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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)