You are on page 1of 47

Dr. Bambang Retnoaji, M.Sc.

Some slide adopted and/or modified from Clarkson University :


http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
2017
Perkembangan embrio

1. Fertilization
2. Cleavage
3. Morulation
4. Blastulation
5. Gastrulation
6. Extraembryonic membrane development
7. Organogenesis
Perkembangan embrio
CELL-CELL AND CELL CONTROL OF GENE
MATRIX INTERACTIONS EXPRESSION

EXTRACELLULAR CONTROL OF
MATRIX CELL DIVISION

CELL SIGNALLING STEM CELLS

CELL MOTILITY
AND SHAPE CHANGE APOPTOSIS
EMBRYOLOGY

LIMBS REPRODUCTIVE
SYSTEM
NERVOUS DIGESTIVE
SYSTEM SYSTEM

CARDIOVASCULAR URINARY
SYSTEM RESPIRATORY SYSTEM
SYSTEM

Cell movement, changing cell interactions, cell division, changing


patterns of gene expression are all quite restricted in the adult but
are rampant in the embryo.
Zebrafish development
ZONA PELLUCIDA
The zona pellucid has many different roles
including in oocyte development, protection
during growth and transport, fertilization,
spermatozoa binding, preventing polyspermy,
blastocyst development, and preventing
premature implantation (ectopic pregnancy).
Human Zona Pellucida
An immunochemistry study[8] of human ZP proteins
expression during folliculogenesis within the ovary
primordial follicle identified the presence of ZP1 and
ZP3 in most follicles (93% and 95%, respectively),
with ZP2 only in 32% of these follicles. The ZP
proteins were also detected in the cytoplasm of
cuboidal granulosa cells. This localization suggested
to the authors that these proteins had been present
since oogenesis.
Zona Pellucida Glycoprotein Features
1. A "ZP domain", which is a signature domain comprised
of approximately 260 amino acid (aa) residues.
2. An N-terminal hydrophobic signal peptide sequence.
3. A potential N- and O-linked glycosylation sites.
4. A C-terminal hydrophobic transmembrane-like domain
(TMD)
5. A potential consensus proprotein convertase (furin)
cleavage site (CFCS) upstream of transmembrane-like
domain (TMD).
Pembelahan pada Kodok (Frog cleavage)
Early division of zygote into multiple cells without increase in
Cleavage size, partitions contents

Morula
solid ball of cells

Zygote

Blastocyst
with blastocoele
cavity
Cleavage Dari satu sel menjadi blastosis

http://faculty.yc.edu/lfrolich/humanbiology/HumanB6.gif
http://www.eikonika.net/v2/imghome/newhome_5.jpg
http://faculty.yc.edu/lfrolich/humanbiology/HumanB6.gif
Perkembangan embrio
1.Mitosis dan pertumbuhan body form
Proliferasi sel dengan cara pembelahan penambahan jumlah
sel, konsekuensi : sel ukuran menjadi kecil.
Pertumbuhan dengan: sintesa matrik ekstra seluler (ECM),
Organela intra sel dan matriks.
Penyebaran atau pelebaran jaringan misal sel epithel atau
membran extraembrional .
2. Pengurangan potensi (Restriction) dan Determinasi
totipotent blastomeresrestricted potency
3. Aktivasi gen dan expresi gen (differential expression) gen-gen
fungsional
Perkembangan embrio
4. Diferensiasi Perubahan sel menghasilkan fenotip yang
spesifik
5. Interaksi antar sel atau jaringan (Cell/tissue interaction)
memfasilitasi terjadinya signals untuk perkembangan
organ/jaringan ( e.g.primary induction-notochord).
6. Pergerakan sel (Cell movement) short or long
migrations, e.g. neural crest cells.
Perkembangan embrio
7. Pembentukan Patern intrinsic blueprint directing
development.
8.pelipatan (Folding) e.g.cephalic, caudal and lateral body
folds body form
9. Morphogenesis "beginning of the shape" cumulative
mechanisms transform internal and external body form.
What is cleavage?
Cleavage is the first phase of embryonic development
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.
3. A larger nucleus to cytoplasmic ratio is optimal for cell function.
Cell division occurs rapidly after fertilization to correct this problem.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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?

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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).

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
Pola Pembelahan Embrio
tergantung pada distribusi tipe yolk telur

1. Pembelahan holoblastik (Holoblastic cleavage): terjadi pada telur tipe


isolecithal (mammals, sea urchins). Seluruh bagian telur mengalami
pembelahan

2. Pembelahan Meroblastik (Meroblastic cleavage): terjadi pada telur


tipe Telolecithal (mammals, sea urchins) dan Centro Lechital. Tidak
semua bagian telur mengalami pembelahan. The egg does not divide
completely at each division.

Ada 2 jenis:
a. Pembelahan Diskoidal (Discoidal cleavage) pembelahan hanya
terjadi pada sebagian kecil sitoplasma yaitu pada kutub animalis
(animal pole). Contoh pada burung.

b. Pembelahan superfisial (Superficial cleavage) pembelahan hanya


terjadi pada sebagian kecil sitoplasma yang berada pada bagian tepi
covers the entire egg. Bagian tengah telur tidak mengalami
pembelahan. Contoh pada insects.
Typical cleavage patterns of isolecithal, mesolecithal,
telolecithal and centrolecithal eggs
Fate map
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
Second polar body
15-30 min after
Frog
fertilization
Point of sperm fusion
with egg determines:

1. Position of grey
Ant Dor crescent
2. Major axes of embryo
3. Position of dorsal lip
4. Position of first cleavage
5-10 min after furrow
fertilization

Ven
Pos
Wilhelm Roux,
1885

Pronuclei will fuse 1.5


- 2 hr after fertilization

L R

30
http://www.md.huji.ac.il/~yisraeli/embryology/embryo.html
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.
http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.
http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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?


http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
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.

http://www.clarkson.edu/class/by310/powerpoint/05%20cleavage.ppt
Clarkson University
http://www.clarkson.edu/class/by310/powerpoint
/05%20cleavage.ppt