Cleavage is the first phase of embryonic development

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Cleavage is the first phase of embryonic
development
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What is cleavage?

• Cleavage is a rapid series of mitotic divisions
  that occur just after fertilization.
• There are two critical reasons why cleavage is so
  important
• Generation of a large number of cells that can
  undergo differentiation and gastrulation to form
  organs.
• 2. Increase in the nucleus / cytoplasmic ratio.
  Eggs need a lot of cytoplasm to support
  embryogenesis. It is difficult or impossible for
  one nucleus to support a huge cytoplasm, and
  oocytes are one of the largest cells that exist.
  One small nucleus just cannot transcribe enough
  RNA to meet the needs of the huge cytoplasm.
• A larger nucleus to cytoplasmic ratio is optimal
  for cell function. Cell
• division occurs rapidly after fertilization to
  correct this problem.

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Cleavage differs from normal mitoses in 2 respects

• Blastomeres do not grow in size between
  successive cell divisions as they do in most
  cells. This leads to a rapid increase in the
  nucleus / cytoplasmic ratio. Cells undergoing
  cleavage have mainly S and M phases of the cell
  cycle (little or no G1 or G2).
• Cleavage occurs very rapidly, and mitosis and
  cytokinesis in each round of cell division are
  complete within an hour. Typical somatic cells
  divide much more slowly (several hours to days)
  and even the fastest cancer cells divide much
  slower than occurs in a zygote during cleavage.
• Cleavage differs in different types of eggs. The
  presence of large amounts of
• yolk alters the cleavage pattern, leading to
  incomplete cleavage that
• characterizes birds and reptiles.
• Two areas of interest
• How does the process of cleavage differ in
  different organisms?
• What mechanisms regulate cleavage?

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Eggs are classified by how much yolk is present

• Isolecithal eggs (iso equal) have a small
  amount of yolk that is equally distributed in the
  cytoplasm (most mammals have isolecithal eggs).
• Mesolecithal eggs (meso middle) have a moderate
  amount of yolk, and the yolk is present mainly in
  the vegetal hemisphere (amphibians have
  mesolecithal eggs).
• Telolecithal eggs (telo end) have a large
  amount of yolk that fills the cytoplasm, except
  for a small area near the animal pole (fish,
  reptiles, and birds).
• Centrolecithal eggs have a lot of yolk that is
  concentrated within the center of the cell
  (insects and arthropods).

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The pattern of cleavage of the zygote depends
upon the pattern of yolk distribution

• Holoblastic cleavage occurs in isolecithal eggs
  (mammals, sea urchins). The entire egg is cleaved
  during each division.
• Meroblastic cleavage occurs when eggs have a lot
  of yolk. The egg does not divide completely at
  each division. Two types
• a. Discoidal cleavage is limited to a small
  disc of cytoplasm at the animal
• pole. All of the yolk filled
  cytoplasm fails to cleave (characteristic of
• telolecithal eggs such as birds).
• b. Superficial cleavage is limited to a
  thin surface area of cytoplasm that
• covers the entire egg. The inside of
  the egg that is filled with yolk fails
• to cleave (centrolecithal eggs such
  as insects).

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Typical cleavage patterns of isolecithal,
mesolecithal, telolecithal and centrolecithal eggs
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Sea urchins have isolecithal eggs and undergo
holoblastic cleavage
Cleavage plane this is the plane in which
cleavage occurs. It is oriented at right angles
to the metaphase plate. In sea urchins, the first
cleavage is meridional. Meridional cleavage runs
from one pole to another (top to bottom), like
the meridian on a globe. The second cleavage is
also meridional. Equatorial cleavage encircles
the zygote like the equator on the globe. The
third cleavage in the sea urchin is equatorial.
This creates an animal and vegetal half.
8
The fourth cleavage is unique. Equal cytokinesis
occurs in the four blastomeres of the animal
pole, giving rise to 8 mesomeres (all the same
size). Unequal cytokinesis occurs in the vegetal
pole. This causes 4 large macromeres and 4 small
micromeres The 5th cleavage is meridional. All
mesomeres divide equally as do the
macromeres. As cleavage progresses, all
blastomeres adhere at the outer surface, but
attachment is lost at the inner surface. The
blastocoel is a cavity formed due to the unequal
adherence of blastomeres.
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Amphibians have mesolecithal eggs and undergo
holoblastic cleavage
Amphibian eggs have a lot of yolk, however, they
are still able to undergo holoblastic
cleavage. The 1st cleavage is meridional, as is
the 2nd. The 3rd cleavage is equatorial. The
cleavage is displaced toward the animal pole due
to the yolk. This results in 4 small animal
blastomeres and 4 large vegetal
blastomeres. Morula (morum mulberry) at the 16
to 32 cell stage the embryo is called a morula
because it looks like a mulberry.
morula
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The blastocoel is displaced to the animal pole in
amphibians
Blastula from the 128 cell stage onward the
amphibian embryo is a blastula. The outer surface
of the amphibian blastula has cells connected by
specialized cell junctions. Tight junctions
create a seal that isolates the outside of an
embryo from the inner layer. Tight junctions
polarize the apical and basal surfaces. The basal
portions of cells start secreting into the
blastocoel. Desmosomes attach the blastomeres
together on the outside. Gap junctions connect
all surface blastomeres.
11
Mammalian eggs have rotational cleavage that is
holoblastic
The mammalian egg is a little slow. It begins to
cleave in the oviduct and continues until it
implants in the wall of the uterus (1 cleavage /
24 hr). Asynchronous cleavage mammalian embryos
are unusual in that they have asynchronous
cleavage. Not all blastomeres divide at the same
time. The first cleavage is meridional, and the
second cleavage is rotational. The 2 blastomeres
divide in different planes (one is equatorial and
one is meridional.
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Mammalian embryos undergo compaction at the 8
cell stage
At first, the blastomeres of mammalian embryos
have a loose arrangement, and touch only at the
basal surfaces. After compaction, blastomeres
adhere tightly, maximizing the area of
contact. During compaction, each blastomere
undergoes polarization. Tight junctions develop
on the outer surface, allowing proteins to
specialize. Cells take up fluids from the uterine
environment and secrete into the blastocoel. Gap
junctions form on the outer cells to aid in
intercellular communication.
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A blastocoel develops as cleavage proceeds to the
32-64 cell stage
After compaction at the 8-16 cell stage, there
are 2 types of blastomeres. Outside blastomeres
are tightly joined and number about 9-14. They
surround 2-7 inside blastomeres that are loosely
joined. Cavitation the outside blastomeres
start to take up fluid from the uterus and pump
it into the center, creating the blastocoel. The
blastocyst is the hallmark of early embryonic
development in mammals.
Inner cell mass this gives rise to the embryo,
and develops from the inside blastomeres
Trophoblast a structure consisting of outside
blastomeres, this contributes to forming the
placenta.
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Embryonic stem cells can be cultured from the
inner cell mass
Cells in the inner cell mass are
undifferentiated, they multiply indefinitely, and
are known as embryonic stem cells. Stem cells are
totipotent they have the potential to form any
tissue. These cells are of great scientific and
medical importance. They can be removed from the
embryo, genes can be introduced into the cells,
and then they can be placed back in the
blastocyst. This is how one constructs transgenic
or knock out mice. The embryonic stem cells are
also used to grow certain types of tissue in
culture. Theoretically, it should be possible to
grow structures such as ears, muscles, nerves,
and skin for transplantation to sick
individuals. Interestingly, if you inject adult,
differentiated cells back into the environment of
the morula or blastula, they become
undifferentiated, and they can redifferentiate to
form many parts of the body.
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Early development and cleavage in humans
How do twins develop?
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Development of monozygotic or identical twins
Monozygotic twins develop from one zygote by
splitting at various stages of development (from
the 2 cell to the blastocyst stage). The stage of
splitting effects the overall structure of the
embryo and extraembryonic membranes. What are
conjoined twins and how do they arise? Where do
fraternal twins come from? Sextuplets?
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Conjoined twins

• are identical twins who develop from a single
  fertilized ovum.
• are always the same sex and race.
• are more often female than male, at a ratio of
  31.
• occur once in 40,000 births but only once in
  200,000 live births.
• may be caused by any number of factors, being
  influenced by genetic
• and environmental conditions.

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Birds, reptiles, and fishes have telolecithal
eggs that completely support embryogenesis they
undergo meroblastic cleavage
In contrast to holoblastic cleavage, where the
entire zygote divides into blastomeres,
meroblastic cleavage leaves a large portion of
the zygote uncleaved. There are 2 types of
meroblastic cleavage, discoidal and superficial.
Discoidal In birds and reptiles, the 1st
cleavage is meridional. It starts at the animal
pole but does not progress far. The 2nd and 3rd
cleavages are also meridional. The 4th cleavage
is equatorial, and it creates a layer of small
cells on top of the huge uncleaved area below
(yolk). Blastoderm when cleavage has progressed
such that there are many blastomeres in the
animal pole, it is a blastoderm. Chicken eggs
have a blastoderm of about 60,000 cells when the
egg is laid.
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The next step in development of telolecithal eggs
is formation of the upper and lower
blastoderm. Epiblast (epi upon) this is the
upper layer and it forms the embryo proper.
Hypoblast (hypo under) this is the bottom
layer that will form the extraembryonic endoderm
that surrounds the yolk. What is the counterpart
in mammals? Blastocoel lies between the 2
layers. Subgerminal space lies between the
hypoblast and yolk.
20
Insects have centrolecithal eggs and undergo
superficial cleavage
Periplasm insect eggs have a superficial area of
cytoplasm that is free from yolk. It surrounds
the entire egg, and cleavage occurs
here. Endoplasm the yolk-rich cytoplasm in the
center of the egg. This area does not undergo
cleavage. Cleavage is a misnomer in insects
because cell division is delayed until after many
rounds of mitosis have been completed.
21
In Drosophila, nuclei start to undergo mitosis
deep within the yolk. No cell division occurs,
and the nuclei slowly migrate out toward to
periphery. A few nuclei are first observed in
the periplasm at the 9 cell division stage. They
quickly become enclosed by a plasma membrane and
become pole cells (primordial germ
cells). Preblastoderm stage (cycles 10 to 13)
Most of the nuclei are present in the periplasm
but no cytokinesis has occurred. Still one big
multinucleated cell! Cellular blastoderm At
about cycle 14, cytokinesis occurs simultaneously
all over the egg. Each nuclei is surrounded by a
plasma membrane to become a cell. This
corresponds to the blastoderm stage of other
embryos.
22
The cell cytoplasm is divided during cytokinesis
Mitosis is followed by cytokinesis, when the
cytoplasm divides equally. A contractile ring
forms beneath the plasma membrane. It contains a
band of actin and myosin filaments. It always
forms in the same place that was occupied by the
metaphase plate. As the actin and myosin
filaments slide by one another, the ring
contracts and pinches the 2 cells apart.
23
Immuno staining of the cortex shows myosin
Cytokinesis is caused by subcortical network of
actin and myosin filaments. These filaments slide
over one another as in muscle, and this causes
contraction and a cleavage furrow to form on the
cell surface. In holoblastic cleavage, the
furrow squeezes around the periphery, like a belt
tightening, to pinch the cell in two. In
meroblastic cleavage, the furrow starts at the
animal pole and progresses into the egg like a
knife. It stops when it reaches the vegetal
portion.
Anti myosin antibodies
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The mitotic spindle determines the orientation of
the cleavage plane
Blastomeres can cleave either equatorially or
meridionally. Cytokinesis usually directly
follows mitosis, except for superficial cleavage.
Cytokinesis invariably occurs in a plane
perpendicular to the axis of the mitotic spindle.
Thus, the spindle orientation controls the
orientation of the contractile ring The proximity
between the egg cortex and the mitotic spindle is
also important for furrow formation. In eggs
where the the outer cortex is displaced from the
spindle (birds and insects), by large amounts of
yolk, the spindle never activates the cleavage
furrow.
How does a blastomere know to divide meridionally
or equatorially?
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Mitotic spindles are oriented with their
axis parallel to the longest available cell
dimension
Mitotic spindles work to keep the cell round in
shape. Experiment It is possible to control how
tightly blastomeres adhere by changing the
concentration of calcium. High calcium
concentrations cause more cell cell attachment.
Low calcium causes minimal attachment. The effect
is likely mediated by adhesion molecules such as
cadherin.
When blastomeres adhere they have a longer axis,
and the mitotic spindle is almost always oriented
parallel to this axis. As the cell becomes more
spherical in low calcium medium, the mitotic
spindle orientation starts to become random.
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How does a cell know when it should divide?
The cyclic activity of a protein dimer controls
the activity of the cell cycle Cyclin dependent
kinase 1 (cdk1) is an enzyme that is always
present in cells. It can phosphorylate other
proteins when it is activated. Cyclins are a
family of proteins that are produced in cyclic
fashion during the cell cycle. Cyclin B is
destroyed shortly after metaphase, but
accumulates slowly thereafter.
M phase promoting factor (MPF) when there is
sufficient cyclin B, it combines with cdk1.
Additional regulatory changes occur such as
phosphorylation of threonine and
dephosphorylation of tyrosine. The active
kinase phosphorylates specific cell proteins that
control mitosis (spindle, nuclear lamins, and
chromosomes). The actual targets of M phase
promoting factor are an area of intense research
interest.
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Timing of cleavage divisions
Normal eukaryotic cells divide slowly, once every
several hours or days. The cell cycle has G1 and
G2 periods. During G1 the cell synthesizes RNA
and other components for cell growth. Cleavage
consists of very rapid successive mitoses. Since
the egg has stored large amounts of RNA and other
material, it does not need G1 or G2. However, as
the number of cells increases, the nucleus /
cytoplasmic ratio also increases. The rate of
cell division slows because the cell now needs to
synthesize its own RNA and grow between
divisions. Thus, G1 and G2 are restored
midblastula transition.
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Midblastula transition is prominent in Drosophila
Nuclei in a Drosophila embryo undergo mitosis
every 9 minutes during the early stage of
development !!! The 1st 10 mitoses are rapid and
synchronous, and only S and M phases exist. After
10 mitoses, the cell cycle increases a little as
RNA must be synthesized before each
division. Midblastula transition After 13
mitoses, the rate slows further, mitoses are
asynchronous, and G1 and G2 reappear. Other
animals, such as mammals and sea urchins,
synthesize RNA throughout cleavage and they have
no midblastula transition.
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How does a blastomere control how fast it divides?
M-phase promoting factor is the critical activity
for initiation of mitosis. During the first 7
mitoses in Drosophila, cyclin B and cdk1
(components of MPF) are constantly present.
During cycles 8-9, cyclin begins to be degraded
after each mitosis. String gene Activates MPF.
This gene is constitutively active during the
first 13 cycles of mitosis. This is because it
is translated from large stores of maternal mRNA.
As the nuclear / cytoplasmic ratio increases,
more string protein is needed to activate MPF in
all of the additional nuclei. Because string
protein synthesis occurs during G1 and G2, the
subsequent mitoses are retarded in each cycle
until normal levels accumulate within the cell.
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What does the product of the string gene do?
The string protein acts as a phosphatase to
remove a phosphate from tyrosine on cdk1. This is
important for activation of cdk1 and allows MPF
activity to initiate mitosis. Similar proteins
are important in human cells.