lecture VI. Making Connections

2. Lecture VI. Making Connections Bio 3411 Monday September 14, 2009

3. T. Woolsey • 3802 North Building • 362-3601 • woolseyt@medicine.wustl.edu Lecture VI. Making Connections

4. Reading NEUROSCIENCE: 4th ed, Chapter 23, pp 577-609 Lecture VI. Making Connections

5. Selected References: Bentley, D., & Caudy, M. (1983). Pioneer axons lose directed growth after selective killing of guidepost cells. Nature, 304(5921), 62-65. Foty, R. A., & Steinberg, M. S. (2004). Cadherin-mediated cell-cell adhesion and tissue segregation in relation to malignancy. Int J Dev Biol, 48(5-6), 397-409. Hayashi, T., & Carthew, R. W. (2004). Surface mechanics mediate pattern formation in the developing retina. Nature, 431(7009), 647-652. Moscona, A., & Moscona, H. (1952). The dissociation and aggregation of cells from organ rudiments of the early chick embryo. J Anat, 86(3), 287-301. Myers, P. Z., & Bastiani, M. J. (1993). Cell-cell interactions during the migration of an identified commissural growth cone in the embryonic grasshopper. J Neurosci, 13(1), 115-126. Sperry, R. W. (1963). Chemoaffinity in the Orderly Growth of Nerve Fiber Patterns and Connections. Proc Natl Acad Sci U S A, 50, 703-710. Tessier-Lavigne, M., & Goodman, C. S. (1996). The molecular biology of axon guidance. Science, 274(5290), 1123-1133. Townes, P. L., & Holtfreter, J. (1955). Directed movements and selective adhesion of embryonic amphibian cells. J Exp Zool, 123, 53–120. Walter, J., Henke-Fahle, S., & Bonhoeffer, F. (1987). Avoidance of posterior tectal membranes by temporal retinal axons. Development, 101(4), 909-913. Wilson, H. (1907). A new method by which sponges may be artificially reared. Science, 23, 161-174. Lecture VI. Making Connections

6. What the last Lecture was about • Programmed cell death (apoptosis) is a physiological mechanism distinct from necrotic cell death. • Apoptosis occurs widely during normal development of the nervous system.  • Isolation of specific molecules involved in promoting growth and survival – “trophism,” e.g., Nerve Growth Factor (NGF).  • What is the “death mechanism” that NGF (and other neruotrophins) inhibit?   • Broader implications: controlled cell death in neuroembryology vs uncontrolled cell growth of cancer. • Gene homologies between organisms - humans and worms (nematodes)  • Molecular models for apoptosis  • How do trophic factors connect to this cell death pathway(s)? Lecture VI. Making Connections

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8. What this Lecture is about • General mechanisms for assembling neurons and groups of neurons • Diffusion vs Contact • Attraction vs Repulsion  • Examples of impacts of contact  • Examples of impacts of diffusion • Specification by growth factors • The chemoaffinity hypothesis Lecture VI. Making Connections

9. Outline of Neurodevelopment Fertilization Embryonic morphogenesis Induction of Neuroectoderm Segmentation 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?) Lecture VI. Making Connections

10. Selective Adhesion Determines Specificity of Tissue and Cellular Associations Lecture VI. Making Connections

11. Selective Aggregation of dissociated embryonic tissues (vertebrate and invertebrate) suggests ancient (surface) Adhesion Molecules Epidermis + Mesoderm 1. Sponges (Wilson, 1907) 2. Amphibians (Townes and Holtfretter, 1955) 3. Chick (Moscona, 1952) (Townes and Holtfretter, 1955) Lecture VI. Making Connections

12. Experimental recreation of morphogenesis by mixing cells expressing low and high levels of one surface adhesion gene (N-cadherin) +4 hrs +24 hrs Green = high N-cadherin Red = low N-cadherin (Foty and Steinberg, 2004) Lecture VI. Making Connections

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16. (Play GFP-Actin Growth Cone Movie) Dr. Andrew Matus Friedrich Miescher Institute, Switzerland Lecture VI. Making Connections

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22. 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. Lecture VI. Making Connections

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24. Observe WT Neuronal Specificity Screen for Mutants of Neuronal Specificity Clone Mutant Genes Identify Factors (Semphorins, Slit, Robo, Commissureless...) Molecules Mediating Axonal Guidance Lecture VI. Making Connections

25. Robo acts as a receptor for a midline repulsive cue Drosophila robo disrupts longitudinal tract formation Lecture VI. Making Connections

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29. Retinotectal Mapping Visualized by Dye Injection in Zebrafish N T D V T D V N (Friche,et al. 2001) Lecture VI. Making Connections

30. Zebrafish ROBO Mutant (astray) Disrupts Midline Retinotectal Axonal Projections WT WT ast ast WT WT WT ast Lecture VI. Making Connections

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32. What this Lecture was about • Grouping neurons and processes • Partner selection • Some genetic foundations/correlates • Systematic organization of connections • Roles of contact and diffusion • Deja vu Lecture VI. Making Connections

33. Sequential Restrictions (Refinements) are the Bases for Development pluripotent, stem cell genetic environmental differentiated Lecture VI. Making Connections

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35. FInis Lecture VI. Making Connections

36. Normal (horizontal gaze palsy) HGPPS (reduced hindbrain volume) (scoliosis) (Jen, et al., 2004) Human ROBO Mutation causes HGPPS (Horizontal Gaze Palsy with Progressive Scoliosis) Lecture VI. Making Connections

37. The Axon Guidance Receptor Gene ROBO1 Is a Candidate Gene for Developmental Dyslexia Katariina Hannula-Jouppi1, Nina Kaminen-Ahola1, Mikko Taipale1,2, Ranja Eklund1, Jaana Nopola Hemmi1,3, Helena Kaariainen4,5, Juha Kere1,6* 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 Lecture VI. Making Connections

38. Physical forces of surface contacts largely determine cell shape: Drosophila cone cell morphology modeled by soap bubbles! WT Drosophila retina Soap bubbles (4 cells) (5 cells) (1 cell) (2 cells) Drosophila Rough eye (Roi) mutants (3 cells) (6 cells) Hayashi & Carthew, (2004) Lecture VI. Making Connections

39. A (T) P (N) L(V) dorsal ventral M(D) nasal temporal Do Molecular Cues Determine the Retinotectal Spatial-topic Map? Optic tectum A (T) D T N M (D) L (V) V P (N) Retina Optic Tectum Lecture VI. Making Connections

40. Subjective “down” Subjective “up” Rotate Eye 180o Retina Optic Tectum (T) V Subjective “down” N T (V) (D) D (N) Retinotectal Map is Preserved Despite Experimental Rotation of the Eye:“Chemaffinity Hypothesis” (T) D T N (V) (D) V (Sperry, 1956) (N) Lecture VI. Making Connections

41. Longitudinal Tracts MP1 aCC Q1 pCC MP1 Q1 MP1 aCC Q1 pCC Identified Neurons Commissural Tracts Grasshopper embryo Q1 Early Embryonic Insect Neurons form a Repeated Segmental Scaffold: Favorable preparation for studying axonal guidance (Meyers and Bastiani, 1993) Lecture VI. Making Connections

42. Pioneer Neurons Create the Early Scaffold of the Adult Nervous System growth cone pioneer neuron guidepost cells Lecture VI. Making Connections selective fasciculation Pioneer neuron and guidepost cells may die after pathway is pioneered, by apoptosis

43. Molecules Mediating Axonal Guidance • Biochemical approach: Friedrich Bonhoeffer, retinotectal culture assay. Observe Neuronal Specificity Functional Assay Fractionate Native Factors Temporal Nasal Purify and Identify Factor (Ephrins...) Lecture VI. Making Connections Nasal Axons Temporal Axons

44. Guidepost Cells Growth Cone Pioneer Neurons Pioneer Neurons and Guidepost Cells guide the initial path of peripheral nervetracts in embryonic grasshopper limbs (Bentley and Caudy, 1983) Lecture VI. Making Connections CT1 Photoablated Control

45. filapodia F-actin Tubulin lamellipodia 2nd Messengers Extracellular Cues Ca+2 Cytoskeletal Rearrangment Intracellular Signaling Pathways GTP cAMP Growth Cones are DynamicSensory Organelles that Guide the Growth of Embryonic Axons • Sensing and Transducing: • Diffusible Cues • Contact-dependent Cues • Trophic Factors • Neurotransmitters (Forscher lab) Lecture VI. Making Connections

46. (Secreted) (sema, slit) (netrin) (fas) (eph) (Membrane Associated) Functional Classes of Axonal Guidance Molecules Molecules may function for both: 1. Selective adhesion 2. Intracellular signaling Lecture VI. Making Connections

47. diffusible repellant diffusible attractant Contact-dependent attractant Contact-dependent repellant selective fasciculation Axonal Guidance Cues Lecture VI. Making Connections (Timing is critical)