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Neuronal Determination and
Differentiation
Cell Differentiation
A human body contains more than 210 major
types of differentiated cells
Cell determination commits a cell to a particular
developmental pathway
-Can only be “seen” by experiment
-Cells are moved to a different location in the
embryo
-If they develop according to their new
position, they are not determined
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Normal
Recipient
After Overt
Differentiation
Determined
(later development)
Tail cells are
transplanted
to head
Tail cells are
transplanted
to head
No donor
Donor
Recipient
Before Overt
Differentiation
Not Determined
(early development)
Tail
Head
Tail cells develop
into head cells in head
Tail cells develop
into tail cells in head
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Cell Differentiation
Cells initiate developmental changes by using
transcriptional factors to change patterns of
gene expression
Cells become committed to follow a particular
developmental pathway in one of two ways:
1) via differential inheritance of cytoplasmic
determinants
2) via cell-cell interactions
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Cell Differentiation
Cytoplasmic determinants
-Female parent provides egg
with macho-1 mRNA
-Encodes a transcription
factor that can activate
expression of muscle- specific
genes
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Cell Differentiation
Induction is the change in the fate of a cell due to
interaction with an adjacent cell
If cells of a frog embryo are separated:
-One pole (“animal pole”) forms ectoderm
-Other pole (“vegetal pole”) forms endoderm
-No mesoderm is formed
If the two pole cells are placed side-by-side, some
animal-pole cells form the mesoderm
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• All cells contain the full genome (which cells do not?)
or the full set of genetic information for the whole
organism.
• Cellular differentiation in vertebrates, therefore, is not
a loss of DNA (as in some insects and nematodes), but
rather, a development of selective gene expression 
specialization.
• How do we know this? Early cloning studies in which
nuclei from cells from later developing tissues were
implanted into enucleated eggs  normal, complete
embryo.
• In the latter part of the 19th century, it was believed
that neurons and glia arise from distinct pressures. In
fact, all cells of the nervous system arise from the same
neuroepithelium.
• Points to portend about cell migration (next
lecture):
1. What is the 1° migration route?
2. What is the purpose of 2° migration?
3. When do they begin to differentiate to their
final structure?
4. Which ones are expected to differentiate
first?
• In the vertebrate, significant neuronal
differentiation occurs once the neurons are
post-mitotic.
• Do neurons divide at birth?
• How about at age 2?
• For the most part, we don’t get new ones.
• In the adult, ongoing neurogenesis is evident
only in the hippocampus and olfactory bulb
(see end of this set of slides).
Two major types of signals for differentiation
•
•
•
Intrinsic cues
Extrinsic cues
Signals inherited from
precursor (present in
the cytoplasm or
nucleus).
Uses transcriptional
activators or
repressors.
May be timed and
automatic (may be
influenced by extrinsic
cues).
• Signals from the environment.
• Distance (hormones).
• Neighboring cells (paracrine) –
can be secreted or membranebound.
• Signaling molecules  surface
receptors  signal
transduction  activates
transcription factors certain
genes are expressed to confer
the phenotype of that
particular cell (e.g., shape, nt
type, how it fires)
Ways to study Neuronal Determination
What is determination? Cell fate…
“What will I be when I grow up?”
1. Transplantation – way to find out if, at certain
developmental time point, cells’ fate is fixed
or is still flexible.
2. Genetic Mutations – how is the normal gene
product important?
- spontaneous mutant (reeler)
- transgenic (knockout)
- mosaic (pt mutation, part normal)
3. Cell/tissue culture
- Isolate or create a controlled system
-cell ablation – way to understand a cell’s fate
when a neighboring cell is removed
(what kind of cue is this?)
Specific Example of Intrinsic/extrinsic cues:
proneuronal and neurogenic genes
1. Invertebrate system:
- proneuronal – neuroprogenitors (neuroblast)
are instructed to help maintain the structural
integrity of the intercellular space (without this
gene, much apoptosis (PCD) would occur),
rather than epidermablasts.
- neurogenic – neuroprogenitors restructure the
# of other neural progenitors (extrinsic signals to
keep certain cells as epidermablasts)
2. Vertebrate system
- Local (extrinsic) signals from the organizer have
already separated neuroectoderm from future
epithelium.
- Proneural genes are used to separate postmitotic neurons (which will do what?) from other
neuroepithelial cells that continue to divide.
-Required for the determination of certain
populations of neural cells.
-Neurogenic genes promote an epithelial
phenotype and inhibit formation of post-mitotic
neurons.
Differentiation of Neurons and Glia
Are also capable of turning other uncommitted cells to
neural precursors.
1. How are specific cell types controlled in a vertebrate
system?
e.g., glial differentiation in vertebrate optic nerve:
expression of the 3 glial cell types is controlled by a
combination of intrinsic and extrinsic signals.
PDGF and NT-3 (growth factors) are secreted by 1 type of
cell (type I astrocytes), which allow another cell type
to continue dividing (w/o these growth factors, it will
stop dividing and immediately differentiate).
This cell is then able to use an “internal clock” (# of cell
divisions) prior to its differentiation.
2. Example from insect:
- switch from neuroblast to glial cell is
controlled by a transcription factor, engrailed,
a DNA binding protein.
Determination of Individual Cell Fate
1. Example of extrinsic cues controlling 2
specific cell types in the PNS.
- Sympatho-adrenal progenitors 
adrenergic neurons vs. endocrine chromaffin
cells, which differ both morphologically and
biochemically.
Glucocorticoids: ligand-receptor complex
translocates to nucleus and then directs
actions of txn factors, which decr neuronal
genes and incr chromaffin genes.
(note that txn factors can decr, as well as activate)
Transmitter switching by
target-derived factors.
A) All sympathetic neurons start as
noradrenergics. Some innervate the sweat
glands and switch their transmitter phenotype
as the sweat gland matures and stop tyrosine
hydroxylase and start choline acetyltransferase
synthesis.
B) The adrenergic-to-cholinergic switch can be
prevented by replacing sweat gland-rich targets
with tissue usually receiving adrenergic
innervation. Conversely, an adrenergic-tocholinergic switch can be accomplished by
transplanting foot pad tissue onto hairy skin,
which is usually innervated by adrenergic
sympathetic neurons.
C) Factors such as LIF and CNTF, found in target
tissues can influence neurotransmitter choice in
cultured sympathetic neurons causing cells that
would differentiate as adrenergic neurons to
become cholinergic.
bFGF/NGF: bFGF secreted by progenitor cells
promotes neuronal development and also
induces expression of NGF receptor – making
cell responsive to NGF.
2. Cell-cell signaling in the insect compound
eye.
Ommatidia: photoreceptor units consisting of
many cell types; i.e., photoreceptor cones.
Progenitors start out completely uncommitted;
cell-cell interactions determine their fate.
Differentiation occurs as a ‘wave’ over the eye –
regulated by txn factors signalled using the
Ras cascade (Ras is ‘molecular switch’ for
photoreceptor vs. cone cells).
SUP (‘seven-up’) is a nuclear receptor protein,
which specifies photoreceptor cells.
Activation of ras pathway is necessary for it to
function:
Ras  generic photoreceptor  subset of
specific outer photoreceptors
(occurs using sup)
Original signal activates the Ras pathway via an
EGF-type receptor.
3. Target-derived signal for (final) neuronal
differentiation.
Final choice of fate for a neuron may come only
after the cell has established contact with its
synaptic target.
Example of a population of neural crest cells all
start out adrenergic (producing NE)
Those reaching their axons out to smooth
muscle remain adrenergic (becoming
sympathetic neurons)
Those making contact with sweat glands switch
their nt production and become cholinergic.
E.g., this experiment has shed some light:
Co-culturing young neurons with sweat gland
tissue could induce.
Transplanting in vivo could cause switch.
Factor involved has not yet been fully clarified.
Differentiation
•Neurons become fixed post
mitotic and specialized
•They develop processes (axons
and dendrites)
•They develop NT-making ability
•They develop electrical
conduction
Schematic diagram of an idealized embryo in cross section
showing pathways of neural crest migration in trunk and
derivatives formed.
Neural crest cells migrate along two
primary pathways:
dorsally under the skin,
ventrally through the sclerotome.
Dorsal migrating cells form pigment
cells,
ventrally migrating cells form dorsal
root and sympathetic ganglia, Schwann
cells, and cells of the adrenal medulla.
Drawn by Mark Selleck.
Trunk neural crest cells migrate in
a segmental fashion.
A) Schematic diagram demonstrating that
neural crest cells migrate through the
sclerotomal portion of the somites, but only
through the rostral half of the sclerotome.
B) In longitudinal section, neural crest cells
(green) can be seen migrating selectively
through the rostral half of each somitic
sclerotome (S).
From Bronner-Fraser (1986).
Differentiation of cranial neural crest cells:
TGF-ß Regulates Expression of Transcription Factors
to Determine the Fate of Cranial Neural Crest Cells
CNC cells give rise to an array of tissue types:
odontoblasts, chondroblasts, osteoblasts, neural tissues, such as sensory neurons
and cranial nerve ganglia etc.
Both ectoderm and endoderm of the branchial arch provide signaling instructions for
the fate specification of these progenitor cells.
Chai et al., 2003
Neuroblast differentiation: Series of GF and transcriptional
regulators affecting neurogenesis of neural crest progenitors.
Neural crest cells can be identified by the expression of FoxD3 and SOX10.
Progenitor cells differentiate into sympathetic, parasympathetic, enteric, or sensory neurons
dependent upon instructive signals encountered at or near the time of egress from the neural
tube.
Extrinsic cues encountered during migration or at sites where neural crest-derived cells
differentiate influence patterns of gene expression.
From Howard, 2004
Specification and differentiation of peripheral autonomic neurons are
dependent upon the interplay between cell extrinsic and cell intrinsic
factors.
Initial instructive cues from the neural tube influence neural crest cells to respond to
BMPs.
Induction of Phox2b and MASH1 is followed by the induction of HAND2 and Phox2a
resulting in expression of pan-neuronal (SCG10, NF) and cell type-specific (TH, DBH,
ChAT, VAChT) genes.
M.J. Howard, 2004
Neuronal & Glial Lineages are derived from common progenitors
A) 2 identified neuroglioblasts in
the Drosophila neuroblast map
(see Fig 7A).
B) Separation of neuronal and glial
sublineages in progenitor 6-4. The
glial regulatory protein, Gmc, is
expressed in 6-4. When this cell
divides into two equally sized
daughter cells, 6-4 G and 6-4 N,
the Inscuteable complex and
Miranda segregate Gmc into 6-4
G, which thereby becomes
specified as glioblast.
C, D) The MNB neuroblast
produces both glial cells and
neurons. The engrailed gene,
which encodes a homeodomain
transcription factor, is required for
glial sublineage. When en function
is reduced by injecting antisense
oligonucleotides, MNB forms only
neurons (D).
Neuronal lineage determined by intrinsic factors:
Mouse Numb is inherited asymmetrically
Asymetrical division of cells.
A) Dividing pair of daughter precursor cells uniformly stained for a proneural
gene.
B) Only one daughter cells inherits the numb protein (green).
C) Double labeling for proneuronal and numb gene (yellow).
D) Asymmetrical distribution in neuronal progenitor cells from cortex, neural
crest, and spinal cord.
Neurons are born: Neurogenesis
• Neurons are born in the ventricular zones
close to the brain ventricles
• Neurons are born mostly prior to birth
• Birth dating studies can determine the time of
the last cell division
• [3H]thymidine or BrdU labeling to determine
“birthdate” of the neurons
• Glia cells proliferate throughout life
Cortical neurons are born consecutively
Neurogenetic timetable for the neocortex, based on long-survival [3H]thymidine
auto-radiography in the rat. SA Bayer & J Altman, 1993
Development of the cerebral cortex.
The ventricular zone (VZ) contains the progenitors of neurons and glia. The first neurons to be
generated establish the preplate (PP); their axons, as well as ingrowing axons from the thalamus,
establish the intermediate zone (IZ). Neurons of cortical layers II–VI establish the cortical plate (CP),
which splits the preplate into the marginal zone (MZ), or future layer I, and the subplate (SP), a
transient population of neurons.At the end, six cortical layers are visible overlying the white matter
(WM) and the subplate has largely disappeared. Neural precursors in the subventricular zone (SVZ)
continue to generate neurons that migrate rostrally into the olfactory bulb, even during postnatal life.
Laminar fate determination in the
cerebral cortex.
A)
Morphogenesis of the mammalian cerebral cortex. Neural
precursors are born in the ventricular layer and migrate
away from the ventricular surface, following tracks
provided by radial glial cells. The first born cells are the
Cajal-Retzius neurons (left). Later born neurons
accumulate in a dense matrix of cells, the cortical plate
(middle). In this plate, neurons are ordered by birth date
in such a way that older neurons (magenta) remain in
deep layers, and younger neurons (blue) migrate through
the deep layers to attain a superficial position (right).
B) If ventricular cells from young donors (which would become
deep cells) are transplanted into an old host, they adapt
to their new environment and develop as superficial
neurons (arrow).
C) In converse heterochronic transplantation (old donor to
young host), transplanted ventricular cells maintain their
laminar fate and become superficial neurons.
D) Layer 4 neurons transplanted into older brains switch their
fate so that it is appropriate for the upper layer neurons.
E) When layer 4 neurons are transplanted into younger hosts,
they end up in layers 4 and 5, but not layer 6.
Is there neurogenesis in the
adult Brain?
Yes there is, in the
Dentate gyrus
Olfactory bulb
Neurogenesis in adult rat hippocampus
One day after BdDU
injection
4 weeks after BrDU
injection
Neuronal marker NeuN
plus
BrDU labeled cells
F. Gage, Salk, San Diego