Lecture VI. Making Connections Bio 3411 Monday September 17, 2012!! 1! Reading NEUROSCIENCE: 5 th ed, pp!507?536! 4 th ed, pp 577-609 Bentley, D., & Caudy, M. (1983). Nature, 304(5921), 62-65. Dickson, B. J. (2002). Science, 298, 1959-1964. Hannula-Jouppi, et al. (2005). PLoS Genet 14, e50. Foty, R. A., & Steinberg, M. S. (2004). Int J Dev Biol, 48(5-6), 397-409. Hayashi, T., & Carthew, R. W. (2004). Nature, 431(7009), 647-652. Jen, J. C., et al. (2004). Science, 304, 1509-1513. Moscona, A., & Moscona, H. (1952). J Anat, 86(3), 287-301. Myers, P. Z., & Bastiani, M. J. (1993). J Neurosci, 13(1), 115-126. Sperry, R. W. (1963). Proc Natl Acad Sci U S A, 50, 703-710. Tessier-Lavigne, M., & Goodman, C. S. (1996). Science, 274(5290), 1123-1133. Townes, P. L., & Holtfreter, J. (1955). J Exp Zool, 128, 53 120. Walter, J., Henke-Fahle, S., & Bonhoeffer, F. (1987). Development, 101(4), 909-913. Wilson, H. (1907). Science, 25, 912-915. (pdfs on course website: [http://www.nslc.wustl.edu/courses/bio3411/bio3411.html] 2 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)? 3! 4! 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 5! 6! 1
Bio 3411 Outline of Neurodevelopment Selective Adhesion Determines Specificity of Tissue and Cellular Associations Fertilization Neurulation Segmentation Embryonic morphogenesis Induction of Neuroectoderm 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?) 7! Selective Aggregation of dissociated embryonic tissues (vertebrate and invertebrate) suggests ancient (surface) Adhesion Molecules 8! Experimental recreation of morphogenesis by mixing cells expressing low and high levels of one surface adhesion gene (N-cadherin) Epidermis + Mesoderm +4 hrs +24 hrs 1. Sponges (Wilson, 1907) 2. Amphibians (Townes and Holtfretter, 1955) 3. Chick (Moscona, 1952) Green = high N-cadherin Red = low N-cadherin (Foty and Steinberg, 2004) 9! Growth Cones are Dynamic Sensory Organelles that Guide the Growth of Embryonic Axons! Sensing and Transducing: Diffusible Cues Contact-dependent Cues Trophic Factors Neurotransmitters Extracellular Cues Intracellular Signaling Pathways 10! 12! Purves 5th ed, p. 510 2nd Messengers Ca+2 GTP Cytoskeletal Rearrangment camp (Forscher lab) 11! 2
Bio 3411 (Play GFP-Actin Growth Cone Movie) Dr. Andrew Matus Friedrich Miescher Institute, Switzerland Purves 5th ed, p. 510 13! Purves 5th ed, p. 511 14! 16! Purves 5th ed, p. 511 15! Functional Classes of Axonal Guidance Molecules! (Secreted)! Effect/ A0rac2on. Repulsion. Proximity. Distant. Contact. Molecules may function for both: 1. Selective adhesion 2. Intracellular signaling! (netrin)! (fas)! (sema, slit)! (eph)! (Membrane Associated)! 17! 18! 3
Purves 5 th ed, p. 517 Purves 5 th ed, p. 514 19! 20! Conserved Structural Classes of Axonal Guidance Molecules: Modular Construction and Multifunctionality! Purves 5 th ed, p. 519 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.! 21! 22! Pioneer Neurons and Guidepost Cells guide the initial path of peripheral nervetracts in embryonic grasshopper limbs! Guidepost Cells Pioneer Neurons Growth Cone CT1 Photoablated Control (Bentley and Caudy, 1983)! 23! 24! 4
Early Embryonic Insect Neurons form a Repeated Segmental Scaffold Molecules Mediating Axonal Guidance! Longitudinal Tracts MP1 Grasshopper embryo acc pcc MP1 MP1 acc pcc Commissural Tracts Identified Neurons (Meyers and Bastiani, 1993) 25! 26! Robo acts as a receptor for a midline repulsive cue Human ROBO Mutation causes HGPPS (Horizontal Gaze Palsy with Progressive Scoliosis) Jen, et al., 2004 Drosophila robo disrupts longitudinal tract formation (horizontal gaze palsy) 27! (reduced hindbrain volume) (scoliosis) 28! Purves 5 th ed, p. 522 Purves 5 th ed, pp. 533-4 29! 30! 5
Purves 5 th p. 531 Retinotectal Mapping Visualized by Dye Injection in Zebrafish! 31! (Friche,et al. 2001) 32! Zebrafish ROBO Mutant (astray) Disrupts Midline Retinotectal Axonal Projections Do Molecular Cues Determine the Retinotectal Spatial-topic Map?!! A anterior D dorsal L (V) M (D) L lateral M medial N nasal P posterior T temporal Optic tectum V ventral D A (T) T V Retina N M (D) P (N) L (V) Optic Tectum A (T) P (N) 33! 34! Molecules Mediating Axonal Guidance! Purves 5 th p. 520 1. Biochemical approach: Friedrich Bonhoeffer, retinotectal culture assay. Observe Neuronal Specificity Functional Assay Temporal Nasal Fractionate Native Factors Purify and Identify Factor (Ephrins...) Temporal Axons Nasal Axons 35! 36! 6
Pioneer Neurons Create the Early Scaffold of the Adult Nervous System growth cone Axonal Guidance Cues diffusible repellant pioneer neuron guidepost cells Pioneer neuron and guidepost cells may die after pathway is pioneered, by apoptosis diffusible attractant Contact-dependent attractant selective fasciculation 37! Contact-dependent repellant selective fasciculation (Timing is critical) 38! What this Lecture was about Sequential Restrictions (Refinements) are the Bases for Development Grouping neurons and processes Partner selection pluripotent, stem cell Some genetic foundations/correlates Systematic organization of connections genetic Roles of contact and diffusion Deja vu environmental differentiated 39! 40! Finis 41! 42! 7