Cleavage is the first phase of embryonic development

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:

1. 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.
 Cleavage differs from normal mitoses in 2 respects

1. 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).
2. 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:

1. How does the process of cleavage differ in different organisms?
2. What mechanisms regulate cleavage?
 Eggs are classified by how much yolk is present

1. Isolecithal eggs (iso = equal) have a small amount of yolk that is equally
distributed in the cytoplasm (most mammals have isolecithal eggs).
2. Mesolecithal eggs (meso = middle) have a moderate amount of yolk, and
the yolk is present mainly in the vegetal hemisphere (amphibians have
mesolecithal eggs).
3. 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).
4. Centrolecithal eggs have a lot of yolk that is concentrated within the
center of the cell (insects and arthropods).
 The pattern of cleavage of the zygote depends upon
the pattern of yolk distribution

1. Holoblastic cleavage: occurs in isolecithal eggs (mammals, sea urchins).

The entire egg is cleaved during each division.
2. 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).

Typical cleavage patterns of isolecithal, mesolecithal,
telolecithal and centrolecithal eggs
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.
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.
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
 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.
 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.
 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.
 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.
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.
Early development and cleavage in humans

How do twins develop?

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?
 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 3:1.
• 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.
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.
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.
 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.
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.
• radial cleavage: holoblastic cleavage that is
typical of deuterostomes and that is characterized
by arrangement of the blastomeres of each upper
tier directly over those of the next lower tier
resulting in radial symmetry around the pole to
pole axis of the embryo
• spiral cleavage: holoblastic cleavage that is
typical of protostomes and that is characterized
by arrangement of the blastomeres of each upper
tier over the cell junctions of the next lower tier
so that the blastomeres spiral around the pole to
pole axis of the embryo
• There are two main types of division pattern that follow on from the two-cell stage.
• The radial cleavage  pattern is the simplest to understand and is shown above.
• This occurs in vertebrates (e.g. mammals like the human) and echinoderms (e.g.
starfish) and hemichordates (e.g. acorn worm) - animals known as deuterostomes.
• The second division occurs also in a vertical plane, but one which is at right angles
to the first division plane and results in 4 cells, more-or-less in the same plane.
• The third division is a horizontal one and gives rise to the 8-cell stage which
consists of two layers of 4 cells each, with the cells of the top layer directly above
the cells of the bottom layer.
• This division is often approximately equal, producing cells of equal size, but in
other cases the top layer of cells may be slightly smaller as the larger vegetal cells
may contain more yolk.
RADIAL CLEAVGE
SPIRAL CLEAVAGE
• In spiral cleavage the divisions, especially from the third upwards, are very unequal.
• The third division creates 4 larger cells called macromeres and 4 smaller cells called
micromeres.
• The macromeres form a layer of 4 cells at the bottom or vegetal pole and the
micromeres are located at the animal pole.
• The macromeres are larger principally because they receive more of the yolk (which
was originally mostly vegetal in position in the egg).
• Even at the 4-cell stage, one of the cells may contain most of the yolk 
and be distinctly larger than the other three.
• The defining feature of spiral cleavage, however, is the fact 
that each successive layer of cells rotates so as to sit in-between the two adjacent cells
of the lower layer, in alternating manner.
• In polychaetes, the first layer of 4 micromeres are rotated approximately 45 degrees
clockwise as viewed from above (from the animal pole), the third layer rotates back 45
degrees anticlockwise.
• Variations exist on these two themes with so-called aberrant
cleavage which is a highly modified form of radial or spiral cleavage
which deviates from the given rules.
• In the annelid worms, polychaetes follow spiral cleavage, but
oligochaetes like the earthworm follow aberrant cleavage which is,
nevertheless, clearly derived from spiral cleavage.
• In the nematodes (round worms), cleavage is so aberrant that it
is virtually impossible to determine whether it is derived from radial or
spiral cleavage.
• However, rotifers ('wheel animalicules', microscope more-or less
worm-like animals with lobes of cilia that appear to rotate like wheels)
exhibit spiral cleavage and as these are relatives of nematodes, it is
reasonable to assume that in nematodes cleavage is aberrant spiral.
• In both radial and spiral cleavage, the early embryo is essentially a
solid ball of cells, called a morula, but already by about the 8-cell
stage a hollow cavity begins to appear in the centre of the morula and
it becomes a hollow ball of cells, called a blastula.
Bilateral cleavage:
• In bilateral cleavage, the blastula can be
cut vertically only along one plane to
get two identical halves, the right and
the left.
• Cleavage activity on one side is
mirrored by activity on the other side.
• In most cases, the plane of bilateral
symmetry is established by the plane of
first cleavage furrow, which is
bilaterally symmetrical. Examples are
found in tunicates, Amphioxus,
amphibians, and higher mammals.
 1. Determinate:
• The fertilized egg forms all the parts of the embryo
by repeated division. Some eggs or ova have, even
before cleavage, different regions earmarked to
form different parts of the embryo. For example, in
the Ascidian eggs the region, which will form the
endoderm is fixed. If this region is dissected out
from a fertilized egg, the embryo formed later will
have no endoderm. Such eggs with predetermined
regions are called mosaic (or determinate) eggs.
• Cleavages in mosaic eggs follow a precise
pattern and each blastomere has its
characteristic position and unalterable fate.
Here cleavage separate different organ
forming regions and are called determinate
or mosaic cleavage. Examples are of
nematodes, annelids, molluses and ascidians,
which show determinate type of cleavage.
 2. Indeterminate:
• In vertebrates the plan of cleavage is less rigid. Here the
fertilized eggs do not have predetermined region. If the region
which normally forms the endoderm removed from a fertilized
sea urchin egg, the embryo formed later will still have the
endoderm. Such eggs are called regulative or indeterminate eggs.
• In these eggs, as there are no predetermined regions and the
cleavages cannot separate such regions, they simply cut the eggs
into segments which have the potential of forming any organ.
This type of cleavage is called indeterminate or regulative
cleavage. Eggs of some groups of invertebrates and of all
vertebrates show indeterminate cleavage.
 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.
 From fertilization to cleavage
• The transition from fertilization to cleavage is caused by
the activation of mitosis promoting factor (MPF).
• Mitosis-promoting factor contains two subunits.
• The large subunit is called cyclin B. It is this component
that shows a periodic behavior, accumulating during S
and then being degraded after the cells have reached M.
• Cyclin B is often encoded by mRNAs stored in the oocyte
cytoplasm, and if the translation of this message is
specifically inhibited, the cell will not enter mitosis.
• The presence of cyclin B depends upon its synthesis and
its degradation. Cyclin B regulates the small subunit of
MPF, the cyclin-dependent kinase.
• This kinase activates mitosis by
phosphorylating several target proteins,
including histones, the nuclear envelope
lamin proteins, and the regulatory subunit
of cytoplasmic myosin.
• This brings about chromatin condensation,
nuclear envelope depolymerization, and the
organization of the mitotic spindle.
• Without cyclin, the cyclin-dependent kinase will not
function.
• The presence of cyclin is controlled by several
proteins that ensure its periodic synthesis and
degradation. In most species studied, the regulators of
cyclin (and thus, of MPF) are stored in the egg
cytoplasm.
• Therefore, the cell cycle is independent of the nuclear
genome for numerous cell divisions.
• These early divisions tend to be rapid and
synchronous. However, as the cytoplasmic
components are used up, the nucleus begins to
synthesize them.
• The embryo now enters the mid-blastula
transition, in which several new phenomena are
added to the biphasic cell divisions of the embryo.
• First, the growth stages (G1 and G2) are added to
the cell cycle, permitting the cells to grow.
• Before this time, the egg cytoplasm was being
divided into smaller and smaller cells, but the total
volume of the organism remained unchanged.
• Xenopus embryos add those phases to the cell
cycle shortly after the twelfth cleavage.
• Drosophila adds G2 during cycle 14 and G1
during cycle 17.
• Second, the synchronicity of cell division is lost,
as different cells synthesize different regulators of
MPF.
• Third, new mRNAs are transcribed.
• Many of these messages encode proteins that will
become necessary for gastrulation.
• If transcription is blocked, cell division will occur
at normal rates and at normal times in many
species, but the embryo will not be able to initiate
gastrulation.
 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
 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?

 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.
 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.
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.
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.
 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.
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.
 Midblastula transition
• The midblastula transition (MBT) refers to a number of
dramatic changes that occur at the midblastula stage in some
embryos and that were first observed in Xenopus.
• The MBT is initiated when nuclear/cytoplasmic ratio reaches
a threshold. As the cytoplasm is divided into more and more
cells, this ratio increases.
• Before MBT
All embryonic cells undergo cell division at the same time.
Zygotic heterochromatin (condensed, hypo-acetylated, H3
methylated).
Only maternal mRNAs are translated.
• MBT Events
Activation of zygotic genes.
Cell cycle lengthened (G1 & G2 added).
Asynchronous cycles of different cells.
Development of cellular motility.
• After MBT
Cell division becomes asynchronous
Embryo begins transcribing its own DNA.
Differential expression of paternal genes begins.
• MBT occurs when a factor in cytoplasm has been
titrated by an increased number of nuclei (high level of
DNA). Increasing DNA levels (via DNA injection or
extra sperm) makes the MBT occur earlier; decreasing
DNA levels (via a haploid Drosophila embryo) makes
the MBT occur later.
• There is activation of transcription when nuclear DNA
(which has increased exponentially during cleavage)
reaches a certain level that titrates out some general
repressor stored in egg cytoplasm. These events are:
The first two events are also seen in the Drosophila embryo
at the blastoderm stage. Not all embryos have a midblastula
transition, however; the sea urchin embryo, for example, has
RNA transcription starting at fertilization, so there is no
dramatic increase in transcription at the blastula stage.