Gastrulation is the first step of morphogenesis
Morphogenesis is the process whereby individual cells undergo complex
movements that generate the organ rudiments. Gastrulation generates the
three basic germ layers from which organs arise.
How do sheets of cells (epithelia) move during gastrulation? 4 methods.
Invagination is the local inward movement of cells from a cavity
Involution is similar, but more dramatic. It is an inward expansion of epithelial
cells around an edge such as the blastpore.
Convergent extension is elongation of an epithelium in one direction while it
shortens in the other direction (stretching taffy). The cells can keep their
relative positions and elongate or they can interdigitate.
Epiboly is spreading movement of an epithelium to a deeper or thinner layer.
How do individual cells move during gastrulation?
4 basic types of cell movement lead to the changes in epithelial sheets that
characterize gastrulation.
1. Migration is the movement of an individual cell over other cells or a
substrate.
2. Intercalation is wedging of cells between their neighbors. Lateral
intercalation involves lateral movements of cells in the same layer between
one another = convergent extension. Radial intercalation involves wedging of
2 different layers. This process often leads to epiboly, the surface area of the
epithelium increases while the thickness decreases.
3. Ingression is the movement of individual cells from an epithelium into an
embryonic cavity.
4. Shape changes are coordinated changes in cell shape that cause an
epithelium to invaginate, buckle or undergo convergent extension.
Embryonic cells are broadly classed as epithelial or mesechymal
Epithelial cells are well-differentiated. They compose skin and line the body
cavities (ie, the digestive tract). They are polarized. Their apical surface
faces out and their basal surface rests on the basement membrane
(extracellular matrix that supports cells). Epithelial cells are closely
connected with adjacent cells by specialized attachments including tight
junctions, gap junctions, and desmosomes.
Mesenchymal cells are
poorly differentiated and
have the potential to
develop into many
different tissues, including
epithelial cells. They have
a leading edge with
lamellipodia, and a trailing
edge. They are not
connected to adjacent
cells but they are in
contact with the
extracellular matrix.
Gastrulation in sea urchins
Primary mesenchymal cells: these cells
change adhesive properties and the large
micromeres start to migrate into the
blastocoel as free mesenchymal cells
(ingression). Mesenchymal cells are loose
cells that can differentiate into many
different organs.
Archenteron: the primitive gut. The
archenteron is formed in several stages. 1.
the vegetal plate invaginates into the
blastocoel, 2. It elongates by convergent
extension. 3. It hooks up with the front and
is pulled forward, and 4. Involution occurs
with movement of cells around the
blastopore and into the archenteron.
What forces drive the process of
gastrulation?
The primitive groove and pit are the site of gastrulation in birds
Crossection of blastoderm (blastula in birds).
Epiblast is the upper layer of epithelial cells, blastocoel is the space below the
epiblast, and hypoblast is the lower layer of epithelial cells.
Epiblast cells
roll over the
primitive ridge
and involute
into the groove.
The cells lose
contact with
one another and
migrate inwards
by ingression =
Mesoderm.
3 germ layers
are established
Gastrulation in a 16 day old human embryo
• The primitive streak and Henson’s node form just as in birds.
• The cell movements are similar
• Cells roll over the primitive ridge and into the groove
• They ingress individually and move out to form discs with the 3 germ layers
• The 3 layers also move laterally to form the extra embryonic endoderm and
mesoderm even though there is no yolk to digest. This is surprising
because mammalian embryos could gastrulate easily by invagination as sea
urchins (they have a placenta and no yolk).
• Instead, gastrulation appears to recapitulate a pattern established by birdlike ancestors and reptiles.
Molecular control of gastrulation and morphogenesis
Does each cell in the blastula have detailed
instructions in the DNA that tell it exactly
where to go during gastrulation?
If an embryo is disaggregated into individual
cells, each should know exactly where to go
to reform a new embryo.
When this experiment is performed, a degree
of reorganization occurs, but it is not
complete. Embryoids: are slightly similar to
embryos but they lack the real organization.
Conclusions:
1. Genes impart only partial instructions for
assembly of the embryo
2. Like cells all stick together, revealing
distinct adhesive properties.
3. The relative positions of aggregates reflect
the relative positions in the embryo (skin
outside, heart inside).
Cell adhesion is the driving force in gastrulation
When cells from an embryo are disaggregated and recombined, they can be
readily ranked according to their ability to form the central portion.
(chondrocytes > heart cells > liver cells is the hierarchical order)
Differential adhesion hypothesis: the cell type with
maximal adhesiveness (chondrocytes) will form a
core that is surrounded by concentric spheres of
cells with progressively lower adhesiveness.
Cell adhesion can be measured by the ‘pancake
test’. When aggregates of different cell types are
subjected to a flattening force (centrifugation to
induce a centrifugal force), the cells that adhere
most tightly form a ball, while those that adhere
more loosely form a flatter, pancake structure.
Cell adhesion is a major factor that regulates
aggregation of like cells and controls position
during morphogenesis.
What regulates how tightly or loosely cells attach?
Cells adhere by cell junctions, cell adhesion molecules,
or substrate adhesion molecules
Cell junctions: large, complex structures that form slowly but generate very
strong and durable connections (tight junctions, desmosomes, and gap
junctions).
Cell adhesion molecules (CAMs): single molecules that traverse cell
membranes and allow cells to adhere to one another. Adhesions form
quickly, they are selective, but they are relatively weak in comparison to cell
junctions.
Substrate adhesion molecules (SAMs): a group that consists of
extracellular matrix molecules and matched receptors that are expressed
on the cell surface.
CAMs firmly anchor adjacent cells to the cytoskeleton
Cell adhesion molecules (CAMs) are glycoproteins with 3 major domains:
The extracellular domain allows one CAM to bind to another on an adjacent
cell. The binding can be to the same type of cell (homotypic) or to a
different cell type (heterotypic).
The transmembrane domain links the CAM to the plasma membrane
through hydrophobic forces.
The cytoplasmic domain is directly connected to the cytoskeleton by linker
proteins. This anchoring is important to prevent lateral diffusion of
adhesion molecules in the membrane.
Three major
types of CAMs
are immuno
globulin-like
CAMs, cadherins,
and lectins.
Neural cell adhesion molecule is typical of
immunoglobulin (IgG)-like CAMs
N-CAM was one of the first to be discovered.
The extracellular domain has IgG like repeats that are thought to allow
binding to other N-CAMs by interdigitation between loops. Insects have IgG
CAMs but no IgG. Thus, IgGs may have evolved from IgG-like CAMs.
Polysialic acid region (PSA): 3 long
carbohydrate chains with negative
charge are attached to the 5th loop. The
overall charge varies on different NCAMs. Large PSA regions induce a large
negative charge which repels cells
(embryonic cells during gastrulation).
Small PSA regions allow attachment due
to low charges, and these are common
on adult cells.
Cadherins mediate calcium-dependent cell adhesion
Cadherins are the most prevalent CAMs in vertebrates. They are rapidly
degraded by proteases in the absence of Ca++. There are 4 major types:
E cadherins in epithelial cells
P cadherins in placenta
N cadherins in neural tissue
L cadherins in liver
Each associates with its own type.
125 kD transmembrane glycoproteins that
bind homotypically using the first 113 AA
The differences in cadherin expression are
responsible for the differential
adhesiveness seen in disaggregated tissue.
Cells that express more cadherin = tissues
that form a ball in the center of cell
aggregates.
Integrins mediate adhesion to ECM
Integrins are a family of
transmembrane glycoproteins that
are composed of 2 chains, a and b.
There are 40 different types of a
chains and 8 types of b chains that
can combine to form a large number
of different integrin molecules.
The a chain has binding sites for
Ca++ and Mg++ which are needed for
integrins to adhere. The 2 subunits
form the site that binds to the RGD
domain on ECM.
The cytoplasmic tail of integrins is
connected to a linker protein that
connects to the cytoskeleton. A
bridge from ECM to cytoskeleton.
CAM expression during gastrulation is correlated with cell fate
Fate map: it is possible to predict which parts of the blastula will develop into
specific structures after gastrulation.
Expression map of CAMs: it is possible to localize expression of CAMs using
in situ hybridization and immunostaining of the blastula.
Cells with different fates express different CAMs. Cells destined to become
neural tissue express high levels of N-CAM. Cells destined for epidermis
express E-cadherin.
The respective cell adhesion
molecules are expressed
before the cells actively start
to form the adult tissue. This
suggests that CAM expression
is important in fate
determination.
During gastrulation, cells go
where their CAMs lead them
Changes in cell adhesion are important for gastrulation
Gastrulation in the sea urchin is initiated by specific changes in cell
adhesion. One of the first steps is ingression of mesenchymal cells from
the vegetal plate into the blastocoel to form the skeleton of spicules.
The mesenchymal cells lose their adhesion to hyaline and the adjacent
nonmesenchymal blastomeres. They start to increase adhesion to the
basement membrane and material within the blastocoel. These changes can
be measured by isolating specific cells and testing adhesion in culture.
E-cadherin is lost from the ingressing cells due to endocytosis of specific
areas where it was expressed. Levels of b-catenin are also reduced on
these cells.
Fibronectin on the inner roof of the blastocoel
is critical for gastrulation
Immunostaining of the blastocoel shows that fibronectin was expressed in
abundance on the inner roof. Fibronectin binds to integrins on the membrane.
Neutralizing antibody to fibronectin was injected into the blastocoel to test the
role of fibronectin. This aborted gastrulation. Since no epidermal cells could
migrate into the blastopore, many cells accumulated on the surface, forming
deep folds. If an unrelated antibody was injected, there was no inhibition.
Fibronectin binds integrins through an RGD sequence. Similar results were
obtained by injecting the tripeptide RGD. Furthermore, blocking the integrin
receptor with injected antibodies also inhibited gastrulation. Fibronectin is
important for contact guidance of migrating cells during gastrulation.
PC12 cells resemble chromaffin cells which can differentiate into neurons.
When they convert to the neural phenotype they express N-CAM and Ncadherin on their cell surface.
When PC12 cells are grown on cells that do not express N-CAM or N-cadherin
(3T3 cells) they retain the undifferentiated chromaffin phenotype.
If the PC12 cells are grown
on the same cells that have
been transfected with NCAM or N-cadherin genes,
they convert to the
neuronal phenotype.
They form long dendrites
and express neuronal
genes.
Differentiation is
accompanied by opening
of calcium channels
Organogenesis in humans is essentially complete after 6-8 weeks
The 5 week old human embryo has a head with rudiments of eyes, ears, and
brain. It also has a trunk with tail and limb buds.
The structures develop rapidly and the organs are basically formed before 8
weeks. The period of histogenesis, when cells acquire functional
specialization, then begins and continues throughout development = fetus.
Organogenesis involves many of the same cell behaviors that occur during
gastrulation.
Neurulation is of scientific and medical interest
Spina bifida: one of the most common birth defects in humans. Due to defects
in closure of the neural tube and malformations of brain and spinal cord. There
are several forms that differ in severity:
Spina bifida occulta: the mildest form is caused by failure of a vertebrate to
fuse dorsally. It causes no pain or neurological disorder. It is very common
and as many as 10% have this minor defect. The only sign of its presence may
be a dimple or a tuft of hair
normal
spina bifida occulta
What forces cause closure of the neural tube?
Closure of the neural tube is mediated by 3
effects:
1.
2.
3.
Apical constriction of neural plate cells
Rapid anteroposterior extension
Cell crawling
1. Apical constriction: a band of
microfilaments in the apical region of neural
plate cells contracts and causes cells to
assume a wedge shape. This causes the
neural plate to bend and form the hinge
regions.
Apical constriction occurs in the midline
throughout the neural plate. It also occurs in
the lateral folds to form the mediolateral
hinge sites.
Spemann’s famous organizer experiment
Spemann originally thought that the donor dorsal lip of the blastpore
differentiated into neural tube and structures of the embryonic axis.
To prove this, he grafted dorsal lip blastopore tissue from a nonpigmented
donor newt onto another gastrula from a heavily pigmented species. When
the embryos developed he found that the embryonic axis was actually
composed of pigmented recipient tissue.
Only a small strip of donor tissue was present in the middle of the neural
plate.
Spemann’s experiments were confirmed in birds
3 conclusions about the organizer:
1.
2.
3.
The dorsal lip of the blastopore developed according to its own fate. It
gastrulated normally in the new location and formed notochord.
The graft dorsalized the host’s ventral mesoderm (converted gut tissue
to kidneys and somites).
The graft acted as a neural inducer. It caused host ectoderm to form a
neural plate and close to become a neural tube.
Axis induction by disinhibition
Early experiments by Spemann studied how the organizer worked. If the cells
of the dorsal lip were killed or crushed, the activity was still present. Activity
could be mimicked partially by changes in pH or ionic strength.
Disinhibition: the normal pattern for ectodermal
development is neurulation. Ventral ectoderm
escapes from this path by producing an inhibitor of
neurulation. The organizer works by inhibiting the
inhibitor.
Cells cultured from ventral ectoderm differentiate
as neurons only if cultures are sparse.
Bone morhogenetic protein (BMP-4) is released by
ventral ectoderm cells. It stimulates formation of
ventral structures and inhibits neurulation if
injected into embryos.
If BMP-4 is blocked, dorsal structures replace
ventral structures (dominant negative mutants or
innactivate the BMP-4 receptor).
Spemann’s organizer (dorsal lip) inactivates BMP-4
Chordin and noggin: the dorsal lip of the blastopore produces 2 proteins
that antagonize the action of BMP-4. Chordin and noggin each bind to BMP4 and prevent it from binding to its receptor. Chordin and noggin have
strong dorsalizing effects. They cause excessive head development and
block ventral differentiation if injected into the embryo.
What turns on chordin and noggin?
Goosecoid: may be the master
regulator that is similar to Spemann’s
organizer. Expression of goosecoid is
limited to the dorsal lip of the
blastopore. It is turned on at the right
place at the right time.
Goosecoid encodes a transcription
factor that can activate chordin gene
expression. Work is continuing to
explore how this gene functions.
Histogenesis is the process by which cells and tissues
acquire functional specialization
embryo
fetus
Cleavage  gastrulation  organogenesis  histogenesis
(2 weeks)
(1 week)
(4 weeks)
(7 months)
Organogenesis: the formation of organ rudiments to establish the basic
body plan.
Histogenesis: differentiation of cells within the organs to form specialized
tissues. Tissues are composed of cells and extracellular material that
perform a specific function.
Each specific tissue develops mainly from one germ layer.
The spinal cord develops a dorsoventral pattern
All nervous functions depend on development of complex connections
between neurons. These circuits start to develop in the embryo.
Alar plate: as neurons and glial cells accumulate in the mantle, they form
ridges on either side of the neural tube (dorsal or afferent columns). These will
develop into afferent nerves that conduct signals to the brain.
Basal plate: accumulation of cells in the ventral region produces basal or
efferent columns. These will develop into efferent neurons that carry signals to
muscles and organs (motor neurons).
The gray matter in the mantle layer is composed of cell bodies of neurons and
the white matter in the marginal layer is composed of myelinated axons. The
floor and roof plates are composed of glial cells. What causes this pattern?
Sonic hedgehog (shh) induces the dorsoventral pattern
Sonic hedgehog (shh) is a gene that
is expressed in the notochord at first
and later in the floor plate. Mice that
lack shh fail to develop floor plates in
the CNS.
Shh is a secreted glycoprotein that
induces a gradient that is high near
the floor plate and progressively
lower in dorsal regions.
Shh initially induces neural plate
cells to form floor plate. Other
signals from the dorsal ectoderm
direct the dorsal columns and the
roof plate.
Different levels of shh appear to
specify different types of neuron
differentiation.
Different concentrations of shh induce distinct types of neurons
In the developing spinal cord, the floor plate produces shh and creates a
concentration gradient. Motor neurons develop closest to the floor plate, type
2 interneurons are next, followed by type 1 interneurons.
To see whether the shh gradient is really important, isolated cells from neural
tubes were cultured in various concentrations of shh. Cells were stained with
antibody specific for floor plate, motor neurons, or type 1 or 2 interneurons.
Bone morphogenetic protein released by the dorsal ectoderm and roof plate
has an analogous function in generating the dorsal columns
Neural crest cells form a variety of tissues
The fate of neural crest cells has been mapped by a number of techniques
(radioactive tracers, transplants from pigmented species to albinos). There
are 2 patterns of migration in the trunk region:
Dorsolateral path: enter skin and form melanocytes
Ventral path: form afferent neurons of dorsal route ganglia, sympathetic
and parasympathetic ganglia, and adrenal medulla
Neural crest cells help to
form addition structures
in the head such as
bones, connective tissue,
eyes, ears, and teeth.
They also help to form
blood vessels and
connective tissue in the
trunk
How do neural crest cells differentiate into many tissues?
Pluripotency hypothesis: each
neural crest cell has the potential
to form any or all structures.
Inductive signals from adjacent
tissue determines their fate.
Selection hypothesis: the neural
crest contains a mixed population
of predetermined cells. Each cell
has only one possible fate and it
migrates according to this fate.
The real truth may lie between these two extremes.
Clonal analysis: when individual neural crest cells are placed in culture, it
is clear that a single cell can give rise to others that differentiate into
multiple cell types (pigment cells and neurons). Premigratory cells have a
wider potential than do the cells that have already started to migrate. They
may become partially differentiated as they migrate.
What are the molecular signals that control
differentiation of neural crest cells?
Extracellular matrix (ECM): neural
crest cells constantly extend
filopodia to feel the ECM.
Pieces of filter were placed in an
embryo at the dorsolateral or
ventral pathways. After the filters
absorbed ECM, they were
removed to a culture dish and
allowed to interact with neural
crest cells.
Dorsolateral ECM induced
melanocytes and yellow pigment
cells. Ventral ECM induced
neurons. No matrix allowed the
cells to remain undifferentiated.
The otic placode forms the inner ear
In humans, the otic placode appears by the third week on both sides of the
rhombencephalon. The otic vesicle is induced by the underlying neural tissue in
the rhombencephalon.
It invaginates to form the otic pit and then pinches off to form the otic vesicle.
Ganglion cells develop from its medial surface.
Labyrinth:the otic vesicle expands unequally and constricts in other areas to
form a complex shape. The cochlea develops to sense sound and the
semicircular canals form to serve as an organ for balance and body position.
Formation of the eye involves reciprocal interactions
Lens placode: the ectoderm invaginates in
response to signals from the optic cup
underneath. It then pinches off as a lens
vesicle. Cells elongate to fill the vesicle and
start to synthesize crystallins.
Optic cup: forms from the neural tube by
invagination. The opening (choroid fissure)
closes forming a round optic cup, an
extension of the brain.
Optic stalk: connection to the brain that is
filled with neurons to form the optic nerve.
Reciprocal interaction: the lens induces the
formation of the optic cup and the cup
regulates formation of the lens. When the
lens from a species with large eyes is
transplanted, it induces an extra large optic
cup and it also does not grow as large as
usual.
Nasal placodes form the olfactory epithelium
In humans, the nasal placodes appear after the 4th week. They are induced
by the underlying telecephalon.
During the fifth week, nasal swellings appear around the placodes which
now become the two nasal pits. The pits start out far apart, but the 2
maxillary swellings grow large and push the pits to the center. The medial
parts of the nasal swellings fuse to form part of the upper lip.
Olfactory epithelium: the original lining of the nasal pit comes to rest on the
roof of the nasal cavity. It forms the epithelium that senses smell and
connects to neurons in the telencephalon. The nasal cavity becomes
continuous with the pharynx.
What can go wrong?
How does skin develop and differentiate?
Epidermis: the largest derivative of ectoderm forms the outer layer of the skin.
It is an epithelium and cells are connected by desmosomes and tight junctions.
The epidermis consists initially of two layers: periderm is a temporary outer
layer and the germanative layer lies below. The germanative layer is composed
of stem cells that divide actively to produce differentiated progeny. The basal
layer develops from the germanative layer (it contains stem cells in the adult).
Spinous layer forms as cells
are squeezed out of the
basal layer. They become
large and differentiate.
Granular layer starts
making keratin granules
Cornified or horny layer is
composed of dead cells that
are filled with keratin
It takes cells about 7 days
for each cell to journey
through the skin
Hair development in humans
The underlying mesenchyme in skin forms a dermis, a layer of connective
tissue just below the epidermis. The dermis induces a variety of epidermal
structures depending on the species (feathers, hair, scales).
Hair bud: hair formation begins as a
small bud that that penetrates the
dermis. It is induced by a group of
mesenchymal cells and the hair bud
then envelopes these cells to form a hair
papilla.
Hair follicle is the entire organ
Sebaceous glands are induced to form
on the side.
The hair shaft is formed when the inner
cells of the follicle start to differentiate
and produce keratin in the form of hair.
The continued production of keratin by
the cells at the base of the shaft causes
the hair to grow longer.
Mammary gland development in humans
mimicks ancestral patterns
In normal development of humans, only one pair of segments in the ridge
survives, and the remaining precursors degenerate. In some individuals, the
other segments of the mammary ridge fail to degenerate, so that accessory
nipples or breasts are formed.
Primates evolved from small creatures
that nursed multiple offspring. An
extended mammary ridge would have
given human ancestors a survival
advantage. Two breasts are obviously
more adaptive for humans who normally
have only one offspring at a time.
Atavism: the occasional and abnormal
persistance of a primitive adult feature in
an evolved species (multiple mammary
glands).
It is easier to modify an older pattern of
development than to develop a totally
new pattern. This idea is a pervasive in
developmental biology.