1. September 14, 2009Lecture VI. Making Connections1

2. Bio 3411 Monday September 14, 2009

3. T. Woolsey 3802 North Building 362-3601 woolseyt@medicine.wustl.edu 3Lecture VI. Making Connections September 14, 2009

4. Reading NEUROSCIENCE: 4 th ed, Chapter 23, pp 577- 609 4Lecture VI. Making Connections September 14, 2009

5. Lecture VI. Making Connections5 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.

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)? September 14, 2009Lecture VI. Making Connections6

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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 September 14, 2009Lecture VI. Making Connections8

9. September 14, 20099Lecture VI. Making Connections 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 Outline of Neurodevelopment

10. Selective Adhesion Determines Specificity of Tissue and Cellular Associations September 14, 200910Lecture VI. Making Connections

11. Selective Aggregation of dissociated embryonic tissues (vertebrate and invertebrate) suggests ancient (surface) Adhesion Molecules September 14, 200911Lecture VI. Making Connections (Townes and Holtfretter, 1955) 1. Sponges (Wilson, 1907) 2. Amphibians (Townes and Holtfretter, 1955) 3. Chick (Moscona, 1952) Epidermis + Mesoderm

12. September 14, 200912Lecture VI. Making Connections 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)

13. September 14, 2009Lecture VI. Making Connections13 Effect/Pro ximity AttractionRepulsion Distant Contact

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16. September 14, 2009Lecture VI. Making Connections16 (Play GFP-Actin Growth Cone Movie) Dr. Andrew Matus Friedrich Miescher Institute, Switzerland

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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. September 14, 200922Lecture VI. Making Connections

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24. Molecules Mediating Axonal Guidance September 14, 200924Lecture VI. Making Connections Screen for Mutants of Neuronal Specificity Clone Mutant Genes Observe WT Neuronal Specificity Identify Factors (Semphorins, Slit, Robo, Commissureless...)

25. September 14, 2009Lecture VI. Making Connections25 Drosophila robo disrupts longitudinal tract formation Robo acts as a receptor for a midline repulsive cue

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29. Retinotectal Mapping Visualized by Dye Injection in Zebrafish September 14, 200929Lecture VI. Making Connections V D N T D V N T (Friche,et al. 2001)

30. Zebrafish ROBO Mutant (astray) Disrupts Midline Retinotectal Axonal Projections September 14, 200930Lecture VI. Making Connections WT astWT astWT ast

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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 September 14, 2009Lecture VI. Making Connections32

33. Sequential Restrictions (Refinements) are the Bases for Development September 14, 200933Lecture VI. Making Connections pluripotent, stem cell differentiated genetic environmental

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35. September 14, 2009Lecture VI. Making Connections35 FInis

36. September 14, 2009Lecture VI. Making Connections36 Human ROBO Mutation causes HGPPS (Horizontal Gaze Palsy with Progressive Scoliosis) HGPPS Normal (reduced hindbrain volume)(scoliosis) (horizontal gaze palsy) (Jen, et al., 2004)

37. 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 September 14, 200937Lecture VI. Making Connections

38. September 14, 200938Lecture VI. Making Connections 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 Drosophila retina WT (4 cells) Soap bubbles Hayashi & Carthew, (2004)

39. September 14, 2009 Do Molecular Cues Determine the Retinotectal Spatial-topic Map? 39Lecture VI. Making Connections 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

40. Retinotectal Map is Preserved Despite Experimental Rotation of the Eye:“Chemaffinity Hypothesis” September 14, 200940Lecture VI. Making Connections (Sperry, 1956) D V TN Retina Optic Tectum (T) (N) (D) (V) Subjective “up” Rotate Eye 180 o N V D T (T) (N) (D) (V) Subjective “down”

41. Early Embryonic Insect Neurons form a Repeated Segmental Scaffold: Favorable preparation for studying axonal guidance September 14, 200941Lecture VI. Making Connections Grasshopper embryo Commissural Tracts Longitudi nal Tracts Identified Neurons Q1 pCC aCC MP1 Q1 MP1 pCC aCC Q1 (Meyers and Bastiani, 1993)

42. Pioneer Neurons Create the Early Scaffold of the Adult Nervous System September 14, 200942Lecture VI. Making Connections pioneer neuron guidepost cells growth cone selective fasciculation Pioneer neuron and guidepost cells may die after pathway is pioneered, by apoptosis

43. Molecules Mediating Axonal Guidance 1.Biochemical approach: Friedrich Bonhoeffer, retinotectal culture assay. 43 TemporalNasal Functional Assay Fractionate Native Factors Observe Neuronal Specificity Purify and Identify Factor (Ephrins...) Temporal Axons Nasal Axons September 14, 2009Lecture VI. Making Connections

44. Pioneer Neurons and Guidepost Cells guide the initial path of peripheral nervetracts in embryonic grasshopper limbs (Bentley and Caudy, 1983) September 14, 200944Lecture VI. Making Connections Pioneer Neurons Guidepost Cells Growth Cone CT1 PhotoablatedControl

45. Growth Cones are Dynamic Sensory Organelles that Guide the Growth of Embryonic Axons September 14, 200945Lecture VI. Making Connections (Forscher lab) Sensing and Transducing: Diffusible Cues Contact-dependent Cues Trophic Factors Neurotransmitters F-actin Tubulin lamellipodia filapodia Extracellular Cues Intracellular Signaling Pathways Cytoskeletal Rearrangment Ca +2 GTP cAMP 2nd Messengers

46. 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 September 14, 200946Lecture VI. Making Connections

47. Axonal Guidance Cues September 14, 200947Lecture VI. Making Connections selective fasciculation diffusible attractant diffusible repellant Contact-dependent attractant Contact-dependent repellant (Timing is critical)