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Outline of Neurodevelopment
Fertilization
Induction of Neuroectoderm
Neurulation
Segmentation
Embryonic morphogenesis
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?)
Physical forces of surface contacts
largely determine cell shape:
Drosophila cone cell morphology
modeled by soap bubbles!
WT
Drosophila retina
(4 cells)
(1 cell)
Soap bubbles
(5 cells)
(2 cells)
(3 cells)
Drosophila
Rough eye (Roi) mutants
(6 cells)
(Hayashi and Carthew, 2004)
Selective Adhesion Determines
Specificity of Tissue and
Cellular Associations
Selective Aggregation of dissociated
embryonic tissues (vertebrate and invertebrate)
suggests ancient (surface) Adhesion Molecules
Epidermis
+
Mesoderm
1. Sponges
(Wilson, 1907)
2. Amphibians
(Townes and
Holtfretter, 1955)
3. Chick
(Moscona, 1952)
(Townes and Holtfretter, 1955)
Experimental recreation of morphogenesis by
mixing cells expressing low and high levels
of one surface adhesion gene (N-cadherin)
+4 hrs
Green = high N-cadherin
Red = low N-cadherin
+24 hrs
(Foty and Steinberg, 2004)
Retinotectal Mapping Visualized
by Dye Injection in Zebrafish
N
T
D
V
T
D
V
N
(Friche,et al. 2001)
Do Molecular Cues Determine
the Retinotectal Spatial-topic Map?
L(V)
Optic tectum
M(D)
dorsal
temporal
ventral
A
P
(T)
(N)
nasal
A
(T)
D
T
N
M
L
(D)
(V)
V
P
Retina
(N)
Optic Tectum
Retinotectal Map is Preserved Despite
Experimental Rotation of the Eye:
“Chemaffinity Hypothesis”
(Sperry, 1956)
(T)
D
T
Subjective “down”
N
Subjective “up”
V
Rotate Eye 180o
(V)
(D)
(N)
Retina
Optic Tectum
(T)
V
Subjective “down”
N
T
D
(V)
(D)
(N)
Early Embryonic Insect Neurons
form a Repeated Segmental Scaffold:
Favorable preparation for studying axonal guidance
Longitudinal
Tracts
MP1
Q1
aCC
pCC
MP1
Q1
MP1
Q1
Grasshopper
embryo
Commissural
Tracts
aCC
pCC
Identified Neurons
Q1
(Meyers and Bastiani, 1993)
Pioneer Neurons Create the Early
Scaffold of the Adult Nervous System
growth cone
pioneer neuron
guidepost cells
selective
fasciculation
Pioneer neuron and guidepost cells
may die after pathway is pioneered,
by apoptosis
Pioneer Neurons and Guidepost Cells
guide the initial path of peripheral nerve
tracts in embryonic grasshopper limbs
Guidepost Cells
CT1 Photoablated
Growth Cone
Pioneer
Neurons
Control
(Bentley and Caudy, 1983)
Growth Cones are Dynamic Sensory Organelles
that Guide the Growth of Embryonic Axons
filapodia
F-actin
Tubulin
lamellipodia
Sensing and Transducing:
• Diffusible Cues
• Contact-dependent Cues
• Trophic Factors
• Neurotransmitters
Extracellular Cues
(Forscher lab)
2nd
Messengers
Ca+2
Intracellular Signaling
Pathways
GTP
cAMP
(Play GFP-Actin Growth Cone Movie)
Dr. Andrew Matus
Friedrich Miescher Institute, Switzerland
Cytoskeletal
Rearrangment
Identification of Molecules Mediating
Axonal Guidance using Model Systems
1. Biochemical approach:
Friedrich Bonhoeffer, retinotectal culture assay.
Observe
Neuronal Specificity
Functional Assay
Fractionate
Native Factors
Temporal
Nasal
Purify and Identify
Factor
(Ephrins...)
Temporal Axons
Nasal Axons
Identification of Molecules Mediating
Axonal Guidance using Model Systems
2. Molecular genetic approach:
Corey Goodman, Drosophila screens for
neurodevelopmental defects.
Observe WT
Neuronal Specificity
Screen for Mutants
of Neuronal Specificity
Clone Mutant Genes
Identify
Factors
(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)
(sema, slit)
(netrin)
(fas)
(eph)
(Membrane Associated)
Molecules may function for both:
1. Selective adhesion
2. Intracellular signaling
Axonal Guidance Cues
diffusible
repellant
diffusible
attractant
Contact-dependent
attractant
Contact-dependent
repellant
selective
fasciculation
(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
WT
WT
WT
ast
ast
WT
ast
WT
(Fricke, et al. 2001)
Robo acts as a receptor for
a midline repulsive cue
Drosophila robo
disrupts longitudinal
tract formation
Human ROBO Mutation causes HGPPS
(Horizontal Gaze Palsy with
Progressive Scoliosis)
HGPPS
Normal
(horizontal gaze palsy)
(reduced hindbrain volume)
(scoliosis)
(Jen, et al., 2004)
Drosophila robo
disrupts longitudinal
tract formation
The Axon Guidance Receptor Gene ROBO1
Is 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,
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
2 European
PLOS Genetics (2005) 1: 0467
Development Proceeds by Progressive
Developmental Restrictions
(pluripotent)
(differentiated)