Lecture VI. Making Connections Bio 3411 Monday September 20, 2010 1Lecture VI. Making Connections

Lecture VI. Making Connections

Bio 3411 Monday

September 20, 2010

September 20, 2010 1Lecture VI. Making Connections

†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.

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ReadingNEUROSCIENCE: 4th ed, Chapter 23, pp

577-609

2Lecture VI. Making ConnectionsSeptember 20, 2010

†(pdfs on course websites: [http://artsci.wustl.edu/~bio3411/] & [http://www.nslc.wustl.edu/courses/Bio3411/bio3411.html]

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)?

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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

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September 20, 2010 7Lecture VI. Making Connections

Fertilization

Embryonic morphogenesis

Induction of Neuroectoderm

Neurulation

Differentiation:1. Formation and placement of neuroblasts2. Axonal outgrowth3. Growth cones, selective migration4. Selective fasciculation5. Target selection6. Synaptogenesis7. Etc…(cell shape, neurotransmitter, ionic channels, receptors)

Adult neuronal plasticity(Activity-dependent?)

Seg

men

tati

on

Outline of Neurodevelopment

Selective Adhesion Determines Specificity of Tissue and Cellular Associations

September 20, 2010 8Lecture VI. Making Connections

Selective Aggregation of dissociated embryonic tissues (vertebrate and invertebrate) suggests ancient (surface)

Adhesion Molecules

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(Townes & Holtfretter, 1955)

1. Sponges (Wilson, 1907)

2. Amphibians (Townes and Holtfretter, 1955)

3. Chick (Moscona, 1952)

Epidermis + Mesoderm

September 20, 2010 10Lecture 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-cadherinRed = low N-cadherin

+4 hrs +24 hrs

(Foty and Steinberg, 2004)

Growth Cones are Dynamic Sensory Organelles that Guide the Growth of Embryonic Axons

September 20, 2010 11Lecture VI. Making Connections

(Forscher lab)

Sensing and Transducing:• Diffusible Cues• Contact-dependent Cues• Trophic Factors• Neurotransmitters

Extracellular Cues

Intracellular SignalingPathways

CytoskeletalRearrangment

Ca+2

GTP

cAMP

2ndMessengers

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September 20, 2010 Lecture VI. Making Connections 13

(Play GFP-Actin Growth Cone Movie)Dr. Andrew Matus

Friedrich Miescher Institute, Switzerland

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Effect/Proximity

Attraction Repulsion

DistantContact

Functional Classes of Axonal Guidance Molecules

(Secreted)

(Membrane Associated)

(netrin)

(sema, slit)

(fas)

(eph)

Molecules may function for both:1. Selective adhesion2. Intracellular signaling

September 20, 2010 18Lecture VI. Making Connections

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September 20, 2010 Lecture VI. Making Connections 20

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.

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September 20, 2010 Lecture VI. Making Connections 23

Pioneer Neurons and Guidepost Cells guide the initial path of peripheral nervetracts in embryonic grasshopper

limbs

(Bentley and Caudy, 1983)

September 20, 2010 24Lecture VI. Making Connections

PioneerNeurons

Guidepost Cells Growth Cone

CT1 Photoablated Control

Early Embryonic Insect Neurons form a Repeated Segmental Scaffold

September 20, 2010 25Lecture VI. Making Connections

Grasshopperembryo

Commissural Tracts

Longitudinal Tracts

Identified Neurons

Q1 pCCaCC

MP1

Q1

Q1

MP1

MP1

pCCaCC

Q1(Meyers and Bastiani, 1993)

Molecules Mediating Axonal Guidance

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September 20, 2010 Lecture VI. Making Connections 27

Drosophila robo disrupts longitudinal

tract formation

Robo acts as a receptor for a midline repulsive cue

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Human ROBO Mutation causes HGPPS(Horizontal Gaze Palsy with Progressive Scoliosis)

(reduced hindbrain volume) (scoliosis)

(horizontal gaze palsy)

Jen, et al., 2004

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Retinotectal Mapping Visualized by Dye Injection in Zebrafish

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(Friche,et al. 2001)

Zebrafish ROBO Mutant (astray)Disrupts Midline Retinotectal Axonal Projections

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September 20, 2010

Do Molecular Cues Determine the Retinotectal Spatial-topic Map?

34Lecture VI. Making Connections

A (T)D

V

T N

Retina

P (N)

M(D)

L(V)

Optic Tectum

Optic tectum

A

P

dorsal

ventral

temporal

nasal

D

T

N

V

M

L

anterior

medial

posterior

lateral

A (T) P (N)

M (D)L (V)

Molecules Mediating Axonal Guidance

1. Biochemical approach: Friedrich Bonhoeffer, retinotectal culture assay.

35

Temporal NasalFunctional Assay

Fractionate Native Factors

ObserveNeuronal Specificity

Purify and IdentifyFactor

(Ephrins...)Temporal AxonsNasal Axons

September 20, 2010 Lecture VI. Making Connections

September 20, 2010 Lecture VI. Making Connections 36

Pioneer Neurons Create the Early Scaffold of the Adult Nervous System

September 20, 2010 37Lecture VI. Making Connections

pioneer neuron

guidepost cells

growth cone

selectivefasciculation

Pioneer neuron and guidepost cells may die after pathway is pioneered,

by apoptosis

Axonal Guidance Cues

September 20, 2010 38Lecture VI. Making Connections

selectivefasciculation

diffusibleattractant

diffusiblerepellant

Contact-dependentattractant

Contact-dependentrepellant

(Timing is critical)

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 20, 2010 Lecture VI. Making Connections 39

Sequential Restrictions (Refinements) are the Bases for Development

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pluripotent, stem cell

differentiated

genetic

environmental

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Finis