Outline of Neurodevelopment

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

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)

DrosophilaRough 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 dissociatedembryonic 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-cadherinRed = low N-cadherin

+4 hrs +24 hrs

(Foty and Steinberg, 2004)

VD

N

T

DV

N

T

(Friche,et al. 2001)

Retinotectal Mapping Visualizedby Dye Injection in Zebrafish

Do Molecular Cues Determine the Retinotectal Spatial-topic Map?

A(T)

D

V

T N

RetinaP(N)

M(D)

L(V)

Optic Tectum

A(T)

P(N)

dorsal ventral

temporal nasal

L(V)

M(D)Optic tectum

D

V

T N

Retina Optic Tectum

(T)

(N)

(D) (V)

Retinotectal Map is Preserved DespiteExperimental Rotation of the Eye:

“Chemaffinity Hypothesis”(Sperry, 1956)

Subjective “up”

Rot

ate

Eye

180

o

N

V

D

T

(T)

(N)

(D) (V)

Subjective “down”

Subjective “down”

Early Embryonic Insect Neurons form a Repeated Segmental Scaffold:

Favorable preparation for studying axonal guidance

Grasshopperembryo

CommissuralTracts

LongitudinalTracts

Identified Neurons

Q1 pCCaCC

MP1

Q1

Q1

MP1

MP1

pCCaCC

Q1

(Meyers and Bastiani, 1993)

Pioneer Neurons Create the Early Scaffold of the Adult Nervous System

pioneer neuron

guidepost cells

growth cone

selectivefasciculation

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

by apoptosis

Pioneer Neurons and Guidepost Cellsguide the initial path of peripheral nervetracts in embryonic grasshopper limbs

PioneerNeurons

Guidepost Cells

(Bentley and Caudy, 1983)

Growth Cone

CT1 Photoablated Control

Growth Cones are Dynamic Sensory Organellesthat 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 SignalingPathways

CytoskeletalRearrangment

Ca+2

GTP

cAMP

2ndMessengers

(Forscher lab)

Identification of Molecules Mediating Axonal Guidance using Model Systems

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

Temporal Nasal

Functional Assay

Fractionate Native Factors

ObserveNeuronal Specificity

Purify and IdentifyFactor

(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 Mutantsof Neuronal Specificity

Clone Mutant Genes

Observe WTNeuronal Specificity

IdentifyFactors

(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 adhesion2. Intracellular signaling

Axonal Guidance Cues

selectivefasciculation

diffusibleattractant

diffusiblerepellant

Contact-dependentattractant

Contact-dependentrepellant

(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 (Sperry’s 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 WT

ast WT ast WT

WT ast

Robo acts as a receptor for a midline repulsive cue

Human ROBO Mutation causes HGPPS(Horizontal Gaze Palsy with

Progressive Scoliosis)

HG

PP

SN

orm

al

(reduced hindbrain volume) (scoliosis)

(horizontal gaze palsy)

(Jen, et al., 2004)

Drosophila robo disrupts longitudinaltract formation

The Axon Guidance Receptor Gene ROBO1Is 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

Development Proceeds by ProgressiveDevelopmental Restrictions

(pluripotent)

(differentiated)