Molecular control of gastrulation and morphogenesis

1
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.
2
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).
3
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 gt heart cells gt liver
cells is the hierarchical order)
Why?
4
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 gt heart cells gt liver
cells is the hierarchical order)
Why? 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?
5
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.
6
3 types of specialized attachments hold embryo
cells together
Tight junctions are regions where membranes of
adjacent cells actually fuse. They encircle the
whole cell and provide a barrier for leakage
between cells (common in the GI tract to prevent
leakage of food out of the gut). The also prevent
membrane proteins from moving freely from apical
to basal regions.
Desmosomes are spot rivets that weld cells
together. A cytoplasmic plaque is connected by
inter cellular protein filaments, and the plaques
are connected to the intermediate filaments
within the cytoplasm (common in cells that are
stretched a lot such as skin). Gap junctions are
small channels between cells that allow for
intercellular communication. They are found in
smooth muscle and allow signals (small ions) to
spread between cells so that all muscle cells
contract as one.
7
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.
8
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
N-CAMs. 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. N-CAMs have isoforms one N-CAM gene can
generate over 100 different molecules by post
transcriptional and post translational
modification. The most common are 120, 140, and
180 kD.
9
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. Catenins are proteins
that link cadherins to the cytoskeleton. If this
linkage is disturbed, cadherins do not work, and
embryonic development is disrupted (disrupt
catenins in neural tissue brain forms
improperly).
10
Lectins bind heterotypically to sugars on the
cell surface
Lectins are the third group of CAMs. They bind
weakly and heterotypically to oligosaccharides of
many types through the large extracellular
domain. Selectins are lectins that are expressed
in endothelial cells. Glycosyltransferases are
lectin-related CAMs. They are enzymes that
transfer monosaccharides to an oligo saccharide
chain on an adjacent cell. In the absence of
monosaccharides, the enzyme links one cell to
another by binding to the oligosaccharide chain
(cant let go). In the presence of mono
saccharides, the binding is lost. A simple way to
regulate cell adhesion by mono saccharides.
11
Substrate adhesion molecules (SAMs) and the
extracellular matrix (ECM)
Spaces between cells are filled with ECM that
consists of 1. Amorphous ground substance a
gel-like material that absorbs water. 2.
Meshwork of fibers that reinforce the ground
substance. The ECM influences cell migration,
cell shape, cell gene expression, and cell
differentiation. Mesenchymal cells are
surrounded by a diffuse ECM. Epithelial cells
rest on a dense sheet of ECM called the basement
membrane.
The ECM is actively secreted by the cells living
there. What molecules compose the ECM?
12
Glycosaminoglycans and proteoglycans form the
amorphous ground substance
Glycosaminoglycan long unbranched polysaccharide
chains composed of repeating units of
disaccharides. One sugar is an amino sugar
(n-acetyl glucosamine) and the other is a uronic
acid (glucuronic acid). The most abundant
glycosaminoglycans are hyaluronic acid,
chondroitin sulfate, heparin, and heparin
sulfate. Proteoglycans glycosaminoglycans are
covalently linked to core proteins The core
proteins have have many side chains of
glycosaminoglycans. They attract Na and water
and expand to form gels that occupy space between
cells. They also bind and selectively release
growth factors.
Glycosaminoglycan (hyaluronic acid)
Proteoglycan
13
Fibrous glycoproteins make up the meshwork of the
ECM
Proteoglycans are carbohydrate with some protein
(gt50 carbohydrate). Glycoproteins are proteins
with some carbohydrate attached (gt 50
protein). ECM consists of 3 major fibrous
glycoproteins Collagen the most abundant
protein in mammals (gt25 total protein). There
are numerous genes that encode different collagen
molecules. Collagen forms very strong fibers that
are abundant in bones, skin, and connective
tissue. Fibronectin a fibrous protein that has
binding sites for cells and other ECM proteins.
It links cells to the ECM. The RGD sequence of
fibronectin (argenine, glycine, aspartate) binds
to cells avidly. Fibronectin is important for
motility. Laminins abundant in basement
membranes where they promote adhesion to many
types of cells. How do cells stick to fibrous
proteins of the ECM?
14
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.
15
Cell surface proteoglycans also link cells to ECM
These molecules often have an extracellular and
intracellular domain. Proteoglycans consist of
glycosaminoglycans linked to a core protein in
the extracellular domain. These interact with
collagen, fibronectin, and other ECM
molecules. Proteoglycans can be released from
the cell by cleavage at protease sensitive
sites. Proteoglycans and integrins have another
important function They are receptors that
transduce signals from the extracellular matrix
to the nucleus.
Syndecan is a common proteoglycan
16
If CAMs and SAMs were important for gastrulation,
their expression might reflect that fact.
1. They might be expressed selectively on
specific cells of the blastula that are destined
to migrate to selected areas to form organs. 2.
Expression might change as cells left the
surface and ingressed or migrated into the
blastocoel. 3. Blocking expression of CAMs or
SAMs might interfere with the normal process of
gastrulation.
17
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
18
If CAMs and SAMs were important for gastrulation,
their expression might reflect that fact.
1. They might be expressed selectively on
specific cells of the blastula that are destined
to migrate to selected areas to form organs. 2.
Expression might change as cells left the
surface and ingressed or migrated into the
blastocoel (changes in CAM drive movement). 3.
Blocking expression of CAMs or SAMs might
interfere with the normal process of gastrulation.
19
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.
20
CAMs promote formation of cell junctions
CAMs allow cells to attach quickly but not
tightly. They allow reversible changes in
adhesion that aid in migration and
intercalation. Cell junctions (tight junctions,
desmosomes, gap junctions) are long term, tight,
cell-cell attachments. They take longer to form.
CAMs facilitate formation of cell junctions by
holding cells in place while the glue sets.
Compaction cells of the blastula become
polarized and form tight junctions which compacts
the surface. Just before compaction, cells
express high levels of E-cadherin where
blastomeres touch (future tight junction). If
blastulas are placed in medium with antibodies to
E-cadherin to disrupt function, no compaction
occurs. Mature tight junctions are associated
with an area of abundant E-cadherin expression
under the cell surface the zonula adherens
(think of a zipper).
21
If CAMs and SAMs were important for gastrulation,
their expression might reflect that fact.
1. They might be expressed selectively on
specific cells of the blastula that are destined
to migrate to selected areas to form organs. 2.
Expression might change as cells left the
surface and ingressed or migrated into the
blastocoel. 3. Blocking expression of CAMs or
SAMs might interfere with the normal process of
gastrulation.
22
Fibrous ECM components provide contact
guidance to migrating cells during gastrulation
The movement of cells during gastrulation may
also depend upon expression of ECM. ECM allows
migrating cells to attach transiently while
moving over the surface. During gastrulation in
amphibians, cells move into the blastocoel and
migrate over the inside of the roof. If a
portion of the roof is cut out and inverted, no
movement of gastrulating cells occurs here. This
suggests that some CAMs or SAMs may be
missing. What molecules would this be?
23
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.
24
CAMs and SAMs are major regulators of cell
movement during gastrulation / morphogenesis. Do
these molecules also directly influence gene
expression or cell differentiation during
morphogenesis?

• During gastrulation cells migrate and are
  rearranged in the developing
• embryo. Many new cell-cell contacts are
  established when cells reach their
• new positions.
• Do specific CAMs on one cell influence how the
  adjacent cell expresses
• genes or undergoes differentiation?
• 2. Would cells have different gene expression
  or differentiation depending upon the different
  types of ECM that they rested upon?

25
Neural differentiation is triggered by N-CAMs or
N-cadherins
N-cadherins and N-CAMs are expressed on
presumptive neural tissue during and after
gastrulation. What would happen if N-cadherin or
N-CAM expression were experimentally abolished?
26
Neural differentiation is triggered by N-CAMs or
N-cadherins
N-cadherins and N-CAMs are expressed on
presumptive neural tissue during and after
gastrulation. What would happen if N-cadherin or
N-CAM expression were experimentally
abolished? Dominant negative mutants of
N-cadherin a mutant that blocks normal function
dominantly (an N-cadherin molecule with the
binding site for other cadherins cut off).
This mutant was injected into a blastomere of
presumptive neural tissue before gastrulation.
The portion of the brain formed by daughter cells
of this blastomere failed to develop. The other
half of the brain that developed from uninjected
blastomeres was normal. N-cadherin is critical
for early brain development. Does N-cadherin
directly influence neural differentiation?
27
PC12 cells resemble chromaffin cells which can
differentiate into neurons. When they convert to
the neural phenotype they express N-CAM and
N-cadherin 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 N-CAM 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
28
CAMs are necessary for neuronal differentiation
Conversion of PC12 cells to the neuronal
phenotype can be inhibited by adding antibodies
that neutralize either N-CAM or N-cadherin on the
3T3 cells. Thus, adhesion through CAMs is
necessary for neuronal differentiation in these
cells. Neuronal differentiation is also
inhibited by drugs that block calcium channels.
This suggests that intracellular signaling by
calcium is an important requirement for neuronal
differentiation.
29
CAMs and SAMs are major regulators of cell
movement during gastrulation / morphogenesis. Do
these molecules also directly influence gene
expression or cell differentiation during
morphogenesis?

• During gastrulation cells migrate and are
  rearranged in the developing
• embryo. Many new cell-cell contacts are
  established when cells reach their
• new positions.
• Do specific CAMs on one cell influence how the
  adjacent cell expresses
• genes or undergoes differentiation?
• 2. Would cells have different gene expression
  or differentiation depending upon the different
  types of ECM that they rested upon?

30
Collagen directly activates epithelial cells to
form stroma
During development, the cornea (outer eye) and
lens (inner eye) interact. The cornea consists
of outer epithelial cells and inner stromal
cells. Formation of the inner layer depends upon
interaction with the underlying lens which is
covered with collagen. This interaction has been
studied in culture. When corneal cells are grown
on top of the lens (i.e., in contact with
collagen), they differentiate to form a lower
layer of stroma. When corneal cells are cultured
on artificial substrate, no stroma forms. When
the cells are cultured on a collagen substrate, a
normal stroma develops. Interaction with
collagen is critical for corneal differentiation.