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Neural Induction
Chapter One
Development of
the Nervous System
Fertilization
Cleavage (Blastula, Gastrula)
Neuronal InductionNeuroblast Formation
Mesodermal Induction
Lateral Inhibition
Axis Formation
Cell Migration
Axon Growth/Target innervation
Differentiation
Functional Nervous System
Cell Lineage
Cell-cell Interactions
Spermann Organizer
Transcription Factors
Homeobox genes
Chemical Gradients
Cell-Cell Interactions
Chemical Gradients
Cell-Cell Interactions
Synaptic Formation
Electrical Differentiation
Cell-Cell Interactions
Trophic Factors
Mechanisms of nervous system
development
Gastrula
(Mesodermal induction via BMP
signal inhibition)
Induction
(Lateral Inhibition via regulation of
transcription factors)
Neuroblast
(Cell migration,
Differentiation)
Functional Nervous System
Questioning
Developmental Biologist started looking:
• Determine the time line of differentiation
into different cell types
• What was determine by cell’s position?
• What part did cell to cell interactions play?
• What was the inducing factor?
• What was required to form neuronal
tissue?
Factors regulating development of the
nervous system
Before
After
• Interactions with neighboring
tissue is required for
ectodermal differentiation
into nervous tissue
• Neuronal lineage arises
during gastrulation
• Isolation of animal cap
before gastrulation gives
origin to epidermal cells.
• If the animal cap is removed
after gastrulation, it will give
origin to neurons.
• These experiments suggest
that tissue arrangements
during gastrulation is
necessary for induction of
nerve tissue
• In Amphibian, the involuting
mesoderm (dorsal lip of the
blastopore) induces neuronal
differentiation of the ectoderm
• Transplant of a dorsal
blastopore gives rise to a new
body axis. Therefore the
dorsal lip of the blastopore
“organize” the formation of a
new body axis. This structure
has received the named of
Spemann organizer
…even at the gastrula stage the head organizer is not an
equipotential entity, but it is subdivided into specialized
inductors although distinct boundaries between then do not
seem to exist….Holtfreter, 1939
• Tissue in the dorsal lip of the
blastopore contain multiple
inducers: subdivision of the
dorsal lip and transplant to
another embryo give origin of
either tails or head-like
structures.
Medial portion
of DLB
Lateral portion
of DLB
Generate head
Generate tail
structures
What chemical factors are involved in
neuronal induction by the mesoderm?
• The use of a expression
cloning system identified
noggin as the first neuronal
inducer – Noggin
• Noggin is a 26 kD protein
expressed in the dorsal lip of
the blastopore during
gastrulation (but not in the
blastula stage)
• Noggin cDNA injection into
UV treated embryos result in
normal development of the
nervous system
• Follistatin signaling has been
identifies as another important
pathway of neuronal induction
• During development follistatin
binds to and inhibits activin (a
TGF-β-like molecule)
• Follistatin is expressed in the
organizer region of the
embryo during gastrulation
• Overexpression of a truncated
form of the activin receptor
result in differentiation of
animal cap cells into neurons
without any neuronal inducing
molecule
• Misexpression of follistatin
causes the formation of a
secondary axis
• These experiments lead to the
idea that neuronal induction
by the mesoderm may involve
inhibition by activin receptors
• Neuronal differentiation of
ectoderm in actively inhibited
by cell-cell interactions
• Dissociation of animal caps
cells causes most cells to
become neurons, whereas
cells maintained as
aggregates develop as
epidermal cells
• So it appears that the
ectoderm is actively inhibited
from becoming neuronal
tissue
Adding BMP4
Blocks dissociated cells from
becoming neural tissue
What chemical factors are involved in
neuronal induction?
• The idea that the ectoderm is
actively inhibited from
differentiating into neuronal
tissue was further proved with
the discovery of chordin
• Chordin overexpression
cause generation of a
secondary axis similar to
transplanting a Spemann
organizer into another embryo
How does chordin induce neuronal
differentiation?
Fly
Sog
Dpp
=
=
Frog
Chordin
BMP-4
•
Studies in Drosophila indicate that
the chordin-like protein sog bindings
to the TGF-β-like receptor dpp
related to the vertebrate genes
bone-morphogenic proteins, BMP-2
and BMP-4
•
Sog and Dpp antagonize each other
in Drosophila
•
Overexpression of sog (ie, chordin)
stimulate neuronal differentiation.
Injection of BMP-4 inhibits neuronal
differentiation.
• The antagonistic action of
sog and dpp lead to the
idea that neuronal inducers
(noggin, follistatin, chordin)
stimulate neuronal
differentiation by inhibiting
BMP-4 signaling
• Overexpression of BMP-4
inhibits neuronal
differentiation of animal
caps treated with noggin,
chordin, or follistatin
• Antisense BMP-4 RNA
causes neuronal
differentiation without any
any neuronal inducing
molecule
Multiple Inducers Selectively Promote
Different Regions of the Nervous System
The Spemann's organizer secretes many proteins that
bind to different growth factors in the extracellular space.
Each one blocks the signaling through their cognate
receptors
Antagonist
Ligands
Mesodermal Induction in Vertebrates
• Experiment in zebra fish have
confirmed that in vertebrates
formation of the neural tube
requires inhibition of BMP
signal
Gene Interactions
• Ectoderm cells start out fairly similar
• Bone Morphogenetic Proteins (BMP)
cause them to form skin and bone
• BMP inhibitors cause them to form
neurons instead
– Examples Chordin and Noggin
Cell-cell Interactions Control Neuroblast
Segregation
• The noggin, chordin,
follistatin molecules only
determine the generation
of a neurogenic region in
the embryo
• The process of neurobalst
formation (ie, neuronal
precursors) has been
studied in Drosophila
where delamination results
in the formation of
precursor neuronal tissue
Delamination and Formation of Ganglion
Mother Cells in Drosophila
• First division of neuroblast
cells generate a ganglion
mother cell (GMC)
• Each GMC generates one pair
of neurons (or glial cells)
Does neuroblast formation require
interaction among neighboring cells?
• Laser ablation of a
delaminating cell causes
another cells to delaminate
• This lead to the idea that
lateral inhibition maybe
responsible for the
neuroblast formation in
Drosophila
• It was found that a family of
transcription factors are
required for this process to
occur
Lateral Inhibition
• Ectodermal cells are actively inhibited from
becoming neuronal cells
• If they are physically separated
• Or if they do not receive inhibition signal
then they will become neuronal cells
•
In Drosophila, segregation of
neuroblast from epidermal cells
requires the expression of bHLH
transcription factors (basic-helixloop-helix transcription factors)
•
bHLH transcription factors
activate transcription by binding
to specific DNA sites called Eboxes (CANNTG)
•
In Drosophila, the achaete scute
family of transcription factors
(ACS) is required for proper
development of neuroblasts.
Because their role in
neuroblasts formation these
genes are called proneural
genes
•
This family of genes were
identified for their effect on the
development of the fly’s bristles
How does the process of lateral inhibition
works?
Some ectodermal
cells express ACS
proneural genes
ACS expression
increases in one
of the cells in
the center of the
cluster
Lateral inhibition
blocks proneural
genes in
neighboring cells
Proneural Genes
• Achaete scute (ACS)
– Transcription factors
• Induce neuronal development
• Once one cell begins expressing ACS,
surrounding cells are laterally inhibited
Lateral Inhibition Requires the
Delta-Notch System
• Delta is the endogenous
ligand for the Notch receptor
(a large transmembrane
protein)
• Low Notch activity induces
neuroblast formation. Thus, in
Notch mutants most of the
cells in the neurogenic region
become neuroblasts
Delta
Y Notch
• Delta mutants undergo
change in same direction
The Delta-Notch Signaling
• Cleavage of the Notch
receptor after binding to
Delta allows for the
diffusion of the Notch tail
to the nucleus and
regulation of transcription
• During development
interactions between Delta
and Notch induce
molecular differences
between neighboring cells
Lateral Inhibition Requires the Delta-Notch
System
• Cells within the proneural
cluster express high
levels of ASC as well as
Delta
• If by chance one cell
express higher levels of
ACS, this cell will express
higher levels of Delta
Ectodermal cell
Notch Activation Results in Cells Adopting
an Epidermal Fate
• Upregulation of Delta
expression in the central cell
will activate Notch in
surrounding cells
• This will lead to release of the
SuH (supressor hairless) from
Notch
• SuH translocate to the
nucleus where it suppresses
neuronal gene expression (ie,
ASC)
Ectodermal cell
Notch Activation Results in Cells Adopting
an Epidermal Fate
• SuH inhibits neuronal gene
expression by regulating the
expression of another set of
bHLH proteins called
enhancer of split complex (Espl)
• E-spl act as repressors of
transcription (notice that ASC
genes are transcriptional
activators)
Ectodermal cell
• E-spl proteins act as
repressors of transcription by
binding to the E-boxes in the
ASC promoter
Tight Control
• Delta/Notch and SuH block neuronal
differentiation in surrounding cells
• This is how lateral inhibition is controlled
• ACS is proneural transcription factor
• SuH is inhibiting transcription factor
• Vertebrate homologs of Drosophila ASC have
been identified (Mash1, Cash1, Xash1, NeuroD)
• These genes act as transcriptional activators
similar to ACS
• Overexpression of the bHLH gene NeuroD in frog
embryos result in significant neuronal formation
throughout the epidermis
Current Model:
Default is epidermis
Fly
Delta
Notch
SuH
ACS
Vertebrates
Wnt
Frizzled
Msx
Sox
Necessary vs. Sufficient
• Necessary = must be present
• Sufficient = all that is needed
• BMP antagonism is necessary and
sufficient to induce a cell to become a
neuron
• Notch/Delta pathway is necessary and
sufficient for inhibition of surrounding cells
so that they do NOT become neurons
Necessary vs. Sufficient
• If you had no BMP antagonism what would
the ectoderm look like?
• If you had only BMP antagonism what
would the ectoderm look like?
• If you had only the Notch/Delta pathway?
• What if you have both BMP antagonism
and the Notch/Delta pathway?
Any Questions?
Read Chapter Two