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Chapter 21.
Development of nervous system
The Nobel Prize in Physiology or
Medicine 1986
“For their discoveries of growth factors"
Stanley Cohen
Rita Levi-Montalcini
1/2 of the prize
1/2 of the prize
USA
Italy and USA
Vanderbilt University
School of Medicine
Nashville, TN, USA
Institute of Cell Biology of
the C.N.R
Rome, Italy
b. 1922
b. 1909
(in Turin, Italy)
The Nobel Prize in Physiology or
Medicine 1906
"in recognition of their work on the
structure of the nervous system"
Camillo Golgi
Santiago Ramón y
Cajal
Italy
Spain
Pavia University
Pavia, Italy
b. 1843
d. 1926
Madrid University
Madrid, Spain
b. 1852
d. 1934
Nerve Nets In Jellyfish
UROCHODATA
Fish (cod)
Amphibian (frog)
Reptile (alligator)
Mammal (shrew)
Mammal (horse)
Mammals brain
Embryonic and Fetal Development of the
Human Brain
Actual Size
Actual Size
Photographs of Human
Fetal Brain Development
Lateral view of the human
brain shown at one-third
size at several stages of
fetal development. Note
the gradual emergence of
gyri and sulci.
Increasing complexity…
Neurodevelopment begins
Neural tube formation,
the first step
Neurulation
Formation of the neural tube
Primary – invagination of the ectoderm
Secondary – hollowing out of a solid cord of cells
Interactions between ectoderm and
mesoderm (chordamesoderm)
Future neural
crest cells
Future neural
plate/tube
Future
epidermis
Neurulation stages
1. Neural plate formation
2. Neural floor plate formation
3. Floor plate and roof of place shaping
4. Formation of the neural groove
5. Tube closure
Neural tube development
(a) At 18 days after
conception the embryo
begins to implant in the
uterine wall. It consists of
3 layers of cells:
endoderm, mesoderm,
and ectoderm. Thickening
of the ectoderm leads to
the development of the
neural plate (inserts). (b)
The neural groove begins
to develop at 20 days.
Neural tube development
(c) At 22 days the
neural groove closes
along the length of the
embryo making a tube.
(d) A few days later 4
major divisions of the
brain are observable –
the telencephalon,
diencephalon,
mesencephalon, and
rhombencephalon.
Neural tube development
Neural Plate Folding
Movement of epidermis towards
midpoint forces edges of neural
plate up and together
Dorsal
Neural plate anchored
to epidermis
Dorsolateral
hinge point
Cells here become
wedge shaped (induced
by notochord)
Epidermis
Neural plate
Neural plate anchored
to notochord
Notochord
Ventral
Medial hinge point
Neural Plate Folding
Neural plate cells at
the medial hinge point
Microtubules
elongate – cells
become columnar
Actin filament bundles
contract – narrowing
the cell apex – like a
drawstring bag
Homophilic aggregation
Red  epidermal cells
Green  mesodermal cells
Blue  neural place cells
Neural Plate Folding
Neural folds meet and adhere
Cells at this junction form neural crest
Notochord
Epidermis
Closure not simultaneous
Neural Crest
Cells
Regional differences
Neural Tube
Cadherins
E
E and P
N
N and P
E and N
Closed tube detaches – change
in adhesion molecule
expression
Neural tube formation
Gli2 KO mice lack floor plate
Shh  gli1,2,3
Shh
Cbl-lacz
Eight phase of Neuronal
development at cellular level
Eight Phases in Embryonic and Fetal
Development at a Cellular Level
1. Mitosis
2. Migration
3. Aggregation and
4. Differentiation
8. Myelination
5. Synaptogenesis 6. Death
7. Rearrangement
1. Mitosis/Proliferation
•Occurs in ventricular zone
•Rate can be 250,000/min
•After mitosis “daughter”
cells become fixed post
mitotic
1. Mitosis/Proliferation:
Neurons and Glia
At early stages, a stem cell
generates neuroblasts. Later, it
undergoes a specific asymmetric
division (the “switch point”) at
which it changes from making
neurons to making glia
2. Migration
Note that
differentiation is
going on as neurons
migrate.
2. Migration
Radial Glia
Radial glial cells
act as guide
wires for the
migration of
neurons
2. Migration
Growth cones crawl forward as they
elaborate the axons training behind them.
Their extension is controlled by cues in
their outside environment that ultimately
direct them toward their appropriate targets.
Growth Cones
The fine threadlike
extensions shown in
red and green are
filopodia, which find
adhesive surfaces and
pull the growth cone
and therefore the
growing axon to the
right.
2. Migration
Growth Cones
Scanning electron micrograph of a growth cone
in culture. On a flat surface growth cones are
very thin. They have numerous filopodia
Ramon y Cajal drew these
growth cones showing their
variable morphology
2. Migration: How Do Neurons “Know”
Where to Go?
There are extrinsic and intrinsic
determinants of neurons’ fate.
A. Extrinsic signals
B. Different sources of extrinsic
signals
C. Generic signal transduction
pathway
D. Intrinsic determinants
3. Differentiation
•Neurons become fixed
post mitotic and
specialized
•They develop processes
(axons and dendrites)
•They develop NTmaking ability
•They develop electrical
conduction
3. Differentiation
Development of the cerebral cortex
The ventricular zone (VZ) contains progenitors of neurons and glia. 1st neurons establish
the preplate (PP); their axons an ingrowing axons from the thalamus establish the
intermediate zone (IZ). Later generated neurons establish layers II-VI. After migration
and differentiation there are 6 cortical layers.
4. Aggregation
Like neurons move
together and form
layers
5. Synaptogenesis
Axons (with
growth cones on
end) form a
synapse with other
neurons or tissue
(e.g. muscle)
5. Synaptogenesis: Attraction to Target
Cells
Target cells release a chemical that
creates a gradient (dots) around them.
Growth cones orient to and follow the
gradient to the cells. The extensions
visible in c are growing out of a sensory
ganglion (left) toward their normal target
tissue. The chemorepellent protein Slit
(red) in an embryo of the fruit fly repels
most axons.
Neuron Death Leads to Synapse
Rearrangement
Release and uptake
of neurotrophic
factors
Neurons receiving
Axonal processes
insufficient neurotropic complete for limited
factor die
neurotrophic factor
6. Neuron Death
•Between 40 and 75
percent of all neurons born
in embryonic and fetal
development do not
survive.
•They fail to make optimal
synapses.
7. Synapse Rearrangement
•Active synapses
likely take up
neurotrophic factor
that maintains the
synapse
•Inactive synapses
get too little trophic
factor to remain
stable
7. Synapse Rearrangement
Time-lapse imaging of synapse elimination
Two neuromuscular junctions (NM1 and
NMJ2) were viewed in vivo on postnatal days
7, 8, and 9.
8. Myelination
Myelination Lasts for up to 30 Years
Brain Weight During Development and
Aging
The second step, neural tube
generates three cell types
Before NT closure…
• Epidermis (disappears after closure)
• Crest cells
• CNS cells
Neural Crest
 Neural crest tissue found ONLY IN
VERTEBRATES.
forms melanocytes, much of the head
skeleton, peripheral glial cells, peripheral
& autonomic nerurons, and chromaffin
cells of the adrenal gland cells.
 Epithelial cells at margin of neural plate
become migratory (“amoeboid”).
Neural Crest
 slug and RhoB genes
 Stop N-cadherin expression
 Migratory pathways
1. Dorsolateral pathway = between
ectoderm and mesoderm; form
melanocytes
2. Ventral pathway = through somite; form
all other neural crest tissues
 “Avoid” the notochord.
Chicken Somite Differentiation
neural tube
somite
sonic hedgehog? notochord
Chicken Somite Differentiation
dermamyotome
sclerotome
Neural Crest Migration
Neural Crest Migration
 Dorsolateral pathway neural crest does not
behave segmentally.
 Ventral pathway neural crest behaves
segmentally.
 Spinal ganglia are segmental; associated
with the somites.
 Dorsal root ganglion forms in the anterior
of each somite.
Neural Crest Migration
 Extracellular matrix molecules “define”
migration pathways.
 Integrins in neural crest cell membranes.
 Ephrin = protein followed by many neural
crest cells
 Most neural crest cells have Eph receptors
(integrins) to follow ephrin pathways.
Neural crest cell migration
• Initiation
Epidermis
Contact with
epidermis induces
Slug expression
Slug induction factors
Neural tube
Slug repressing factors
• Path finding
Notochord
ECM components involved in Axolotl crest cell migration
Source of crest cells Matrix components
Cranial
Fibronectin,Tenascin
laminin-heparansulphate
proteolglycan
Trunk
Peanutagglutinin - binding protein
Ephrins
Neural Crest Migration
 Anterior-Posterior regions of neural crest.
 Is neural crest cell fate determined before
migration?
 Seems to be little determination before
migration, except…
between the anterior to posterior regions.
Neural Crest Fate Map
Neural Crest
 Cranial (Cephalic) - head region
forms cranial neurons, face skeleton, tooth primordia,
thymus
 Trunk - posterior to the head
melanocytes, peripheral neurons
Cardiac - near somites 1-3; large heart arteries and
some throat skeleton
near somites 18-24 - chromaffin cells of adrenal gland
 Vagal and Sacral – Parasympathetic ganglion in gut
Origin of neural crest cells
• Neural crest cells can form from the dorsal side of
the closed neural tube
• Epidermal and neural plate/tube interactions
may generate crest cells
• Co-culturing plate and epidermal cells
produces crest cells
•Culturing plate cells with BMP4 or BMP7 also
produces crest cells
Neural crest cell differentiation
NGF – cells now
unable to respond to
glucocorticoids
FGF promotes
competency to respond
to NGF signal
Pluripotent
neural crest cell
Bipotent
precursor
NGF-competent
cell
Sympathetic neuron
Chromaffin
precursor cell
Chromaffin cell
Glucocorticoids inhibit
neural differentiation
Glucocorticoids promote
accumulation of chromaffinspecific enzymes
Formation of brain
Olfactory lobes
Cortex, Hippocampus
Cerebrum
Telencephalon
Forebrain
(Prosencephalon)
Retina
Epithalamus
Thalamus
Hypothalamus
Diencephalon
Mesencephalon
Midbrain
Metencephalon
Cerebellum
Midbrain
(Mesencephalon)
Hindbrain
(Rhombencephalon)
Pons/Medulla
Myelencephalon
Inhibitory HLHs may
limit effects of proneural
genes to specific tissues
Hindbrain segmented
(rhombomeres) – isolated
‘territories’ of neurons
Proneural genes
Neural precursor genes
Neurogenin
bHLH
NeuroD
bHLH
Determination
Differentiation
Neurons
Neuron
‘Birthday’ –
final division
Differentiated neurons make
functional neural networks
The Growth Cone
Growth
cone
A retinal ganglion cell sending out its axon
The cell is stained with fluorescent antibodies against the adhesion
molecule N-CAM
The Growth Cone
Key determinant of axonal growth
Axon
Node of
Ranvier
Schwann
cell
Axon
hillock
dendrites
Filopodium –
actin bundles
• Extension and retraction of filopodia
• Cytoskeletal-dependent
• Adhesion of filipodia – allows
growth cone to pull the cell
Microtubules
Lamellipodium –
actin network
• Filipodia find path
GTP-binding Proteins and
The Growth Cone
Attractant
SGBPs Rac1
and CDC42
N-WASP – actin
depolymerisation
Repellent
SGBP RhoA
mlc phosphatasepolymerization
Growth cone
collapse
Motor Neurons
 Axons grow to specific muscles.
 Growth Cone = region of extension of
the axon near its tip
 Guided by chemoattraction and
chemorepulsion
 Guidepost Cells = isolated cells secreting
a chemoattractant
Motor Neurons
 Long-range chemoattractants &
chemorepulsants = diffusable
substances released by cells forming a
gradient
 E.g., netrins (a) & semaphorins (r)
 Short-range chemoattractants &
chemorepulsants = gradient of a
substance within cells (a tissue) or an
extracellular matrix
 E.g., ligands & cadherins
Neuronal Apoptosis
 NGF (neuronal growth factor) =
chemoattractant & essential for the
survival of neurons
 neurotrophins (e.g., NGF)
 Trk receptor proteins in neurons bind
to neurotrophins.
 “Competition” for neurotrophins
secreted by target tissues.
 Too little neurotrophin = apoptosis
Commisural Growth Cones
 Commisural neuron axons grow to the floor
plate.
It produces a chemoattractive netrin-1
gradient.
 Semaphorins (chemorepellants) then
produced cause the axon to grow dorsally
to the other side.
Neurogenesis in neural tube
Tissue Organization of CNS
Differentiation…
Neuron or glial cell
Spinal Cord
Cells migrate into
intermediate zone
Division only in
germinal zone
Late ‘birthday’
External granule zone
contains external
germinal layer forming
granular cells that migrate
to the granular layer
Early ‘birthday’
Cerebellum
Purkinje neurons have a
complex cluster of
dendrites forming up to
100,000 synaptic
connections
Germinal layer
Cerebral cortex
Marginal Zone
Intermediate zone
External granule layer
Purkinje layer
Granule layer
Cortical plate
Subventricular zone
Ventricular germinal zone / Ependymal layer
Lamina dissecans
Neopallial cortex –
eventually 6 layers of
neurons with
different functions
Heavily myelinated
axonal layer – white
matter
Neuron Types and The Neural Tube
Sensory neurons
Shh from
notochord
ventralized
cells
Shh from floor
plate instructs
precursors to
become motor
neurons
Transplant piece of floor
plate causes nearby
neurons to become
motor neurons
Shh induces motor neuron-specific Islet-1 TF
Neuron Types and The Neural Tube
Roof plate
Association
neuron
Commisural
neuron
Dorsal root
ganglion neuron
LH2a, b
Islet-1
Lmx1
Lim 1, 2
Sulcus
limitans
Lim 3
Ventral
interneuron
Islet 1, 2
Floor plate
Notochord
Neuronal types
Motor
neuron
•Gene expression domains in the neural tube
• These domains define neuronal populations
• Neuronal populations form columns parallel to anterior-posterior axis
Target Specificity and Hox Genes
Cranial ganglia
Cranial neurons arise from
migrating crest cells
Brancial Arch
Visceral arches
Neural tube
Rhombomere
2
1
3
Crest cells ‘inherit’ the neural
tube’s Hoxb expression
pattern
4
2
5
6
3
7
4
Ectoderm
This pattern establishes
boundaries regulating which
arch each ganglion will
innervate
Spinal Cord
Mesenchyme
Hoxb1 Migrating crest cells
Hoxb2 induce same Hoxb
pattern in ectoderm
Hoxb3
Hoxb4
Gene Expression and Motor Neuron Target
Pools of neurons
innervating a single muscle
Cervical
All motor neurons express Islet-1
and Islet-2. Alone these specify
body wall muscle targets
Forelimb
Thoracic
Hindlimb
Lateral motor column
Medial motor column
Column of Terni
Time
Isl-2
Isl-1
Column of Terni
Lim-3
Isl-2
Isl-1
Medial motor column
Isl-2
Isl-1
Medial motor column
Lateral motor column
Lim-1
Isl-2
Isl-1
Lateral motor column
Transient Lim-1 expression
determines neurons innervating
limb muscles
Column of Terni motor neurons project into sympathetic ganglia
Medial motor column neurons extend into axial muscles
Lateral motor column neurons project into limb muscles
How neurons know where to go?
Contacts Between Retina and Visual Cortex
Each side of the cortex is contacted by axons from both eyes
(binocular vision)
From Retina to Brain:
•Neurons from retina contact specific sites within brain
•Harrison (1910) – Pioneer neurons used as migration highway
•Pioneer neurons can migrate short distances in developing embryo
•Pioneer neurons may die after being used as a guide
•Neuron migration stages
Pathway Selection
Neuronal activity independent
Target selection
Address selection
Pathway Selection
Collagen IV
Physical Cues (Stereotropism)
Strips of extracellular
matrix components
layered on the floor
of the culture dish
Grooves
Existing Channels
Cultured neurons
Adhesive Gradients (Haptotaxis)
‘Differential adhesive specificity hypothesis’
Laminin
Uneven distribution of e.g. laminin and fibronectin (support
outgrowth) or GAGs (inhibit outgrowth)
Retinal glial cells downregulate laminin, and neurons downregulate
integrins, once optic tectum innervated
Pathway Selection: Labelled Pathways
Fascicles – axon bundles.
Formation of these is
dependent on expression of
proteins such as fasciclin I
Axon fascicles
Laminin unlikely to be specific
7-4 Neuroblast progeny
Q1
Q2
C
G
Q5
Q6
Destroy any bundle other
than P1/P2 and G finds its
target
Destroy P1/P2 and G tries
in vain to find its target
Q1 & Q2 migrate laterally
until they meet dorsal
midline dmp2 fascicle –
then turn to posterior
C & G ignore dmp2. C heads to
posterior at X1 and X2, G
continues to P1 and P2 then
heads to the anterior
dMP2
X1 P1
X2 P2
Pathway Selection: Netrins
• Netrins involved in attraction and repulsion
Unc-6
• C. elegans genes
Netrin homologue
Unc-5
Netrin receptors
Unc-40
• Attract commisural neurons, repel trochlear neurons
Pathway Selection: Diffusible Gradients
First proposed in 1892 (Ramon y Cajal)
Netrin-1 and netrin-2 soluble signals directing commisural neuron outgrowth
Commissural neuron
Neurons
Netrin-2
gradient
COS cells
Netrin-1
gradient
Netrin-2 synthesised in
lower region of spinal cord
(but not floor plate)
Commissural neurons,
cultured in the
presence of COS cells
do not extend axons
Netrin-2 may act to attract
neurons to netrin-1 (expressed
by floor plate cells)
Axon extension from
commissural neurons
cultured with COS cells
transfected with netrin1 (or netrin-2) gene
Commisural Axon Growth
netrin
notochord
Pathway Selection: Netrins - Signalling
Attraction and repulsion also dependent on signalling ‘state’ of neuron
Netrin added
Add KT5720
Remove Ca2+
Netrin attracts
No netrin effect
Netrin repels
KT5720 blocks cAMP-dependent signalling
cAMP levels block RhoA-mediated growth cone collapse?
Pathway Selection: Growth Cone
Repulsion
Semaphorins found in a variety of
animals
Ti1 axon progresses towards spinal
cord until it encounters G-Sema-1
band at which point it re-orientates
untils filipodia contact Cx1
Semaphorin III (collapsin) causes
growth cone collapse – prevents
dorsal root ganglia from innervating
ventral spinal cord
Limb
Ti1
G-Sema-1
expression
band
Cx1
Spinal cord
Some dorsal root gangla do
innervate ventral spinal cord –
collapsin-insensitive
Neuropilin – part of the semaphorin
receptor complex
Some semaphorins (e.g. G-Sema-I) can act as attractants
Commisural Axon Growth
semaphorin
notochord
Target Selection
Retinal cells are ‘mapped’
onto the optic tectum
Establishment of retinal layers – N-cadherin
Directed axonal growth – N-CAM and laminin
Entry into optic nerve – N-CAM (laminin?
cadherin?) position-specific fasciculation
Cross or Turn? – repulsion? Fasciculation specificity?
Homing – laminin? Global cues
Arrival at target – loss of laminin and loss
of sensitivity to laminin
Signals in optic tectum innervation
Topographic map – position-specific
inhibitors. Other signals?
Target Selection: Signals
• Ephrins bind to Eph receptors (receptor tyrosine kinases)
• Ephrins are repulsive or attractive depending on EphrinEph receptor combination
• Gradient of RAGS and ELF-1 from posterior tectum
• RAGS and ELF-1 repel temporal retinal axons
• RAGS and ELF-1 expression dependent upon expression
of Engrailed
• Expressing Engrailed in anterior tectum results in
RAGS/ELF-1 expression throughout tectum and failure of
temporal axons to contact any part of tectum
Address Selection:
A Matter of Life and Death
• Muscle cells initially innervated
by >1 neuron
• Mature muscle cells contacted by just
one axon
• Post-partum all but one of the
axons retract
• Active axons suppress their
immediate neighbours