Induction and Neurulation
Neuroembryology
1. Early Stage: blastula – a hollow ball of cells.
Sphere in amphibians
Flat sheet of cells in birds, mammals.
Up to this point, egg is partitioned (whole egg is
about the same size; cells divide smaller).
[show amphibian pictures]
Induction - Definition
1. Dictionary - Production of an effect elsewhere than at
the original locus of activity.
2. Developmental Biology text - The ability of one tissue to
influence the fate of nearby cells, presumably by a
chemical signal (Purves and Lichtman, 1985)
3. Developmental Biology text - The process whereby an
inducing tissue interacts with a responding tissue,
causing the tissue to differentiate (Oppenheimer and
Lefevre Jr., 1984)
Some Terminology
• Totipotent cell - a cell that has the ability to form a
complete organism through embryogenesis (e.g. the
zygote, one of the first two blastomeres formed
during cleavage in frogs or mammals).
• Pleuripotent cell - a cell that has the ability to
differentiate into any type of tissue if exposed to the
appropriate chemical signals (e.g. stem cells derived
from the inner cell mass of an early mammalian
embryo).
• Multipotent cell - a cell that has the ability to
differentiate into a limited number of tissue types if
exposed to the appropriate chemical signals (e.g.
stem cells obtained from adult tissues)
There is a conserved pattern in
the early development of animals
•Egg cells of all animals are polarized: an
‘animal’ pole and a ’vegetal’ pole.
•Latter contains the nourishing yolk.
•After fertilization, many cell divisions
occur, forming the blastula = a hollow ball
with an inner cavity = blastocele.
•Cells at the animal pole  epidermis and
nervous system.
•Cells at the vegetal pole  Gut.
•Cells in between these 2 poles 
mesodermal derivatives (muscles, skeleton.
•The rearrangement of this collection of
cells is called gastrulation.
•Gastrulation will give rise to 3 germ layers:
ectoderm, mesoderm, endoderm. [Will
elaborate on this later in these slides].
Fig. 1-12 (previous slide)
After a sufficient # of divisions, when the
blastula forms, there are cells on the inside of
the ball, called the inner cell mass, that
actually produce the embryo, whereas cells on
the exterior of the ball make the placenta and
other extra-embryonic membranes.
Note that the primitive streak runs along the A-P
axis of the embryo and the ectoderm laying
above the mesoderm cells  neural plate,
then the neural tube.
2. Gastrula: blastopore – invagination.
Gastrulation: The process by which the simple
blastula is transformed to the more complex 3layered organization. Ultimately gives rise to 3
different tissue layers.
Cells of the mesoderm and endoderm move into
the inside of the embryo, often at a single
invagination region, called the blastopore.
Eventually, blastocele is obliterated and a new
cavity, a primative gut forms (archenteron).
Some cells from inner and/or outer layer detach to
form the intermediate layer, called the
‘mesoderm’.
Note the involuting cells that were originally on
the surface of the embryo to the interior,
called the involuting marginal zone (IMZ) 
muscle and bone.
The first of the IMZ cells to migrate the furthest
will  anterior portion of the animal (head).
The later of the IMZ cells to migrate (the least)
will  posterior portion of the animal.
At this point, the neural plate of the vertebrate
embryo still resembles the rest of the surface
ectoderm.
Shortly after its
formation, the neural
plate begins to fold in on
itself to form a tube-like
structure – the neural
tube.
Neural crest cells, which
arise from the junction
between the neural tube
and the ectoderm, will
eventually form the
neurons and glia of the
PNS.
Neurulation
• Neurulation is the formation of the vertebrate
nervous system in embryos.
• The notochord induces the formation of the
CNS by signaling the ectoderm above it to
form the thick and flat neural plate.
• The neural plate then folds in on itself to form
the neural tube, which will then later
differentiate into the spinal cord and brain.
Neurulation (cont’d)
• Different portions of the neural tube then
form by 2 different processes in different
species:
1. Primary Neurulation – the neural plate
creases inward until the edges come into
contact and then fuse.
2. Secondary Neurulation – the tube forms by
hollowing out of the interior of a solid
precursor
Germ Layers:
Endoderm  gut and major digestive organs
Mesoderm  muscle, skeleton, connective
tissue, CV, and urogenital systems.
Ectoderm  skin and nervous system.
3. Neurula – The Next Stage of Development =
neurulation.
Embryo at this stage is called ‘neurula’
a. Formation of neural groove cranially from dorsal
blastopore or Hansen’s Node.
b. Ectoderm lateral to this groove thickens  the
neural plate.
c. Edges of the plate form distinctive ridges: neural
folds. These will ultimately meet and fuse 
neural tube  brain and s.c.
At the same time, the notochord and pamites
(masses of mesoderm incorporated into
connective tissue of spinal column) located
lateral to the 1st indication of segmentation.
d. The PNS arises from a distinct group of
precursor cells called the ‘neural crest’. The
neural crest arises from the neural plate, but
separates from it to form bands that run
dorsalaterally to the neural tube  gives rise to
spinal and autonomic ganglia, glial cells of PNS,
and several non-neural tissues:
- melanocytes, chromaffin cells, cartillage,
blood-forming cells, connective tissue covering
brain and spinal cord (DAP mater), parts of facial
bone (drives home the point that some tissues
(bone and connective tissue) come from >1
embryonic layer)
Origin of PNS Cells
• From neural tube:
– All motor neurons of somatic nervous system
– Preganglionic neurons of autonomic system
• From neural crest:
– Sensory nerves and associated ganglia
– Postganglionic neurons of autonomic system
Neural Crest Cells
• Induced by organizing cells of notochord
• Main functional groups:
– Cranial neural crest:
• Bones and connective tissue of face
• Tooth primordia
• Thymus, parathyroid, thyroid glands
• Sensory cranial neurons
• Parasympathetic ganglia and nerves
• Parts of the heart (cardiac neural crest)
Neural Crest Cells
•
•
•
•
A group of cells, which breaks away from the closing
neural tube and populate the periphery (as
opposed to the CNS, which will develop from the
tube).
Neural crest will supply all neurons of the PNS and a
variety of other peripheral structures, ranging from
melanocytes to craniofacial bones to cells of the adrenals.
This population of cells separates from the neural plate
shortly after the fusion of the neural folds, and streams of
dividing cells begin their journey through the embryo.
The expression of which genes are turned off during this
migratory stage?
And, this occurs for individual cells as well as for groups of
cells
Neural Crest Cells
• For neural crest cells, migratory pathway is
particularly important in cellular determination, as
location (or path) controls the availability of inducing
factors for particular cell fates.
Making Cells
The use of chimeras has been invaluable in the study of
individual cell fates.
What is a chimera?
Cells with a different genome; e.g., chick/quail mix –
heterochromatin marker not found in chick.
[3H] thymidine labeling has helped in the delineation of
migratory pathways and development potential of
neural crest cells.
Neural Crest Cells
Interaction:
Neural crest migration/movement is rigid and occurs in
a ventral (1st cells give rise to ventral structures) - todorsal order in the head.
Migratory pathways are linked to neuronal fate.
What is the mostly likely result of transplant
experiments when early cells will switch their fate?
Extrinsic cues  ?
Whether these come from the pathway itself or the
final destination (target-derived cues) is not clear.
As in the CNS, the earlier the cell is, the more
pleuripotent it is (the more flexibility of fate).
Neural Crest Cells
• Main functional groups:
• The stream of neural crest cells migrates via a
ventral route to form:
– Trunk neural crest:
• Melanocytes (via the dorsal route)
• Sensory neurons (DRG)
• Sympathetic ganglia and nerves (ANS)
• Medulla of adrenal glands (chromaffin cells)
Note that they migrate segmentally (sclerotome) – only in
the rostral compartment.
Neural Crest Cells
• Migration:
– Epithelial to mesenchyme transition
– Migrational pathways are established by juxtacrine
signals:
• Fibronection, laminin in ECM + integrins
• Ephrin proteins: Restrict movement
• Contact inhibition
• Use of existing structures
– Migration ceases when these signals are reversed
Migration of Neural Crest Cells
Unlike cells in the CNS, which migrate radially along
glial fibers, neural crest cells “crawl along”
independently (like fibroblasts).
Motility is promoted by integrins – bind cell surface to
ECM (how does this contrast with cadherins?)
Prominent ECM components along neural crest cell
pathway: fibronectin, laminin, collagen.
The ECM provides attractive (permissive) cues for
movements, as well as a substrate on which to bind.
A set of repulsive cues in neighboring structures keeps
cells in their precise migratory pathway
Migration of Neural Crest Cells
As the cells reach their destination, the expression of
cadherins is once again activated (had been
repressed during free movement)  cells aggregate
into ganglia when they undergo terminal neuronal
and glial differentiation.
Side bar: retroviral labeling:
A cell can be labelled permanently and heritably by
injection with a retrovirus carrying a gene (e.g., βgalactosidase)  incorporated into cells’ DNA and
then expressed.
A substance, which will turn blue from the action of the
enzyme, can then be introduced in a histochemical
test.
Neural Crest Cells
• Differentiation:
– Largely based on location along neural tube and their
migration route:
Neural Crest Cells
• Differentiation:
– Migration routes along trunk:
– Ventral pathway: cells move through anterior portion of
somite toward ventral side of embryo
• Cells become: sensory neurons, sympathetic ganglia,
medulla of adrenal gland
– Dorsolateral pathway: cells move between epidermis and
somite
• Cells become: melanocytes
• Basic organization of the PNS is established by the migratory
pathways of the neural crest cells
Neural Crest Cells
• Differentiation:
– How do they know what to become?
– Most cells are pleuripotent- fate determined by position
– Paracrine factors play a role
• Example: Endothelin-3 and Wnt
– Some exceptions: only NC cells from head make bone
– Individual cells may differentiate early in migration
Differentiation of Neurons
• Within nerve tube:
– Dorsal Interneurons
– Ventral Motor neurons
Differentiation of Neurons
• Motor neurons:
– Tissues they innervate depends on:
– Anterior-posterior location along the nerve tube
– When the cells were “born”
e. Sensory Placodes: arises from separate thickenings of
the ectoderm in the head region  central ganglia and
cranial sense organs (e.g., ear, lens)
f. Emergence of the Vertebrate brain from the neural
tube.
Rigid and disproportionate cell proliferation in neural
tube  swellings: vessicles:
Proencephalon  cerebral hemispheres
Mesencephalon  midbrain
Rhombencephalon  brainstem and cerebellum.
Most proliferation of nerve cell precursors occur inner 
outer surface of the neural tube.
(from ventricular zone outward)
There is also a rostral-caudal progression of maturation in
the neural tube.
Formation of the Neural Tube
•
Secondary Neurulation
1. Occurs beyond the caudal neuropore
2. lumbar and tail region
3. Exclusive mechanism for fish
4. Starts with formation of medullary cord
5. Cavitation of cord to form hollow tube
Secondary Neurulation
Differentiation of Neural Tube
• Major morphological changes: differentiation of
brain vesicles and spinal cord
• Differentiation of neural tube cells
• Development of peripheral nervous system
Differentiation of the Neural Tube
• Neural tube must maintain dorsal-ventral polarity
– Sensory neurons- dorsal
– Motor neurons- ventral
• Accomplished by “inductive cascades”
– Dorsal: BMPs from epidermisRoof plate cells in
neural tubeTGF-B cascadeCell differentiation
– Ventral: Sonic hedgehog from notochord and retinoic
acid from somitesFloor plate cells of neural
tubeshh gradientCell differentiation
Differentiation of the Neural Tube
• Histological changes
1. Neural tube initially a single layer of cells: germinal
epithelium
2. Cells are called neural stem cells
•
•
Neurons
Glial Cells: Myelin sheath
Neural Induction and the Search for
the “Organizing Principle”
• Formation of the neural plate is signaled from
the underlying notochord.
• The lens placode is stimulated by cells in the
optic cups (evaginations of proencephalons
which will become retina and optic nerve).
• What causes this induction?
In the whole embryo, research on an
‘organizer’ has been conducted for the last
100 yrs.
Era of the Quest for the Neural Inducer(s)
EVERYTHING induces neural tissue–
High pH, Low pH
Divalent cations
Nucleic acids
Dead tissues
Organic chemicals
Sterols
Methylene Blue!
Transfilter experiments suggest diffusible
molecule(s)
Neural response programmed into dorsal
ectoderm?
Suggests that neural induction is “permissive”
rather than instructive.
So…..what is the neural inducer?
Primary Induction
Fibronectin ECM on ectodermal cells
Presumptive
Ectoderm
Integrins on
mesodermal
cells
Presumptive
Endoderm
Dorsal Blastopore Lip
Presumptive
Mesoderm
Gastrulation in Amphibians
Normal Dorsal
Blastopore Lip
Hans Spemann &
Hilde Mangold
1924
“Organizer” and Mesoderm Induction
Presumptive Ectoderm
Presumptive Endoderm
Xnr protein
concentration
“Morphogen”
Synergistic
1 – tissue capable
of inducing a
stimulus
2 – tissue competent
of responding to
stimulus
Y
Inductive Event:
= cell surface receptors
= intracellular signaling
pathway
O
= target genes
• Spemann found that a split early embryo will
form 2 completely independent ones, as long as
the dorsal lip of the blastopore was included in
each piece.
• This was considered the 1st “center of
differentiation”.
• KEY EXPERIMENTS:
i. Induction of 2nd neural plate when dorsal lip of
blastopore transplanted, which  notochord.
The ectoderm of host that would have formed
skin, instead, formed neural structures. But, it
was discovered that a number of tissues could
induce complex structures at this embryonic
stage.
Artificial Inducers and Non-specific
Activators
• Perhaps the inducers were not instructional,
but permissive in their actions.
Perhaps, the ectoderm is pre-programmed to
form neural tissue and needs little impetus to
do so (?)
MORE RECENT EXPERIMENTS (past decade):
Have focused on identifying genes, which are only
expressed by organizer tissues.
How could a potential inducer be tested?
Detect transcribed mRNA for that inducer in
organizer tissue by in situ hybridization.
The inducer itself gives rise to mesodermal tissues.
Going back to an earlier stage, it was shown that
isolated ectoderm, exposed to the appropriate
inducing signals, could be induced to form
organizer tissue (like blastopore dorsal lip) and
gives rise to mesoderm.
• In recent yrs, scientists discovered polypeptide
growth factors, originally identified as hormone
regulators of the reproductive system, which
could induce ectoderm to form mesoderm.
• These peptides, such as activin and inhibin, are
related to TGF-β.
• These peptides bind to tyr kinase receptors
(which dimerize, autophosphorylate, and start
intracellular signaling cascades).
• Important experimental tests of these peptides
led to greater understanding of the functions of
the neural organizer.
• To determine if native embryonic tissue is
induced by activin (or other PGFs), scientists
inhibited (disabled) the signal by truncating the
receptor  mesodermal inducer was blocked;
embryo developed with only axial structures  in
isolated ectodermal tissue, neural tissue was
induced!
• This suggested that the elusive neural inducers
may block the activity of other molecules, which
might cause the ectoderm to form other tissues
(i.e., mesoderm or epidermis).
• This may explain why so many random
(frequently toxic) substances could cause
ectoderm to become neural.
• They could have disrupted an active process,
which allows the tissue not to become neural, but
to become epidermal instead.
• Again, what happens?
• TGF-β-like PGF (such as BMP-2 and BMP-4) are
produced by ectoderm in the embryo.
• Neural inducers block these PGFs: to induce
neural tissue.
• [In nature, the neural inducer is not a damaged
receptor, but a specific inhibitor].
• Candidates studied: fallistatin, noggin, chordin
are expressed by organizer tissue, block
formation of epidermis and induce formation of
neural tissue.
• However, are these really essential for neural
induction in nature? (Just because they can do it
doesn’t guarantee that they do in the natural
state.
(Planar)
BMP inhibitors
(vertical)
• One way to test: gene knock-out studies in
mice.
• Follistatin mutant  normal development.
Follistatin is not essential in nature.
• Studies are continuing regarding the other 2.
• Note: the nervous system induced by these
substances is rather primitive, suggesting that
the other factors come into play in nature.
General Morphogenesis
Morphogenesis is the formation of ordered form
and structure
-Animals achieve it through changes in:
-Cell division
-Cell shape and size
-Cell death
-Cell migration
-Plants use these except for cell migration
69
General Morphogenesis
Cell division
-The orientation of the mitotic spindle
determines the plane of cell division in
eukaryotic cells
-If spindle is centrally located, two equalsized daughter cells will result
-If spindle is off to one side, two unequal
daughter cells will result
70
General Morphogenesis
Cell shape and size
-In animals, cell differentiation is
accomplished by profound changes in cell size
and shape
-Nerve cells develop long processes (axons)
-Skeletal muscles cells are large and
multinucleated
71
General Morphogenesis
Cell death
-Necrosis is accidental cell death
-Apoptosis is programmed cell death
-Is required for normal development in
animals
-“Death program” pathway consists of:
-Activator, inhibitor and apoptotic
protease
all
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General Morphogenesis
Cell migration
-Cell movement involves both adhesion and loss
of adhesion between cells and substrate
-Cell-to-cell interactions are often mediated
through cadherins
-Cell-to-substrate interactions often involve
complexes between integrins and the
extracellular matrix (ECM)
73
Cadherins and cell adhesion
Early Neural Morphogenesis
• The focus underlying the change in shape and
movements during gastrulation and
neurulation have been an active area of
research.
• Two Known Mechanisms:
1. Coordinated growth; change in cell shape
and cell movement.
2. Differential cell adhesion
1. Cells become more flask-shaped  inward
curvature of neural plate.
• Elongation at the floor of neural plate  elastic
sheet is pulled to form a tube (convergent
extension).
• Rate of growth varies from ant  post to
promote the formation of vessicles and flexures.
• There is still much to be understood regarding
how these processes are mediated at the cellular
level.
• Note: at this stage, the embryo has already
established a degree of polarity: anterior
behaves differently from posterior; dorsal
behaves differently from ventral.
• One way that positional polarity (and
information) can be signaled is through gradients
of substances.
2. The role of differential cell adhesion was first
demonstrated when it was found that amphibian
embryonic tissue dissociated to separate cells at
high pH and would re-aggregate when pH was
returned to normal.
Cadherins: a family of cell-surface glycoproteins,
which appear to play a role in differential cell
adhesion.
Cells containing the same class of cadherins will
interact and aggregate.
These form adhesive junctions and interact with
force-generating, actin-based cytoskeletal
network to assist in mediating movements.
Differential
cell adhesion
• E.g., N-cadherins (neural) vs. E-cadherins
(epidermal).
• These 2 types probably mediate the sorting.
• Cadherins can be expressed at precise times
they are needed during neural development.
• Alternatively, they can be turned off at
opportune times as well.
• E.g., migrating cells of newly-forming neural
crest (or of mesenchyme), which decreases
cadherins during migrating phase. Then,
increased cadherins expression when they
stop migrating and differentiating.
Tripeptide binding sequence
…arginine-glycine-aspartate…
Homophilic binding
Protease
cleaves
Talin;
binding/
uncoupling
with actin
Integrins - Binding to
extracellular matrix
Cadherins – Ca++ dependent
Neural induction is not a switch. It is a gradual process,
as are all determination mechanisms